U.S. patent application number 12/607017 was filed with the patent office on 2011-06-23 for preparation of 7-dehydrocholesterol and/or the biosynthetic intermediates and/or secondary products thereof in transgenic organisms.
This patent application is currently assigned to ORGANOBALANCE GMBH. Invention is credited to Christine Lang, Markus Veen.
Application Number | 20110151509 12/607017 |
Document ID | / |
Family ID | 7713290 |
Filed Date | 2011-06-23 |
United States Patent
Application |
20110151509 |
Kind Code |
A1 |
Lang; Christine ; et
al. |
June 23, 2011 |
PREPARATION OF 7-DEHYDROCHOLESTEROL AND/OR THE BIOSYNTHETIC
INTERMEDIATES AND/OR SECONDARY PRODUCTS THEREOF IN TRANSGENIC
ORGANISMS
Abstract
The present invention relates to a method for preparing
7-dehydrocholesterol and/or the biosynthetic intermediates and/or
secondary products thereof by culturing organisms, in particular
yeasts. Furthermore, the invention relates to the preparation of
the nucleic acid constructs required for preparing the genetically
modified organisms and to said genetically modified organisms, in
particular yeasts, themselves.
Inventors: |
Lang; Christine; (Berlin,
DE) ; Veen; Markus; (Berlin, DE) |
Assignee: |
ORGANOBALANCE GMBH
Berlin
BE
|
Family ID: |
7713290 |
Appl. No.: |
12/607017 |
Filed: |
October 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10503044 |
Jul 29, 2004 |
7608421 |
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PCT/EP2003/000592 |
Jan 22, 2003 |
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12607017 |
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Current U.S.
Class: |
435/52 ;
435/254.2; 435/320.1 |
Current CPC
Class: |
C12N 9/0006 20130101;
C12N 9/0073 20130101; C12P 33/00 20130101; C12Y 201/01041 20130101;
C12N 15/52 20130101; C12N 9/0004 20130101; C12N 9/90 20130101; C12Y
114/19 20130101; C12N 15/81 20130101; C12N 9/1007 20130101; C12N
9/0071 20130101; C12Y 503/03005 20130101; C12Y 114/1307 20130101;
C12Y 101/01088 20130101; C12Y 114/13132 20130101; C12N 9/001
20130101; C12Y 103/01072 20130101 |
Class at
Publication: |
435/52 ;
435/320.1; 435/254.2 |
International
Class: |
C12P 33/00 20060101
C12P033/00; C12N 15/63 20060101 C12N015/63; C12N 1/19 20060101
C12N001/19 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 29, 2002 |
DE |
10203352.8 |
Claims
1. A method for preparing 7-dehydrocholesterol and/or the
biosynthetic intermediates and/or secondary products thereof by
culturing organisms which, compared to the wild type, have an
increased activity of at least one of the activities selected from
the group consisting of .DELTA.8-.DELTA.7-isomerase activity,
.DELTA.5-desaturase activity and .DELTA.24-reductase activity.
2. The method of claim 1, wherein the organisms, compared to the
wild type, have an increased activity of at least two of the
activities selected from the group consisting of
.DELTA.8-.DELTA.7-isomerase activity, .DELTA.5-desaturase activity
and .DELTA.24-reductase activity.
3. The method of claim 1, wherein the organisms, compared to the
wild type, have an increased .DELTA.8-.DELTA.7-isomerase activity,
.DELTA.5-desaturase activity and .DELTA.24-reductase activity.
4. The method of claim 1, wherein the .DELTA.8-.DELTA.7-isomerase
activity is increased by increasing, compared to the wild type,
gene expression of a nucleic acid encoding a
.DELTA.8-.DELTA.7-isomerase.
5. The method of claim 4, wherein gene expression is increased by
introducing into the organism one or more nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase.
6. The method of claim 5, wherein nucleic acids are introduced,
which encode proteins comprising the amino acid sequence SEQ. ID.
NO. 2 or a sequence derived from this sequence by substitution,
insertion or deletion of amino acids, which is at least 30%
identical at the amino acid level with the sequence SEQ. ID. NO. 2,
and having the enzyme property of a
.DELTA.8-.DELTA.7-isomerase.
7. The method of claim 6, which comprises introducing a nucleic
acid comprising the sequence SEQ. ID. NO. 1.
8. The method of claim 1, wherein the .DELTA.5-desaturase activity
is increased by increasing, compared to the wild type, gene
expression of a nucleic acid encoding a .DELTA.5-desaturase.
9. The method of claim 8, wherein gene expression is increased by
introducing into the organism one or more nucleic acids encoding a
.DELTA.5-desaturase.
10. The method of claim 9, wherein nucleic acids are introduced,
which encode proteins comprising the amino acid sequence SEQ. ID.
NO. 4 or a sequence derived from this sequence by substitution,
insertion or deletion of amino acids, which is at least 30%
identical at the amino acid level with the sequence SEQ. ID. NO. 4,
and having the enzyme property of a .DELTA.5-desaturase.
11. The method of claim 10, which comprises introducing a nucleic
acid comprising the sequence SEQ. ID. NO. 3.
12. The method of claim 1, wherein the .DELTA.24-reductase activity
is increased by increasing, compared to the wild type, gene
expression of a nucleic acid encoding a .DELTA.24-reductase.
13. The method of claim 12, wherein gene expression is increased by
introducing into the organism one or more nucleic acids encoding a
.DELTA.24-reductase.
14. The method of claim 13, wherein nucleic acids are introduced,
which encode proteins comprising the amino acid sequence SEQ. ID.
NO. 6 or a sequence derived from this sequence by substitution,
insertion or deletion of amino acids, which is at least 30%
identical at the amino acid level with the sequence SEQ. ID. NO. 6,
and having the enzymic property of a .DELTA.24-reductase.
15. The method of claim 14, which comprises introducing a nucleic
acid comprising the sequence SEQ. ID. NO. 5.
16. The method of claim 1, wherein the organisms, compared to the
wild type, additionally have a reduced activity of at least one of
the activities selected from the group consisting of
C24-methyltransferase activity and .DELTA.22-desaturase
activity.
17. The method of claim 16, wherein the organisms, compared to the
wild type, have a reduced C24-methyltransferase activity and a
reduced .DELTA.22-desaturase activity.
18. The method of claim 16, wherein the C24-methyltransferase
activity is reduced by reducing, compared to the wild type, gene
expression of a nucleic acid encoding a C24-methyltransferase.
19. The method of claim 18, wherein an organism is used, which has
no functional C24-methyltransferase gene.
20. The method of claim 16, wherein the .DELTA.22-desaturase
activity is reduced by reducing, compared to the wild type, gene
expression of a nucleic acid encoding a .DELTA.22-desaturase.
21. The method of claim 20, wherein an organism is used, which has
no functional .DELTA.22-desaturase gene.
22. The method of claim 1, wherein the organisms additionally have,
compared to the wild type, an increased activity of at least one of
the activities selected from the group consisting of
HMG-CoA-reductase activity, lanosterol C14-demethylase activity,
squalene-epoxidase activity, squalene-synthetase activity and
sterol-acyltransferase activity.
23. The method of claim 22, wherein the organisms additionally
have, compared to the wild type, an increased lanosterol
C14-demethylase activity and an increased HMG-CoA-reductase
activity.
24. The method of claim 22, wherein the lanosterol C14-demethylase
activity is increased by increasing, compared to the wild type,
gene expression of a nucleic acid encoding a lanosterol
C14-demethylase.
25. The method of claim 24, wherein gene expression is increased by
introducing into the organism one or more nucleic acids encoding a
lanosterol C14-demethylase.
26. The method of claim 25, wherein nucleic acids are introduced,
which encode proteins comprising the amino acid sequence SEQ. ID.
NO. 8 or a sequence derived from this sequence by substitution,
insertion or deletion of amino acids, which is at least 30%
identical at the amino acid level with the sequence SEQ. ID. NO. 8,
and having the enzymic property of a lanosterol
C14-demethylase.
27. The method of claim 26, which comprises introducing a nucleic
acid comprising the sequence SEQ. ID. NO. 7.
28. The method of claim 22, wherein the HMG-CoA-reductase activity
is increased by increasing, compared to the wild type, gene
expression of a nucleic acid encoding an HMG-CoA reductase.
29. The method of claim 28, wherein gene expression is increased by
introducing into the organism a nucleic acid construct comprising a
nucleic acid which encodes an HMG-CoA reductase and whose
expression in said organism, in comparison with the wild type, is
subject to a reduced regulation.
30. The method of claim 29, wherein the nucleic acid construct
contains a promoter which, in comparison with the wild-type
promoter, is subjected to a reduced regulation in the organism.
31. The method of claim 29, wherein the HMG-CoA reductase-encoding
nucleic acid used is a nucleic acid whose expression in the
organism, in comparison with the orthologous nucleic acid intrinsic
to said organism, is subject to a reduced regulation.
32. The method of claim 31, wherein the HMG-CoA reductase-encoding
nucleic acid used is a nucleic acid which encodes the catalytic
region of said HMG-CoA reductase.
33. The method of claim 32, wherein nucleic acids are introduced,
which encode proteins comprising the amino acid sequence SEQ. ID.
NO. 10 or a sequence derived from this sequence by substitution,
insertion or deletion of amino acids, which is at least 30%
identical at the amino acid level with the sequence SEQ. ID. NO.
10, and having the enzyme property of a HMG-CoA reductase.
34. The method of claim 33, which comprises introducing a nucleic
acid comprising the sequence SEQ. ID. NO. 9.
35. The method of claim 22, wherein an organism is used which,
compared to the wild type, additionally has an increased
squalene-epoxidase activity.
36. The method of claim 35, wherein the squalene-epoxidase activity
is increased by increasing, compared to the wild type, gene
expression of a nucleic acid encoding a squalene epoxidase.
37. The method of claim 36, wherein gene expression is increased by
introducing into the organism one or more nucleic acids encoding a
squalene epoxidase.
38. The method of claim 37, wherein nucleic acids are introduced,
which encode proteins comprising the amino acid sequence SEQ. ID.
NO. 12 or a sequence derived from this sequence by substitution,
insertion or deletion of amino acids, which is at least 30%
identical at the amino acid level with the sequence SEQ. ID. NO.
12, and having the enzyme property of a squalene epoxidase.
39. The method of claim 38, which comprises introducing a nucleic
acid comprising the sequence SEQ. ID. NO. 11.
40. The method of claim 1, wherein the organism used is yeast.
41. The method of claim 1, which comprises harvesting the organism,
after culturing, and then isolating 7-dehydrocholesterol and/or the
biosynthetic intermediates and/or secondary products thereof from
said organism.
42. A nucleic acid construct, comprising at least one nucleic acid
selected from the group consisting of nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase, nucleic acids encoding a
.DELTA.5-desaturase and nucleic acids encoding a
.DELTA.24-reductase, which are functionally linked with one or more
regulatory signals ensuring transcription and translation in
organisms.
43. A nucleic acid construct of claim 42, additionally comprising
at least one nucleic acid selected from the group consisting of
nucleic acids encoding an HMG-CoA reductase, nucleic acids encoding
a lanosterol C14-demethylase, nucleic acids encoding a squalene
epoxidase, nucleic acids encoding a squalene synthetase and nucleic
acids encoding a sterol acyltransferase, which are functionally
linked with one or more regulatory signals ensuring transcription
and translation in organisms.
44. A combination of nucleic acid constructs, which comprises at
least one nucleic acid construct selected from the groups A to C A
nucleic acid construct comprising nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase, which are functionally linked to one
or more regulatory signals ensuring transcription and translation
in organisms, B nucleic acid construct comprising nucleic acids
encoding a .DELTA.5-desaturase, which are functionally linked to
one or more regulatory signals ensuring transcription and
translation in organisms and C nucleic acid construct comprising
nucleic acids encoding a .DELTA.24-reductase, which are
functionally linked to one or more regulatory signals ensuring
transcription and translation in organisms, and at least one
nucleic acid construct selected from the groups D to H D nucleic
acid construct comprising nucleic acids encoding an HMG-CoA
reductase, which are functionally linked to one or more regulatory
signals ensuring transcription and translation in organisms, E
nucleic acid construct comprising nucleic acids encoding a
lanosterol C14-demethylase, which are functionally linked to one or
more regulatory signals ensuring transcription and translation in
organisms, F nucleic acid construct comprising nucleic acids
encoding a squalene epoxidase, which are functionally linked to one
or more regulatory signals ensuring transcription and translation
in organisms, G nucleic acid construct comprising nucleic acids
encoding a squalene synthetase, which are functionally linked to
one or more regulatory signals ensuring transcription and
translation in organisms, H nucleic acid construct comprising
nucleic acids encoding a sterol acyltransferase, which are
functionally linked to one or more regulatory signals ensuring
transcription and translation in organisms.
45. The nucleic acid construct of claim 42, wherein the regulatory
signals comprise one or more promoters and one or more terminators,
which ensure transcription and translation in organisms.
46. The nucleic acid construct of claim 42, wherein regulatory
signals ensuring transcription and translation in yeasts are
used.
47. A genetically modified organism, wherein the genetic
modification increases at least one of the activities selected from
the group consisting of .DELTA.8-.DELTA.7-isomerase activity,
.DELTA.5-desaturase activity and .DELTA.24-reductase activity,
compared to a wild type.
48. The genetically modified organism of claim 47, wherein the
increase of at least one of the activities is caused by an increase
in gene expression of at least one nucleic acid selected from the
group consisting of nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase, nucleic acids encoding a
.DELTA.5-desaturase and nucleic acids encoding a
.DELTA.24-reductase, compared to the wild type.
49. The genetically modified organism of claim 48, which contains
two or more nucleic acids encoding a .DELTA.8-.DELTA.7-isomerase
and/or two or more nucleic acids encoding a .DELTA.5-desaturase
and/or two or more nucleic acids encoding a
.DELTA.24-reductase.
50. The genetically modified organism of claim 47, wherein the
genetic modification additionally reduces at least one of the
activities selected from the group consisting of
C24-methyltransferase activity and .DELTA.22-desaturase activity
compared to a wild type.
51. The genetically modified organism of claim 50, wherein the
reduction in at least one of the activities is caused by a
reduction in gene expression of at least one nucleic acid selected
from the group consisting of nucleic acids encoding a
C24-methyltransferase and nucleic acids encoding a
.DELTA.22-desaturase, compared to the wild type.
52. The genetically modified organism of claim 51, which has no
functional C24-methyltransferase gene and/or .DELTA.22-desaturase
gene.
53. The genetically modified organism of claim 47, wherein the
genetic modification additionally increases at least one of the
activities selected from the group consisting of HMG-CoA-reductase
activity, lanosterol C14-demethylase activity, squalene-epoxidase
activity, squalene-synthetase activity and sterol-acyltransferase
activity, compared to a wild type.
54. The genetically modified organism of claim 53, wherein the
increase in at least one of the activities is caused by an increase
in gene expression of at least one nucleic acid selected from the
group consisting of nucleic acids encoding an HMG-CoA-reductase
activity, nucleic acids encoding a lanosterol C14-demethylase,
nucleic acids encoding a squalene epoxidase, nucleic acids encoding
a squalene synthetase and nucleic acids encoding a sterol
acyltransferase, compared to the wild type.
55. The genetically modified organism of claim 54, which contains
two or more nucleic acids encoding an HMG-CoA reductase and/or two
or more nucleic acids encoding a lanosterol C14-demethylase and/or
two or more nucleic acids encoding a squalene epoxidase and/or two
or more nucleic acids encoding a squalene synthetase and/or two or
more nucleic acids encoding a sterol acyltransferase.
56. The genetically modified organism of claim 47, which, compared
to the wild type, has an increased content of 7-dehydrocholesterol
and/or the biosynthetic intermediates and/or secondary products
thereof.
57. The genetically modified organism of claim 47, wherein the
organism used is yeast.
58. The use of a genetically modified organism as claimed in claim
47 for preparing 7-dehydrocholesterol and/or the biosynthetic
intermediates and/or secondary products thereof.
59. The combination of nucleic acid constructs of claim 44, wherein
the regulatory signals comprise one or more promoters and one or
more terminators, which ensure transcription and translation in
organisms.
60. The combination of nucleic acid constructs of claim 44, wherein
regulatory signals ensuring transcription and translation in yeasts
are used.
Description
[0001] The present invention relates to a method for preparing
7-dehydrocholesterol and/or the biosynthetic intermediates and/or
secondary products thereof by culturing organisms, in particular
yeasts. Furthermore, the invention relates to the preparation of
the nucleic acid constructs required for preparing the genetically
modified organisms and to said genetically modified organisms, in
particular yeasts, themselves.
[0002] 7-Dehydrocholesterol, also referred to as
cholesta-5,7-dienol or provitamin D3, its biosynthetic
intermediates of the sterol metabolism, such as, for example,
zymosterol, farnesol, geraniol, squalene, lanosterol,
cholesta-5,7,24-trienol and cholesta-5,7,22,24-tetraenol and its
biosynthetic secondary products of the sterol metabolism, such as
vitamin D.sub.3 and cholesterol, are compounds of high economic
value.
[0003] 7-Dehydrocholesterol is economically important especially
for obtaining vitamin D.sub.3 from 7-dehydrocholesterol via UV
irradiation.
[0004] Squalene is used as building block for the synthesis of
terpenes. It is used in hydrogenated form as squalane in
dermatology and cosmetics and also in various derivatives as an
ingredient of skin and haircare products.
[0005] Furthermore, sterols such as zymosterol and lanosterol can
be utilized economically, lanosterol being pivotal as crude and
synthesis material for the chemical synthesis of saponins and
steroid hormones. Due to its good skin penetration and spreading
properties, lanosterol serves as emulsifier and active substance in
skin creams.
[0006] An economic method for preparing 7-dehydrocholesterol and/or
the biosynthetic intermediates and/or secondary products thereof is
therefore of great importance.
[0007] Particularly economic methods are biotechnological methods
utilizing organisms which have been optimized by genetic
modification and which produce 7-dehydrocholesterol and/or the
biosynthetic intermediates and/or secondary products thereof.
[0008] While the sterol metabolism in bacteria, fungi, yeasts and
some insects essentially goes from zymosterol via fecosterol,
episterol, ergosta-5,7-dienol and
ergosta-5,7,22,24-tetraen-3.beta.-ol to ergosterol (provitamin
D.sub.2), the sterol metabolism in mammals essentially goes from
zymosterol via cholesta-7,24-dienol, lathosterol to
7-dehydrocholesterol (provitamin D.sub.3).
[0009] 7-Dehydrocholesterol (provitamin D.sub.3) is converted to
cholesterol by 7-dehydrocholesterol reductase and cholesterol is
converted to steroid hormones, corticoids and bile acids, such as
progesterone, testosterone, estradiol, aldosterone, cortisone and
cholate.
[0010] Some genes of the 7-dehydrocholesterol metabolism in mammals
are known and have been cloned, such as, for example,
nucleic acids encoding a human .DELTA.8-.DELTA.7-isomerase (also
referred to as emopamil-binding protein (EBP)), ACCESSION
NM.sub.--006579, and a murine .DELTA.8-.DELTA.7-isomerase
(Braverman, N. et al., (1999): Mutations in the gene encoding
3.beta.-hydroxysteroid-.DELTA.8,.DELTA.7-isomerase cause X-linked
dominant Conradi-Hunermann syndrome. Nat. Genet. 22(3), 291-294),
nucleic acids encoding a human .DELTA.5-desaturase (also referred
to as sterol C5-desaturase), ACCESSION AB016247 and a murine
.DELTA.5-desaturase (Nishi, S. et al., (2000): cDNA cloning of the
mammalian sterol C5-desaturase and the expression in yeast mutant.
Biochim. Biophys. Acta 1490(1-2), 106-108), nucleic acids encoding
a human .DELTA.24-reductase (also referred to as
24-dehydrocholesterol reductase (DHCR24)), ACCESSION
NM.sub.--014762 and a murine .DELTA.24-reductase (Waterham, H. R.
et al. (2001): Mutations in the 3.beta.-hydroxysterol
.DELTA.24-reductase gene cause desmosterolosis, an autosomal
recessive disorder of cholesterol biosynthesis. Am. J. Hum. Genet.
69(4), 685-694) and nucleic acids encoding a human sterol
acyltransferase (Chang, C. C. et al., Molecular cloning and
functional expression of human acyl-coenzyme A:cholesterol
acyltransferase cDNA in mutant Chinese hamster ovary cells, J.
Biol. Chem. 1993, Oct. 5; 268(28):20747-55) and a murine sterol
acyltransferase (Uelmen, P. J.: Tissue-specific expression and
cholesterol regulation of acylcoenzyme A:cholesterol
acyltransferase (ACAT) in mice. Molecular cloning of mouse ACAT
cDNA, chromosomal localization, and regulation of ACAT in vivo and
in vitro, J. Biol. Chem. 1995 Nov. 3; 270(44):26192-201).
[0011] The genes of the ergosterol metabolism in yeast are
essentially known and have been cloned, such as, for example,
nucleic acids encoding a .DELTA.8-.DELTA.7-isomerase (ERG2)
(Ashman, W. H. et al. (1991): Cloning and disruption of the yeast
C-8 sterol isomerase gene. Lipids. August; 26(8):628-32), Nucleic
acids encoding a .DELTA.5-desaturase (ERG3) (Arthington, B. A. et
al. (1991): Cloning, disruption and sequence of the gene encoding
yeast C-5 sterol desaturase. Gene. June 15; 102(1):39-44), nucleic
acids encoding a .DELTA.24-reductase (ERG 4) (Lai, M. H. et al.,
(1994): The identification of a gene family in the Saccharomyces
cerevisiae ergosterol biosynthesis pathway. Gene. March 11;
140(1):41-9), nucleic acids encoding an HMG-CoA reductase (HMG)
(Bason M. E. et al, (1988) Structural and functional conservation
between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A
reductases, the rate-limiting enzyme of sterol biosynthesis. Mol
Cell Biol 8:3797-3808, nucleic acids encoding a truncated HMG-CoA
reductase (t-HMG) (Polakowski T, Stahl U, Lang C. (1998)
Overexpression of a cytosolic hydroxymethylglutaryl-CoA reductase
leads to squalene accumulation in yeast. Appl Microbiol Biotechnol.
January; 49(1):66-71, nucleic acids encoding a lanosterol
C14-demethylase (ERG11) (Kalb V F, Loper J C, Dey C R, Woods C W,
Sutter T R (1986) Isolation of a cytochrome P-450 structural gene
from Saccharomyces cerevisiae. Gene 45(3):237-45, nucleic acids
encoding a squalene synthetase (ERG9) (Jennings, S. M., (1991):
Molecular cloning and characterization of the yeast gene for
squalene synthetase. Proc Natl Acad Sci USA. July 15;
88(14):6038-42), nucleic acids encoding a sterol acyltransferase
(SAT1) and (SAT2) (Yang, H.: Sterol esterification in yeast: a
two-gene process. Science. 1996 May 31; 272(5266):1353-6) and a
further sterol acyltransferase (J. Biol. Chem. 1996, Sep. 27;
271(39):24157-63), nucleic acids encoding a squalene epoxidase
(ERG1) (Jandrositz, A., et al (1991) The gene encoding squalene
epoxidase from Saccharomyces cerevisiae: cloning and
characterization. Gene 107:155-160), nucleic acids encoding a
C24-methyltransferase (ERG6) (Hardwick, K. G. et al.: SED6 is
identical to ERG6, and encodes a putative methyltransferase
required for ergosterol synthesis. Yeast. February; 10(2):265-9)
and nucleic acids encoding a .DELTA.22-desaturase (ERG5) (Skaggs,
B. A. et al: Cloning and characterization of the Saccharomyces
cerevisiae C-22 sterol desaturase gene, encoding a second
cytochrome P-450 involved in ergosterol biosynthesis, Gene. 1996
Feb. 22; 169(1):105-9).
[0012] Furthermore, methods are known whose aim is an increase in
the content of specific intermediates and final products of the
sterol metabolism in yeasts and fungi.
[0013] EP 486 290 discloses that the content of squalene and other
specific sterols such as, for example, zymosterol, in yeasts can be
increased by increasing the rate of expression of HMG-CoA reductase
and simultaneously interrupting the metabolic pathway of zymosterol
C24-methyltransferase (ERG6) and ergosta-5,7,24(28)-trienol
22-dehydrogenase (ERG9).
[0014] From T. Polakowski, Molekularbiologische Beeinflussung des
Ergosterolstoffwechsels der Hefe Saccharomyces cerevisiae
[Influencing the ergosterol metabolism of the yeast Saccharomyces
cerevisiae by molecular biological means], Shaker Verlag Aachen,
1999, pages 59 to 66, it is known that increasing the rate of
expression of HMG-CoA reductase alone, without interrupting the
downstream metabolic flow as in EP 486 290, merely leads to a
slight increase in the content of early sterols and of squalene,
while the content of later sterols such as ergosterol does not
change substantially and, in the case of ergosterol, even
tendentially decreases.
[0015] WO 99/16886 describes a method for preparing ergosterol in
yeasts which overexpress a combination of genes tHMG, ERG9, SAT1
and ERG1.
[0016] Tainaka et al., J, Ferment. Bioeng. 1995, 79, 64-66, further
describe that overexpression of ERG11 (lanosterol C14-demethylase)
leads to accumulation of 4,4-dimethylzymosterol but not of
ergosterol. Compared to the wild type, the transformant showed an
increase in the zymosterol content by a factor of from 1.1 to 1.47,
depending on fermentation conditions.
[0017] Avruch et al, Can. J. Biochem 1976, 54(7), 657-665 and Xu et
al, Biochem. Biophys. Res. Commun. 1988, 155(1), 509-517 describe
that it is possible to detect, apart from zymosterol, also traces
of cholesterol by specifically inhibiting C24-methyltransferase and
also by a mutation in the gene locus erg6 in S. cerevisiae.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 illustrates the vector denoted pUG6-tHMG.
[0019] FIG. 2 illustrates the vector denoted pUG6-ERG1.
[0020] FIG. 3 illustrates the vector denoted pUG6-ERG11.
[0021] FIG. 4 illustrates the vectors denoted pFlat3 and
pFlat1.
[0022] FIG. 5a illustrates the vectors denoted pFlat1-EBP and
pFlat3-EBP.
[0023] FIG. 5b illustrates the vector denoted pFlat3-SC5D.
[0024] FIG. 5c illustrates the vector denoted pFlat1-Ebp.
[0025] FIG. 5d illustrates the vector denoted pFlat4-D24R.
[0026] FIG. 6 illustrates the vector denoted pFlat4.
[0027] FIG. 7 illustrates the vector denoted pFlat4-ERG4.
[0028] It is an object of the present invention to provide a method
for preparing 7-dehydrocholesterol and/or the biosynthetic
intermediates and/or secondary products thereof, which method has
advantageous properties such as a higher product yield.
[0029] We have found that this object is achieved by a method for
preparing 7-dehydrocholesterol and/or the biosynthetic
intermediates and/or secondary products thereof, in which organisms
are cultured which have, compared to the wild type, an increased
activity of at least one of the activities selected from the group
consisting of .DELTA.8-.DELTA.7-isomerase activity,
.DELTA.5-desaturase activity and .DELTA.24-reductase activity.
[0030] An increased activity compared to the wild type means, in
the case of the starting organism not having said activity, that
said activity is caused. In the case of the starting organism
already having said activity, an increased activity compared to the
wild type means an activity increased by a percentage.
[0031] .DELTA.8-.DELTA.7-Isomerase activity means the enzyme
activity of a .DELTA.8-.DELTA.7-isomerase, also referred to as
.DELTA.8-.DELTA.7-sterol isomerase.
[0032] A .DELTA.8-.DELTA.7-isomerase means a protein which has the
enzyme activity of converting zymosterol to
cholesta-7,24-dienol.
[0033] Accordingly, .DELTA.8-.DELTA.7-isomerase activity means the
amount of zymosterol converted or the amount of
cholesta-7,24-dienol formed by the protein
.DELTA.8-.DELTA.7-isomerase in a particular time.
[0034] In the case of an increased .DELTA.8-.DELTA.7-isomerase
activity compared to the wild type, thus the amount of zymosterol
converted or the amount of cholesta-7,24-dienol formed by the
protein .DELTA.8-.DELTA.7-isomerase in a particular time is
increased in comparison with the wild type.
[0035] This increase in .DELTA.8-.DELTA.7-isomerase activity is
preferably at least 5%, further preferably at least 20%, further
preferably at least 50%, further preferably at least 100%, more
preferably at least 300%, still more preferably at least 500%, in
particular at least 600%, of the .DELTA.8-.DELTA.7-isomerase
activity of the wild type.
[0036] .DELTA.5-Desaturase activity means the enzyme activity of a
.DELTA.5-desaturase, also referred to as lathosterol 5-desaturase
or sterol C5-desaturase.
[0037] A .DELTA.5-desaturase means a protein which has the enzyme
activity of converting cholesta-7,24-dienol to
cholesta-5,7,24-trienol.
[0038] Accordingly, .DELTA.5-desaturase activity means the amount
of cholesta-7,24-dienol converted or the amount of
cholesta-5,7,24-trienol formed by the protein .DELTA.5-desaturase
in a particular time.
[0039] In the case of an increased .DELTA.5-desaturase activity
compared to the wild type, thus the amount of cholesta-7,24-dienol
converted or the amount of cholesta-5,7,24-trienol formed by the
protein .DELTA.5-desaturase in a particular time is increased in
comparison with the wild type.
[0040] This increase in .DELTA.5-desaturase activity is preferably
at least 5%, further preferably at least 20%, further preferably at
least 50%, further preferably at least 100%, more preferably at
least 300%, still more preferably at least 500%, in particular at
least 600%, of the .DELTA.5-desaturase activity of the wild
type.
[0041] .DELTA.24-Reductase activity means the enzyme activity of a
.DELTA.24-reductase, also referred to as 24-dehydrocholesterol
reductase.
[0042] A .DELTA.24-reductase means a protein which has the enzyme
activity of converting the double bond between C24 and C25 of
cholesterol compounds to a single bond, for example converting
cholesta-5,7,24-trienol to 7-dehydrocholesterol or zymosterol to
lathosterol or cholesta-7,24-dienol to cholesta-7-enol.
[0043] Accordingly, .DELTA.24-reductase activity means preferably
the amount of cholesta-5,7,24-trienol converted or the amount of
7-dehydrocholesterol formed by the protein .DELTA.24-reductase in a
particular time.
[0044] In the case of an increased .DELTA.24-reductase activity
compared to the wild type, thus the amount of
cholesta-5,7,24-trienol converted or the amount of
7-dehydrocholesterol formed by the protein .DELTA.24-reductase in a
particular time is increased in comparison with the wild type.
[0045] This increase in .DELTA.24-reductase activity is preferably
at least 5%, further preferably at least 20%, further preferably at
least 50%, further preferably at least 100%, more preferably at
least 300%, still more preferably at least 500%, in particular at
least 600%, of the .DELTA.24-reductase activity of the wild
type.
[0046] A wild type means the corresponding not genetically modified
starting organism. Preferably and, in particular in those cases in
which the organism or the wild type cannot be classified
unambiguously, wild type means a reference organism for increasing
the .DELTA.8-.DELTA.7-isomerase activity, increasing the
.DELTA.5-desaturase activity, increasing the .DELTA.24-reductase
activity, reducing the C24-methyltransferase activity described
below, reducing the .DELTA.22-desaturase activity described below,
increasing the HMG-CoA-reductase activity described below,
increasing the lanosterol C14-demethylase activity described below,
increasing the squalene-epoxidase activity described below,
increasing the squalene-synthetase activity described below and
increasing the sterol-acyltransferase activity described below and
also for increasing the content of 7-dehydrocholesterol and/or of
the biosynthetic intermediates and/or secondary products thereof.
This reference organism is preferably the yeast strain
Saccharomyces cerevisiae AH22.
[0047] In the method of the invention, organisms are cultured
which, compared to the wild type, have an increased activity of at
least one of the activities selected from the group consisting of
.DELTA.8-.DELTA.7-isomerase activity, .DELTA.5-desaturase activity
and .DELTA.24-reductase activity.
[0048] In a preferred embodiment, organisms are cultured which,
compared to the wild type, have an increased
.DELTA.8-.DELTA.7-isomerase activity, .DELTA.5-desaturase activity
or .DELTA.24-reductase activity.
[0049] In a particularly preferred embodiment of the method of the
invention, the organisms have, compared to the wild type, an
increased activity of at least two of the activities selected from
the group consisting of .DELTA.8-.DELTA.7-isomerase activity,
.DELTA.5-desaturase activity and .DELTA.24-reductase activity.
[0050] Particularly preferred combinations are
.DELTA.8-.DELTA.7-isomerase activity and .DELTA.5-desaturase
activity, increased in comparison to the wild type,
.DELTA.8-.DELTA.7-isomerase activity and .DELTA.24-reductase
activity, increased in comparison to the wild type, and
.DELTA.5-desaturase activity and .DELTA.24-reductase activity,
increased in comparison with the wild type.
[0051] In a very particularly preferred embodiment of the method of
the invention, the organisms have, compared to the wild type, an
increased .DELTA.8-.DELTA.7-isomerase activity, .DELTA.5-desaturase
activity and .DELTA.24-reductase activity.
[0052] The .DELTA.8-.DELTA.7-isomerase activity,
.DELTA.5-desaturase activity and .DELTA.24-reductase activity and
also the HMG-CoA-reductase activity, lanosterol C14-demethylase
activity, squalene-epoxidase activity, squalene-synthetase activity
and sterol-acyltransferase activity, which are described below, may
be increased independently of one another in various ways, for
example by eliminating inhibiting regulatory mechanisms at the
expression and protein level or by increasing, compared to the wild
type, gene expression of the corresponding nucleic acids, i.e.
nucleic acids encoding a .DELTA.8-.DELTA.7-isomerase,
.DELTA.5-desaturase, .DELTA.24-reductase, HMG-CoA reductase,
lanosterol C14-demethylase, squalene epoxidase, squalene synthetase
or sterol acyltransferase.
[0053] Likewise, gene expression of the corresponding nucleic acid
may be increased compared to the wild type in various ways, for
example by inducing the appropriate genes by activators, i.e. by
inducing the .DELTA.8-.DELTA.7-isomerase gene, the
.DELTA.5-desaturase gene, the .DELTA.24-reductase gene, the
HMG-CoA-reductase gene, the lanosterol C14-demethylase gene, the
squalene-epoxidase gene, the squalene-synthetase gene or the
sterol-acyltransferase gene by activators, or by introducing one or
more gene copies of the appropriate nucleic acids, i.e. by
introducing one or more nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase, .DELTA.5-desaturase,
.DELTA.24-reductase, HMG-CoA reductase, lanosterol C14-demethylase,
squalene epoxidase, squalene synthetase or sterol acyltransferase
into the organism.
[0054] Increasing the gene expression of a nucleic acid encoding a
.DELTA.8-.DELTA.7-isomerase, .DELTA.5-desaturase,
.DELTA.24-reductase, HMG-CoA reductase, lanosterol C14-demethylase,
squalene epoxidase, squalene synthetase or sterol acyltransferase
means according to the invention also manipulation of the
expression of endogenous .DELTA.8-.DELTA.7-isomerases,
.DELTA.5-desaturases, .DELTA.24-reductases, HMG-CoA reductases,
lanosterol C14-demethylases, squalene epoxidases, squalene
synthetases or sterol acyltransferases, which are intrinsic to the
organism, in particular to the yeasts.
[0055] This may be achieved, for example, by modifying the promoter
DNA sequence of genes coding for .DELTA.8-.DELTA.7-isomerase,
.DELTA.5-desaturase, .DELTA.24-reductase, HMG-CoA reductase,
lanosterol C14-demethylase, squalene epoxidase, squalene synthetase
or sterol acyltransferase. Such a modification which causes an
increased rate of expression of the relevant gene may be carried
out, for example, by deleting or inserting DNA sequences.
[0056] As described above, it is possible to modify expression of
the endogenous .DELTA.8-.DELTA.7-isomerase, .DELTA.5-desaturase,
.DELTA.24-reductase, HMG-CoA reductase, lanosterol C14-demethylase,
squalene epoxidase, squalene synthetase or sterol acyltransferase
by applying exogenous stimuli. This may be carried out using
particular physiological conditions, i.e. by applying foreign
substances.
[0057] Furthermore, a modified or increased expression of
endogenous .DELTA.8-.DELTA.7-isomerase, .DELTA.5-desaturase,
.DELTA.24-reductase, HMG-CoA reductase, lanosterol C14-demethylase,
squalene epoxidase, squalene synthetase or sterol acyltransferase
genes may be achieved by interaction of a regulatory protein which
is not present in the untransformed organism with the promoter of
said genes.
[0058] A regulator of this type may be a chimeric protein which
consists of a DNA-binding domain and a transcriptional activator
domain, as described, for example, in WO 96/06166.
[0059] In a preferred embodiment, the .DELTA.8-.DELTA.7-isomerase
activity is increased compared to the wild type by increasing the
gene expression of a nucleic acid encoding a
.DELTA.8-.DELTA.7-isomerase.
[0060] In a further preferred embodiment, gene expression of a
nucleic acid encoding a .DELTA.8-.DELTA.7-isomerase is increased by
introducing into the organism one or more nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase.
[0061] For this purpose, it is possible to use in principle any
.DELTA.8-.DELTA.7-isomerase gene, i.e. any nucleic acid encoding a
.DELTA.8-.DELTA.7-isomerase.
[0062] In the case of genomic .DELTA.8-.DELTA.7-isomerase nucleic
acid sequences from eukaryotic sources, which contain introns,
preferably already processed nucleic acid sequences such as the
corresponding cDNAs are to be used, if the host organism is unable
to or cannot be enabled to express the appropriate
.DELTA.8-.DELTA.7-isomerase.
[0063] Examples of .DELTA.8-.DELTA.7-isomerase genes are nucleic
acids encoding a murine .DELTA.8-.DELTA.7-isomerase (nucleic acid:
Seq. ID. No. 1, protein: Seq. ID. No. 2) or a human
.DELTA.8-.DELTA.7-isomerase (nucleic acid: Seq. ID. No. 3, protein:
Seq. ID. No. 4) (Braverman, N. et al., (1999): Mutations in the
gene encoding
[0064] 3.beta.-hydroxysteroid-.DELTA.8,.DELTA.7-isomerase cause
X-linked dominant Conradi-Hunermann syndrome, Nat. Genet. 22(3),
291-294), or else nucleic acids encoding proteins which have the
activity of a .DELTA.8-.DELTA.7-isomerase, for example due to a
broad substrate specificity, such as, for example, nucleic acids
encoding a C8-isomerase Saccharomyces cerevisiae (ERG2) (Nucleic
acid: Seq. ID. No. 5, protein: Seq. ID. No. 6) (Ashman, W. H. et
al. (1991): Cloning and disruption of the yeast C-8 sterol
isomerase gene. Lipids. August; 26(8):628-32).
[0065] In this preferred embodiment, thus at least one further
.DELTA.8-.DELTA.7-isomerase gene is present in the transgenic
organisms of the invention, compared to the wild type.
[0066] The number of .DELTA.8-.DELTA.7-isomerase genes in the
transgenic organisms of the invention is at least two, preferably
more than two, particularly preferably more than three and very
particularly preferably more than five.
[0067] All of the nucleic acids mentioned in the description may
be, for example, an RNA sequence, DNA sequence or cDNA
sequence.
[0068] Preferred .DELTA.8-.DELTA.7-isomerase genes are nucleic
acids encoding proteins which have a high substrate specificity for
zymosterol. Therefore, preference is given in particular to
.DELTA.8-.DELTA.7-isomerase genes and to the corresponding
.DELTA.8-.DELTA.7-isomerases of mammals and to the functional
equivalents thereof.
[0069] Accordingly, preference is given to using in the
above-described method nucleic acids which encode proteins
comprising the amino acid sequence SEQ. ID. NO. 2 or a sequence
derived from this sequence by substitution, insertion or deletion
of amino acids, which is at least 30%, preferably at least 50%,
more preferably at least 70%, still more preferably at least 90%,
most preferably at least 95%, identical at the amino acid level
with the sequence SEQ. ID. NO. 2, and having the enzyme property of
a .DELTA.8-.DELTA.7-isomerase.
[0070] The sequence SEQ. ID. NO. 2 represents the amino acid
sequence of Mus musculus .DELTA.8-.DELTA.7-isomerase.
[0071] Further examples of .DELTA.8-.DELTA.7-isomerases and
.DELTA.8-.DELTA.7-isomerase genes can readily be found, for
example, for various organisms whose genomic sequence is known by
comparing the homology of the amino acid sequences or the
corresponding backtranslated nucleic acid sequences from databases
with the SeQ ID. NO. 2.
[0072] The Homo sapiens .DELTA.8-.DELTA.7-isomerase (Seq. ID. No.
4), for example, is 74% identical to the Mus musculus
.DELTA.8-.DELTA.7-isomerase (Seq. ID. No. 2).
[0073] Further examples of .DELTA.8-.DELTA.7-isomerases and
.DELTA.8-.DELTA.7-isomerase genes can furthermore readily be found
for various organisms whose genomic sequence is unknown, for
example starting from the sequence SEQ. ID. No. 1, by hybridization
techniques and PCR techniques in a manner known per se.
[0074] The term "substitution" means in the description the
replacement of one or more amino acids by one or more amino acids.
Preference is given to carrying out "conservative" replacements in
which the replacing amino acid has a similar property to that of
the original amino acid, for example replacement of Glu by Asp, Gln
by Asn, Val by Ile, Leu by Ile, Ser by Thr.
[0075] A deletion is the replacement of an amino acid by a direct
bond. Preferred positions for deletions are the polypeptide termini
and the linkages between the individual protein domains.
[0076] Insertions are introductions of amino acids into the
polypeptide chain, with a direct bond formally being replaced by
one or more amino acids.
[0077] Identity between two proteins means identity of the amino
acids over the in each case entire length of the protein, in
particular the identity which is calculated by comparison with the
aid of the Lasergene software from DNASTAR Inc., Madison, Wis.
(USA), using the Clustal method (Higgins D G, Sharp P M. Fast and
sensitive multiple sequence alignments on a microcomputer. Comput
Appl. Biosci. 1989 April; 5(2):151-1) and setting the following
parameters: TABLE-US-00001 Multiple alignment parameter: Gap
penalty 10 Gap length penalty 10 Pairwise alignment parameter:
K-tuple 1 Gap penalty 3 Window 5 Diagonals saved 5
[0078] Accordingly, a protein which is at least 30% identical at
the amino acid level with the sequence SEQ. ID. NO. 2 means a
protein which is at least 30% identical when comparing its sequence
with the sequence SEQ. ID. NO. 2, in particular according to the
above program algorithm with the above set of parameters.
[0079] In a further, particularly preferred embodiment, the
.DELTA.8-.DELTA.7-isomerase activity is increased by introducing
into organisms nucleic acids which encode proteins comprising the
amino acid sequence of Mus musculus .DELTA.8-.DELTA.7-isomerase
(SEQ. ID. NO. 2).
[0080] Suitable nucleic acid sequences can be obtained, for
example, by backtranslating the polypeptide sequence according to
the genetic code.
[0081] Preference is given to using for this those codons which are
frequently used according to the organism-specific codon usage.
Said codon usage can readily be determined on the basis of computer
analyses of other known genes of the organisms in question.
[0082] If the protein is to be expressed in yeast, for example, it
is often advantageous to use the codon usage of yeast for
backtranslation.
[0083] In a particularly preferred embodiment, a nucleic acid
comprising the sequence SEQ. ID. NO. 1 is introduced into the
organism.
[0084] The sequence SEQ. ID. NO. 1 represents the Mus musculus cDNA
which encodes the .DELTA.8-.DELTA.7-isomerase of the sequence SEQ
ID NO. 2.
[0085] Furthermore, all of the .DELTA.8-.DELTA.7-isomerase genes
mentioned above can be prepared in a manner known per se by
chemical synthesis from the nucleotide building blocks, for example
by fragment condensation of individual overlapping complementary
nucleic acid building blocks of the double helix. The chemical
synthesis of oligonucleotides may be carried out, for example, in a
known manner according to the phosphoramidite method (Voet, Voet,
2nd edition, Wiley Press New York, pages 896-897). Annealing of
synthetic oligonucleotides and filling-in of gaps with the aid of
the Klenow fragment of DNA polymerase and the ligation reactions
and also general cloning methods are described in Sambrook et al.
(1989), Molecular cloning: A laboratory manual, Cold Spring Harbor
Laboratory Press.
[0086] In a preferred embodiment, the .DELTA.5-desaturase activity
is increased compared to the wild type by increasing the gene
expression of a nucleic acid encoding a .DELTA.5-desaturase.
[0087] In a further preferred embodiment, gene expression of a
nucleic acid encoding a .DELTA.5-desaturase is increased by
introducing into the organism one or more nucleic acids encoding a
.DELTA.5-desaturase.
[0088] For this purpose, it is possible to use in principle any
.DELTA.5-desaturase gene, i.e. any nucleic acid encoding a
.DELTA.5-desaturase.
[0089] In the case of genomic .DELTA.5-desaturase nucleic acid
sequences from eukaryotic sources, which contain introns,
preferably already processed nucleic acid sequences such as the
corresponding cDNAs are to be used, if the host organism is unable
to or cannot be enabled to express the appropriate
.DELTA.5-desaturase.
[0090] Examples of .DELTA.5-desaturase genes are nucleic acids
encoding a murine .DELTA.5-desaturase (nucleic acid: Seq. ID. No.
7, protein: Seq. ID. No. 8) or a human .DELTA.5-desaturase (nucleic
acid: Seq. ID. No. 9, protein: Seq. ID. No. 10) (Nishi, S. et al.,
(2000): cDNA cloning of the mammalian sterol C5-desaturase and the
expression in yeast mutant. Biochim. Biophys. Acta, 1490, (1-2),
106-108), or else nucleic acids encoding proteins which have the
activity of a .DELTA.5-desaturase, for example due to a broad
substrate specificity, such as, for example, nucleic acids encoding
a Saccharomyces cerevisiae C5-desaturase (ERG3) (nucleic acid: Seq.
ID. No. 11, protein: Seq. ID. No. 12), (Arthington, B. A. et al.
(1991): Cloning, disruption and sequence of the gene encoding yeast
C-5 sterol desaturase. Gene. June 15; 102(1):39-44).
[0091] In this preferred embodiment, thus at least one further
.DELTA.5-desaturase gene is present in the transgenic organisms of
the invention, compared to the wild type.
[0092] The number of .DELTA.5-desaturase genes in the transgenic
organisms of the invention is at least two, preferably more than
two, particularly preferably more than three and very particularly
preferably more than five.
[0093] Preferred .DELTA.5-desaturase genes are nucleic acids
encoding proteins which have a high substrate specificity for
cholesta-7,24-dienol. Therefore, preference is given in particular
to .DELTA.5-desaturase genes and to the corresponding
.DELTA.5-desaturases of mammals and to the functional equivalents
thereof.
[0094] Accordingly, preference is given to using in the
above-described method nucleic acids which encode proteins
comprising the amino acid sequence SEQ. ID. NO. 8 or a sequence
derived from this sequence by substitution, insertion or deletion
of amino acids, which is at least 30%, preferably at least 50%,
more preferably at least 70%, still more preferably at least 90%,
most preferably at least 95%, identical at the amino acid level
with the sequence SEQ. ID. NO. 8, and having the enzyme property of
a .DELTA.5-desaturase.
[0095] The sequence SEQ. ID. NO. 8 represents the amino acid
sequence of Mus musculus .DELTA.5-desaturase.
[0096] Further examples of .DELTA.5-desaturase and
.DELTA.5-desaturase genes can readily be found, for example, for
various organisms whose genomic sequence is known by comparing the
homology of the amino acid sequences or the corresponding
backtranslated nucleic acid sequences from databases with the SeQ
ID. NO. 2.
[0097] The Homo sapiens .DELTA.5-desaturase (Seq. ID. No. 10), for
example, is 84% identical to Mus musculus .DELTA.5-desaturase (Seq.
ID. No. 8).
[0098] Further examples of .DELTA.5-desaturases and
.DELTA.5-desaturase genes can furthermore readily be found for
various organisms whose genomic sequence is unknown, for example
starting from the sequence SEQ. ID. No. 7, by hybridization
techniques and PCR techniques in a manner known per se.
[0099] Accordingly, a protein which is at least 30% identical at
the amino acid level with the sequence SEQ. ID. NO. 8 means a
protein which is at least 30% identical when comparing its sequence
with the sequence SEQ. ID. NO. 8, in particular according to the
above program algorithm with the above set of parameters.
[0100] In a further, particularly preferred embodiment, the
.DELTA.5-desaturase activity is increased by introducing into
organisms nucleic acids which encode proteins comprising the amino
acid sequence of Mus musculus .DELTA.5-desaturase (SEQ. ID. NO.
8).
[0101] Suitable nucleic acid sequences can be obtained, for
example, by backtranslating the polypeptide sequence according to
the genetic code.
[0102] Preference is given to using for this those codons which are
frequently used according to the organism-specific codon usage.
Said codon usage can readily be determined on the basis of computer
analyses of other known genes of the organisms in question.
[0103] If the protein is to be expressed in yeast, for example, it
is often advantageous to use the codon usage of yeast for
backtranslation.
[0104] In a particularly preferred embodiment, a nucleic acid
comprising the sequence SEQ. ID. NO. 7 is introduced into the
organism.
[0105] The sequence SEQ. ID. NO. 7 represents the Mus musculus cDNA
which encodes the .DELTA.5-desaturase of the sequence SEQ ID NO.
8.
[0106] Furthermore, all of the .DELTA.5-desaturase genes mentioned
above can be prepared in a manner known per se by chemical
synthesis from the nucleotide building blocks, for example by
fragment condensation of individual overlapping complementary
nucleic acid building blocks of the double helix. The chemical
synthesis of oligonucleotides may be carried out, for example, in a
known manner according to the phosphoramidite method (Voet, Voet,
2nd edition, Wiley Press New York, pages 896-897). Annealing of
synthetic oligonucleotides and filling-in of gaps with the aid of
the Klenow fragment of DNA polymerase and the ligation reactions
and also general cloning methods are described in Sambrook et al.
(1989), Molecular cloning: A laboratory manual, Cold Spring Harbor
Laboratory Press.
[0107] In a preferred embodiment, the .DELTA.24-reductase activity
is increased compared to the wild type by increasing the gene
expression of a nucleic acid encoding a .DELTA.24-reductase.
[0108] In a further preferred embodiment, gene expression of a
nucleic acid encoding a .DELTA.24-reductase is increased by
introducing into the organism one or more nucleic acids encoding a
.DELTA.24-reductase.
[0109] For this purpose, it is possible to use in principle any
.DELTA.24-reductase gene, i.e. any nucleic acid encoding a
.DELTA.24-reductase.
[0110] In the case of genomic .DELTA.24-reductase nucleic acid
sequences from eukaryotic sources, which contain introns,
preferably already processed nucleic acid sequences such as the
corresponding cDNAs are to be used, if the host organism is unable
to or cannot be enabled to express the appropriate
.DELTA.24-reductase.
[0111] Examples of .DELTA.24-reductase genes are nucleic acids
encoding a murine .DELTA.24-reductase (nucleic acid: Seq. ID. No.
13, protein: Seq. ID. No. 14) or a human .DELTA.24-reductase
(nucleic acid: Seq. ID. No. 15, protein: Seq. ID. No. 16)
(Waterham, H. R. et al.: Mutations in the 3.beta.-Hydroxysterol
.DELTA.24-Reductase Gene Cause Desmosterolosis, an Autosomal
Recessive Disorder of Cholesterol Biosynthesis, Am. J. Hum. Genet.
69 (4), 685-694 (2001)), or else nucleic acids encoding proteins
which have the activity of a .DELTA.24-reductase, for example due
to a broad substrate specificity, such as, for example, nucleic
acids encoding a Saccharomyces cerevisiae .DELTA.24-reductase
(ERG4) (nucleic acid: Seq. ID. No. 17, protein: Seq. ID. No. 18)
(Lai, M. H. et al., (1994): The identification of a gene family in
the Saccharomyces cerevisiae ergosterol biosynthesis pathway. Gene.
March 11; 140(1):41-9).
[0112] In this preferred embodiment, thus at least one further
.DELTA.24-reductase gene is present in the transgenic organisms of
the invention, compared to the wild type.
[0113] The number of .DELTA.24-reductase genes in the transgenic
organisms of the invention is at least two, preferably more than
two, particularly preferably more than three and very particularly
preferably more than five.
[0114] Preferred .DELTA.24-reductase genes are nucleic acids
encoding proteins which have a high substrate specificity for
cholesta-5,7,24-trienol. Therefore, preference is given in
particular to .DELTA.24-reductase genes and to the corresponding
.DELTA.24-reductase of mammals and to the functional equivalents
thereof.
[0115] Accordingly, preference is given to using in the
above-described method nucleic acids which encode proteins
comprising the amino acid sequence SEQ. ID. NO. 14 or a sequence
derived from this sequence by substitution, insertion or deletion
of amino acids, which is at least 30%, preferably at least 50%,
more preferably at least 70%, still more preferably at least 90%,
most preferably at least 95%, identical at the amino acid level
with the sequence SEQ. ID. NO. 14, and having the enzyme property
of a .DELTA.24-reductase.
[0116] The sequence SEQ. ID. NO. 14 represents the amino acid
sequence of Mus musculus .DELTA.24-reductase.
[0117] Further examples of .DELTA.24-reductases and
.DELTA.24-reductase genes can readily be found, for example, for
various organisms whose genomic sequence is known by comparing the
homology of the amino acid sequences or the corresponding
backtranslated nucleic acid sequences from databases with the SeQ
ID. NO. 14.
[0118] The Homo sapiens .DELTA.24-reductase (Seq. ID. No. 16), for
example, is 96% identical to Mus musculus .DELTA.24-reductase (Seq.
ID. No. 14).
[0119] Further examples of .DELTA.24-reductases and
.DELTA.24-reductase genes can furthermore readily be found for
various organisms whose genomic sequence is unknown, for example
starting from the sequence SEQ. ID. No. 13, by hybridization
techniques and PCR techniques in a manner known per se.
[0120] Accordingly, a protein which is at least 30% identical at
the amino acid level with the sequence SEQ. ID. NO. 14 means a
protein which is at least 30% identical when comparing its sequence
with the sequence SEQ. ID. NO. 14, in particular according to the
above program algorithm with the above set of parameters.
[0121] In a further, particularly preferred embodiment, the
.DELTA.24-reductase activity is increased by introducing into
organisms nucleic acids which encode proteins comprising the amino
acid sequence of Mus musculus .DELTA.24-reductase (SEQ. ID. NO.
14).
[0122] Suitable nucleic acid sequences can be obtained, for
example, by backtranslating the polypeptide sequence according to
the genetic code.
[0123] Preference is given to using for this those codons which are
frequently used according to the organism-specific codon usage.
Said codon usage can readily be determined on the basis of computer
analyses of other known genes of the organisms in question.
[0124] If the protein is to be expressed in yeast, for example, it
is often advantageous to use the codon usage of yeast for
backtranslation.
[0125] In a particularly preferred embodiment, a nucleic acid
comprising the sequence SEQ. ID. NO. 13 is introduced into the
organism.
[0126] The sequence SEQ. ID. NO. 13 represents the Mus musculus
genomic DNA which encodes the .DELTA.24-reductase of the sequence
SEQ ID NO. 14.
[0127] Furthermore, all of the .DELTA.24-reductase genes mentioned
above can be prepared in a manner known per se by chemical
synthesis from the nucleotide building blocks, for example by
fragment condensation of individual overlapping complementary
nucleic acid building blocks of the double helix. The chemical
synthesis of oligonucleotides may be carried out, for example, in a
known manner according to the phosphoramidite method (Voet, Voet,
2nd edition, Wiley Press New York, pages 896-897). Annealing of
synthetic oligonucleotides and filling-in of gaps with the aid of
the Klenow fragment of DNA polymerase and the ligation reactions
and also general cloning methods are described in Sambrook et al.
(1989), Molecular cloning: A laboratory manual, Cold Spring Harbor
Laboratory Press.
[0128] In a further preferred embodiment of the method of the
invention, organisms are cultured which have, compared to the wild
type, a reduced activity of at least one of the activities selected
from the group consisting of C24-methyltransferase activity and
.DELTA.22-desaturase activity in addition to the above-described
genetic modifications.
[0129] In a further particularly preferred embodiment, organisms
are cultured which have, compared to the wild type, a reduced
C24-methyltransferase activity and a reduced .DELTA.22-desaturase
activity in addition to the above-described genetic
modifications.
[0130] A reduced activity means both the reduced and the complete
elimination of said activity. Reducing an activity therefore also
comprises a reduction in the amount of the corresponding protein in
the organism up to a complete absence of the corresponding protein,
which can be tested, for example, via missing detectability of the
corresponding enzyme activity or missing immunological
detectability of the corresponding proteins.
[0131] A C24-methyltransferase activity means the enzyme activity
of a C24-methyltransferase.
[0132] A C24-methyltransferase means a protein which has the enzyme
activity of converting zymosterol to fecosterol
(ergosta-8,24(28)dienol).
[0133] Accordingly, C24-methyltransferase activity means the amount
of zymosterol converted or the amount of fecosterol formed by the
protein C24-methyltransferase in a particular time.
[0134] In the case of a reduced C24-methyltransferase activity
compared to the wild type, thus the amount of zymosterol converted
or the amount of fecosterol formed by the protein
C24-methyltransferase in a particular time is reduced in comparison
with the wild type.
[0135] The C24-methyltransferase activity is reduced preferably to
at least 90%, further preferably to at least 70%, further
preferably to at least 50%, further preferably to at least 30%,
more preferably to at least 10%, still more preferably to at least
5%, in particular to 0%, of the C24-methyltransferase activity of
the wild type. Therefore, particular preference is given to
eliminating the C24-methyltransferase activity in the organism.
[0136] .DELTA.22-desaturase activity means the enzyme activity of a
.DELTA.22-desaturase.
[0137] A .DELTA.22-desaturase means a protein which has the enzyme
activity of converting ergosta-5,7-dienol to
ergosta-5,7,22,24-tetraen-3.beta.-ol.
[0138] Accordingly, .DELTA.22-desaturase activity means the amount
of ergosta-5,7-dienol converted or the amount of
ergosta-5,7,22,24-tetraen-3.beta.-ol formed by the protein
.DELTA.22-desaturase in a particular time.
[0139] In the case of a reduced .DELTA.22-desaturase activity
compared to the wild type, thus the amount of ergosta-5,7-dienol
converted or the amount of ergosta-5,7,22,24-tetraen-3.beta.-ol
formed by the protein .DELTA.22-desaturase in a particular time is
reduced in comparison with the wild type.
[0140] The .DELTA.22-desaturase activity is reduced preferably to
at least 90%, further preferably to at least 70%, further
preferably to at least 50%, further preferably to at least 30%,
more preferably to at least 10%, still more preferably to at least
5%, in particular to 0%, of the .DELTA.22-desaturase activity of
the wild type. Therefore, particular preference is given to
eliminating the .DELTA.22-desaturase activity in the organism.
[0141] The reduction in C24-methyltransferase activity and/or
.DELTA.22-desaturase activity may be carried out independently of
one another by different cell-biological mechanisms, for example by
inhibiting the corresponding activity at the protein level, for
example by adding inhibitors of the corresponding enzymes or by
reducing gene expression of the corresponding nucleic acids
encoding a C24-methyltransferase or .DELTA.22-desaturase, compared
to the wild type.
[0142] In a particularly preferred embodiment of the method of the
invention, the C24-methyltransferase activity and/or the
.DELTA.22-desaturase activity are reduced compared to the wild type
by reducing the gene expression of the corresponding nucleic acids
encoding a C24-methyltransferase or .DELTA.22-desaturase.
[0143] Likewise, gene expression of the nucleic acids encoding a
C24-methyltransferase or .DELTA.22-desaturase may be reduced
compared to the wild type in various ways, for example by
a) introducing nucleic acid sequences which can be transcribed to
an antisense nucleic acid sequence which is capable of inhibiting
the C24-methyltransferase activity and/or .DELTA.22-desaturase
activity, for example by inhibiting the expression of endogenous
C24-methyltransferase and/or .DELTA.22-desaturase activity, b)
overexpression of homologous C24-methyltransferase nucleic acid
sequences and/or .DELTA.22-desaturase nucleic acid sequences, which
leads to cosuppression, c) introducing nonsense mutations into the
endogene by means of introducing RNA/DNA oligonucleotides into the
organism, d) introducing specific DNA-binding factors, for example
factors of the zinc finger transcription factor type, which cause a
reduction in gene expression or e) generating knockout mutants, for
example with the aid of T-DNA mutagenesis or homologous
recombination.
[0144] In a preferred embodiment of the method of the invention,
gene expression of the nucleic acids encoding a
C24-methyltransferase or .DELTA.22-desaturase is reduced by
generating knockout mutants, particularly preferably by homologous
recombination.
[0145] Therefore, preference is given to using an organism which
has no functional C24-methyltransferase gene and/or
.DELTA.22-desaturase gene.
[0146] In a preferred embodiment, knockout mutants are generated,
i.e. the C24-methyltransferase-gene target locus and/or the
.DELTA.22-desaturase-gene target locus are deleted with
simultaneous integration of an expression cassette containing at
least one of the nucleic acids described above or below, which
encode a protein whose activity is increased in comparison with the
wild type, by homologous recombination.
[0147] For this purpose, it is possible to use nucleic acid
constructs which, in addition to the expression cassettes described
below which contain promoter, coding sequence and, where
appropriate, terminator and in addition to a selection marker at
the 3' and 5' ends, described below, contain nucleic acid sequences
which are identical to nucleic acid sequences at the start and the
end of the gene to be deleted.
[0148] After selection by recombinase systems, the selection marker
may preferably be removed again, for example via loxP signals at
the 3' and 5' ends of the selection marker, using a Cre recombinase
(Cre-loxP system).
[0149] In the preferred organism Saccharomyces cerevisiae, the
C24-methyltransferase gene is the gene ERG6 (SEQ. ID. NO. 19). SEQ.
ID. NO. 20 represents the corresponding Saccharomyces cerevisiae
C24-methyltransferase (Hardwick, K. G. et al.: SED6 is identical to
ERG6, and encodes a putative methyltransferase required for
ergosterol synthesis. Yeast. February; 10(2):265-9).
[0150] In the preferred organism Saccharomyces cerevisiae, the
.DELTA.22-desaturase gene is the gene ERG5 (SEQ. ID. NO. 21). SEQ.
ID. NO. 22 represents the corresponding Saccharomyces cerevisiae
.DELTA.22-desaturase (Skaggs, B. A. et al: Cloning and
characterization of the Saccharomyces cerevisiae C-22 sterol
desaturase gene, encoding a second cytochrome P-450 involved in
ergosterol biosynthesis, Gene. 1996 Feb. 22; 169(1):105-9).
[0151] In a further preferred embodiment of the method of the
invention, organisms are cultured which have, in addition to the
above-described modifications, an increased activity of at least
one of the activities selected from the group consisting of
HMG-CoA-reductase activity, lanosterol-C14-demethylase activity,
squalene-epoxidase activity, squalene-synthetase activity and
sterol-acyltransferase activity, compared to the wild type.
[0152] HMG-CoA-reductase activity means the enzyme activity of an
HMG-CoA reductase (3-hydroxy-3-methylglutaryl-coenzyme-A
reductase).
[0153] HMG-CoA reductase means a protein which has the enzyme
activity of converting 3-hydroxy-3-methylglutaryl-coenzyme A to
mevalonate.
[0154] Accordingly, HMG-CoA-reductase activity means the amount of
3-hydroxy-3-methylglutaryl-coenzyme A converted or the amount of
mevalonate formed by the protein HMG-CoA reductase in a particular
time.
[0155] In the case of an increased HMG-CoA-reductase activity
compared to the wild type, thus the amount of
3-hydroxy-3-methylglutaryl-coenzyme A converted or the amount of
mevalonate formed by the protein HMG-CoA reductase in a particular
time is increased in comparison with the wild type.
[0156] This increase in HMG-CoA-reductase activity is preferably at
least 5%, further preferably at least 20%, further preferably at
least 50%, further preferably at least 100%, more preferably at
least 300%, still more preferably at least 500%, in particular at
least 600%, of the HMG-CoA-reductase activity of the wild type.
[0157] Lanosterol C14-demethylase activity means the enzyme
activity of a lanosterol C14-demethylase.
[0158] Lanosterol C14-demethylase means a protein which has the
enzyme activity of converting lanosterol to
4,4-dimethylcholesta-8,14,24-trienol.
[0159] Accordingly, lanosterol C14-demethylase activity means the
amount of lanosterol converted or the amount of
4,4-dimethylcholesta-8,14,24-trienol formed by the protein
lanosterol C14-demethylase in a particular time.
[0160] In the case of an increased lanosterol C14-demethylase
activity compared to the wild type, thus the amount of lanosterol
converted or the amount of 4,4-dimethylcholesta-8,14,24-trienol
formed by the protein lanosterol C14-demethylase in a particular
time is increased in comparison with the wild type.
[0161] This increase in lanosterol C14-demethylase activity is
preferably at least 5%, further preferably at least 20%, further
preferably at least 50%, further preferably at least 100%, more
preferably at least 300%, still more preferably at least 500%, in
particular at least 600%, of the lanosterol C14-demethylase
activity of the wild type.
[0162] Squalene-epoxidase activity means the enzyme activity of a
squalene epoxidase.
[0163] Squalene epoxidase means a protein which has the enzyme
activity of converting squalene to squalene epoxide.
[0164] Accordingly, squalene-epoxidase activity means the amount of
squalene converted or the amount of squalene epoxide formed by the
protein squalene epoxidase in a particular time.
[0165] In the case of an increased squalene-epoxidase activity
compared to the wild type, thus the amount of squalene converted or
the amount of squalene epoxide formed by the protein squalene
epoxidase in a particular time is increased in comparison with the
wild type.
[0166] This increase in squalene-epoxidase activity is preferably
at least 5%, further preferably at least 20%, further preferably at
least 50%, further preferably at least 100%, more preferably at
least 300%, still more preferably at least 500%, in particular at
least 600%, of the squalene-epoxidase activity of the wild
type.
[0167] Squalene-synthetase activity means the enzyme activity of a
squalene synthetase.
[0168] Squalene synthetase means a protein which has the enzyme
activity of converting farnesyl-pyrophosphate to squalene.
[0169] Accordingly, squalene-synthetase activity means the amount
of farnesyl-pyrophosphate converted or the amount of squalene
formed by the protein squalene synthetase in a particular time.
[0170] In the case of an increased squalene-synthetase activity
compared to the wild type, thus the amount of
farnesyl-pyrophosphate converted or the amount of squalene formed
by the protein squalene synthetase in a particular time is
increased in comparison with the wild type.
[0171] This increase in squalene-synthetase activity is preferably
at least 5%, further preferably at least 20%, further preferably at
least 50%, further preferably at least 100%, more preferably at
least 300%, still more preferably at least 500%, in particular at
least 600%, of the squalene-synthetase activity of the wild
type.
[0172] Sterol-acyltransferase activity means the enzyme activity of
a sterol acyltransferase.
[0173] Sterol acyltransferase means a protein which has the enzyme
activity of converting 7-dehydrocholesterol to corresponding
acetylated 7-dehydrocholesterol.
[0174] Accordingly, sterol-acyltransferase activity means the
amount of 7-dehydrocholesterol converted or the amount of
acetylated 7-dehydrocholesterol formed by the protein sterol
acyltransferase in a particular time.
[0175] In the case of an increased sterol-acyltransferase activity
compared to the wild type, thus the amount of 7-dehydrocholesterol
converted or the amount of acetylated 7-dehydrocholesterol formed
by the protein sterol acyltransferase in a particular time is
increased in comparison with the wild type.
[0176] This increase in sterol-acyltransferase activity is
preferably at least 5%, further preferably at least 20%, further
preferably at least 50%, further preferably at least 100%, more
preferably at least 300%, still more preferably at least 500%, in
particular at least 600%, of the sterol-acyltransferase activity of
the wild type.
[0177] In a preferred embodiment, the HMG-CoA-reductase activity is
increased compared to the wild type by increasing the gene
expression of a nucleic acid encoding an HMG-CoA reductase.
[0178] In a particularly preferred embodiment of the method of the
invention, gene expression of a nucleic acid encoding an HMG-CoA
reductase is increased by introducing into the organism a nucleic
acid construct comprising an HMG-CoA reductase-encoding nucleic
acid whose expression in said organism is subject to a reduced
regulation, in comparison with the wild type.
[0179] A reduced regulation in comparison with the wild type means
a reduced regulation and, preferably, no regulation at the
expression or protein level, in comparison with the above-defined
wild type.
[0180] The reduced regulation may be achieved preferably by a
promoter which is functionally linked with the coding sequence in
the nucleic acid construct and which is subject to a reduced
regulation in the organism, in comparison with the wild-type
promoter.
[0181] For example, the medium ADH promoter in yeast is subject
only to a reduced regulation and is therefore particularly
preferred as promoter in the above-described nucleic acid
construct.
[0182] This promoter fragment of the ADH12s promoter, also referred
to as ADH1 hereinbelow, exhibits nearly constitutive expression
(Ruohonen L, Penttila M, Keranen S. (1991) Optimization of Bacillus
.alpha.-amylase production by Saccharomyces cerevisiae. Yeast.
May-June; 7(4):337-462; Lang C, Looman A C. (1995) Efficient
expression and secretion of Aspergillus niger RH5344
polygalacturonase in Saccharomyces cerevisiae. Appl Microbiol
Biotechnol. December; 44(1-2):147-56) so that transcriptional
regulation no longer proceeds via intermediates of ergosterol
biosynthesis.
[0183] Other preferred promoters with reduced regulation are
constitutive promoters such as, for example, the yeast TEF1
promoter, the yeast GPD promoter or the yeast PGK promoter (Mumberg
D, Muller R, Funk M. (1995) Yeast vectors for the controlled
expression of heterologous proteins in different genetic
backgrounds. Gene. 1995 Apr. 14; 156(1):119-22; Chen C Y, Oppermann
H, Hitzeman R A. (1984) Homologous versus heterologous gene
expression in the yeast, Saccharomyces cerevisiae. Nucleic Acids
Res. December 11; 12(23):8951-70).
[0184] In a further preferred embodiment, reduced regulation can be
achieved by using as an HMG-CoA reductase-encoding nucleic acid a
nucleic acid whose expression in the organism is subject to a
reduced regulation, in comparison with the orthologous nucleic acid
intrinsic to said organism.
[0185] Particular preference is given to using as an HMG-CoA
reductase-encoding nucleic acid a nucleic acid which encodes only
the catalytic region of HMG-CoA reductase (truncated (t-) HMG-CoA
reductase). This nucleic acid (t-HMG), described in EP 486 290 and
WO 99/16886 encodes only the catalytically active part of HMG-CoA
reductase, with the membrane domain responsible for regulation at
the protein level missing. This nucleic acid is thus subject to a
reduced regulation, in particular in yeast, and leads to an
increase in gene expression of HMG-CoA reductase.
[0186] In a particularly preferred embodiment, nucleic acids are
introduced, preferably via the above-described nucleic acid
construct, which encode proteins comprising the amino acid sequence
SEQ. ID. NO. 24 or a sequence derived from this sequence by
substitution, insertion or deletion of amino acids, which is at
least 30% identical at the amino acid level to the sequence SEQ ID.
NO. 24, and having the enzyme property of an HMG-CoA reductase.
[0187] The sequence SEQ ID NO. 24 is the amino acid sequence of the
truncated HMG-CoA reductase (t-HMG).
[0188] Further examples of HMG-CoA reductases and thus also of the
t-HMG-CoA reductases reduced to the catalytic region or of the
coding genes can readily be found, for example, for various
organisms whose genomic sequence is known by comparing the homology
of the amino acid sequences or of the corresponding backtranslated
nucleic acid sequences from databases with the sequence SEQ ID. No.
24.
[0189] Further examples of HMG-CoA reductases and thus also of the
t-HMG-CoA reductases reduced to the catalytic region and of the
coding genes can furthermore readily be found for various organisms
whose genomic sequence is unknown by hybridization techniques and
PCR techniques in a manner known per se, for example starting from
the sequence SEQ ID NO. 23.
[0190] Particular preference is given to using as a truncated
HMG-CoA reductase-encoding nucleic acid a nucleic acid comprising
the sequence SEQ ID NO. 23.
[0191] In a particularly preferred embodiment, the reduced
regulation is achieved by using as an HMG-CoA reductase-encoding
nucleic acid a nucleic acid whose expression in the organism is
subject to a reduced regulation, in comparison with the orthologous
nucleic acid intrinsic to said organism, and by using a promoter
which is subject to a reduced regulation in said organism, in
comparison with the wild-type promoter.
[0192] In a preferred embodiment, the lanosterol C14-demethylase
activity is increased compared to the wild type by increasing the
gene expression of a nucleic acid encoding a lanosterol
C14-demethylase.
[0193] In a further preferred embodiment, gene expression of a
nucleic acid encoding a lanosterol C14-demethylase is increased by
introducing into the organism one or more nucleic acids encoding a
lanosterol C14-demthylase.
[0194] For this purpose, it is possible to use in principle any
lanosterol C14-demethylase gene (ERG11), i.e. any nucleic acids
encoding a lanosterol C14-demethylase. In the case of genomic
lanosterol C14-demethylase nucleic acid sequences from eukaryotic
sources, which contain introns, already processed nucleic acid
sequences such as the corresponding cDNAs are to be used
preferably, if the host organism is unable to or cannot be enabled
to express the appropriate lanosterol C14-demethylase.
[0195] Examples of lanosterol C14-demethylase genes are nucleic
acids encoding a lanosterol C14-demethylase of Saccharomyces
cerevisiae (Kalb V F, Loper J C, Dey C R, Woods C W, Sutter T R
(1986) Isolation of a cytochrome P-450 structural gene from
Saccharomyces cerevisiae. Gene 45(3):237-45), Candida albicans
(Lamb D C, Kelly D E, Baldwin B C, Gozzo F, Boscott P, Richards W
G, Kelly S L (1997) Differential inhibition of Candida albicans
CYP51 with azole antifungal stereoisomers. FEMS Microbiol Lett
149(1):25-30), Homo sapiens (Stromstedt M, Rozman D, Waterman M R.
(1996) The ubiquitously expressed human CYP51 encodes lanosterol 14
.alpha.-demethylase, a cytochrome P450 whose expression is
regulated by oxysterols. Arch Biochem Biophys 1996 May 1;
329(1):73-81c) or Rattus norvegicus, Aoyama Y, Funae Y, Noshiro M,
Horiuchi T, Yoshida Y. (1994) Occurrence of a P450 showing high
homology to yeast lanosterol 14-demethylase (P450(14DM)) in the rat
liver. Biochem Biophys Res Commun. June 30; 201(3):1320-6).
[0196] In this preferred embodiment, thus at least one further
lanosterol C14-demethylase gene is present in the transgenic
organisms of the invention, compared to the wild type.
[0197] The number of C14-demethylase genes in the transgenic
organisms of the invention is at least two, preferably more than
two, particularly preferably more than three and very particularly
preferably more than five.
[0198] Preference is given to using in the above-described method
nucleic acids which encode proteins comprising the amino acid
sequence SEQ. ID. NO. 26 or a sequence derived from this sequence
by substitution, insertion or deletion of amino acids, which is at
least 30%, preferably at least 50%, more preferably at least 70%,
still more preferably at least 90%, most preferably at least 95%,
identical at the amino acid level with the sequence SEQ. ID. NO.
26, and having the enzyme property of a lanosterol
C14-demethylase.
[0199] The sequence SEQ. ID. NO. 26 represents the amino acid
sequence of Saccharomyces cerevisiae lanosterol
C14-demethylase.
[0200] Further examples of lanosterol C14-demethylases and
lanosterol C14-demethylase genes can readily be found, for example,
for various organisms whose genomic sequence is known by comparing
the homology of the amino acid sequences or the corresponding
backtranslated nucleic acid sequences from databases with the SeQ
ID. NO. 26.
[0201] Further examples of lanosterol C14-demethylases and
lanosterol C14-demethylase genes can furthermore readily be found
for various organisms whose genomic sequence is unknown, for
example starting from the sequence SEQ. ID. No. 25, by
hybridization techniques and PCR techniques in a manner known per
se.
[0202] Accordingly, a protein which is at least 30% identical at
the amino acid level with the sequence SEQ. ID. NO. 26 means a
protein which is at least 30% identical when comparing its sequence
with the sequence SEQ. ID. NO. 26, in particular according to the
above program algorithm with the above set of parameters.
[0203] In another preferred embodiment, nucleic acids are
introduced into organisms, which encode proteins comprising the
amino acid sequence of Saccharomyces cerevisiae lanosterol
C14-demethylase (SEQ. ID. NO. 26).
[0204] Suitable nucleic acid sequences can be obtained, for
example, by backtranslating the polypeptide sequence according to
the genetic code.
[0205] Preference is given to using for this those codons which are
frequently used according to the organism-specific codon usage.
Said codon usage can readily be determined on the basis of computer
analyses of other known genes of the organisms in question.
[0206] If the protein is to be expressed in yeast, for example, it
is often advantageous to use the codon usage of yeast for the
backtranslation.
[0207] In a particularly preferred embodiment, a nucleic acid
comprising the sequence SEQ. ID. NO. 25 is introduced into the
organism.
[0208] The sequence SEQ. ID. NO. 25 represents the genomic DNA of
Saccharomyces cerevisiae (ORF S0001049), which encodes the
lanosterol C14-demethylase of the sequence SEQ ID NO. 26.
[0209] Furthermore, all of the lanosterol C14-demethylase genes
mentioned above can be prepared in a manner known per se by
chemical synthesis from the nucleotide building blocks, for example
by fragment condensation of individual overlapping complementary
nucleic acid building blocks of the double helix. The chemical
synthesis of oligonucleotides may be carried out, for example, in a
known manner according to the phosphoramidite method (Voet, Voet,
2nd edition, Wiley Press New York, pages 896-897). Annealing of
synthetic oligonucleotides and filling-in of gaps with the aid of
the Klenow fragment of DNA polymerase and the ligation reactions
and also general cloning methods are described in Sambrook et al.
(1989), Molecular cloning: A laboratory manual, Cold Spring Harbor
Laboratory Press.
[0210] In a preferred embodiment, the squalene-epoxidase activity
is increased compared to the wild type by increasing the gene
expression of a nucleic acid encoding a squalene epoxidase.
[0211] In a further preferred embodiment, gene expression of a
nucleic acid encoding a squalene epoxidase is increased by
introducing into the organism one or more nucleic acids encoding a
squalene epoxidase.
[0212] For this purpose, it is possible to use in principle any
squalene-epoxidase gene (ERG1), i.e. any nucleic acids encoding a
squalene epoxidase. In the case of genomic squalene epoxidase
nucleic acid sequences from eukaryotic sources, which contain
introns, already processed nucleic acid sequences such as the
corresponding cDNAs are to be used preferably, if the host organism
is unable to or cannot be enabled to express the appropriate
squalene epoxidase.
[0213] Examples of nucleic acids encoding a squalene epoxidase are
nucleic acids encoding a squalene epoxidase of Saccharomyces
cerevisiae (Jandrositz, A., et al (1991) The gene encoding squalene
epoxidase from Saccharomyces cerevisiae: cloning and
characterization. Gene 107:155-160, of Mus musculus (Kosuga K, Hata
S, Osumi T, Sakakibara J, Ono T. (1995) Nucleotide sequence of a
cDNA for mouse squalene epoxidase, Biochim Biophys Acta, February
21; 1260(3):345-8b), of Rattus norvegicus (Sakakibara J, Watanabe
R, Kanai Y, Ono T. (1995) Molecular cloning and expression of rat
squalene epoxidase. J Biol Chem January 6; 270(1):17-20c) or of
Homo sapiens (Nakamura Y, Sakakibara J, Izumi T, Shibata A, Ono T.
(1996) Transcriptional regulation of squalene epoxidase by sterols
and inhibitors in HeLa cells, J. Biol. Chem. 1996, Apr. 5;
271(14):8053-6).
[0214] In this preferred embodiment, thus at least one further
squalene epoxidase is present in the transgenic organisms of the
invention, compared to the wild type.
[0215] The number of squalene-epoxidase genes in the transgenic
organisms of the invention is at least two, preferably more than
two, particularly preferably more than three and very particularly
preferably more than five.
[0216] Preference is given to using in the above-described method
nucleic acids which encode proteins comprising the amino acid
sequence SEQ. ID. NO. 28 or a sequence derived from this sequence
by substitution, insertion or deletion of amino acids, which is at
least 30%, preferably at least 50%, more preferably at least 70%,
still more preferably at least 90%, most preferably at least 95%,
identical at the amino acid level with the sequence SEQ. ID. NO.
28, and having the enzyme property of a squalene epoxidase.
[0217] The sequence SEQ. ID. NO. 28 represents the amino acid
sequence of Saccharomyces cerevisiae squalene epoxidase.
[0218] Further examples of squalene epoxidases and
squalene-epoxidase genes can readily be found, for example, for
various organisms whose genomic sequence is known by comparing the
homology of the amino acid sequences or the corresponding
backtranslated nucleic acid sequences from databases with the SeQ
ID. NO. 28.
[0219] Further examples of squalene epoxidases and
squalene-epoxidase genes can furthermore readily be found for
various organisms whose genomic sequence is unknown, for example
starting from the sequence SEQ. ID. No. 27, by hybridization
techniques and PCR techniques in a manner known per se.
[0220] In another preferred embodiment, nucleic acids are
introduced into organisms, which encode proteins comprising the
amino acid sequence of Saccharomyces cerevisiae squalene epoxidase
(SEQ. ID. NO. 28).
[0221] Suitable nucleic acid sequences can be obtained, for
example, by backtranslating the polypeptide sequence according to
the genetic code.
[0222] Preference is given to using for this those codons which are
frequently used according to the organism-specific codon usage.
Said codon usage can readily be determined on the basis of computer
analyses of other known genes of the organisms in question.
[0223] If the protein is to be expressed in yeast, for example, it
is often advantageous to use the codon usage of yeast for
backtranslation.
[0224] In a particularly preferred embodiment, a nucleic acid
comprising the sequence SEQ. ID. NO. 27 is introduced into the
organism.
[0225] The sequence SEQ. ID. NO. 27 represents the genomic DNA of
Saccharomyces cerevisiae (ORF YGR175C), which encodes the squalene
epoxidase of the sequence SEQ ID NO. 28.
[0226] Furthermore, all of the squalene-epoxidase genes mentioned
above can be prepared in a manner known per se by chemical
synthesis from the nucleotide building blocks, for example by
fragment condensation of individual overlapping complementary
nucleic acid building blocks of the double helix. The chemical
synthesis of oligonucleotides may be carried out, for example, in a
known manner according to the phosphoramidite method (Voet, Voet,
2nd edition, Wiley Press New York, pages 896-897). Annealing of
synthetic oligonucleotides and filling-in of gaps with the aid of
the Klenow fragment of DNA polymerase and the ligation reactions
and also general cloning methods are described in Sambrook et al.
(1989), Molecular cloning: A laboratory manual, Cold Spring Harbor
Laboratory Press.
[0227] In a preferred embodiment, the squalene-synthetase activity
is increased compared to the wild type by increasing the gene
expression of a nucleic acid encoding a squalene synthetase.
[0228] In a further preferred embodiment, gene expression of a
nucleic acid encoding a squalene synthetase is increased by
introducing into the organism one or more nucleic acids encoding a
squalene synthetase.
[0229] For this purpose, it is possible to use in principle any
squalene-synthetase gene (ERG9), i.e. any nucleic acids encoding a
squalene synthetase. In the case of genomic squalene synthetase
nucleic acid sequences from eukaryotic sources, which contain
introns, already processed nucleic acid sequences such as the
corresponding cDNAs are to be used preferably, if the host organism
is unable to or cannot be enabled to express the appropriate
squalene synthetase.
[0230] Examples of nucleic acids encoding a squalene synthetase are
nucleic acids encoding a Saccharomyces cerevisiae squalene
synthetase (ERG9) (Jennings, S. M., (1991): Molecular cloning and
characterization of the yeast gene for squalene synthetase. Proc
Natl Acad Sci USA. July 15; 88(14):6038-42), nucleic acids encoding
a Botryococcus braunii Okada squalene synthetase (Devarenne, T. P.
et al.: Molecular characterization of squalene synthetase from the
green microalga Botryococcus braunii, raceB, Arch. Biochem.
Biophys. 2000, Jan. 15, 373(2):307-17), nucleic acids encoding a
Potato tuber squalene synthetase (Yoshioka H. et al.: cDNA cloning
of sesquiter penecyclase and squalene synthase, and expression of
the genes in potato tuber infected with Phytophthora infestans,
Plant. Cell. Physiol. 1999, September; 40(9):993-8) and nucleic
acids encoding a Glycyrrhiza glabra squalene synthetase (Hayashi,
H. et al.: Molecular cloning and characterization of two cDNAs for
Glycyrrhiza glabra squalene synthase, Biol. Pharm. Bull. 1999,
September; 22(9):947-50).
[0231] In this preferred embodiment, thus at least one further
squalene-synthetase gene is present in the transgenic organisms of
the invention, compared to the wild type.
[0232] The number of squalene-synthetase genes in the transgenic
organisms of the invention is at least two, preferably more than
two, particularly preferably more than three and very particularly
preferably more than five.
[0233] Preference is given to using in the above-described method
nucleic acids which encode proteins comprising the amino acid
sequence SEQ. ID. NO. 30 or a sequence derived from this sequence
by substitution, insertion or deletion of amino acids, which is at
least 30%, preferably at least 50%, more preferably at least 70%,
still more preferably at least 90%, most preferably at least 95%,
identical at the amino acid level with the sequence SEQ. ID. NO.
30, and having the enzyme property of a squalene synthetase.
[0234] The sequence SEQ. ID. NO. 30 represents the amino acid
sequence of Saccharomyces cerevisiae squalene synthetase
(ERG9).
[0235] Further examples of squalene synthetases and
squalene-synthetase genes can readily be found, for example, for
various organisms hose genomic sequence is known by comparing the
homology of the amino acid sequences or the corresponding
backtranslated nucleic acid sequences from databases with the SeQ
ID. NO. 30.
[0236] Further examples of squalene synthetases and
squalene-synthetase genes can furthermore readily be found for
various organisms whose genomic sequence is unknown, for example
starting from the sequence SEQ. ID. No. 29, by hybridization
techniques and PCR techniques in a manner known per se.
[0237] In another preferred embodiment, nucleic acids are
introduced into organisms, which encode proteins comprising the
amino acid sequence of Saccharomyces cerevisiae squalene synthetase
(SEQ. ID. NO. 30).
[0238] Suitable nucleic acid sequences can be obtained, for
example, by backtranslating the polypeptide sequence according to
the genetic code.
[0239] Preference is given to using for this those codons which are
frequently used according to the organism-specific codon usage.
Said codon usage can readily be determined on the basis of computer
analyses of other known genes of the organisms in question.
[0240] If the protein is to be expressed in yeast, for example, it
is often advantageous to use the codon usage of yeast for the
backtranslation.
[0241] In a particularly preferred embodiment, a nucleic acid
comprising the sequence SEQ. ID. NO. 29 is introduced into the
organism.
[0242] The sequence SEQ. ID. NO. 29 represents the genomic DNA of
Saccharomyces cerevisiae (ORF YHR190W), which encodes the squalene
synthetase of the sequence SEQ ID NO. 30.
[0243] Furthermore, all of the squalene-synthetase genes mentioned
above can be prepared in a manner known per se by chemical
synthesis from the nucleotide building blocks, for example by
fragment condensation of individual overlapping complementary
nucleic acid building blocks of the double helix. The chemical
synthesis of oligonucleotides may be carried out, for example, in a
known manner according to the phosphoramidite method (Voet, Voet,
2nd edition, Wiley Press New York, pages 896-897). Annealing of
synthetic oligonucleotides and filling-in of gaps with the aid of
the Klenow fragment of DNA polymerase and the ligation reactions
and also general cloning methods are described in Sambrook et al.
(1989), Molecular cloning: A laboratory manual, Cold Spring Harbor
Laboratory Press.
[0244] In a preferred embodiment, the sterol-acyltransferase
activity is increased compared to the wild type by increasing the
gene expression of a nucleic acid encoding a sterol
acyltransferase.
[0245] In a further preferred embodiment, gene expression of a
nucleic acid encoding a sterol acyltransferase is increased by
introducing into the organism one or more nucleic acids encoding a
sterol acyltransferase.
[0246] For this purpose, it is possible to use in principle any
sterol-acyltransferase gene (SAT1 or SAT2), i.e. any nucleic acids
encoding a sterol acyltransferase.
[0247] In the case of genomic sterol acyltransferase nucleic acid
sequences from eukaryotic sources, which contain introns, already
processed nucleic acid sequences such as the corresponding cDNAs
are to be used preferably, if the host organism is unable to or
cannot be enabled to express the appropriate sterol
acyltransferase.
[0248] Examples of nucleic acids encoding a sterol acyltransferase
are nucleic acids encoding a Saccharomyces cerevisiae sterol
acyltransferase (SAT1) or (SAT2) (Yang, H.: Sterol esterification
in yeast: a two-gene process. Science. 1996 May 31;
272(5266):1353-6), a further nucleic acid encoding a further
Saccharomyces cerevisiae sterol acyltransferase (J. Biol. Chem.
1996, Sep. 27; 271(39):24157-63), nucleic acids encoding a human
sterol acyltransferase (Chang, C. C. et al., Molecular cloning and
functional expression of human acyl-coenzyme A:cholesterol
acyltransferase cDNA in mutant Chinese hamster ovary cells, J.
Biol. Chem. 1993, Oct. 5; 268(28):20747-55) and nucleic acids
encoding a murine sterol acyltransferase (Uelmen, P. J.:
Tissue-specific expression and cholesterol regulation of
acylcoenzyme A:cholesterol acyltransferase (ACAT) in mice.
Molecular cloning of mouse ACAT cDNA, chromosomal localization, and
regulation of ACAT in vivo and in vitro, J. Biol. Chem. 1995 Nov.
3; 270(44):26192-201).
[0249] In this preferred embodiment, thus at least one further
sterol-acyltransferase gene is present in the transgenic organisms
of the invention, compared to the wild type.
[0250] The number of sterol-acyltransferase genes in the transgenic
organisms of the invention is at least two, preferably more than
two, particularly preferably more than three and very particularly
preferably more than five.
[0251] Preference is given to using in the above-described method
nucleic acids which encode proteins comprising the amino acid
sequence SEQ. ID. NO. 32 or SEQ ID NO. 50 or a sequence derived
from these sequences by substitution, insertion or deletion of
amino acids, which is at least 30%, preferably at least 50%, more
preferably at least 70%, still more preferably at least 90%, most
preferably at least 95%, identical at the amino acid level with the
sequence SEQ. ID. NO. 32 or SEQ. ID. NO. 50, and having the enzyme
property of a sterol acyltransferase.
[0252] The sequence SEQ. ID. NO. 32 represents the amino acid
sequence of Saccharomyces cerevisiae sterol acyltransferase
SAT1.
[0253] The sequence SEQ. ID. NO. 50 represents the amino acid
sequence Saccharomyces cerevisiae sterol acyltransferase SAT2.
[0254] SAT 1 and SAT2 differ from one another by a different
substrate specificity.
[0255] Further examples of sterol acyltransferases and
sterol-acyltransferase genes can readily be found, for example, for
various organisms whose genomic sequence is known by comparing the
homology of the amino acid sequences or the corresponding
backtranslated nucleic acid sequences from databases with the SeQ
ID. NO. 32 or 50.
[0256] Further examples of sterol acyltransferase and
sterol-acyltransferase genes can furthermore readily be found for
various organisms whose genomic sequence is unknown, for example
starting from the sequence SEQ. ID. No. 31 or 49, by hybridization
techniques and PCR techniques in a manner known per se.
[0257] In another preferred embodiment, nucleic acids are
introduced into organisms, which encode proteins comprising the
amino acid sequence of Saccharomyces cerevisiae sterol
acyltransferase SAT1 (SEQ. ID. NO. 32) or Saccharomyces cerevisiae
sterol acyltransferase SAT2 (SEQ. ID. NO. 50).
[0258] Suitable nucleic acid sequences can be obtained, for
example, by backtranslating the polypeptide sequence according to
the genetic code.
[0259] Preference is given to using for this those codons which are
frequently used according to the organism-specific codon usage.
Said codon usage can readily be determined on the basis of computer
analyses of other known genes of the organisms in question.
[0260] If the protein is to be expressed in yeast, for example, it
is often advantageous to use the codon usage of yeast for the
backtranslation.
[0261] In a particularly preferred embodiment, a nucleic acid
comprising the sequence SEQ. ID. NO. 31 or 49 is introduced into
the organism.
[0262] The sequence SEQ. ID. NO. 31 represents the genomic DNA of
Saccharomyces cerevisiae (ORF YNR019W), which encodes the sterol
acyltransferase SAT1 of the sequence SEQ ID NO. 32.
[0263] The sequence SEQ. ID. NO. 49 represents the genomic DNA of
Saccharomyces cerevisiae (ORF YCR048W), which encodes the sterol
acyltransferase SAT2 of the sequence SEQ ID NO. 50.
[0264] Furthermore, all of the sterol-acyltransferase genes
mentioned above can be prepared in a manner known per se by
chemical synthesis from the nucleotide building blocks, for example
by fragment condensation of individual overlapping complementary
nucleic acid building blocks of the double helix. The chemical
synthesis of oligonucleotides may be carried out, for example, in a
known manner according to the phosphoramidite method (Voet, Voet,
2nd edition, Wiley Press New York, pages 896-897). Annealing of
synthetic oligonucleotides and filling-in of gaps with the aid of
the Klenow fragment of DNA polymerase and the ligation reactions
and also general cloning methods are described in Sambrook et al.
(1989), Molecular cloning: A laboratory manual, Cold Spring Harbor
Laboratory Press.
[0265] According to the invention, organisms mean, for example,
bacteria, in particular bacteria of the genus Bacillus, Escherichia
coli, Lactobacillus spec. or Streptomyces spec.,
for example yeasts, in particular yeasts of the genus Saccharomyces
cerecisiae, Pichia pastoris or Klyveromyces spec. for example
fungi, in particular fungi of the genus Aspergillus spec.,
Penicillium spec. or Dictyostelium spec. and also, for example,
insect cell lines, which are capable, either as wild type or owing
to previous genetic modification, of producing zymosterol and/or
the biosynthetic intermediates and/or secondary products
thereof.
[0266] Particularly preferred organisms are yeasts, in particular
those of the species Saccharomyces cerevisiae, in particular the
yeast strains Saccharomyces cerevisiae AH22, Saccharomyces
cerevisiae GRF, Saccharomyces cerevisiae DBY747 and Saccharomyces
cerevisiae BY4741.
[0267] In the case of yeasts as organisms or genetically modified
organisms, it is possible, as mentioned above, to increase at least
one of the activities selected from the group consisting of
.DELTA.8-.DELTA.7-isomerase activity, .DELTA.5-desaturase activity
and .DELTA.24-reductase activity by overexpressing the
corresponding nucleic acids.
[0268] The overexpression may be carried out both homologously by
introducing nucleic acids intrinsic to yeast and heterologously by
introducing nucleic acids from other organisms, in particular
mammals, or natural or artificial variants derived therefrom into
the yeast. Preference is given to using mammalian genes in yeasts,
since these genes have a better substrate specificity with respect
to 7-dehydrocholesterol.
[0269] The .DELTA.8-.DELTA.7-isomerase activity,
.DELTA.5-desaturase activity, .DELTA.24-reductase activity,
C24-methyltransferase activity, .DELTA.22-desaturase activity,
HMG-CoA-reductase activity, lanosterol-C14-demethylase activity,
squalene-epoxidase activity, squalene-synthetase activity and
sterol-acyltransferase activity of the genetically modified
organism of the invention and of the reference organism is
determined under the following conditions:
[0270] The activity of HMG-CoA reductase is determined as described
in Th. Polakowski, Molekularbiologische Beeinflussung des
Ergosterolstoffwechsels der Hefe Saccharomyces cerevisiae
[influencing the ergosterol metabolism of the yeast Saccharomyces
cerevisiae by molecular biological means], Shaker-Verlag, Aachen
1999, ISBN 3-8265-6211-9, beschrieben.
[0271] According to this, 10.sup.9 yeast cells of a 48 h culture
are harvested by centrifugation (3500.times.g, 5 min) and washed in
2 ml of buffer I (100 mM potassium phosphate buffer, pH 7.0). The
cell pellet is taken up in 500 .mu.l of buffer 1 (cytosolic
proteins) or 2 (100 mM potassium phosphate buffer pH 7.0; 1% Triton
X-100) (total proteins), and 1 .mu.l of 500 mM PMSF in isopropanol
is added. 500 .mu.l of glass beads (d=0.5 mm) are added to the
cells and the cells are disrupted by vortexing 5.times. for one
minute each. The liquid between the glass beads is transferred to a
new Eppendorf vessel. Cell debris and membrane components are
removed by centrifugation (14000.times.g; 15 min).
[0272] The supernatant is transferred to a new Eppendorf vessel and
represents the protein fraction.
[0273] The activity of HMG-CoA reductase is determined by measuring
NADPH+H.sup.+ consumption during the reduction of
3-hydroxy-3-methylglutaryl-CoA which is added as substrate.
[0274] In a 1000 .mu.l assay mixture, 20 .mu.l of yeast protein
isolate are combined with 910 .mu.l of buffer I; 50 .mu.l of 0.1 M
DTT and 10 .mu.l of 16 mM NADPH+H.sup.+. The mixture is adjusted to
30.degree. C. and measured in a spectrophotometer at 340 nm for 7.5
min. The decrease in NADPH, which is measured over this period, is
the rate of degradation without addition of substrate and is taken
into account as background.
[0275] Subsequently, substrate (10 .mu.l of 30 mM HMG-CoA) is
added, and measurement continues for another 7.5 min. The
HMG-CoA-reductase activity is calculated by determining the
specific rate of NADPH degradation.
[0276] The activity of lanosterol C14-demethylase is determined as
described in Omura, T. and Sato, R. (1964) The carbon monoxide
binding pigment in liver microsomes. J. Biol. Chem. 239, 2370-2378.
In this assay, the amount of P450 enzyme as holoenzyme with bound
heme can be semi-quantified. The (active) holoenzyme (with heme)
can be reduced by CO and only the CO-reduced enzyme has an
absorption maximum at 450 nm. Thus the absorption maximum at 450 nm
is a measure for lanosterol C14-demethylase activity.
[0277] The activity is determined by diluting a microsomal fraction
(4-10 mg/ml protein in 100 mM potassium phosphate buffer) 1:4 so
that the protein concentration used in the assay is 2 mg/ml. The
assay is carried out directly in a cuvette.
[0278] A spatula tipful of dithionite (S.sub.2O.sub.4Na.sub.2) is
added to the microsomes. The baseline is recorded in the 380-500 nm
region in a spectrophotometer.
[0279] Subsequently, approx. 20-30 CO bubbles are passed through
the sample. The absorption is then measured in the same region. The
absorption level at 450 nm corresponds to the amount of P450 enzyme
in the assay mixture.
[0280] The activity of squalene epoxidase is determined as
described in Leber R, Landl K, Zinser E, Ahorn H, Spok A, Kohlwein
S D, Turnowsky F, Daum G. (1998) Dual localization of squalene
epoxidase, Erg1p, in yeast reflects a relationship between the
endoplasmic reticulum and lipid particles, Mol. Biol. Cell. 1998,
February; 9(2):375-86.
[0281] In this method, a total volume of 500 .mu.l contains from
0.35 to 0.7 mg of microsomal protein or from 3.5 to 75 .mu.g of
lipid-particle protein in 100 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1
mM FAD, 3 mM NADPH, 0.1 mM squalene 2,3-epoxidase cyclase inhibitor
U18666A, 32 .mu.M [.sup.3H]squalene dispersed in 0.005% Tween
80.
[0282] The assay is carried out at 30.degree. C. After 10 minutes
of pretreatment, the reaction is started by adding squalene and
stopped after 15, 30 or 45 min by lipid extraction with 3 ml of
chloroform/methanol (2:1 vol/vol) and 750 .mu.l of 0.035%
MgCl.sub.2.
[0283] The lipids are dried under nitrogen and redissolved in 0.5
ml of chloroform/methanol (2:1 vol/vol). For thin layer
chromatography, portions are applied to a Silica Gel 60 plate (0.2
mm) and fractionated using chloroform as eluent. The positions
containing [.sup.3H]2,3-oxidosqualene and [.sup.3H]squalene were
scraped off and quantified in a scintillation counter.
[0284] The .DELTA.8-.DELTA.7-isomerase activity is determined, with
a slight modification, as described in Silve S. et al.:
Emopamil-binding Protein, a Mammalian Protein That Binds a Series
of Structurally Diverse Neuroprotective Agents, Exhibits 8-7 Sterol
Isomerase Activity in Yeast. J Biol Chem 1996 Sep. 13;
271(37):22434-40:
[0285] Microsomes prepared from a culture volume of 10 ml are
incubated in the presence of 75 .mu.M cholesta-8-en-3-ol at
30.degree. C. for 3 h. The sterols are then extracted with 4 times
5 ml of hexane and purified. Aliquots are analyzed by means of
GC/MS.
[0286] The .DELTA.5-desaturase activity is determined, with slight
modification, as described in Nishi, S. et al. (2000): cDNA cloning
of the mammalian sterol C5-desaturase and the expression in yeast
mutant. Biochim. Biophys. Acta1490(1-2),106-108:
[0287] Microsomes prepared from a culture volume of 10 ml are
incubated in the presence of 75 .mu.M lathosterol and 2 mM NADH at
30.degree. C. for 3 h. The sterols are then extracted with 4 times
5 ml of hexane and purified. Aliquots are analyzed by means of
GC/MS.
[0288] The .DELTA.24-reductase activity can be determined as
described below:
[0289] Microsomes prepared from a culture volume of 10 ml are
incubated in the presence of 75 .mu.M cholesta-5,7,24-trienol at
30.degree. C. for 3 h. The sterols are then extracted with 4 times
5 ml of hexane and purified. Aliquots are analyzed by means of
GC/MS.
[0290] The C24-methyltransferase activity can be determined as
described below:
80% of the protein Erg6p (C24-methyltransferase) are detectable in
lipid particles in the yeast (Athenstaedt K, Zweytick D, Jandrositz
A, Kohlwein S D, Daum G: Identification and characterization of
major lipid particle proteins of the yeast Saccharomyces
cerevisiae. J. Bacteriol. 1999 October; 181(20):6441-8). The enzyme
activity is determined by preparing lipid particles from a culture
volume (48 h) of 100 ml (according to a method described in
Athenstaedt K, Zweytick D, Jandrositz A, Kohlwein S D, Daum G:
Identification and characterization of major lipid particle
proteins of the yeast Saccharomyces cerevisiae. J. Bacteriol. 1999
October; 181(20):6441-8).
[0291] The protein content is determined by a Biorad enzyme assay
and 3 mg of protein are used in a volume of 500 .mu.l for each
assay mixture. 50 .mu.M [methyl-.sup.3H.sub.3]-S-adenosylmethionine
and 50 .mu.M zymosterol are added to the assay mixture which is
then incubated at 35.degree. C. for 10 min. Subsequently, the same
volume (500 .mu.l) of chloroform/methanol (4:1) is added and the
sterols are then extracted.
[0292] The proportion of zymosterol with incorporated
[methyl-.sup.3H.sub.3]-S-adenosylmethionine can be determined by
means of scintillation measurement, since chloroform/methanol
extraction extracts only lipid-soluble substances. For
quantification, the radioactive decays are likewise determined for
50 .mu.M [methyl-.sup.3H.sub.3]-S-adenosylmethionine by means of
scintillation measurement.
[0293] This method is a modification of the method described in Nes
W D, Guo D, Zhou W.: Substrate-based inhibitors of the
(S)-adenosyl-L-methionine:.DELTA.24(25)- to .DELTA.24(28)-sterol
methyl transferase from Saccharomyces cerevisiae, Arch. Biochem.
Biophys. 1997 Jun. 1; 342(1):68-81.
[0294] The activity of .DELTA.22-desaturase (ERG5p) can be
determined as described below:
[0295] Various concentrations of Ergosta-5,7-dienol, purified from
S. cerevisiae erg5 mutants (Parks et al, 1985. Yeast sterols.yeast
mutants as tools for the study of sterol metabolism. Methods
Enzymol. 111:333-346) and 50 .mu.g of dilauroylphosphatidylcholine
are mixed and treated with ultrasound until a white suspension is
formed. Prepared microsomes are added (1 ml) (3 mg/ml protein).
NADPH (1 mM final concentration) is added to the assay mixture to
start the enzyme reaction. The mixture is incubated at 37.degree.
C. for 20 min. The reaction is stopped by adding 3 ml of methanol
and sterols are hydrolyzed by adding 2 ml of 60% (wt/vol) KOH in
water. The mixture is incubated at 90.degree. C. for 2 h. After
cooling, the mixture is extracted three times with 5 ml of hexane
and concentrated in a rotary evaporator. Subsequently, the sterols
are silylated with bis(trimethylsilyl)trifluoroacetamide (50 .mu.l
in 50 .mu.l toluene) at 60.degree. C. for 1 h. The sterols are
analyzed by gas chromatography-mass spectrometry (GC-MS) (for
example Model VG 12-250 gas chromatograph-mass spectrometer; VG
Biotech, Manchester, United Kingdom). The resultant
.DELTA.22-desaturated intermediate can be identified depending on
the amount of substrate used. Microsomes which are not incubated
with substrate serve as reference.
[0296] This method is a modification of the method described in
Lamb et al: Purification, reconstitution, and inhibition of
cytochrome P-450 sterol .DELTA.22-desaturase from the pathogenic
fungus Candida glabrata. Antimicrob Agents Chemother. 1999 July;
43(7):1725-8.
[0297] The squalene-synthetase activity can be determined as
described below:
[0298] The assays contain 50 mM MOPS, pH 7.2, 10 mM MgCl.sub.2, 1%
(v/v) Tween-80, 10% (v/v) 2-propanol, 1 mM DTT, 1 mg/mL BSA, NADPH,
FPP (or PSPP) and microsomes (protein content 3 mg) in a total
volume of 200 .mu.l in glass tubes. The reaction mixtures
containing the radioactive substrate [1-.sup.3H]FPP (15-30
mCi/.mu.mol) are incubated at 30.degree. C. for 30 min and one
volume of 1:1 (v/v) 40% aqueous KOH:methanol is added to the
suspension mixture. Liquid NaCl is added to saturate the solution
and 2 ml of naphtha containing 0.5% (v/v) squalene are likewise
added.
[0299] The suspension is vortexed for 30 s. In each case 1 ml of
the naphtha layer is applied to a packed 0.5.times.6 cm aluminum
column (80-200 mesh, Fisher) using a Pasteur pipette. The column
has been pre-equilibrated with 2 ml of naphtha containing 0.5%
(v/v) squalene. The column is then eluted with 5.times.1 ml of
toluene containing 0.5% (v/v) squalene. Squalene radioactivity is
measured in Cytoscint (ICN) scintillation cocktail in a
scintillation counter (Beckman).
[0300] This method is a modification of the method described in
Radisky et al., Biochemistry. 2000 Feb. 22; 39(7):1748-60, Zhang et
al. (1993) Arch. Biochem. Biophys. 304, 133-143 and Poulter, C. D.
et al. (1989) J. Am. Chem. Soc. 111, 3734-3739.
[0301] The sterol-acyltransferase activity can be determined as
described below:
[0302] A 200 ml main culture is inoculated at 1% strength from a 20
ml preculture which has been incubated for two days and is
incubated in complete medium overnight. The cells are harvested and
then washed in two volumes of HP buffer (100 mM potassium phosphate
buffer, pH 7.4; 0.5 mM EDTA; 1 mM glutathione; 20 .mu.M leupeptin;
64 .mu.M benzamidine; 2 mM PMSF) and resuspended in HP buffer.
[0303] After adding 1 g of glass beads, the cells are disrupted by
vortexing 8 times for one minute each. The supernatant is
ultracentrifuged at 105000.times.g. The pellet is taken up in 1 ml
of ACAT buffer (100 mM potassium phosphate buffer pH7.4; 1 mM
glutathione).
[0304] The enzyme assay is carried out in a volume of 500 .mu.l.
The substrate ergosterol is taken up in 62.5 ml of 0.5.times.ACAT
buffer with vigorous vortexing. 250 .mu.l of this solution are used
as substrate in the assay. To this, 20 .mu.l of protein extract, 50
.mu.l of water and 130 .mu.l of 0.5.times.ACAT buffer are
added.
[0305] The mixture is incubated at 37.degree. C. for 15 min.
Subsequently, 50 .mu.l of 14C-oleoyl-CoA (600000 dpm) are added and
the reaction is stopped after one minute by adding 4 ml of
chloroform/methanol (2:1). To this, 500 .mu.l of H.sub.2O are
added. The phases are separated by briefly centrifuging the
suspension at 2000.times.g. The lower phase is evaporated to
dryness in a pear-shaped flask and redissolved in 100 .mu.l of
chloroform/methanol (4:1) and applied to a TLC plate (silica gel 60
F254). The TLC is carried out using petroleum ether/diethyl
ether/acetic acid 90:10:1 as eluent. The spots of the steryl ester
fractions are cut out and the number of radioactive decays is
determined in a scintillation column. The enzyme activity can be
determined via the amount of sterile ester-bound 14C-oleoyl-CoA
molecules.
[0306] In a preferred embodiment of the method of the invention
7-dehydrocholesterol and/or the biosynthetic intermediates and/or
intermediates thereof are prepared by culturing organisms, in
particular yeasts, which have, compared to the wild type, an
increased activity of at least one of the activities selected from
the group consisting of .DELTA.8-.DELTA.7-isomerase activity,
.DELTA.5-desaturase activity and .DELTA.24-reductase activity and
which have additionally a reduced activity of at least one of the
activities selected from the group consisting of
C24-methyltransferase activity and .DELTA.22-desaturase activity
and which have additionally an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and an
increased squalene-epoxidase activity.
[0307] In other preferred embodiments of the method of the
invention, 7-dehydrocholesterol and/or the biosynthetic
intermediates and/or secondary products thereof are prepared by
culturing organisms, in particular yeasts, which have, compared to
the wild type,
an increased .DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity and an
increased .DELTA.5-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity and an increased
.DELTA.24-reductase activity, an increased .DELTA.5-desaturase
activity and an increased .DELTA.24-reductase activity, an
increased .DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity and an increased .DELTA.24-reductase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity and a
reduced C24-methyltransferase activity, an increased
.DELTA.5-desaturase activity and a reduced C24-methyltransferase
activity, an increased .DELTA.24-reductase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity and a reduced C24-methyltransferase
activity, increased .DELTA.8-.DELTA.7-isomerase activity, an
increased .DELTA.24-reductase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.5-desaturase
activity, an increased .DELTA.24-reductase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity and a reduced
.DELTA.22-desaturase activity, an increased .DELTA.5-desaturase
activity and a reduced .DELTA.22-desaturase activity, an increased
.DELTA.24-reductase activity and a reduced .DELTA.22-desaturase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity, an
increased .DELTA.5-desaturase activity and a reduced
.DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.24-reductase activity and a reduced .DELTA.22-desaturase
activity, an increased .DELTA.5-desaturase activity, an increased
.DELTA.24-reductase activity and a reduced .DELTA.22-desaturase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity, an
increased .DELTA.5-desaturase activity, an increased
.DELTA.24-reductase activity and a reduced .DELTA.22-desaturase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity, a
reduced .DELTA.22-desaturase activity and a reduced
C24-ethyltransferase activity, an increased .DELTA.5-desaturase
activity, a reduced .DELTA.22-desaturase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, a reduced .DELTA.22-desaturase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.24-reductase activity, a reduced .DELTA.22-desaturase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity and an increased
HMG-CoA-reductase activity, an increased .DELTA.5-desaturase
activity and an increased HMG-CoA-reductase activity, an increased
.DELTA.24-reductase activity and an increased HMG-CoA-reductase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity, an
increased HMG-CoA-reductase activity and an increased
.DELTA.5-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
HMG-CoA-reductase activity and an increased .DELTA.24-reductase
activity, an increased .DELTA.5-desaturase activity, an increased
HMG-CoA-reductase activity and an increased .DELTA.24-reductase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity, an
increased .DELTA.5-desaturase activity, an increased
HMG-CoA-reductase activity and an increased .DELTA.24-reductase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity, an
increased HMG-CoA-reductase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.5-desaturase
activity, an increased HMG-CoA-reductase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.24-reductase
activity, an increased HMG-CoA-reductase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased HMG-CoA-reductase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.24-reductase activity, an increased HMG-CoA-reductase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, an increased HMG-CoA-reductase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
HMG-CoA-reductase activity and a reduced .DELTA.22-desaturase
activity, an increased .DELTA.5-desaturase activity, an increased
HMG-CoA-reductase activity and a reduced .DELTA.22-desaturase
activity, an increased .DELTA.24-reductase activity, an increased
HMG-CoA-reductase activity and a reduced .DELTA.22-desaturase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity, an
increased .DELTA.5-desaturase activity, an increased
HMG-CoA-reductase activity and a reduced .DELTA.22-desaturase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity, an
increased .DELTA.24-reductase activity, an increased
HMG-CoA-reductase activity and a reduced .DELTA.22-desaturase
activity, an increased .DELTA.5-desaturase activity, an increased
.DELTA.24-reductase activity, an increased HMG-CoA-reductase
activity and a reduced .DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, an increased HMG-CoA-reductase activity and a reduced
.DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, a reduced
.DELTA.22-desaturase activity, an increased HMG-CoA-reductase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.5-desaturase activity, a reduced .DELTA.22-desaturase
activity, an increased HMG-CoA-reductase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
HMG-CoA-reductase activity and a reduced C24-methyltransferase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity, an
increased .DELTA.5-desaturase activity, a reduced
.DELTA.22-desaturase activity, an increased HMG-CoA-reductase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.24-reductase activity, a reduced .DELTA.22-desaturase
activity, an increased HMG-CoA-reductase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.5-desaturase
activity, an increased .DELTA.24-reductase activity, a reduced
.DELTA.22-desaturase activity, an increased HMG-CoA-reductase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
HMG-CoA-reductase activity and a reduced C24-methyltransferase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity and an
increased lanosterol-C14-demethylase activity, an increased
.DELTA.5-desaturase activity and an increased
lanosterol-C14-demethylase activity, an increased
.DELTA.24-reductase activity and an increased
lanosterol-C14-demethylase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
lanosterol-C14-demethylase activity and an increased
.DELTA.5-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
lanosterol-C14-demethylase activity and an increased
.DELTA.24-reductase activity, an increased .DELTA.5-desaturase
activity, an increased lanosterol-C14-demethylase activity and an
increased .DELTA.24-reductase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased
lanosterol-C14-demethylase activity and an increased
.DELTA.24-reductase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.5-desaturase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.24-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.24-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.5-desaturase
activity, an increased .DELTA.24-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
lanosterol-C14-demethylase activity and a reduced
.DELTA.22-desaturase activity, an increased .DELTA.5-desaturase
activity, an increased lanosterol-C14-demethylase activity and a
reduced .DELTA.22-desaturase activity, an increased
.DELTA.24-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
.DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased
lanosterol-C14-demethylase activity and a reduced
.DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.24-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
.DELTA.22-desaturase activity, an increased .DELTA.5-desaturase
activity, an increased .DELTA.24-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
.DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced .DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, a reduced
.DELTA.22-desaturase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.5-desaturase
activity, a reduced .DELTA.22-desaturase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, a reduced .DELTA.22-desaturase
activity, an increased lanosterol-C14-demethylase activity and a
reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.24-reductase activity, a reduced .DELTA.22-desaturase
activity, an increased lanosterol-C14-demethylase activity and a
reduced C24-methyltransferase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
lanosterol-C14-demethylase activity and an increased
HMG-CoA-reductase activity, an increased .DELTA.5-desaturase
activity, an increased lanosterol-C14-demethylase activity and an
increased HMG-CoA-reductase activity, an increased
.DELTA.24-reductase activity, an increased
lanosterol-C14-demethylase activity and an increased
HMG-CoA-reductase activity, an increased AB-A7-isomerase activity,
an increased HMG-CoA-reductase activity, an increased
lanosterol-C14-demethylase activity and an increased
.DELTA.5-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity and an increased .DELTA.24-reductase activity, an
increased .DELTA.5-desaturase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity and an increased .DELTA.24-reductase activity, an
increased .DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and an
increased .DELTA.24-reductase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.5-desaturase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced C24-methyltransferase activity, an increased
.DELTA.24-reductase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.24-reductase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced C24-methyltransferase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, an increased HMG-CoA-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity and a reduced .DELTA.22-desaturase activity, an increased
.DELTA.5-desaturase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced .DELTA.22-desaturase activity, an increased
.DELTA.24-reductase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced .DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced .DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.24-reductase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced .DELTA.22-desaturase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, an increased HMG-CoA-reductase activity and a reduced
.DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, an increased HMG-CoA-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
.DELTA.22-desaturase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, a reduced
.DELTA.22-desaturase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.5-desaturase activity, a reduced .DELTA.22-desaturase
activity, an increased HMG-CoA-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, a reduced .DELTA.22-desaturase
activity, an increased HMG-CoA-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.24-reductase activity, a reduced .DELTA.22-desaturase
activity, an increased HMG-CoA-reductase activity, an increased
lanosterol-C14-demethylase activity and a reduced
C24-methyltransferase activity, an increased .DELTA.5-desaturase
activity, an increased .DELTA.24-reductase activity, a reduced
.DELTA.22-desaturase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity and a
reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity, an increased squalene-epoxidase activity and a reduced
C24-methyltransferase activity, or an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity, an increased squalene-epoxidase activity and a reduced
C24-methyltransferase activity.
[0308] In further particularly preferred embodiments of the method
of the invention, 7-dehydrocholesterol and/or the biosynthetic
intermediates and/or secondary products thereof are prepared by
culturing organisms, in particular yeasts, which have, compared to
the wild type, an increased .DELTA.8-.DELTA.7-isomerase activity,
an increased .DELTA.5-desaturase activity, an increased
.DELTA.24-reductase activity, a reduced .DELTA.22-desaturase
activity, an increased HMG-CoA-reductase activity, an increased
lanosterol-C14-demethylase activity and an increased
squalene-epoxidase activity,
[0309] an increased .DELTA.8-.DELTA.7-isomerase activity, an
increased .DELTA.5-desaturase activity, an increased
.DELTA.24-reductase activity, an increased HMG-CoA-reductase
activity, an increased lanosterol-C14-demethylase activity, an
increased squalene-epoxidase activity and a reduced
C24-methyltransferase activity,
an increased .DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity, an increased squalene-epoxidase activity and a reduced
C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity, an increased squalene-epoxidase activity, an increased
squalene-synthetase activity and a reduced C24-methyltransferase
activity, an increased .DELTA.8-.DELTA.7-isomerase activity, an
increased .DELTA.5-desaturase activity, an increased
.DELTA.24-reductase activity, a reduced .DELTA.22-desaturase
activity, an increased HMG-CoA-reductase activity, an increased
lanosterol-C14-demethylase activity, an increased
squalene-epoxidase activity, an increased sterol-acyltransferase
activity and a reduced C24-methyltransferase activity, an increased
.DELTA.8-.DELTA.7-isomerase activity, an increased
.DELTA.5-desaturase activity, an increased .DELTA.24-reductase
activity, a reduced .DELTA.22-desaturase activity, an increased
HMG-CoA-reductase activity, an increased lanosterol-C14-demethylase
activity, an increased squalene-epoxidase activity, an increased
squalene-synthetase activity, an increased sterol-acyltransferase
activity and a reduced C24-methyltransferase activity.
[0310] Biosynthetic 7-dehydrocholesterol intermediates mean all
compounds which appear as intermediates during 7-dehydrocholesterol
biosynthesis in the organism used, preferably the compounds
mevalonate, farnesyl pyrophosphate, geraniol pyrophosphate,
squalene epoxide, 4-dimethylcholesta-8,14,24-trienol,
4,4-dimethylzymosterol, squalene, farnesol, geraniol, lanosterol,
zymosterol, lathosterol, cholesta-7,24-dienol and
cholesta-5,7,24-trienol.
[0311] Biosynthetic secondary products of zymosterol mean all
compounds which can be derived biosynthetically from
7-dehydrocholesterol in the organism used, i.e. for which
7-dehydrocholesterol appears as an intermediate. These may be
compounds which the organism used produces naturally from
7-dehydrocholesterol, such as, for example, cholesterol or vitamin
D3 in mammals. However, they also mean compounds which can be
produced in the organism from 7-dehydrocholesterol only by
introducing genes and enzyme activities of other organisms for
which the starting organism has no orthologous gene.
[0312] It is possible, for example, to prepare secondary products
from 7-dehydrocholesterol, which are naturally present only in
mammals, by introducing mammalian genes into yeast:
[0313] Introducing a human or murine nucleic acid encoding a human
or murine .DELTA.-7-reductase enables the yeast to produce
cholesterol.
[0314] Under UV irradiation, vitamin D.sub.3 (cholecalciferol) is
produced from 7-dehydrocholesterol via provitamin D.sub.3 by
rearrangement.
[0315] Therefore, the biosynthetic secondary products of
7-dehydrocholesterol mean in particular provitamin D3, vitamin
D.sub.3 (cholecalciferol) and/or cholesterol.
[0316] Preferred biosynthetic secondary products are provitamin
D.sub.3 and in particular vitamin D.sub.3.
[0317] The compounds prepared in the method of the invention may be
used in biotransformations, chemical reactions and for therapeutic
purposes, for example for producing vitamin D.sub.3 from
7-dehydrocholesterol via UV irradiation, or for producing steroid
hormones via biotransformation starting from cholesta-7,24-dienol
or cholesta-5,7,24-trienol.
[0318] In the inventive method for preparing 7-dehydrocholesterol
and/or the biosynthetic intermediates and/or secondary products
thereof, the step of culturing the genetically modified organisms,
also referred to as transgenic organisms hereinbelow, is preferably
followed by harvesting said organisms and isolating
7-dehydrocholesterol and/or the biosynthetic intermediates and/or
secondary products thereof from said organisms.
[0319] The organisms are harvested in a manner known per se and
appropriate for the particular organism. Microorganisms such as
bacteria, mosses, yeasts and fungi or plant cells which are
cultured in liquid media by fermentation may be removed, for
example, by centrifugation, decanting or filtration.
[0320] 7-Dehydrocholesterol and/or the biosynthetic intermediates
and/or secondary products thereof are isolated from the harvested
biomass together or each compound is harvested separately in a
manner known per se, for example by extraction and, where
appropriate, further chemical or physical purification processes
such as, for example, precipitation methods, crystallography,
thermal separation methods such as rectification methods or
physical separation methods such as, for example,
chromatography.
[0321] The transgenic organisms, in particular yeasts, are
preferably prepared either by transforming the starting organisms,
in particular yeasts, with a nucleic acid construct containing at
least one nucleic acid selected from the group consisting of
nucleic acids encoding a .DELTA.8-.DELTA.7-isomerase, nucleic acids
encoding a .DELTA.5-desaturase and nucleic acids encoding a
.DELTA.24-reductase which are functionally linked with one or more
regulatory signals ensuring transcription and translation in
organisms. In this embodiment, the transgenic organisms are
prepared using a nucleic acid construct.
[0322] In a particularly preferred embodiment, the above-described
nucleic acid construct additionally contains at least one nucleic
acid selected from the group consisting of nucleic acids encoding
an HMG-CoA-reductase activity, nucleic acids encoding a
lanosterol-C14-demethylase, nucleic acids encoding a squalene
epoxidase, nucleic acids encoding a squalene synthetase and nucleic
acids encoding a sterol acyltransferase which are functionally
linked to one or more regulatory signals ensuring transcription and
translation in organisms.
[0323] However, the transgenic organisms may also preferably be
prepared by transforming the starting organisms, in particular
yeasts, with at least one nucleic acid construct selected from the
group consisting of nucleic acid constructs containing nucleic
acids encoding a .DELTA.8-.DELTA.7-isomerase, nucleic acid
construct containing nucleic acids encoding a .DELTA.5-desaturase
and nucleic acid construct containing nucleic acids encoding a
.DELTA.24-reductase which nucleic acids are in each case
functionally linked to one or more regulatory signals ensuring
transcription and translation in organisms. In this embodiment, the
transgenic organisms are prepared using individual nucleic acid
constructs or a combination of nucleic acid constructs.
[0324] In a particularly preferred embodiment, the above-described
combination of nucleic acid constructs additionally comprises at
least one nucleic acid construct selected from the group consisting
of nucleic acid construct containing nucleic acids encoding an
HMG-CoA-reductase activity, nucleic acid construct containing
nucleic acids encoding a lanosterol-C14-demethylase, nucleic acid
construct containing nucleic acids encoding a squalene epoxidase,
nucleic acid construct containing nucleic acids encoding a squalene
synthetase and nucleic acid construct containing nucleic acids
encoding a sterol acyltransferase which nucleic acids are in each
case functionally linked to one or more regulatory signals ensuring
transcription and translation in organisms.
[0325] Nucleic acid constructs in which the encoding nucleic acid
sequence is functionally linked to one or more regulatory signals
ensuring transcription and translation in organisms, in particular
in yeasts, are also referred to as expression cassettes
hereinbelow.
[0326] Examples of nucleic acid constructs containing said
expression cassette are vectors and plasmids.
[0327] Accordingly, the invention further relates to nucleic acid
constructs, in particular nucleic acid constructs functioning as
expression cassettes, which contain at least one nucleic acid
selected from the group consisting of nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase, nucleic acids encoding a
.DELTA.5-desaturase and nucleic acids encoding a
.DELTA.24-reductase which are functionally linked to one or more
regulatory signals ensuring transcription and translation in
organisms.
[0328] In a preferred embodiment, said nucleic acid construct
additionally comprises at least one nucleic acid selected from the
group consisting of nucleic acids encoding an HMG-CoA-reductase
activity, nucleic acids encoding a lanosterol-C14-demethylase,
nucleic acids encoding a squalene epoxidase, nucleic acids encoding
a squalene synthetase and nucleic acids encoding a sterol
acyltransferase which are functionally linked to one or more
regulatory signals ensuring transcription and translation in
organisms.
[0329] As an alternative, it is also possible to prepare the
transgenic organisms of the invention by transformation with
individual nucleic acid constructs or with a combination of nucleic
acid constructs, said combination comprising at least one nucleic
acid construct selected from the groups A to C
A nucleic acid construct comprising nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase, which are functionally linked to one
or more regulatory signals ensuring transcription and translation
in organisms, B nucleic acid construct comprising nucleic acids
encoding a .DELTA.5-desaturase, which are functionally linked to
one or more regulatory signals ensuring transcription and
translation in organisms and C nucleic acid construct comprising
nucleic acids encoding a .DELTA.24-reductase, which are
functionally linked to one or more regulatory signals ensuring
transcription and translation in organisms, and at least one
nucleic acid construct selected from the groups D to H D nucleic
acid construct comprising nucleic acids encoding an HMG-CoA
reductase, which are functionally linked to one or more regulatory
signals ensuring transcription and translation in organisms, E
nucleic acid construct comprising nucleic acids encoding a
lanosterol C14-demethylase, which are functionally linked to one or
more regulatory signals ensuring transcription and translation in
organisms, F nucleic acid construct comprising nucleic acids
encoding a squalene epoxidase, which are functionally linked to one
or more regulatory signals ensuring transcription and translation
in organisms, G nucleic acid construct comprising nucleic acids
encoding a squalene synthetase, which are functionally linked to
one or more regulatory signals ensuring transcription and
translation in organisms, H nucleic acid construct comprising
nucleic acids encoding a sterol acyltransferase, which are
functionally linked to one or more regulatory signals ensuring
transcription and translation in organisms.
[0330] The regulatory signals preferably contain one or more
promoters which ensure transcription and translation in organisms,
in particular in yeasts.
[0331] The expression cassettes include regulatory signals, i.e.
regulatory nucleic acid sequences, which control expression of the
coding sequence in the host cell. According to a preferred
embodiment, an expression cassette comprises upstream, i.e. at the
5' end of the coding sequence, a promoter and downstream, i.e. at
the 3' end, a terminator and, where appropriate, further regulatory
elements which are operatively linked to the coding sequence for at
least one of the above-described genes located in between.
[0332] Operative linkage means the sequential arrangement of
promoter, coding sequence, where appropriate, terminator and, where
appropriate, further regulatory elements in such a way that each of
the regulatory elements can properly carry out its function in the
expression of the coding sequence.
[0333] The preferred nucleic acid constructs, expression cassettes
and plasmids for yeasts and fungi and methods for preparing
transgenic yeasts and also the transgenic yeasts themselves are
described by way of example below.
[0334] A suitable promoter of the expression cassette is in
principle any promoter which is able to control the expression of
foreign genes in organisms, in particular in yeasts.
[0335] Preference is given to using in particular a promoter which
is subject to reduced regulation in yeast, such as, for example,
the medium ADH promoter.
[0336] This promoter fragment of the ADH12s promoter, also referred
to as ADH1 hereinbelow, exhibits nearly constitutive expression
(Ruohonen L, Penttila M, Keranen S. (1991) Optimization of Bacillus
.alpha.-amylase production by Saccharomyces cerevisiae. Yeast.
May-June; 7(4):337-462; Lang C, Looman A C. (1995) Efficient
expression and secretion of Aspergillus niger RH5344
polygalacturonase in Saccharomyces cerevisiae. Appl Microbiol
Biotechnol. December; 44(1-2):147-56) so that transcriptional
regulation no longer proceeds via intermediates of ergosterol
biosynthesis.
[0337] Other preferred promoters with reduced regulation are
constitutive promoters such as, for example, the yeast TEF1
promoter, the yeast GPD promoter or the yeast PGK promoter (Mumberg
D, Muller R, Funk M. (1995) Yeast vectors for the controlled
expression of heterologous proteins in different genetic
backgrounds. Gene. 1995 Apr. 14; 156(1):119-22; Chen C Y, Oppermann
H, Hitzeman R A. (1984) Homologous versus heterologous gene
expression in the yeast, Saccharomyces cerevisiae. Nucleic Acids
Res. December 11; 12(23):8951-70).
[0338] The expression cassette may also contain inducible
promoters, in articular a chemically inducible promoter which can
be used to control expression of the nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase, .DELTA.5-desaturase,
.DELTA.24-reductase, HMG-CoA-reductase, lanosterol-C14-demethylase,
squalene epoxidase, squalene synthetase or sterol acyltransferase
in the organism at a particular time.
[0339] Promoters of this kind, such as, for example, the yeast Cupl
promoter (Etcheverry T. (1990) Induced expression using yeast
copper metallothionein promoter. Methods Enzymol. 1990;
185:319-29), the yeast Gall-10 promoter (Ronicke V, Graulich W,
Mumberg D, Muller R, Funk M. (1997) Use of conditional promoters
for expression of heterologous proteins in Saccharomyces
cerevisiae, Methods Enzymol. 283:313-22) or the yeast Pho5 promoter
(Bajwa W, Rudolph H, Hinnen A. (1987) PHO5 upstream sequences
confer phosphate control on the constitutive PHO3 gene. Yeast. 1987
March; 3(1):33-42), may be used, for example.
[0340] A suitable terminator of the expression cassette is in
principle any terminator which is able to control the expression of
foreign genes in organisms, in particular in yeasts.
[0341] Preference is given to the tryptophan terminator of yeasts
(TRP1 terminator).
[0342] An expression cassette is preferably prepared by fusing a
suitable promoter with the above-described nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase, .DELTA.5-desaturase,
.DELTA.24-reductase, HMG-CoA-reductase, lanosterol-C14-demethylase,
squalene epoxidase, squalene synthetase or sterol acyltransferase
and, where appropriate, a terminator according to common
recombination and cloning techniques as described, for example, in
T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1989) and in T. J. Silhavy, M. L. Berman and L. W.
Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M.
et al., Current Protocols in Molecular Biology, Greene Publishing
Assoc. and Wiley-Interscience (1987).
[0343] The nucleic acids of the invention may be prepared
synthetically or obtained naturally or may contain a mixture of
synthetic and natural nucleic acid components and may also comprise
various heterologous gene sections of various organisms.
[0344] As described above, preference is given to synthetic
nucleotide sequences with codons which are preferred by yeasts.
These codons which are preferred by yeasts may be determined from
codons which have the highest frequency in proteins and which are
expressed in most of the interesting yeast species.
[0345] When preparing an expression cassette, it is possible to
manipulate various DNA fragments in order to obtain a nucleotide
sequence which expediently can be read in the correct direction and
is provided with a correct reading frame. The DNA fragments may be
linked to one another by attaching adaptors or linkers to said
fragments.
[0346] Expediently, the promoter and terminator regions may be
provided in the direction of transcription with a linker or
polylinker which contains one or more restriction sites for
inserting this sequence. Normally, the linker has from 1 to 10,
mostly from 1 to 8, preferably from 2 to 6, restriction sites.
Generally, the linker is, within the regulatory regions, less than
100 bp, frequently less than 60 bp, but at least 5 bp, in length.
The promoter may be both native or homologous and non-native or
heterologous to the host organism. The expression cassette
preferably includes in the 5'-3' direction of transcription the
promoter, a coding nucleic acid sequence or a nucleic acid
construct and a region for transcriptional termination. Various
termination regions can be exchanged with one another randomly.
[0347] It is furthermore possible to use manipulations which
provide appropriate restriction cleavage sites or which remove
excess DNA or restriction cleavage sites. In those cases for which
insertions, deletions or substitutions such as, for example,
transitions and transversions are suitable, in vitro mutagenesis,
primer repair, restriction or ligation can be used.
[0348] In suitable manipulations such as, for example, restriction,
"chewing-back" or filling-in of protruding ends to form "blunt
ends", complementary fragment ends may be provided for
ligation.
[0349] The invention further relates to the use of the
above-described nucleic acids, the above-described nucleic acid
constructs or the above-described proteins for preparing transgenic
organisms, in particular yeasts.
[0350] Preferably, said transgenic organisms, in particular yeasts,
have an increased content of 7-dehydrocholesterol and/or of the
biosynthetic intermediates and/or secondary products thereof
compared to the wild type.
[0351] Therefore, the invention further relates to the use of the
above-described nucleic acids or the nucleic acid constructs of the
invention for increasing the content of 7-dehydrocholesterol and/or
of the biosynthetic intermediates and/or secondary products thereof
in organisms.
[0352] The above-described proteins and nucleic acids may be used
for producing 7-dehydrocholesterol and/or the biosynthetic
intermediates and/or secondary products thereof in transgenic
organisms.
[0353] The transfer of foreign genes into the genome of an
organism, in particular of yeast, is referred to as
transformation.
[0354] For this purpose, methods known per se can be used for
transformation, in particular in yeasts.
[0355] Examples of suitable methods for transforming yeasts are the
LiAC method as described in Schiestl R H, Gietz R D. (1989) High
efficiency transformation of intact yeast cells using single
stranded nucleic acids as a carrier, Curr Genet. December; 16
(5-6):339-46, electroporation as described in Manivasakam P,
Schiestl R H. (1993) High efficiency transformation of
Saccharomyces cerevisiae by electroporation. Nucleic Acids Res.
September 11; 21(18):4414-5, and the preparation of protoplasts, as
described in Morgan A J. (1983) Yeast strain improvement by
protoplast fusion and transformation, Experientia Suppl.
46:155-66
[0356] The construct to be expressed is preferably cloned into a
vector, in particular into plasmids which are suitable for
transforming yeasts, such as, for example, the vector systems Yep24
(Naumovski L, Friedberg E C (1982) Molecular cloning of eucaryotic
genes required for excision repair of UV-irradiated DNA: isolation
and partial characterization of the RAD3 gene of Saccharomyces
cerevisiae. J Bacteriol October; 152(1):323-31), Yep13 (Broach J R,
Strathern J N, Hicks J B. (1979) Transformation in yeast:
development of a hybrid cloning vector and isolation of the CAN1
gene. Gene. 1979 December; 8(1):121-33), the pRS series of vectors
(Centromer and Episomal) (Sikorski R S, Hieter P. (1989) A system
of shuttle vectors and yeast host strains designed for efficient
manipulation of DNA in Saccharomyces cerevisiae. Genetics. May;
122(1):19-27) and the vector systems YCp19 or pYEXBX.
[0357] Accordingly, the invention furthermore relates to vectors,
in particular plasmids, which comprise the above-described nucleic
acids, nucleic acid constructs or expression cassettes.
[0358] The invention further relates to a method for preparing
genetically modified organisms by functionally introducing an
above-described nucleic acid or an above-described nucleic acid
construct into the starting organism.
[0359] The invention further relates to said genetically modified
organisms, the genetic modification increasing at least one of the
activities selected from the group consisting of
.DELTA.8-.DELTA.7-isomerase activity, .DELTA.5-desaturase activity
and .DELTA.24-reductase activity, compared to a wild type.
[0360] Preferably, at least one of the activities is increased by
increasing the gene expression of at least one nucleic acid
selected from the group consisting of nucleic acids encoding a
.DELTA.8-.DELTA.7-isomerase, nucleic acids encoding a
.DELTA.5-desaturase and nucleic acids encoding a
.DELTA.24-reductase.
[0361] Preferably, gene expression of the above-described nucleic
acids is increased by increasing in the organism the copy number of
the nucleic acids encoding a .DELTA.8-.DELTA.7-isomerase, encoding
a .DELTA.5-desaturase and/or encoding a .DELTA.24-reductase.
[0362] Accordingly, the invention preferably relates to an
above-described genetically modified organism which contains two or
more nucleic acids encoding a .DELTA.8-.DELTA.7-isomerase and/or
two or more nucleic acids encoding a .DELTA.5-desaturase and/or two
or more nucleic acids encoding a .DELTA.24-reductase.
[0363] In a preferred embodiment, the genetically modified organism
has, compared to the wild type, in addition to the above-described
genetic modifications a reduced activity of at least one of the
activities selected from the group consisting of
C24-methyltransferase activity and .DELTA.22-desaturase
activity.
[0364] The reduction of at least one of the activities is
preferably caused by reducing, compared to the wild type, gene
expression of at least one nucleic acid selected from the group
consisting of nucleic acids encoding a C24-methyltransferase and
nucleic acids encoding a .DELTA.22-desaturase.
[0365] A particularly preferred genetically modified organism has,
apart from the above-described genetic modifications, no functional
C24-methyltransferase gene and/or .DELTA.22-desaturase gene.
[0366] Particular preference is given to above-mentioned
genetically modified organisms in which the genetic modification
additionally increases at least one of the activities selected from
the group consisting of HMG-CoA-reductase activity,
lanosterol-C14-demethylase activity, squalene-epoxidase activity,
squalene-synthetase activity and sterol-acyltransferase activity
compared to a wild type.
[0367] Preferably, at least one of these activities is increased,
as mentioned above, by increasing, compared to the wild type, gene
expression of at least one nucleic acid selected from the group
consisting of nucleic acids encoding an HMG-CoA-reductase activity,
nucleic acids encoding a lanosterol-C14-demethylase, nucleic acids
encoding a squalene epoxidase, nucleic acids encoding a squalene
synthetase and nucleic acids encoding a sterol acyltransferase.
[0368] Preferably, gene expression of at least one nucleic acid
selected from the group consisting of nucleic acids encoding an
HMG-CoA-reductase activity, nucleic acids encoding a
lanosterol-C14-demethylase, nucleic acids encoding a squalene
epoxidase, nucleic acids encoding a squalene synthetase and nucleic
acids encoding a sterol acyltransferase is increased compared to
the wild type by increasing in the organism the copy number of at
least one nucleic acid selected from the group consisting of
nucleic acids encoding an HMG-CoA-reductase activity, nucleic acids
encoding a lanosterol-C14-demethylase, nucleic acids encoding a
squalene epoxidase, nucleic acids encoding a squalene synthetase
and nucleic acids encoding a sterol acyltransferase.
[0369] Accordingly, the invention preferably relates to an
above-described genetically modified organism which contains two or
more of at least one nucleic acid selected from the group
consisting of nucleic acids encoding an HMG-CoA-reductase activity,
nucleic acids encoding a lanosterol-C14-demethylase, nucleic acids
encoding a squalene epoxidase, nucleic acids encoding a squalene
synthetase and nucleic acids encoding a sterol acyltransferase.
[0370] In particular, the invention preferably relates to a
genetically modified organism which contains, in addition to the
above-described genetic modifications, two or more nucleic acids
encoding an HMG-CoA-reductase and/or two or more nucleic acids
encoding a lanosterol-C14-demethylase and/or two or more nucleic
acids encoding a squalene epoxidase and/or two or more nucleic
acids encoding a squalene synthetase and/or two or more nucleic
acids encoding a sterol acyltransferase.
[0371] The above-described genetically modified organisms have,
compared to the wild type, an increased content of
7-dehydrocholesterol and/or of the biosynthetic intermediates
and/or secondary products thereof.
[0372] Accordingly, the invention relates to an above-described
genetically modified organism which, compared to the wild type, has
an increased content of 7-dehydrocholesterol and/or of the
biosynthetic intermediates and/or secondary products thereof.
[0373] Preferred genetically modified organisms are yeasts or fungi
which have been genetically modified according to the invention, in
particular yeasts which have been genetically modified according to
the invention, in particular the yeast species Saccharomyces
cerevisiae which has been genetically modified according to the
invention, in particular the genetically modified yeast strains
Saccharomyces cerevisiae AH22, Saccharomyces cerevisiae GRF,
Saccharomyces cerevisiae DBY747 and Saccharomyces cerevisiae
BY4741.
[0374] In the scope of the present invention, increasing the
content of 7-dehydrocholesterol and/or of the biosynthetic
intermediates and/or secondary products thereof preferably means
the artificially acquired ability to produce biosynthetically an
increased amount of at least one of these compounds mentioned above
in the genetically modified organism compared to the genetically
unmodified organism.
[0375] Accordingly, as mentioned at the beginning, wild type
preferably means the genetically unmodified organism, but in
particular the reference organism mentioned above.
[0376] An increased content of 7-dehydrocholesterol and/or of the
biosynthetic intermediates and/or secondary products thereof in
comparison with the wild type means in particular the increase in
the content of at least one of the abovementioned compounds in the
organism by at least 50%, preferably 100%, more preferably 200%,
particularly preferably 400%, in comparison with the wild type.
[0377] The content of at least one of the mentioned compounds is
preferably determined according to analytical methods known per se
and preferably refers to those compartments of the organism, in
which sterols are produced.
[0378] The invention is illustrated by the following examples but
is not limited to them:
I. GENERAL EXPERIMENTAL CONDITIONS
1. Restriction
[0379] Restriction of the plasmids (1 to 10 .mu.g) was carried out
in 30 .mu.l reaction mixtures. For this purpose, the DNA was taken
up in 24 .mu.l of H.sub.20 and admixed with 3 .mu.l of the
appropriate buffer, 1 ml of BSA (bovine serum albumin) and 2 .mu.l
of enzyme. The enzyme concentration was 1 unit/.mu.l or 5
units/.mu.l, depending on the amount of DNA. In some cases, 1 .mu.l
of RNase was added to the reaction mixture in order to degrade the
tRNA. The restriction mixture was incubated at 37.degree. C. for 2
hours. The restriction was monitored using a minigel.
2. Gel Electrophoreses
[0380] The gel electrophoreses were carried out in minigel or wide
minigel apparatuses. The minigels (approx. 20 ml, 8 pockets) and
the wide minigels (50 ml, 15 or 30 pockets) consisted of 1%
strength agarose in TAE. The running buffer used was
1.times.TAE.
[0381] After adding 3 .mu.l of stop solution, the samples (10
.mu.l) were applied. A-DNA cut with HindIII (bands at: 23.1 kb; 9.4
kb; 6.6 kb; 4.4 kb; 2.3 kb; 2.0 kb; 0.6 kb) served as standard. For
fractionation, a voltage of 80 V was applied for 45 to 60 min.
Thereafter, the gel was stained in ethidium bromide solution and
documented under UV light using the INTAS video documentation
system or photographed using an orange filter.
3. Gel Elution
[0382] The desired fragments were isolated by means of gel elution.
The restriction mixture was applied to several pockets of a minigel
and fractionated. Only .lamda.-HindIII and a "sacrifice lane" were
stained in ethidium bromide solution, examined under UV light, and
the desired fragment was marked. This prevented the DNA of the
remaining pockets from being damaged by ethidium bromide and UV
light. Putting the stained and unstained gel slices side by side
made it possible to excise the desired fragment from the unstained
gel slice on the basis of the marking. The agarose slice with the
fragment to be isolated was introduced into a dialysis tube, sealed
in air-bubble-free together with a small amount of TAE buffer and
introduced into the BioRad minigel apparatus. The running buffer
was 1.times.TAE and the voltage was 100 V for 40 min. Afterward,
the polarity was switched for 2 min in order to redissolve DNA
sticking to the dialysis tube. The buffer in the dialysis tube,
which contained the DNA fragments, was transferred to reaction
vessels and subjected to ethanol precipitation. For this purpose,
1/10 volume of 3M sodium acetate, tRNA (1 .mu.l per 50 .mu.l of
solution) and 2.5 volumes of ice-cold 96% strength ethanol were
added to the DNA solution. The mixture was incubated at -20.degree.
C. for 30 min and then removed by centrifugation at 12 000 rpm,
4.degree. C., 30 min. The DNA pellet was dried and taken up in 10
to 50 .mu.l of H.sub.20 (depending on the amount of DNA).
4. Klenow Treatment
[0383] The Klenow treatment fills in protruding ends of DNA
fragments, resulting in blunt ends. Per 1 .mu.g of DNA, the
following reaction mixture was pipetted:
DNA.times..times.pellet+.times.11.times..mu..times..times.1.times..times.-
H2.times.0+.times.1.5.times..times.10.times.Klenow.times..times.buffer+.ti-
mes.1.times..mu..times..times.I.times..times.0.1.times..times.M.times..tim-
es.DTT+.times.1.times..mu..times..times.I.times..times.nucleotide.times..t-
imes.(dNTP.times..times.2.times..times.mM)+.times.1.times..mu..times..time-
s.I.times..times.Klenow.times..times.polymerase.times..times.(1.times..tim-
es.unit/.mu..times..times.I) 25
[0384] The DNA should be from an ethanol precipitation, in order to
prevent contaminations from inhibiting the Klenow polymerase. The
reaction mixture was incubated at 37.degree. C. for 30 min, and the
reaction was stopped by incubating for another 5 min at 70.degree.
C. The DNA was recovered from the reaction mixture by ethanol
precipitation and taken up in 10 .mu.l of H.sub.20.
5. Ligation
[0385] The DNA fragments to be ligated were combined. The final
volume of 13.1 .mu.l contained approx. 0.5 .mu.l of DNA with a
vector/insert ratio of 1:5. The sample was incubated at 70.degree.
C. for 45 seconds, cooled to room temperature (approx. 3 min) and
then incubated on ice for 10 min. The ligation buffers were then
added: 2.6 .mu.l of 500 mM Tris-HCl pH 7.5 and 1.3 .mu.l of 100 mM
MgCl.sub.2, followed by incubation on ice for a further 10 min.
After adding 1 .mu.l of 500 mM DTT and 1 .mu.l of 10 mM ATP and
another 10 min on ice, 1 .mu.l of ligase (1 unit/pl) was added. The
whole treatment should be carried out as free from vibrations as
possible so that adjoining DNA ends are not separated again. The
ligation was carried out at 14.degree. C. over night.
6. Transformation of E. coli
[0386] Competent Escherichia coli (E. coli) NM522 cells were
transformed with the DNA of the ligation mixture. A reaction
mixture containing 50 .mu.g of the pScL3 plasmids and a reaction
mixture without DNA were run as positive control and zero control,
respectively. For each transformation mixture, 100 .mu.l of 8% PEG
solution, 10 .mu.l of DNA and 200 .mu.l of competent cells (E. coli
NM522) were pipetted into a benchtop-centrifuge tube. The reaction
mixtures were put on ice for 30 min and agitated occasionally.
[0387] Then the heat shock was carried out: 1 min at 42.degree. C.
For regeneration, 1 ml of LB medium was added to the cells and the
suspension was incubated on a shaker at 37.degree. C. for 90 min.
In each case, 100 .mu.l of the undiluted reaction mixtures, a 1:10
dilution and a 1:100 dilution were plated on LB+ampicillin plates
and incubated at 37.degree. C. over night.
7. Plasmid Isolation from E. coli (Miniprep)
[0388] E. coli colonies were grown in 1.5 ml of LB+ampicillin
medium in benchtop-centrifuge tubes at 37.degree. C. and 120 rpm
over night. On the next day, the cells were removed by
centrifugation at 5000 rpm and 4.degree. C. for 5 min and the
pellet was taken up in 50 .mu.l of TE buffer. 100 .mu.l of 0.2 N
NaOH, 1% SDS solution were added to and mixed with each reaction
mixture, and the mixture was put on ice for 5 min (lysis of the
cells). Then, 400 .mu.l of Na acetate/NaCl solution (230 .mu.l of
H.sub.20, 130 .mu.l of 3 M sodium acetate, 40 .mu.l of 5M NaCl)
were added, the reaction mixture was mixed and put on ice for a
further 15 min (protein precipitation). After centrifugation at 11
000 rpm for 15 minutes, the supernatant containing the plasmid DNA
was transferred to an Eppendorf vessel. If the supernatant was not
completely clear, centrifugation was repeated. 360 .mu.l of
ice-cold isopropanol were added to the supernatant and the reaction
mixture was incubated at -20.degree. C. for 30 min (DNA
precipitation). The DNA was removed by centrifugation (15 min, 12
000 rpm, 4.degree. C.), the supernatant was discarded, the pellet
was washed in 100 .mu.l of ice-cold 96% strength ethanol, incubated
at -20.degree. C. for 15 min and again removed by centrifugation
(15 min, 12 000 rpm, 4.degree. C.). The pellet was dried in a Speed
Vac and then taken up in 100 .mu.l of H.sub.20. The plasmid DNA was
characterized by restriction analysis. For this purpose, 10 .mu.l
of each reaction mixture were restriction-digested and fractionated
gel-electrophoretically in a wide minigel (see above).
8. Plasmid Preparation from E. coli (Maxiprep)
[0389] In order to isolate larger amounts of plasmid DNA, the
maxiprep method was carried out. Two flasks with 100 ml of
LB+ampicillin medium were inoculated with a colony or with 100
.mu.l of a frozen culture which carries the plasmid to be isolated
and incubated at 37.degree. C. and 120 rpm over night. On the next
day, the culture (200 ml) was transferred to a GSA beaker and
centrifuged at 4000 rpm (2600.times.g) for 10 min. The cell pellet
was taken up in 6 ml of TE buffer. The cell wall was digested by
adding 1.2 ml of lysozyme solution (20 mg/ml of TE buffer) and
incubated at room temperature for 10 min. Subsequently, the cells
were lysed with 12 ml of a 0.2 N NaOH, 1% SDS solution, followed by
incubation at room temperature for another 5 min. The proteins were
precipitated by adding 9 ml of a cooled 3 M sodium acetate solution
(pH 4.8) and incubation on ice for 15 minutes. After centrifugation
(GSA: 13 000 rpm (27 500.times.g), 20 min, 4.degree. C.), the
supernatant containing the DNA was transferred to a new GSA beaker
and the DNA was precipitated with 15 ml of ice-cold isopropanol and
incubation at -20.degree. C. for 30 min. The DNA pellet was washed
in 5 ml of ice-cold ethanol and dried in air (approx. 30-60 min).
Thereafter, it was taken up in 1 ml of H.sub.2O. The plasmid was
checked by restriction analysis. The concentration was determined
by applying dilutions to a minigel. The salt content was reduced by
microdialysis (pore size 0.025 .mu.m) for 30-60 minutes.
9. Transformation of Yeast
[0390] For the transformation of yeast, a preculture of the strain
Saccharomyces cerevisiae AH22 was prepared. A flask containing 20
ml of YE medium was inoculated with 100 .mu.l of the frozen culture
and incubated at 28.degree. C. and 120 rpm over night. The main
culture was carried out under the same conditions in flasks
containing 100 ml of YE medium which was inoculated with 10 .mu.l,
20 .mu.l or 50 .mu.l of the preculture.
9.1 Preparation of Competent Cells
[0391] On the next day, the cells in the flasks were counted by
means of a Thoma chamber and the flask containing from
3-5.times.10.sup.7 cells/ml was chosen for the subsequent
procedure. The cells were harvested by centrifugation (GSA: 5000
rpm (4000.times.g) 10 min). The cell pellet was taken up in 10 ml
of TE buffer and distributed into two benchtop-centrifuged tubes (5
ml each). The cells were removed by centrifugation at 6000 rpm for
3 min and then washed twice with in each case 5 ml of TE buffer.
The cell pellet was then taken up in 330 .mu.l of lithium acetate
buffer per 10.sup.9 cells, transferred to a sterile 50 ml
Erlenmeyer flask and agitated at 28.degree. C. for one hour. As a
result, the cells were competent for transformation.
9.2 Transformation
[0392] For each transformation mixture, 15 .mu.l of herring sperm
DNA (10 mg/ml), 10 .mu.l of the DNA to be transformed (approx. 0.5
.mu.g) and 330 .mu.l of competent cells were pipetted into a
benchtop-centrifuged tube and incubated at 28.degree. C. for 30 min
(without agitation). Then, 700 .mu.l of 50% PEG 6000 were added and
the suspension was incubated at 28.degree. C. for another hour,
without agitation. This was followed by a heat shock at 42.degree.
C. for 5 min. 100 .mu.l of the suspension were plated on selection
medium (YNB, Difco) in order to select for leucine prototrophy. In
the case of selection for G418 resistance, the cells are
regenerated after the heat shock (see under 9.3 Regeneration
phase).
9.3 Regeneration Phase
[0393] Since the selection marker is the resistance to G418, the
cells needed time to express the resistance gene. 4 ml of YE medium
were added to the transformation mixtures which were then incubated
on the shaker (120 rpm) at 28.degree. C. over night. On the next
day, the cells were removed by centrifugation (6000 rpm, 3 min),
taken up in 1 ml YE medium, and 100 .mu.l or 200 .mu.l thereof were
plated on YE+G418 plates. The plates were incubated at 28.degree.
C. for several days.
10. PCR Reaction Conditions
[0394] The reaction conditions for the polymerase chain reaction
must be optimized in each individual case and do not apply
absolutely to each reaction mixture. Thus it is possible, inter
alia, to vary the amount of DNA used, the salt concentrations and
the melting temperature. For our task, it proved advantageous to
combine in an Eppendorf vessel which was suitable for use in a
thermocycler the following substances: 5 .mu.l of Super buffer, 811
of dNTPs (0.625 .mu.M each), 5' primer, 3' primer and 0.2 .mu.g of
template DNA, dissolved in enough water so as to result in a total
volume of 50 .mu.l for the PCR reaction mixture, were added to 2
.mu.l of (=0.1 U) Super Taq polymerase. The reaction mixture was
briefly centrifuged and overlaid with a drop of oil. Between 37 and
40 cycles were chosen for amplification.
II. EXAMPLES
Example 1
[0395] Expression and overexpression of a truncated HMG-CoA
reductase, a squalene epoxidase (ERG1) and/or a
lanosterol-C14-demethylase (ERG11), partially with deletion of ERG5
and ERG6 in S. cerevisiae GRF18 and GRFura3, respectively.
1.1 Preparation of the Plasmids pFlat1 and pFlat3 and pFlat4
[0396] The expression vector pFlat3 was prepared by linearizing the
plasmid YEp24 (Naumovski L, Friedberg E C (1982) Molecular cloning
of eucaryotic genes required for excision repair of UV-irradiated
DNA: isolation and partial characterization of the RAD3 gene of
Saccharomyces cerevisiae. J Bacteriol October; 152(1):323-31) via
restriction with SphI and a 900 by SphI fragment of the vector
pPT2B (Lang C, Looman A C. (1995) Efficient expression and
secretion of Aspergillus niger RH5344 polygalacturonase in
Saccharomyces cerevisiae. Appl Microbiol Biotechnol. December;
44(1-2): 147-56) which contains the ADH1 promoter and the TRP1
terminator of the yeast Saccharomyces cerevisiae and a
multiple-cloning site of the vector pUC19 (Yanisch-Perron C, Vieira
J, Messing J. (1985) Improved M13 phage cloning vectors and host
strains: nucleotide sequences of the M13 mp18 and pUC19 vectors.
Gene. 1985; 33(1): 103-19) was integrated.
[0397] The multiple-cloning site was extended by a polylinker
containing the restriction sites NotI and XhoI. The polylinker was
integrated via the SalI cleavage site of the vector. The resulting
plasmid is denoted pFlat1.
[0398] The vector pFlat3 was prepared by linearizing the vector
pFlat1 by the enzyme NcoI and blunt-ending it by means of Klenow
treatment. This was followed by integrating a BamHI fragment which
had been blunt-ended by means of Klenow-polymerase treatment and
which contains the yeast LEU2 gene and originates from the plasmid
YDpL (Berben, G., Dumont, J., Gilliquet, V., Bolle, P. A. and
Hilger F. (1991) The YDp Plasmids: a Uniform Set of Vectors Bearing
Versatile Disruption Cassettes for Saccharomyces cerevisiae. Yeast
7: 475-477).
[0399] The vector pFlat4 was prepared by linearizing the vector
pFlat1 by the enzyme NcoI and blunt-ending it by means of Klenow
treatment. This was followed by integrating a BamHI fragment which
had been blunt-ended by means of Klenow-polymerase treatment and
which contains the yeast HIS3 gene and originates from plasmid YDpH
(Berben et al., 1991).
1.2 Integration of ERG1, ERG11, ERG4, ERG2 or ERG3 or of the
.DELTA.24-Reductase Gene into the Vectors pFLat1, pFlat3 and
pFlat4
[0400] First, a NotI restriction cleavage site was inserted at the
5'-coding side of the genes ERG1, ERG11, ERG4, .DELTA.24-reductase,
ERG2 or ERG3 and an XhoI restriction cleavage site was inserted at
the 3'-coding side of said genes by means of PCR and the
corresponding coding regions were amplified. Subsequently, the
amplicons were treated with the restriction enzymes NotI and XhoI.
The plasmids pFlat1, pFlat3 and pFlat4 were treated in parallel
with enzymes NotI and XhoI. The cleaved amplicons were then
integrated into the cleaved plasmids via ligation using T4 ligase.
FIG. 7 depicts as an example the plasmid pFLAT-3-ERG4.
[0401] Primer sequences for cloning ERG1, ERG11, ERG2, ERG3, ERG4,
.DELTA.24-reductase: TABLE-US-00002 Primer ERG1-5' (SEQ. ID. No.
51):
CTGCGGCCGC ATCATGTCTG CTGTTAACGT TGC Primer ERG1-3' (SEQ. ID. No.
52): TTCTCGAGTT AACCAATCAA CTCACCAAAC Primer ERG11-5' (SEQ. ID. No.
53): CTGCGGCCGCAGGATGTCTGCTACCAAGTCAATCG Primer ERG11-3' (SEQ. ID.
No. 54):
ATCTCGAGCTTAGATCTTTTGTTCTGGATTTCTC Primer ERG2-5' (SEQ. ID. No.
55): CTGCGGCCGCACCATGAAGTTTTT000ACT CC Primer ERG2-3' (SEQ. ID. No.
56): TTCTCGAGTTAGAACTTTTTGTTTTGCAACAAG Primer ERG3-5' (SEQ. ID. No.
57):
CTGCGGCCGCAATATGGATTTGGTCTTAGAAGTCG Primer E RG3-3' (SEQ. ID. No.
58): AACTCGAGTCAGTTGTTCTTCTTGGTATTTG Primer ERG4-5' (SEQ. ID. No.
59): CTGCGGCCGCACTATGGCAAAGGATAATAGTGAG Primer ERG4-3' (SEQ. ID.
No. 60):
[0402] TTCTCGAGCTAGAAAACATAAGGAATAAAGAC Primer ti24R-5' (SEQ. ID.
No. 47): CTGCGGCCGCAAGATGGAG000GCCGTGTCGC Primer .DELTA.24R-3'
(SEQ. ID. No. 48) AACTCGAGTCAGTGCCTTGCCGCCTTGC 1.3 Preparation of
the Integration Vectors pUG6-tHMG, pUG6-ERG1, pUG6-ERG11 1.3.1
pUG6-tHMG
[0403] The DNA sequence for the expression cassette composed of
ADH1-promoter-tHMG-tryptophan-terminator was isolated from the
vector YepH2 (Polakowski, T., Stahl, U., Lang, C. (1998):
Overexpression of a cytosolic HMG-CoA reductase in yeast leads to
squalene accumulation. Appl. Microbiol. Biotechnol. 49: 66-71) by
restriction with the enzymes EcoRV and Bsp68I (NruI) by using
standard methods. The DNA fragment obtained was cloned with blunt
ends into the EcoRV cleavage site of the vector pUG6 (Guldener, U
et al. (1996): A new efficient gene disruption cassette for
repeated use in budding yeast, Nucleic Acids Res. July 1;
24(13):2519-24), resulting in the vector denoted pUG6-tHMG (FIG.
1).
1.3.2 pUG6-ERG1
[0404] The DNA sequence for the expression cassette composed of
ADH1-promoter-ERG1-tryptophan-terminator was isolated from the
vector pFlat3-ERG1 by restriction with the enzymes NheI and Bsp68I
(NruI), using standard methods. After Klenow treatment, the DNA
fragment obtained was cloned with blunt ends into the EcoRV
cleavage site of the vector pUG6 (Guldener, U et al. (1996): A new
efficient gene disruption cassette for repeated use in budding
yeast, Nucleic Acids Res. July 1; 24(13):2519-24), resulting in the
vector denoted pUG6-ERG1 (FIG. 2).
1.3.3 pUG6-ERG11
[0405] The DNA sequence for the expression cassette composed of
ADH1-promotor-ERG11-tryptophan-terminator was isolated from the
vector pFlat3-ERG11 by restriction with the enzymes EcoRV and
Bsp68I (NruI) using standard methods. The DNA fragment obtained was
cloned with blunt ends into the EcoRV cleavage site of the vector
pUG6 (Guldener, U et al. (1996): A new efficient gene disruption
cassette for repeated use in budding yeast, Nucleic Acids Res. July
1; 24(13):2519-24), resulting in the vector denoted pUG6-ERG11
(FIG. 3).
1.4. Integrative Transformation of the Expression Cassettes into
the Yeast Strains GRF or GRFura3
[0406] After plasmid isolation, fragments of the vectors pUG6-tHMG,
pUG6-ERG1 and pUG6-ERG11 were amplified by means of PCR in such a
way that the resulting fragments consist of the following
components: loxP-kanMX-loxP-ADH1 promoter-target gene-tryptophan
terminator, with target gene meaning tHMG, ERG1 and, ERG11 and
kanMX respectively, meaning a kanamycin-resistance gene.
[0407] The selected primers were oligonucleotide sequences which
contain in the annealing region the sequences beyond the cassettes
to be amplified of the vector pUG6-target gene and which contain at
the 5' and 3' protruding ends in each case 40 base pairs of the 5'
or 3' sequence of the integration locus. This ensures that on the
one hand the entire fragment, including KanMX and target gene, is
amplified and, on the other hand, this fragment can then be
transformed into yeast and be integrated by homologous
recombination into the target gene locus of the yeast. Depending on
the target gene locus in the yeast, the following oligonucleotide
sequences were used as primers:
[0408] For integration at the URA3 gene locus: TABLE-US-00003 For
integration at the URA3 gene locus: URA3-Crelox-5' (SEQ. ID. No.
33): 5'-ATGTCGAAAG CTACATATAA GGAACGTGCT GCATCTCATC CCAGCTGAAG
CTTCGTACGC-3' URA3-Crelox-3' (SEQ. ID. No. 34): 5'-TTAGTTTTGC
TGGCCGCATC TTCTCAAATA TGCTTCCCAG GCATAGGCCA CTAGTGGATC TG-3' For
integration at the LEU2 gene locus: LEU2-Crelox-5' (SEQ. ID. No.
35): 5'-GAATACTCAG GTATCGTAAG ATGCAAGAGT TCGAATCTCT CCAGCTGAAG
CTTCGTACGC-3' LEU2-Crelox-3' (SEQ. ID. No. 36): 5'-TCTACCCTAT
GAACATATTC CATTTTGTAA TTTCGTGTCG GCATAGGCCA CTAGTGGATC TG-3'
[0409] For integration at the HIS3 gene locus: TABLE-US-00004
HIS3-Crelox-5' (SEQ. ID. No. 37): 5'-ATGACAGAGC AGAAACCCCT
AGTAAAGCGT ATTACAAATG CCAGCTGAAG CTTCGTACGC-3' HIS3-Crelox-3' (SEQ.
ID. No. 38): 5'-CTACATAAGA ACACCTTTGG TGGAGGGAAC ATCGTTGGTA
GCATAGGCCA CTAGTGGATC TG-3'
[0410] For integration at the ERG6 gene locus: TABLE-US-00005
ERG6-Crelox-5' (SEQ. ID. No. 39): 5'-ATGAGTGAAA CAGAATTGAG
AAAAAGACAG GCCCAATTCA CCAGCTGAAG CTTCGTACGC-3' ERG6-Crelox-3' (SEQ.
ID. No. 40): 5'-TTATTGAGTT GCTTCTTGGG AAGTTTGGGA GGGGGTTTCG
GCATAGGCCA CTAGTGGATC TG-3'
[0411] For integration at the ERG5 gene locus: TABLE-US-00006
ERG5-Crelox-5' (SEQ. ID. No. 41): 5'-ATGAGTTCTG TCGCAGAAAA
TATAATACAA CATGCCACTC CCAGCTGAAG CTTCGTACGC-3' ERG5-Crelox-3' (SEQ.
ID. No. 42): 5'-TTATTCGAAG ACTTCTCCAG TAATTGGGTC TCTCTTTTTG
GCATAGGCCA CTAGTGGATC TG-3'
[0412] The resistance to Geneticin (G418) served as selection
marker. The resulting strains contained a copy of the particular
target gene (tHMG, ERG1 or ERG11) under the control of the ADH
promoter and the tryptophan terminator. At the same time, it was
possible to delete the particular gene of the target locus by
integrating the expression cassette. In order to subsequently
remove again the gene for G418 resistance, the resultant yeast
strain was transformed with the cre recombinase-containing vector
pSH47 (Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann J H.
(1996) A new efficient gene disruption cassette for repeated use in
budding yeast. Nucleic Acids Res. July 1; 24(13):2519-24). This
vector caused the expression of cre recombinase in the yeast, and,
as a consequence, the sequence region within the two loxP sequences
was removed by recombination, and this in turn resulted in only one
of the two loxP sequences and the ADH1 promoter-target
gene-tryptophan terminator expression cassette remaining in the
target gene locus.
[0413] As a consequence, the yeast strain loses its G418 resistance
again and is therefore suitable for integrating or removing further
genes by means of this "cre-lox" system into or from said yeast
strain. The vector pSH47 can then be removed selectively by
cultivation on FOA medium.
[0414] Thus it is possible to integrate a plurality of target genes
successively into the yeast strain under the control of the ADH1
promoter and tryptophan terminator at various target loci.
[0415] First, a target gene is integrated at the URA3 locus or a
ura3 strain is used in order to render the yeast strain
uracil-auxotrophic, since the vector pSH47 contains a URA3 gene for
selection of uracil-prototrophic strains. FIG. 4 shows an example
of the method.
[0416] This method produced the yeast integration and deletion
strains listed in Table 1, with, in a manner known per se, the gene
in lower-case letters representing a deletion and the gene in
capital letters representing an integration.
TABLE-US-00001 TABLE 1 Modification No. Strain name No. Strain name
compared to GRF yeast strain I GRFtH1 ura3, tHMG:leu2 II GRFth1e1
ERG1:ura3, tHMG:leu2 III GRFtH1E11 ura3, tHMG:leu2, ERG11:his3 IV
GRFtH1E1E11 ERG1:ura3, tHMG:leu2, ERG11:his3 V GRFtH1E1E11erg5erg6
ura3, tHMG:leu2, ERG1:erg6, ERG11:erg5 VI GRFtH1erg5erg6 ura3,
tHMG:leu2, erg5, erg6
[0417] The yeast strains were cultured in a culture volume of 20 ml
in WMVIII medium at 28.degree. C. and 160 rpm for 48 hours.
Subsequently, 500 .mu.l of this preculture were transferred to a 50
ml main culture of the same medium and cultured in a baffled flask
at 28.degree. C. and 160 rpm for 3 days.
[0418] After 3 days, the sterols and squalene were extracted (Parks
L W, Bottema C D, Rodriguez R J, Lewis T A. (1985) Yeast sterols:
yeast mutants as tools for the study of sterol metabolism. Methods
Enzymol. 1985; 111:333-46) and analyzed by means of gas
chromatography. The following values were obtained (see Table
2).
TABLE-US-00002 Content of sterols 1 to 11 in [peak area/gTS] No.
Strain name 1 2 3 4 5 6 7 8 9 10 11 I GRFtH1 9.9 0.8 0.3 1.2 1.1
1.0 0.0 0.0 0.0 0.0 4.7 II GRFtH1E1 6.8 1.9 0.4 1.5 2.2 2.1 0.0 0.0
0.0 0.0 6.9 III GRFtH1E11 9.9 0.4 0.7 2.3 1.9 1.9 0.0 0.0 0.0 0.0
5.0 IV GRFtH1E1E11 6.0 1.2 0.9 3.0 2.3 2.2 0.0 0.0 0.0 0.0 7.2 V
GRFtH1E1E11 5.8 0.8 0.4 23.1 0.0 0.0 0.0 0.0 11.8 0.0 0.0 erg5erg6
VI GRFtH1erg5erg6 9.9 0.8 0.3 12.6 0.0 0.0 0.0 0.0 7.1 0.0 0.0 1 =
Squalene 2 = Lanosterol 3 = Dimethylzymosterol 4 = Zymosterol 5 =
Fecosterol 6 = Episterol 7 = Cholesta-7,24-dienol 8 =
Cholesta-8-enol 9 = Cholesta-5,7,24 trienol 10 =
7-Dehydrocholesterol 11 = Ergosterol
Example 2
Expression of the Heterologous Gene Encoding a
.DELTA.8-.DELTA.7-Isomerase (Ebp) from Mice (Mus musculus) in
Yeast
[0419] The cDNA sequence of Mus musculus
.DELTA.8-.DELTA.7-isomerase (Moebius, F. F., Soellner, K. E. M.,
Fiechter, B., Huck, C. W., Bonn, G., Glossmann, H. (1999):
Histidine-77, Glutamic Acid123, Threonine126, Asparagine194, and
Tryptophan197 of Human Emopamil Protein Are Required for in Vivo
Sterol .DELTA.8-.DELTA.7 Isomerisation. Biochem. 38, 1119-1127) was
amplified by PCR from the cDNA clone IMAGp998A22757 (Host: E. coli
DH10B) of the Deutsches Resourcenzentrum fur Genomforschung [German
resource center for genome research] GmbH (Berlin).
[0420] The primers used here are the DNA oligomers Ebp-5' (SEQ. ID.
No. 43) and Ebp-3' (SEQ. ID. No. 44). The DNA fragment obtained was
treated with restriction enzymes NotI and XhoI and then integrated
into the vectors pFlat3 and pFlat1 (FIG. 4) which likewise been
treated with the enzymes NotI and XhoI beforehand by means of a
ligase reaction. The resulting vectors pFlat1-EBP and pFlat3-EBP
(FIG. 5a) contain the EBP gene under the control of the ADH
promoter and the tryptophan terminator.
[0421] The expression vector pFlat3-EBP was then transformed into
the yeast strains I to VI of Table 1 from Example 1 and also into
the GRFura3 strain. The yeast strains obtained in this way were
then cultured in a culture volume of 20 ml in WMVIII medium at
28.degree. C. and 160 rpm for 48 hours. Subsequently, 500 .mu.l of
this preculture were transferred to a 50 ml main culture of the
same medium and cultured in a baffled flask at 28.degree. C. and
160 rpm for 3 days.
[0422] The sterols were extracted after 3 days and analyzed by
means of gas chromatography, as described in Example 1. The
influence of the expression of a Mus musculus
.DELTA.8-.DELTA.7-isomerase in combination with the expression of
the transcriptionally deregulated intrinsic yeast genes tHMG and/or
ERG1 and/or ERG11 and/or deletion of the intrinsic yeast genes ERG6
and ERG5 is listed in Table 3. The abbreviations have the following
meanings: -=decrease; 0=no change; /=not present; +, ++, +++,
++++=concentrated to highly concentrated.
TABLE-US-00003 Influence of the genetic modifications on the sterol
content compared to the GRF yeast strain No. Strain name 1 2 3 4 5
6 7 8 9 10 11 VII GRFtH1 0 0 0 0 0 0 / / / / 0 pFlat3-Ebp VIIII
GRFtH1E1 0 0 0 - 0 0 + / / / 0 pFlat3-Ebp IX GRFtH1E11pFlat3- 0 0 0
- 0 0 + / / / 0 Ebp X GRFtH1E1E11 pFlat3- 0 0 0 - 0 0 + / / / 0 Ebp
XI GRFtH1E1E11erg5erg6 0 0 0 - / / + / ++ / / pFlat3-Ebp XII
GRFtH1erg5erg6 0 0 0 - / / + / + / / pFlat3-Ebp 1 = Squalene 2 =
Lanosterol 3 = Dimethylzymosterol 4 = Zymosterol 5 = Fecosterol 6 =
Episterol 7 = Cholesta-7,24-dienol 8 = Cholesta-8-enol 9 =
Cholesta-5,7,24 trienol 10 = 7-Dehydrocholesterol 11 =
Ergosterol
Example 3
Expression of the Heterologous Gene Encoding a .DELTA.5-Desaturase
(Sc5d) from Mice (Mus musculus) in Yeast
[0423] The cDNA sequence of Mus musculus .DELTA.5-desaturase
(Nishi, S., Hideaki, N., Ishibashi, T. (2000): cDNA cloning of the
mammalian sterol C5-desaturase and the expression in yeast mutant.
Biochim. Biophys. A 1490, 106-108) was amplified by PCR from the
cDNA clone IMAGp998K144618 (Host: E. coli DH10B) of the Deutsches
Resourcenzentrum fur Genomforschung [German resource center for
genome research] GmbH (Berlin). The primers used here are the DNA
oligomers Sc5d-5' (SEQ. ID. No. 45) and Sc5d-3' (SEQ. ID. No. 46).
The DNA fragment obtained was treated with restriction enzymes NotI
and XhoI and then integrated into the vector pFlat3 (FIG. 4) which
likewise had been treated with the enzymes NotI and XhoI
beforehand, by means of a ligase reaction. The resulting vector
pFlat3-SC5D (FIG. 5b) contains the SC5D gene under the control of
the ADH promoter and the tryptophan terminator.
[0424] The expression vector pFlat3-SC5D was then transformed into
the yeast strains I to VI of Table 1 from Example 1 and also into
the GRFura3 strain. The yeast strains obtained in this way were
then cultured in a culture volume of 20 ml in WMVIII medium at
28.degree. C. and 160 rpm for 48 hours. Subsequently, 500 .mu.l of
this preculture were transferred to a 50 ml main culture of the
same medium and cultured in a baffled flask at 28.degree. C. and
160 rpm for 3 days.
[0425] The sterols were extracted after 3 days and analyzed by
means of gas chromatography, as described in Example 1. The
influence of the expression of a Mus musculus .DELTA.5-desaturase
in combination with the expression of the transcriptionally
deregulated intrinsic yeast genes tHMG and/or ERG1 and/or ERG11
and/or deletion of the intrinsic yeast genes ERG6 and ERG5 is
listed in Table 4. The abbreviations have the following meanings:
-=decrease; 0=no change; /=not present; +, ++, +++,
++++=concentrated to highly concentrated.
TABLE-US-00004 TABLE 4 Influence of the genetic modifications on
the sterol content compared to the GRF yeast strain No. Strain name
1 2 3 4 5 6 7 8 9 10 11 XIII GRFtH1 pFlat3-Sc5d 0 0 0 0 0 0 / / / /
0 XIV GRFtH1E1 pFlat3-Sc5d 0 0 0 - 0 0 / / + / 0 XV GRFtH1E11 0 0 0
- 0 0 / / + / 0 pFlat3-Sc5d XVI GRFtH1E1E11 0 0 0 - 0 0 / / + / 0
pFlat3-Sc5d XVII GRFtH1E1E11erg5erg6 0 - 0 -- / / / / +++ + /
pFlat3-Sc5d XVIII GRFtH1erg5erg6 0 0 0 -- / / / / ++ / /
pFlat3-Sc5d 1 = Squalene 2 = Lanosterol 3 = Dimethylzymosterol 4 =
Zymosterol 5 = Fecosterol 6 = Episterol 7 = Cholesta-7,24-dienol 8
= Cholesta-8-enol 9 = Cholesta-5,7,24 trienol 10 =
7-Dehydrocholesterol 11 = Ergosterol
Example 4
Expression of the Heterologous Gene Encoding a .DELTA.24-Reductase
(D24R) from Mice (Mus musculus) in Yeast
[0426] The cDNA sequence of Mus musculus .DELTA.24-reductase
(Waterham, H. R., Koster, J., Romeijn, G. J., Hennekam, R. C.,
Vreken, P., Andersson, H. C., FitzPatrick, D. R., Kelley, R. I. and
Wanders, R. J., Mutations in the 3.beta.-Hydroxysterol
.DELTA.24-Reductase Gene Cause Desmosterolosis, an Autosomal
Recessive Disorder of Cholesterol Biosynthesis, Am. J. Hum. Genet.
69 (4), 685-694 (2001)) was amplified by PCR from the cDNA clone
IMAGp998K179532 (Host: E. coli DH10B) of the Deutsches
Resourcenzentrum fur Genomforschung [German resource center for
genome research] GmbH (Berlin).
[0427] The primers used here are the DNA oligomers D24R-5' (SEQ.
ID. No. 47) and D24R-3' (SEQ. ID. No. 48). The DNA fragment
obtained was treated with restriction enzymes NotI and XhoI and
then integrated into the vector pFlat4 (FIG. 6) which likewise had
been treated with the enzymes NotI and XhoI beforehand, by means of
a ligase reaction. The resulting vector pFlat4-D24R (FIG. 5d)
contains the D24R gene under the control of the ADH1 promoter and
the tryptophan terminator.
[0428] The expression vector pFlat4-D24R was then transformed into
the yeast strains I to VI of Table 1 from Example 1 and also into
the GRFura3 strain. The yeast strains obtained in this way were
then cultured in a culture volume of 20 ml in WMVIII medium at
28.degree. C. and 160 rpm for 48 hours. Subsequently, 500 .mu.l of
this preculture were transferred to a 50 ml main culture of the
same medium and cultured in a baffled flask at 28.degree. C. and
160 rpm for 3 days.
[0429] The sterols were extracted after 3 days and analyzed by
means of gas chromatography, as described in Example 1. The
influence of the expression of a Mus musculus .DELTA.24-reductase
in combination with the expression of the transcriptionally
deregulated intrinsic yeast genes tHMG and/or ERG1 and/or ERG11
and/or deletion of the intrinsic yeast genes ERG6 and ERG5 is
listed in Table 5. The abbreviations have the following meanings:
-=decrease; 0=no change; /=not present; +, ++, +++,
++++=concentrated to highly concentrated.
TABLE-US-00005 TABLE 5 Influence of the genetic modifications on
the sterol content compared to the GRF yeast strain No. Strain name
1 2 3 4 5 6 7 8 9 10 11 XIX GRFtH1 0 0 0 0 0 0 / / / / 0
pFlat4-D24R XX GRFtH1E1 0 - - - 0 0 / / / + 0 pFlat4-D24R XXI
GRFtH1E11 pFlat4- 0 0 0 - 0 0 / + / + 0 D24R XXII GRFtH1E1E11
pFlat4- 0 0 0 - 0 0 / + / + 0 D24R XXIII GRFtH1E1E11erg5erg6 0 - -
-- / / 0 + + +++ / pFlat4-D24R XXIV GRFtH1erg5erg6 0 - - - / / 0 +
+ ++ / pFlat4-D24R 1 = Squalene 2 = Lanosterol 3 =
Dimethylzymosterol 4 = Zymosterol 5 = Fecosterol 6 = Episterol 7 =
Cholesta-7,24-dienol 8 = Cholesta-8-enol 9 = Cholesta-5,7,24
trienol 10 = 7-Dehydrocholesterol 11 = Ergosterol
Example 5
Coexpression of the Heterologous Genes Encoding a
.DELTA.8-.DELTA.7-Isomerase (Ebp) from Mice (Mus musculus) and a
C5-Desaturase (Sc5d) from Mice (Mus musculus) in Yeast
[0430] The expression vectors pFlat1-EBP (from Example 2) and
pFlat3-SC5D (from Example 3) were transformed into the yeast
strains I to VI of Table 1 of Example 1 and also into the GRFura3
strain. The yeast strains obtained in this way were then cultured
in a culture volume of 20 ml in WMVIII medium at 28.degree. C. and
160 rpm for 48 hours. Subsequently, 500 .mu.l of this preculture
were transferred to a 50 ml main culture of the same medium and
cultured in a baffled flask at 28.degree. C. and 160 rpm for 3
days.
[0431] The sterols were extracted after 3 days and analyzed by
means of gas chromatography, as described in Example 1. The
influence of the expression of a .DELTA.8-.DELTA.7-isomerase and a
Mus musculus C5-desaturase in combination with the expression of
the transcriptionally deregulated intrinsic yeast genes tHMG and/or
ERG1 and/or ERG11 and/or deletion of the intrinsic yeast genes ERG6
and ERG5 is listed in Table 6. The abbreviations have the following
meanings: -=decrease; 0=no change; /=not present; +, ++, +++,
++++=concentrated to highly concentrated.
TABLE-US-00006 TABLE 6 Influence of the genetic modifications on
the sterol content compared to the GRF yeast strain No. Strain name
1 2 3 4 5 6 7 8 9 10 11 VVX GRFtH1 pFlat3-Ebp/ 0 0 0 - 0 0 / / + /
0 pFlat1-Sc5d XXVI GRFtH1E1 pFlat3-Ebp/ 0 - 0 -- 0 0 / / + / 0
pFlat1-Sc5d XXVII GRFtH1E11 pFlat3- 0 0 0 -- 0 0 / / + / 0
Ebp/pFlat1-Sc5d XXVIII GRFtH1E1E11 pFlat3- 0 - - -- 0 0 / / ++ / 0
Ebp/pFlat1-Sc5d XXIX GRFtH1E1E11erg5erg6 0 - 0 -- / / / / +++ + /
pFlat3-Ebp/pFlat1- Sc5d XXX GRFtH1erg5erg6 0 0 0 - / / / / ++ + /
pFlat3-Ebp/pFlat1- Sc5d 1 = Squalene 2 = Lanosterol 3 =
Dimethylzymosterol 4 = Zymosterol 5 = Fecosterol 6 = Episterol 7 =
Cholesta-7,24-dienol 8 = Cholesta-8-enol 9 = Cholesta-5,7,24
trienol 10 = 7-Dehydrocholesterol 11 = Ergosterol
Example 6
Coexpression of the Heterologous Genes Encoding a
.DELTA.8-.DELTA.7-Isomerase (Ebp) from Mice (Mus musculus) Encoding
a C5-Desaturase (Sc5d) from Mice (Mus musculus) and a
.DELTA.24-Reductase from Mice (Mus musculus) in Yeast
[0432] The expression vectors pFlat1-EBP (from Example 2) and
pFlat3-SC5D (from Example 3) and pFlat4-D24R (from Example 4) were
transformed into the yeast strains I to VI of Table 1 of Example 1
and also into the GRFura3 strain. The yeast strains obtained in
this way were then cultured in a culture volume of 20 ml in WMVIII
medium at 28.degree. C. and 160 rpm for 48 hours. Subsequently, 500
.mu.l of this preculture were transferred to a 50 ml main culture
of the same medium and cultured in a baffled flask at 28.degree. C.
and 160 rpm for 3 days.
[0433] The sterols were extracted after 3 days and analyzed by
means of gas chromatography, as described in Example 1. The
influence of the expression of a .DELTA.8-.DELTA.7-isomerase, a Mus
musculus C5-desaturase and a Mus musculus .DELTA.24-reductase in
combination with the expression of the transcriptionally
deregulated intrinsic yeast genes tHMG and/or ERG1 and/or ERG11
and/or deletion of the intrinsic yeast genes ERG6 and ERG5 is
listed in Table 7. The abbreviations have the following meanings:
-=decrease; 0=no change; /=not present; ++, +++, ++++=concentrated
to highly concentrated.
TABLE-US-00007 TABLE 7 Influence of the genetic modifications on
the sterol content compared to the GRF yeast strain No. Strain name
1 2 3 4 5 6 7 8 9 10 11 XXXI GRFtH1 pFlat3-Ebp/ 0 0 0 - 0 0 / / / +
0 pFlat1-Sc5d/pFlat4- D24R XXXII GRFtH1E1 pFlat3-Ebp/ 0 - 0 -- 0 0
/ / / + 0 pFlat1-Sc5d/pFlat4- D24R XXXIII GRFtH1E11 pFlat3- 0 0 0
-- 0 0 / / / + 0 Ebp/pFlat1-Sc5d/ pFlat4-D24R XXXIV GRFtH1E1E11
pFlat3- 0 - - -- 0 0 / / / + 0 Ebp/pFlat1-Sc5d/ pFlat4-D24R XXXV
GRFtH1E1E11erg5erg6 0 - 0 --- / / / / + ++++ / pFlat3-Ebp/pFlat1-
Sc5d/pFlat4-D24R XXXVI GRFtH1erg5erg6 0 0 0 - / / / / ++ +++ /
pFlat3-Ebp/pFlat1- Sc5d/pFlat4-D24R 1 = Squalene 2 = Lanosterol 3 =
Dimethylzymosterol 4 = Zymosterol 5 = Fecosterol 6 = Episterol 7 =
Cholesta-7,24-dienol 8 = Cholesta-8-enol 9 = Cholesta-5,7,24
trienol 10 = 7-Dehydrocholesterol 11 = Ergosterol
Sequence CWU 1
1
601693DNAMus musculusCDS(1)..(693) 1atg acc acc aat acg gtc ccc ttg
cac ccg tac tgg ccc agg cac ctg 48Met Thr Thr Asn Thr Val Pro Leu
His Pro Tyr Trp Pro Arg His Leu1 5 10 15aag ctg gac aac ttc gtg cct
aat gac ctc ccg act tcg cat atc ctg 96Lys Leu Asp Asn Phe Val Pro
Asn Asp Leu Pro Thr Ser His Ile Leu 20 25 30gtt ggc ctc ttc tcc atc
tct ggg ggc cta att gtg atc acg tgg ctg 144Val Gly Leu Phe Ser Ile
Ser Gly Gly Leu Ile Val Ile Thr Trp Leu 35 40 45ttg tct agc cga gct
tcc gtc gtc cca ctt gga gct ggg cgg cga ctg 192Leu Ser Ser Arg Ala
Ser Val Val Pro Leu Gly Ala Gly Arg Arg Leu 50 55 60gcc ttg tgc tgg
ttt gct gtg tgt acc ttc att cac ctt gtg atc gag 240Ala Leu Cys Trp
Phe Ala Val Cys Thr Phe Ile His Leu Val Ile Glu65 70 75 80ggc tgg
ttc tct ctc tac aat ggc atc ctt tta gaa gac caa gcc ttc 288Gly Trp
Phe Ser Leu Tyr Asn Gly Ile Leu Leu Glu Asp Gln Ala Phe 85 90 95tta
tcc caa ctc tgg aaa gag tat tcc aag gga gat agc cga tat atc 336Leu
Ser Gln Leu Trp Lys Glu Tyr Ser Lys Gly Asp Ser Arg Tyr Ile 100 105
110ctt agt gac agc ttc gtc gtc tgt atg gag act gtc aca gct tgt ctc
384Leu Ser Asp Ser Phe Val Val Cys Met Glu Thr Val Thr Ala Cys Leu
115 120 125tgg gga cca ctc agc cta tgg gta gtg att gcc ttt ctc cgc
caa cag 432Trp Gly Pro Leu Ser Leu Trp Val Val Ile Ala Phe Leu Arg
Gln Gln 130 135 140ccc ttc cgc ttt gtc cta cag ctt gtg gtg tct atg
ggc cag ata tac 480Pro Phe Arg Phe Val Leu Gln Leu Val Val Ser Met
Gly Gln Ile Tyr145 150 155 160ggg gat gtg ctg tac ttc ctg aca gag
cta cac gaa gga ctc cag cat 528Gly Asp Val Leu Tyr Phe Leu Thr Glu
Leu His Glu Gly Leu Gln His 165 170 175ggg gag ata ggc cac ccc gtt
tat ttc tgg ttc tat ttt gtt ttc ctg 576Gly Glu Ile Gly His Pro Val
Tyr Phe Trp Phe Tyr Phe Val Phe Leu 180 185 190aat gct gta tgg ttg
gtg ata cca agc atc ctt gtg ctt gat gcc ata 624Asn Ala Val Trp Leu
Val Ile Pro Ser Ile Leu Val Leu Asp Ala Ile 195 200 205aag cat ctc
act agt gcc cag agc gtg ctg gac agc aaa gtc atg aaa 672Lys His Leu
Thr Ser Ala Gln Ser Val Leu Asp Ser Lys Val Met Lys 210 215 220att
aag agc aag cat aac taa 693Ile Lys Ser Lys His Asn225 2302230PRTMus
musculus 2Met Thr Thr Asn Thr Val Pro Leu His Pro Tyr Trp Pro Arg
His Leu1 5 10 15Lys Leu Asp Asn Phe Val Pro Asn Asp Leu Pro Thr Ser
His Ile Leu 20 25 30Val Gly Leu Phe Ser Ile Ser Gly Gly Leu Ile Val
Ile Thr Trp Leu 35 40 45Leu Ser Ser Arg Ala Ser Val Val Pro Leu Gly
Ala Gly Arg Arg Leu 50 55 60Ala Leu Cys Trp Phe Ala Val Cys Thr Phe
Ile His Leu Val Ile Glu65 70 75 80Gly Trp Phe Ser Leu Tyr Asn Gly
Ile Leu Leu Glu Asp Gln Ala Phe 85 90 95Leu Ser Gln Leu Trp Lys Glu
Tyr Ser Lys Gly Asp Ser Arg Tyr Ile 100 105 110Leu Ser Asp Ser Phe
Val Val Cys Met Glu Thr Val Thr Ala Cys Leu 115 120 125Trp Gly Pro
Leu Ser Leu Trp Val Val Ile Ala Phe Leu Arg Gln Gln 130 135 140Pro
Phe Arg Phe Val Leu Gln Leu Val Val Ser Met Gly Gln Ile Tyr145 150
155 160Gly Asp Val Leu Tyr Phe Leu Thr Glu Leu His Glu Gly Leu Gln
His 165 170 175Gly Glu Ile Gly His Pro Val Tyr Phe Trp Phe Tyr Phe
Val Phe Leu 180 185 190Asn Ala Val Trp Leu Val Ile Pro Ser Ile Leu
Val Leu Asp Ala Ile 195 200 205Lys His Leu Thr Ser Ala Gln Ser Val
Leu Asp Ser Lys Val Met Lys 210 215 220Ile Lys Ser Lys His Asn225
2303693DNAHomo sapiensCDS(1)..(693) 3atg act acc aac gcg ggc ccc
ttg cac cca tac tgg cct cag cac cta 48Met Thr Thr Asn Ala Gly Pro
Leu His Pro Tyr Trp Pro Gln His Leu1 5 10 15aga ctg gac aac ttt gta
cct aat gac cgc ccc acc tgg cat ata ctg 96Arg Leu Asp Asn Phe Val
Pro Asn Asp Arg Pro Thr Trp His Ile Leu 20 25 30gct ggc ctc ttc tct
gtc aca ggg gtc tta gtc gtg acc aca tgg ctg 144Ala Gly Leu Phe Ser
Val Thr Gly Val Leu Val Val Thr Thr Trp Leu 35 40 45ttg tca ggt cgt
gct gcg gtt gtc cca ttg ggg act tgg cgg cga ctg 192Leu Ser Gly Arg
Ala Ala Val Val Pro Leu Gly Thr Trp Arg Arg Leu 50 55 60tcc ctg tgc
tgg ttt gca gtg tgt ggg ttc att cac ctg gtg atc gag 240Ser Leu Cys
Trp Phe Ala Val Cys Gly Phe Ile His Leu Val Ile Glu65 70 75 80ggc
tgg ttc gtt ctc tac tac gaa gac ctg ctt gga gac caa gcc ttc 288Gly
Trp Phe Val Leu Tyr Tyr Glu Asp Leu Leu Gly Asp Gln Ala Phe 85 90
95tta tct caa ctc tgg aaa gag tat gcc aag gga gac agc cga tac atc
336Leu Ser Gln Leu Trp Lys Glu Tyr Ala Lys Gly Asp Ser Arg Tyr Ile
100 105 110ctg ggt gac aac ttc aca gtg tgc atg gaa acc atc aca gct
tgc ctg 384Leu Gly Asp Asn Phe Thr Val Cys Met Glu Thr Ile Thr Ala
Cys Leu 115 120 125tgg gga cca ctc agc ctg tgg gtg gtg atc gcc ttt
ctc cgc cag cat 432Trp Gly Pro Leu Ser Leu Trp Val Val Ile Ala Phe
Leu Arg Gln His 130 135 140ccc ctc cgc ttc att cta cag ctt gtg gtc
tct gtg ggc cag atc tat 480Pro Leu Arg Phe Ile Leu Gln Leu Val Val
Ser Val Gly Gln Ile Tyr145 150 155 160ggg gat gtg ctc tac ttc ctg
aca gag cac cgc gac gga ttc cag cac 528Gly Asp Val Leu Tyr Phe Leu
Thr Glu His Arg Asp Gly Phe Gln His 165 170 175gga gag ctg ggc cac
cct ctc tac ttc tgg ttt tac ttt gtc ttc atg 576Gly Glu Leu Gly His
Pro Leu Tyr Phe Trp Phe Tyr Phe Val Phe Met 180 185 190aat gcc ctg
tgg ctg gtg ctg cct gga gtc ctt gtg ctt gat gct gtg 624Asn Ala Leu
Trp Leu Val Leu Pro Gly Val Leu Val Leu Asp Ala Val 195 200 205aag
cac ctc act cat gcc cag agc acg ctg gat gcc aag gcc aca aaa 672Lys
His Leu Thr His Ala Gln Ser Thr Leu Asp Ala Lys Ala Thr Lys 210 215
220gcc aag agc aag aag aac tga 693Ala Lys Ser Lys Lys Asn225
2304230PRTHomo sapiens 4Met Thr Thr Asn Ala Gly Pro Leu His Pro Tyr
Trp Pro Gln His Leu1 5 10 15Arg Leu Asp Asn Phe Val Pro Asn Asp Arg
Pro Thr Trp His Ile Leu 20 25 30Ala Gly Leu Phe Ser Val Thr Gly Val
Leu Val Val Thr Thr Trp Leu 35 40 45Leu Ser Gly Arg Ala Ala Val Val
Pro Leu Gly Thr Trp Arg Arg Leu 50 55 60Ser Leu Cys Trp Phe Ala Val
Cys Gly Phe Ile His Leu Val Ile Glu65 70 75 80Gly Trp Phe Val Leu
Tyr Tyr Glu Asp Leu Leu Gly Asp Gln Ala Phe 85 90 95Leu Ser Gln Leu
Trp Lys Glu Tyr Ala Lys Gly Asp Ser Arg Tyr Ile 100 105 110Leu Gly
Asp Asn Phe Thr Val Cys Met Glu Thr Ile Thr Ala Cys Leu 115 120
125Trp Gly Pro Leu Ser Leu Trp Val Val Ile Ala Phe Leu Arg Gln His
130 135 140Pro Leu Arg Phe Ile Leu Gln Leu Val Val Ser Val Gly Gln
Ile Tyr145 150 155 160Gly Asp Val Leu Tyr Phe Leu Thr Glu His Arg
Asp Gly Phe Gln His 165 170 175Gly Glu Leu Gly His Pro Leu Tyr Phe
Trp Phe Tyr Phe Val Phe Met 180 185 190Asn Ala Leu Trp Leu Val Leu
Pro Gly Val Leu Val Leu Asp Ala Val 195 200 205Lys His Leu Thr His
Ala Gln Ser Thr Leu Asp Ala Lys Ala Thr Lys 210 215 220Ala Lys Ser
Lys Lys Asn225 2305669DNASaccharomyces cerevisiaeCDS(1)..(669) 5atg
aag ttt ttc cca ctc ctt ttg ttg att ggt gtt gta ggc tac att 48Met
Lys Phe Phe Pro Leu Leu Leu Leu Ile Gly Val Val Gly Tyr Ile1 5 10
15atg aac gta ttg ttc act acc tgg ttg cca acc aat tac atg ttc gat
96Met Asn Val Leu Phe Thr Thr Trp Leu Pro Thr Asn Tyr Met Phe Asp
20 25 30cca aaa act ttg aac gaa ata tgt aac tcg gtg att agc aaa cac
aac 144Pro Lys Thr Leu Asn Glu Ile Cys Asn Ser Val Ile Ser Lys His
Asn 35 40 45gca gca gaa ggt tta tcc act gaa gac ctg tta cag gat gtc
aga gac 192Ala Ala Glu Gly Leu Ser Thr Glu Asp Leu Leu Gln Asp Val
Arg Asp 50 55 60gca ctt gcc tct cat tac ggg gac gaa tac atc aac agg
tac gtc aaa 240Ala Leu Ala Ser His Tyr Gly Asp Glu Tyr Ile Asn Arg
Tyr Val Lys65 70 75 80gaa gaa tgg gtc ttc aac aat gct ggt ggt gcg
atg ggc caa atg atc 288Glu Glu Trp Val Phe Asn Asn Ala Gly Gly Ala
Met Gly Gln Met Ile 85 90 95atc cta cac gct tcc gta tcc gag tac tta
att cta ttc gga acc gct 336Ile Leu His Ala Ser Val Ser Glu Tyr Leu
Ile Leu Phe Gly Thr Ala 100 105 110gtt ggt act gaa ggg cac aca ggt
gtt cac ttt gct gac gac tat ttt 384Val Gly Thr Glu Gly His Thr Gly
Val His Phe Ala Asp Asp Tyr Phe 115 120 125acc atc tta cat ggt acg
caa atc gca gca ttg cca tat gcc act gaa 432Thr Ile Leu His Gly Thr
Gln Ile Ala Ala Leu Pro Tyr Ala Thr Glu 130 135 140gcc gaa gtt tac
act cct ggt atg act cat cac ttg aag aag gga tac 480Ala Glu Val Tyr
Thr Pro Gly Met Thr His His Leu Lys Lys Gly Tyr145 150 155 160gcc
aag caa tac agc atg cca ggt ggt tcc ttt gcc ctt gaa ttg gct 528Ala
Lys Gln Tyr Ser Met Pro Gly Gly Ser Phe Ala Leu Glu Leu Ala 165 170
175caa ggc tgg att cca tgt atg ttg cca ttc ggg ttt ttg gac act ttc
576Gln Gly Trp Ile Pro Cys Met Leu Pro Phe Gly Phe Leu Asp Thr Phe
180 185 190tcc agt act ctt gat tta tac act cta tat aga act gtc tac
ctg act 624Ser Ser Thr Leu Asp Leu Tyr Thr Leu Tyr Arg Thr Val Tyr
Leu Thr 195 200 205gcc agg gac atg ggt aag aac ttg ttg caa aac aaa
aag ttc taa 669Ala Arg Asp Met Gly Lys Asn Leu Leu Gln Asn Lys Lys
Phe 210 215 2206222PRTSaccharomyces cerevisiae 6Met Lys Phe Phe Pro
Leu Leu Leu Leu Ile Gly Val Val Gly Tyr Ile1 5 10 15Met Asn Val Leu
Phe Thr Thr Trp Leu Pro Thr Asn Tyr Met Phe Asp 20 25 30Pro Lys Thr
Leu Asn Glu Ile Cys Asn Ser Val Ile Ser Lys His Asn 35 40 45Ala Ala
Glu Gly Leu Ser Thr Glu Asp Leu Leu Gln Asp Val Arg Asp 50 55 60Ala
Leu Ala Ser His Tyr Gly Asp Glu Tyr Ile Asn Arg Tyr Val Lys65 70 75
80Glu Glu Trp Val Phe Asn Asn Ala Gly Gly Ala Met Gly Gln Met Ile
85 90 95Ile Leu His Ala Ser Val Ser Glu Tyr Leu Ile Leu Phe Gly Thr
Ala 100 105 110Val Gly Thr Glu Gly His Thr Gly Val His Phe Ala Asp
Asp Tyr Phe 115 120 125Thr Ile Leu His Gly Thr Gln Ile Ala Ala Leu
Pro Tyr Ala Thr Glu 130 135 140Ala Glu Val Tyr Thr Pro Gly Met Thr
His His Leu Lys Lys Gly Tyr145 150 155 160Ala Lys Gln Tyr Ser Met
Pro Gly Gly Ser Phe Ala Leu Glu Leu Ala 165 170 175Gln Gly Trp Ile
Pro Cys Met Leu Pro Phe Gly Phe Leu Asp Thr Phe 180 185 190Ser Ser
Thr Leu Asp Leu Tyr Thr Leu Tyr Arg Thr Val Tyr Leu Thr 195 200
205Ala Arg Asp Met Gly Lys Asn Leu Leu Gln Asn Lys Lys Phe 210 215
2207900DNAMus musculusCDS(1)..(900) 7atg gac ctg gtt ctc agt gcc
gcc gat tac tac ttc ttc act ccg tat 48Met Asp Leu Val Leu Ser Ala
Ala Asp Tyr Tyr Phe Phe Thr Pro Tyr1 5 10 15gta tat cca gcc acg tgg
ccc gag gac aac atc atc cga caa act att 96Val Tyr Pro Ala Thr Trp
Pro Glu Asp Asn Ile Ile Arg Gln Thr Ile 20 25 30agc ctc ctg att gtc
aca aac ctg ggt gct tac att ctc tac ttc ttc 144Ser Leu Leu Ile Val
Thr Asn Leu Gly Ala Tyr Ile Leu Tyr Phe Phe 35 40 45tgt gca acc ctc
agc tat tat ttt gtc tat gat cat tcc tta atg aaa 192Cys Ala Thr Leu
Ser Tyr Tyr Phe Val Tyr Asp His Ser Leu Met Lys 50 55 60cac cca cag
ttt tta aag aac caa gtc tcg cgt gag atc gtg ttc act 240His Pro Gln
Phe Leu Lys Asn Gln Val Ser Arg Glu Ile Val Phe Thr65 70 75 80gtc
aag tct ttg cct tgg atc agc atc ccc acc gtc tca cta ttc ctg 288Val
Lys Ser Leu Pro Trp Ile Ser Ile Pro Thr Val Ser Leu Phe Leu 85 90
95ctg gag ctg agg ggt tac agc aaa ctc tac gat gac atc gga gac ttt
336Leu Glu Leu Arg Gly Tyr Ser Lys Leu Tyr Asp Asp Ile Gly Asp Phe
100 105 110cca aat ggc tgg att cat ctc atg gtt agc gtc gta tcc ttc
ctc ttt 384Pro Asn Gly Trp Ile His Leu Met Val Ser Val Val Ser Phe
Leu Phe 115 120 125ttc aca gac atg ttg atc tac agg att cat agg ggc
ctg cac cac aga 432Phe Thr Asp Met Leu Ile Tyr Arg Ile His Arg Gly
Leu His His Arg 130 135 140ctg gtc tac aag cgc ata cat aaa cca cat
cat att tgg aag atc ccc 480Leu Val Tyr Lys Arg Ile His Lys Pro His
His Ile Trp Lys Ile Pro145 150 155 160acg ccg ttt gca agt cat gct
ttt cac cct gtg gac ggc ttc ctt cag 528Thr Pro Phe Ala Ser His Ala
Phe His Pro Val Asp Gly Phe Leu Gln 165 170 175agt ctg cct tac cat
ata tac ccc ttt gtc ttt cca ctg cac aag gtg 576Ser Leu Pro Tyr His
Ile Tyr Pro Phe Val Phe Pro Leu His Lys Val 180 185 190gtc tac tta
ggt tta tat gtc ttg gtt aat gtc tgg aca att tct att 624Val Tyr Leu
Gly Leu Tyr Val Leu Val Asn Val Trp Thr Ile Ser Ile 195 200 205cat
gat ggt gat ttt cgg gtt ccc cag atc tta agg cca ttt att aac 672His
Asp Gly Asp Phe Arg Val Pro Gln Ile Leu Arg Pro Phe Ile Asn 210 215
220ggg tca gct cac cac aca gac cac cac atg ttc ttt gac tat aac tat
720Gly Ser Ala His His Thr Asp His His Met Phe Phe Asp Tyr Asn
Tyr225 230 235 240gga cag tat ttc aca ttg tgg gat aga att gga ggc
tct ttt aaa cat 768Gly Gln Tyr Phe Thr Leu Trp Asp Arg Ile Gly Gly
Ser Phe Lys His 245 250 255cct tcc tct ttt gaa ggg aaa gga cca cat
agt tac gtg aag aac atg 816Pro Ser Ser Phe Glu Gly Lys Gly Pro His
Ser Tyr Val Lys Asn Met 260 265 270aca gaa aaa gaa tct aac agc ttt
gca gaa aac ggc tgt aaa ggc aaa 864Thr Glu Lys Glu Ser Asn Ser Phe
Ala Glu Asn Gly Cys Lys Gly Lys 275 280 285aaa gta agc aat gga gag
ttt aca aag aat aag tag 900Lys Val Ser Asn Gly Glu Phe Thr Lys Asn
Lys 290 2958299PRTMus musculus 8Met Asp Leu Val Leu Ser Ala Ala Asp
Tyr Tyr Phe Phe Thr Pro Tyr1 5 10 15Val Tyr Pro Ala Thr Trp Pro Glu
Asp Asn Ile Ile Arg Gln Thr Ile 20 25 30Ser Leu Leu Ile Val Thr Asn
Leu Gly Ala Tyr Ile Leu Tyr Phe Phe 35 40 45Cys Ala Thr Leu Ser Tyr
Tyr Phe Val Tyr Asp His Ser Leu Met Lys 50 55 60His Pro Gln Phe Leu
Lys Asn Gln Val Ser Arg Glu Ile Val Phe Thr65 70 75 80Val Lys Ser
Leu Pro Trp Ile Ser Ile Pro Thr Val Ser Leu Phe Leu 85 90 95Leu Glu
Leu Arg Gly Tyr Ser Lys Leu Tyr Asp Asp Ile Gly Asp Phe 100 105
110Pro Asn Gly Trp Ile His Leu Met Val Ser Val Val Ser Phe Leu Phe
115 120 125Phe Thr Asp Met Leu Ile Tyr Arg Ile His Arg Gly Leu His
His Arg 130 135 140Leu Val Tyr Lys Arg Ile His Lys Pro His His Ile
Trp Lys Ile Pro145 150 155 160Thr Pro Phe Ala Ser His Ala Phe His
Pro Val Asp Gly Phe Leu Gln
165 170 175Ser Leu Pro Tyr His Ile Tyr Pro Phe Val Phe Pro Leu His
Lys Val 180 185 190Val Tyr Leu Gly Leu Tyr Val Leu Val Asn Val Trp
Thr Ile Ser Ile 195 200 205His Asp Gly Asp Phe Arg Val Pro Gln Ile
Leu Arg Pro Phe Ile Asn 210 215 220Gly Ser Ala His His Thr Asp His
His Met Phe Phe Asp Tyr Asn Tyr225 230 235 240Gly Gln Tyr Phe Thr
Leu Trp Asp Arg Ile Gly Gly Ser Phe Lys His 245 250 255Pro Ser Ser
Phe Glu Gly Lys Gly Pro His Ser Tyr Val Lys Asn Met 260 265 270Thr
Glu Lys Glu Ser Asn Ser Phe Ala Glu Asn Gly Cys Lys Gly Lys 275 280
285Lys Val Ser Asn Gly Glu Phe Thr Lys Asn Lys 290 2959900DNAHomo
sapiensCDS(1)..(900) 9atg gat ctt gta ctc cgt gtt gca gat tac tat
ttt ttt aca cca tac 48Met Asp Leu Val Leu Arg Val Ala Asp Tyr Tyr
Phe Phe Thr Pro Tyr1 5 10 15gtg tat cca gcc aca tgg cca gaa gat gac
atc ttc cga caa gct att 96Val Tyr Pro Ala Thr Trp Pro Glu Asp Asp
Ile Phe Arg Gln Ala Ile 20 25 30agt ctt ctg att gta aca aat gtt ggt
gct tac atc ctt tat ttc ttc 144Ser Leu Leu Ile Val Thr Asn Val Gly
Ala Tyr Ile Leu Tyr Phe Phe 35 40 45tgt gca aca ctg agc tat tat ttt
gtc ttc gat cat gca tta atg aaa 192Cys Ala Thr Leu Ser Tyr Tyr Phe
Val Phe Asp His Ala Leu Met Lys 50 55 60cat cca caa ttt tta aag aat
caa gtc cgt cga gag att aag ttt act 240His Pro Gln Phe Leu Lys Asn
Gln Val Arg Arg Glu Ile Lys Phe Thr65 70 75 80gtc cag gca ttg cca
tgg ata agt att ctt act gtt gca ctg ttc ttg 288Val Gln Ala Leu Pro
Trp Ile Ser Ile Leu Thr Val Ala Leu Phe Leu 85 90 95ctg gag ata aga
ggt tac agc aaa tta cat gat gac cta gga gag ttt 336Leu Glu Ile Arg
Gly Tyr Ser Lys Leu His Asp Asp Leu Gly Glu Phe 100 105 110cca tat
gga ttg ttt gaa ctt gtc gtt agt ata ata tct ttc ctc ttt 384Pro Tyr
Gly Leu Phe Glu Leu Val Val Ser Ile Ile Ser Phe Leu Phe 115 120
125ttc act gac atg ttc atc tac tgg att cac aga ggc ctt cat cat aga
432Phe Thr Asp Met Phe Ile Tyr Trp Ile His Arg Gly Leu His His Arg
130 135 140ctg gta tat aag cgc cta cat aaa cct cac cat att tgg aag
att cct 480Leu Val Tyr Lys Arg Leu His Lys Pro His His Ile Trp Lys
Ile Pro145 150 155 160act cca ttt gca agt cat gct ttt cac cct att
gat ggc ttt ctt cag 528Thr Pro Phe Ala Ser His Ala Phe His Pro Ile
Asp Gly Phe Leu Gln 165 170 175agt cta cct tac cat ata tac cct ttt
atc ttt cca tta cac aag gtg 576Ser Leu Pro Tyr His Ile Tyr Pro Phe
Ile Phe Pro Leu His Lys Val 180 185 190gtt tat tta agt ctg tac atc
ttg gtt aat atc tgg aca att tcc att 624Val Tyr Leu Ser Leu Tyr Ile
Leu Val Asn Ile Trp Thr Ile Ser Ile 195 200 205cat gac ggt gat ttt
cgt gtc ccc caa atc tta cag cca ttt att aat 672His Asp Gly Asp Phe
Arg Val Pro Gln Ile Leu Gln Pro Phe Ile Asn 210 215 220ggc tca gct
cat cat aca gac cac cat atg ttc ttt gac tat aat tat 720Gly Ser Ala
His His Thr Asp His His Met Phe Phe Asp Tyr Asn Tyr225 230 235
240gga caa tat ttc act ttg tgg gat agg att ggc ggc tca ttc aaa aat
768Gly Gln Tyr Phe Thr Leu Trp Asp Arg Ile Gly Gly Ser Phe Lys Asn
245 250 255cct tca tcc ttt gag ggg aag gga ccg ctc agt tat gtg aag
gag atg 816Pro Ser Ser Phe Glu Gly Lys Gly Pro Leu Ser Tyr Val Lys
Glu Met 260 265 270aca gag gga aag cgc agc agc cct tca gga aat ggc
tgt aag aat gaa 864Thr Glu Gly Lys Arg Ser Ser Pro Ser Gly Asn Gly
Cys Lys Asn Glu 275 280 285aaa tta ttc aat gga gag ttt aca aag act
gaa tag 900Lys Leu Phe Asn Gly Glu Phe Thr Lys Thr Glu 290
29510299PRTHomo sapiens 10Met Asp Leu Val Leu Arg Val Ala Asp Tyr
Tyr Phe Phe Thr Pro Tyr1 5 10 15Val Tyr Pro Ala Thr Trp Pro Glu Asp
Asp Ile Phe Arg Gln Ala Ile 20 25 30Ser Leu Leu Ile Val Thr Asn Val
Gly Ala Tyr Ile Leu Tyr Phe Phe 35 40 45Cys Ala Thr Leu Ser Tyr Tyr
Phe Val Phe Asp His Ala Leu Met Lys 50 55 60His Pro Gln Phe Leu Lys
Asn Gln Val Arg Arg Glu Ile Lys Phe Thr65 70 75 80Val Gln Ala Leu
Pro Trp Ile Ser Ile Leu Thr Val Ala Leu Phe Leu 85 90 95Leu Glu Ile
Arg Gly Tyr Ser Lys Leu His Asp Asp Leu Gly Glu Phe 100 105 110Pro
Tyr Gly Leu Phe Glu Leu Val Val Ser Ile Ile Ser Phe Leu Phe 115 120
125Phe Thr Asp Met Phe Ile Tyr Trp Ile His Arg Gly Leu His His Arg
130 135 140Leu Val Tyr Lys Arg Leu His Lys Pro His His Ile Trp Lys
Ile Pro145 150 155 160Thr Pro Phe Ala Ser His Ala Phe His Pro Ile
Asp Gly Phe Leu Gln 165 170 175Ser Leu Pro Tyr His Ile Tyr Pro Phe
Ile Phe Pro Leu His Lys Val 180 185 190Val Tyr Leu Ser Leu Tyr Ile
Leu Val Asn Ile Trp Thr Ile Ser Ile 195 200 205His Asp Gly Asp Phe
Arg Val Pro Gln Ile Leu Gln Pro Phe Ile Asn 210 215 220Gly Ser Ala
His His Thr Asp His His Met Phe Phe Asp Tyr Asn Tyr225 230 235
240Gly Gln Tyr Phe Thr Leu Trp Asp Arg Ile Gly Gly Ser Phe Lys Asn
245 250 255Pro Ser Ser Phe Glu Gly Lys Gly Pro Leu Ser Tyr Val Lys
Glu Met 260 265 270Thr Glu Gly Lys Arg Ser Ser Pro Ser Gly Asn Gly
Cys Lys Asn Glu 275 280 285Lys Leu Phe Asn Gly Glu Phe Thr Lys Thr
Glu 290 295111098DNASaccharomyces cerevisiaeCDS(1)..(1098) 11atg
gat ttg gtc tta gaa gtc gct gac cat tat gtc tta gac gac ttg 48Met
Asp Leu Val Leu Glu Val Ala Asp His Tyr Val Leu Asp Asp Leu1 5 10
15tac gct aaa gtt ctg ccc gct tcg ttg gca gct aat att cct gtc aag
96Tyr Ala Lys Val Leu Pro Ala Ser Leu Ala Ala Asn Ile Pro Val Lys
20 25 30tgg cag aaa ttg cta ggg ttg aac agt ggg ttc agc aat tct acg
att 144Trp Gln Lys Leu Leu Gly Leu Asn Ser Gly Phe Ser Asn Ser Thr
Ile 35 40 45ttg cag gag act ttg aac tcc aag aat gcc gtc aaa gaa tgt
aga agg 192Leu Gln Glu Thr Leu Asn Ser Lys Asn Ala Val Lys Glu Cys
Arg Arg 50 55 60ttc tac ggg cag gtg cca ttc ctg ttt gat atg tcg acg
acg tct ttt 240Phe Tyr Gly Gln Val Pro Phe Leu Phe Asp Met Ser Thr
Thr Ser Phe65 70 75 80gca tcg cta ttg cct cgt tcc agc atc ttg aga
gaa ttc ctc tca cta 288Ala Ser Leu Leu Pro Arg Ser Ser Ile Leu Arg
Glu Phe Leu Ser Leu 85 90 95tgg gtt att gtt acg atc ttt ggt tta cta
ctt tac tta ttc acg gct 336Trp Val Ile Val Thr Ile Phe Gly Leu Leu
Leu Tyr Leu Phe Thr Ala 100 105 110agt ctc agc tac gtg ttt gtg ttt
gac aag tcg att ttc aac cat cct 384Ser Leu Ser Tyr Val Phe Val Phe
Asp Lys Ser Ile Phe Asn His Pro 115 120 125cgt tac ttg aaa aac caa
atg gca atg gaa atc aag ttg gca gtc agt 432Arg Tyr Leu Lys Asn Gln
Met Ala Met Glu Ile Lys Leu Ala Val Ser 130 135 140gct atc cca tgg
atg tcg atg ttg acc gtt cca tgg ttt gtt atg gaa 480Ala Ile Pro Trp
Met Ser Met Leu Thr Val Pro Trp Phe Val Met Glu145 150 155 160ttg
aac ggc cat tct aaa cta tac atg aag att gat tat gaa aac cac 528Leu
Asn Gly His Ser Lys Leu Tyr Met Lys Ile Asp Tyr Glu Asn His 165 170
175ggt gta agg aag ctc att atc gag tac ttc act ttc atc ttt ttc act
576Gly Val Arg Lys Leu Ile Ile Glu Tyr Phe Thr Phe Ile Phe Phe Thr
180 185 190gat tgc ggt gtg tat tta gcg cac aga tgg ttg cat tgg cca
agg gtc 624Asp Cys Gly Val Tyr Leu Ala His Arg Trp Leu His Trp Pro
Arg Val 195 200 205tac cgt gct ctg cac aag cct cat cac aag tgg ctg
gtc tgc aca cct 672Tyr Arg Ala Leu His Lys Pro His His Lys Trp Leu
Val Cys Thr Pro 210 215 220ttc gca tct cat tct ttc cat cct gta gac
ggg ttt ttg caa tcc atc 720Phe Ala Ser His Ser Phe His Pro Val Asp
Gly Phe Leu Gln Ser Ile225 230 235 240tcg tac cac atc tac cca ttg
att ctg cca tta cac aag gtt tct tat 768Ser Tyr His Ile Tyr Pro Leu
Ile Leu Pro Leu His Lys Val Ser Tyr 245 250 255ttg att ctg ttc act
ttt gtt aac ttt tgg act gtt atg att cat gac 816Leu Ile Leu Phe Thr
Phe Val Asn Phe Trp Thr Val Met Ile His Asp 260 265 270ggt caa tac
cta tca aac aat cct gcc gtc aac ggt act gcc tgc cac 864Gly Gln Tyr
Leu Ser Asn Asn Pro Ala Val Asn Gly Thr Ala Cys His 275 280 285acg
gtt cac cat cta tat ttc aac tac aac tac ggt caa ttc acc act 912Thr
Val His His Leu Tyr Phe Asn Tyr Asn Tyr Gly Gln Phe Thr Thr 290 295
300ctg tgg gac aga cta ggg ggt tct tac cgt aga cca gat gac tca ttg
960Leu Trp Asp Arg Leu Gly Gly Ser Tyr Arg Arg Pro Asp Asp Ser
Leu305 310 315 320ttt gat cct aag tta aga gat gct aag gag acc tgg
gac gct caa gtt 1008Phe Asp Pro Lys Leu Arg Asp Ala Lys Glu Thr Trp
Asp Ala Gln Val 325 330 335aag gaa gtt gaa cat ttc atc aag gag gtc
gaa ggt gat gat aat gat 1056Lys Glu Val Glu His Phe Ile Lys Glu Val
Glu Gly Asp Asp Asn Asp 340 345 350aga atc tat gaa aac gac cca aat
acc aag aag aac aac tga 1098Arg Ile Tyr Glu Asn Asp Pro Asn Thr Lys
Lys Asn Asn 355 360 36512365PRTSaccharomyces cerevisiae 12Met Asp
Leu Val Leu Glu Val Ala Asp His Tyr Val Leu Asp Asp Leu1 5 10 15Tyr
Ala Lys Val Leu Pro Ala Ser Leu Ala Ala Asn Ile Pro Val Lys 20 25
30Trp Gln Lys Leu Leu Gly Leu Asn Ser Gly Phe Ser Asn Ser Thr Ile
35 40 45Leu Gln Glu Thr Leu Asn Ser Lys Asn Ala Val Lys Glu Cys Arg
Arg 50 55 60Phe Tyr Gly Gln Val Pro Phe Leu Phe Asp Met Ser Thr Thr
Ser Phe65 70 75 80Ala Ser Leu Leu Pro Arg Ser Ser Ile Leu Arg Glu
Phe Leu Ser Leu 85 90 95Trp Val Ile Val Thr Ile Phe Gly Leu Leu Leu
Tyr Leu Phe Thr Ala 100 105 110Ser Leu Ser Tyr Val Phe Val Phe Asp
Lys Ser Ile Phe Asn His Pro 115 120 125Arg Tyr Leu Lys Asn Gln Met
Ala Met Glu Ile Lys Leu Ala Val Ser 130 135 140Ala Ile Pro Trp Met
Ser Met Leu Thr Val Pro Trp Phe Val Met Glu145 150 155 160Leu Asn
Gly His Ser Lys Leu Tyr Met Lys Ile Asp Tyr Glu Asn His 165 170
175Gly Val Arg Lys Leu Ile Ile Glu Tyr Phe Thr Phe Ile Phe Phe Thr
180 185 190Asp Cys Gly Val Tyr Leu Ala His Arg Trp Leu His Trp Pro
Arg Val 195 200 205Tyr Arg Ala Leu His Lys Pro His His Lys Trp Leu
Val Cys Thr Pro 210 215 220Phe Ala Ser His Ser Phe His Pro Val Asp
Gly Phe Leu Gln Ser Ile225 230 235 240Ser Tyr His Ile Tyr Pro Leu
Ile Leu Pro Leu His Lys Val Ser Tyr 245 250 255Leu Ile Leu Phe Thr
Phe Val Asn Phe Trp Thr Val Met Ile His Asp 260 265 270Gly Gln Tyr
Leu Ser Asn Asn Pro Ala Val Asn Gly Thr Ala Cys His 275 280 285Thr
Val His His Leu Tyr Phe Asn Tyr Asn Tyr Gly Gln Phe Thr Thr 290 295
300Leu Trp Asp Arg Leu Gly Gly Ser Tyr Arg Arg Pro Asp Asp Ser
Leu305 310 315 320Phe Asp Pro Lys Leu Arg Asp Ala Lys Glu Thr Trp
Asp Ala Gln Val 325 330 335Lys Glu Val Glu His Phe Ile Lys Glu Val
Glu Gly Asp Asp Asn Asp 340 345 350Arg Ile Tyr Glu Asn Asp Pro Asn
Thr Lys Lys Asn Asn 355 360 365131557DNAMus musculusCDS(1)..(1557)
13atg gag ccc gcc gtg tcg ctg gcc gtg tgc gcg ctg ctc ttt ctg ctc
48Met Glu Pro Ala Val Ser Leu Ala Val Cys Ala Leu Leu Phe Leu Leu1
5 10 15tgg gtg cga gtg aag ggg ttg gag ttc gtt ctc atc cac cag cgc
tgg 96Trp Val Arg Val Lys Gly Leu Glu Phe Val Leu Ile His Gln Arg
Trp 20 25 30gtg ttc gtg tgc ctc ttc ttg ctg ccg ctc tcg ctc atc ttc
gat atc 144Val Phe Val Cys Leu Phe Leu Leu Pro Leu Ser Leu Ile Phe
Asp Ile 35 40 45tac tac tac gtg cgc gcc tgg gtg gtg ttc aag ctg agc
agt gcg ccg 192Tyr Tyr Tyr Val Arg Ala Trp Val Val Phe Lys Leu Ser
Ser Ala Pro 50 55 60cgc ctg cac gag cag cgc gtg cgg gac atc cag aaa
cag gtc cgg gaa 240Arg Leu His Glu Gln Arg Val Arg Asp Ile Gln Lys
Gln Val Arg Glu65 70 75 80tgg aag gaa cag ggc agt aag acc ttc atg
tgc acg ggg cgc cca ggc 288Trp Lys Glu Gln Gly Ser Lys Thr Phe Met
Cys Thr Gly Arg Pro Gly 85 90 95tgg ctc act gtc tcg ctg cga gtc gga
aag tac aag aag acc cat aag 336Trp Leu Thr Val Ser Leu Arg Val Gly
Lys Tyr Lys Lys Thr His Lys 100 105 110aac atc atg atc aac ctg atg
gac atc ctg gag gtg gac acc aag aaa 384Asn Ile Met Ile Asn Leu Met
Asp Ile Leu Glu Val Asp Thr Lys Lys 115 120 125cag att gtt cga gtg
gag ccc ttg gtg tct atg ggt cag gtg aca gct 432Gln Ile Val Arg Val
Glu Pro Leu Val Ser Met Gly Gln Val Thr Ala 130 135 140ttg ctg aac
tcc att ggc tgg acc ctg cct gtg ttg cct gag ctt gat 480Leu Leu Asn
Ser Ile Gly Trp Thr Leu Pro Val Leu Pro Glu Leu Asp145 150 155
160gac ctc aca gtg ggg ggc ctg atc atg ggc aca ggc atc gag tca tcg
528Asp Leu Thr Val Gly Gly Leu Ile Met Gly Thr Gly Ile Glu Ser Ser
165 170 175tcc cac aag tat ggc ctg ttc caa cac att tgc act gcc tac
gag ctg 576Ser His Lys Tyr Gly Leu Phe Gln His Ile Cys Thr Ala Tyr
Glu Leu 180 185 190atc ctg gca gac ggc agc ttt gtg cgc tgc aca ccg
tct gaa aac tca 624Ile Leu Ala Asp Gly Ser Phe Val Arg Cys Thr Pro
Ser Glu Asn Ser 195 200 205gac ctg ttc tat gcc gtg ccc tgg tcc tgt
ggg acc ctg ggc ttc ctg 672Asp Leu Phe Tyr Ala Val Pro Trp Ser Cys
Gly Thr Leu Gly Phe Leu 210 215 220gtg gct gcc gag atc cgg atc atc
ccg gcc aag aag tat gtc aag ctg 720Val Ala Ala Glu Ile Arg Ile Ile
Pro Ala Lys Lys Tyr Val Lys Leu225 230 235 240cgg ttt gag cct gtt
cgg ggc ctg gag gcc atc tgt gaa aaa ttc acc 768Arg Phe Glu Pro Val
Arg Gly Leu Glu Ala Ile Cys Glu Lys Phe Thr 245 250 255cgc gag tcc
cag cgg ctg gag aac cac ttc gtg gaa ggg ttg ctg tac 816Arg Glu Ser
Gln Arg Leu Glu Asn His Phe Val Glu Gly Leu Leu Tyr 260 265 270tcc
ctg gat gag gct gtg gct gtc atc atg aca ggg gtc atg acg gac 864Ser
Leu Asp Glu Ala Val Ala Val Ile Met Thr Gly Val Met Thr Asp 275 280
285gac gta gag tcc agc aag ctg aat agc att ggc agt tac tac aag ccc
912Asp Val Glu Ser Ser Lys Leu Asn Ser Ile Gly Ser Tyr Tyr Lys Pro
290 295 300tgg ttc ttc aag cat gtg gag aac tac ctg aag aca aac cgg
gag ggc 960Trp Phe Phe Lys His Val Glu Asn Tyr Leu Lys Thr Asn Arg
Glu Gly305 310 315 320ctc gaa tac att ccc ctg aga cac tac tac cac
cga cac acg cgc agc 1008Leu Glu Tyr Ile Pro Leu Arg His Tyr Tyr His
Arg His Thr Arg Ser 325 330 335atc ttc tgg gag ctc cag gac atc atc
cct ttc ggc aac aac ccc atc 1056Ile Phe Trp Glu Leu Gln Asp Ile Ile
Pro Phe Gly Asn Asn Pro Ile 340 345 350ttc cgc tac ctc ttc ggc tgg
atg gtg cct ccc aag atc tcc ctc ctg 1104Phe Arg Tyr Leu Phe Gly Trp
Met Val Pro Pro Lys Ile Ser Leu Leu
355 360 365aag ctg acc cag ggc gag acg cta cgc aag ctg tac gag cag
cac cac 1152Lys Leu Thr Gln Gly Glu Thr Leu Arg Lys Leu Tyr Glu Gln
His His 370 375 380gtg gtg cag gac atg ctg gtg ccc atg aag tgc atg
tca cag gcc ctg 1200Val Val Gln Asp Met Leu Val Pro Met Lys Cys Met
Ser Gln Ala Leu385 390 395 400cat acc ttc caa aat gac atc cac gtc
tac ccc atc tgg ctg tgc cca 1248His Thr Phe Gln Asn Asp Ile His Val
Tyr Pro Ile Trp Leu Cys Pro 405 410 415ttc atc ctg ccc agc cag cca
gga cta gtg cat ccc aag gga gat gaa 1296Phe Ile Leu Pro Ser Gln Pro
Gly Leu Val His Pro Lys Gly Asp Glu 420 425 430gca gag ctc tac gtg
gac atc ggg gca tac ggg gag cca cgt gtg aag 1344Ala Glu Leu Tyr Val
Asp Ile Gly Ala Tyr Gly Glu Pro Arg Val Lys 435 440 445cac ttc gag
gcc agg tcc tgc atg agg cag ctg gag aag ttt gtg cgg 1392His Phe Glu
Ala Arg Ser Cys Met Arg Gln Leu Glu Lys Phe Val Arg 450 455 460agt
gtg cac ggg ttc caa atg tta tac gcc gat tgc tat atg aac cgc 1440Ser
Val His Gly Phe Gln Met Leu Tyr Ala Asp Cys Tyr Met Asn Arg465 470
475 480gag gaa ttc tgg gag atg ttc gat ggc tcc ttg tac cac aag ctg
cgc 1488Glu Glu Phe Trp Glu Met Phe Asp Gly Ser Leu Tyr His Lys Leu
Arg 485 490 495aag cag ctg ggc tgc cag gac gcc ttc cct gag gtg tac
gac aag atc 1536Lys Gln Leu Gly Cys Gln Asp Ala Phe Pro Glu Val Tyr
Asp Lys Ile 500 505 510tgc aag gcg gca agg cac tga 1557Cys Lys Ala
Ala Arg His 51514518PRTMus musculus 14Met Glu Pro Ala Val Ser Leu
Ala Val Cys Ala Leu Leu Phe Leu Leu1 5 10 15Trp Val Arg Val Lys Gly
Leu Glu Phe Val Leu Ile His Gln Arg Trp 20 25 30Val Phe Val Cys Leu
Phe Leu Leu Pro Leu Ser Leu Ile Phe Asp Ile 35 40 45Tyr Tyr Tyr Val
Arg Ala Trp Val Val Phe Lys Leu Ser Ser Ala Pro 50 55 60Arg Leu His
Glu Gln Arg Val Arg Asp Ile Gln Lys Gln Val Arg Glu65 70 75 80Trp
Lys Glu Gln Gly Ser Lys Thr Phe Met Cys Thr Gly Arg Pro Gly 85 90
95Trp Leu Thr Val Ser Leu Arg Val Gly Lys Tyr Lys Lys Thr His Lys
100 105 110Asn Ile Met Ile Asn Leu Met Asp Ile Leu Glu Val Asp Thr
Lys Lys 115 120 125Gln Ile Val Arg Val Glu Pro Leu Val Ser Met Gly
Gln Val Thr Ala 130 135 140Leu Leu Asn Ser Ile Gly Trp Thr Leu Pro
Val Leu Pro Glu Leu Asp145 150 155 160Asp Leu Thr Val Gly Gly Leu
Ile Met Gly Thr Gly Ile Glu Ser Ser 165 170 175Ser His Lys Tyr Gly
Leu Phe Gln His Ile Cys Thr Ala Tyr Glu Leu 180 185 190Ile Leu Ala
Asp Gly Ser Phe Val Arg Cys Thr Pro Ser Glu Asn Ser 195 200 205Asp
Leu Phe Tyr Ala Val Pro Trp Ser Cys Gly Thr Leu Gly Phe Leu 210 215
220Val Ala Ala Glu Ile Arg Ile Ile Pro Ala Lys Lys Tyr Val Lys
Leu225 230 235 240Arg Phe Glu Pro Val Arg Gly Leu Glu Ala Ile Cys
Glu Lys Phe Thr 245 250 255Arg Glu Ser Gln Arg Leu Glu Asn His Phe
Val Glu Gly Leu Leu Tyr 260 265 270Ser Leu Asp Glu Ala Val Ala Val
Ile Met Thr Gly Val Met Thr Asp 275 280 285Asp Val Glu Ser Ser Lys
Leu Asn Ser Ile Gly Ser Tyr Tyr Lys Pro 290 295 300Trp Phe Phe Lys
His Val Glu Asn Tyr Leu Lys Thr Asn Arg Glu Gly305 310 315 320Leu
Glu Tyr Ile Pro Leu Arg His Tyr Tyr His Arg His Thr Arg Ser 325 330
335Ile Phe Trp Glu Leu Gln Asp Ile Ile Pro Phe Gly Asn Asn Pro Ile
340 345 350Phe Arg Tyr Leu Phe Gly Trp Met Val Pro Pro Lys Ile Ser
Leu Leu 355 360 365Lys Leu Thr Gln Gly Glu Thr Leu Arg Lys Leu Tyr
Glu Gln His His 370 375 380Val Val Gln Asp Met Leu Val Pro Met Lys
Cys Met Ser Gln Ala Leu385 390 395 400His Thr Phe Gln Asn Asp Ile
His Val Tyr Pro Ile Trp Leu Cys Pro 405 410 415Phe Ile Leu Pro Ser
Gln Pro Gly Leu Val His Pro Lys Gly Asp Glu 420 425 430Ala Glu Leu
Tyr Val Asp Ile Gly Ala Tyr Gly Glu Pro Arg Val Lys 435 440 445His
Phe Glu Ala Arg Ser Cys Met Arg Gln Leu Glu Lys Phe Val Arg 450 455
460Ser Val His Gly Phe Gln Met Leu Tyr Ala Asp Cys Tyr Met Asn
Arg465 470 475 480Glu Glu Phe Trp Glu Met Phe Asp Gly Ser Leu Tyr
His Lys Leu Arg 485 490 495Lys Gln Leu Gly Cys Gln Asp Ala Phe Pro
Glu Val Tyr Asp Lys Ile 500 505 510Cys Lys Ala Ala Arg His
515151551DNAHomo sapiensCDS(1)..(1551) 15atg gag ccc gcc gtg tcg
ctg gcc gtg tgc gcg ctg ctc ttc ctg ctg 48Met Glu Pro Ala Val Ser
Leu Ala Val Cys Ala Leu Leu Phe Leu Leu1 5 10 15tgg gtg cgc ctg aag
ggg ctg gag ttc gtg ctc atc cac cag cgc tgg 96Trp Val Arg Leu Lys
Gly Leu Glu Phe Val Leu Ile His Gln Arg Trp 20 25 30gtg ttc gtg tgc
ctc ttc ctc ctg ccg ctc tcg ctt atc ttc gat atc 144Val Phe Val Cys
Leu Phe Leu Leu Pro Leu Ser Leu Ile Phe Asp Ile 35 40 45tac tac tac
gtg cgc gcc tgg gtg gtg ttc aag ctc agc agc gct ccg 192Tyr Tyr Tyr
Val Arg Ala Trp Val Val Phe Lys Leu Ser Ser Ala Pro 50 55 60cgc ctg
cac gag cag cgc gtg cgg gac atc cag aag cag gtg cgg gaa 240Arg Leu
His Glu Gln Arg Val Arg Asp Ile Gln Lys Gln Val Arg Glu65 70 75
80tgg aag gag cag ggt agc aag acc ttc atg tgc acg ggg cgc cct ggc
288Trp Lys Glu Gln Gly Ser Lys Thr Phe Met Cys Thr Gly Arg Pro Gly
85 90 95tgg ctc act gtc tca cta cgt gtc ggg aag tac aag aag aca cac
aaa 336Trp Leu Thr Val Ser Leu Arg Val Gly Lys Tyr Lys Lys Thr His
Lys 100 105 110aac atc atg atc aac ctg atg gac att ctg gaa gtg gac
acc aag aaa 384Asn Ile Met Ile Asn Leu Met Asp Ile Leu Glu Val Asp
Thr Lys Lys 115 120 125cag att gtc cgt gtg gag ccc ttg gtg acc atg
ggc cag gtg act gcc 432Gln Ile Val Arg Val Glu Pro Leu Val Thr Met
Gly Gln Val Thr Ala 130 135 140ctg ctg acc tcc att ggc tgg act ctc
ccc gtg ttg cct gag ctt gat 480Leu Leu Thr Ser Ile Gly Trp Thr Leu
Pro Val Leu Pro Glu Leu Asp145 150 155 160gac ctc aca gtg ggg ggc
ttg atc atg ggc aca ggc atc gag tca tca 528Asp Leu Thr Val Gly Gly
Leu Ile Met Gly Thr Gly Ile Glu Ser Ser 165 170 175tcc cac aag tac
ggc ctg ttc caa cac atc tgc act gct tac gag ctg 576Ser His Lys Tyr
Gly Leu Phe Gln His Ile Cys Thr Ala Tyr Glu Leu 180 185 190gtc ctg
gct gat ggc agc ttt gtg cga tgc act ccg tcc gaa aac tca 624Val Leu
Ala Asp Gly Ser Phe Val Arg Cys Thr Pro Ser Glu Asn Ser 195 200
205gac ctg ttc tat gcc gta ccc tgg tcc tgt ggg acg ctg ggt ttc ctg
672Asp Leu Phe Tyr Ala Val Pro Trp Ser Cys Gly Thr Leu Gly Phe Leu
210 215 220gtg gcc gct gag atc cgc atc atc cct gcc aag aag tac gtc
aag ctg 720Val Ala Ala Glu Ile Arg Ile Ile Pro Ala Lys Lys Tyr Val
Lys Leu225 230 235 240cgt ttc gag cca gtg cgg ggc ctg gag gct atc
tgt gcc aag ttc acc 768Arg Phe Glu Pro Val Arg Gly Leu Glu Ala Ile
Cys Ala Lys Phe Thr 245 250 255cac gag tcc cag cgg cag gag aac cac
ttc gtg gaa ggg ctg ctc tac 816His Glu Ser Gln Arg Gln Glu Asn His
Phe Val Glu Gly Leu Leu Tyr 260 265 270tcc ctg gat gag gct gtc att
atg aca ggg gtc atg aca gat gag gca 864Ser Leu Asp Glu Ala Val Ile
Met Thr Gly Val Met Thr Asp Glu Ala 275 280 285gag ccc agc aag ctg
aat agc att ggc aat tac tac aag ccg tgg ttc 912Glu Pro Ser Lys Leu
Asn Ser Ile Gly Asn Tyr Tyr Lys Pro Trp Phe 290 295 300ttt aag cat
gtg gag aac tat ctg aag aca aac cga gag ggc ctg gag 960Phe Lys His
Val Glu Asn Tyr Leu Lys Thr Asn Arg Glu Gly Leu Glu305 310 315
320tac att ccc ttg aga cac tac tac cac cgc cac acg cgc agc atc ttc
1008Tyr Ile Pro Leu Arg His Tyr Tyr His Arg His Thr Arg Ser Ile Phe
325 330 335tgg gag ctc cag gac atc atc ccc ttt ggc aac aac ccc atc
ttc cgc 1056Trp Glu Leu Gln Asp Ile Ile Pro Phe Gly Asn Asn Pro Ile
Phe Arg 340 345 350tac ctc ttt ggc tgg atg gtg cct ccc aag atc tcc
ctc ctg aag ctg 1104Tyr Leu Phe Gly Trp Met Val Pro Pro Lys Ile Ser
Leu Leu Lys Leu 355 360 365acc cag ggt gag acc ctg cgc aag ctg tac
gag cag cac cac gtg gtg 1152Thr Gln Gly Glu Thr Leu Arg Lys Leu Tyr
Glu Gln His His Val Val 370 375 380cag gac atg ctg gtg ccc atg aag
tgc ctg cag cag gcc ctg cac acc 1200Gln Asp Met Leu Val Pro Met Lys
Cys Leu Gln Gln Ala Leu His Thr385 390 395 400ttc caa aac gac atc
cac gtc tac ccc atc tgg ctg tgt ccg ttc atc 1248Phe Gln Asn Asp Ile
His Val Tyr Pro Ile Trp Leu Cys Pro Phe Ile 405 410 415ctg ccc agc
cag cca ggc cta gtg cac ccc aaa gga aat gag gca gag 1296Leu Pro Ser
Gln Pro Gly Leu Val His Pro Lys Gly Asn Glu Ala Glu 420 425 430ctc
tac atc gac att gga gca tat ggg gag ccg cgt gtg aaa cac ttt 1344Leu
Tyr Ile Asp Ile Gly Ala Tyr Gly Glu Pro Arg Val Lys His Phe 435 440
445gaa gcc agg tcc tgc atg agg cag ctg gag aag ttt gtc cgc agc gtg
1392Glu Ala Arg Ser Cys Met Arg Gln Leu Glu Lys Phe Val Arg Ser Val
450 455 460cat ggc ttc cag atg ctg tat gcc gac tgc tac atg aac cgg
gag gag 1440His Gly Phe Gln Met Leu Tyr Ala Asp Cys Tyr Met Asn Arg
Glu Glu465 470 475 480ttc tgg gag atg ttt gat ggc tcc ttg tac cac
aag ctg cga gag aag 1488Phe Trp Glu Met Phe Asp Gly Ser Leu Tyr His
Lys Leu Arg Glu Lys 485 490 495ctg ggt tgc cag gac gcc ttc ccc gag
gtg tac gac aag atc tgc aag 1536Leu Gly Cys Gln Asp Ala Phe Pro Glu
Val Tyr Asp Lys Ile Cys Lys 500 505 510gcc gcc agg cac tga 1551Ala
Ala Arg His 51516516PRTHomo sapiens 16Met Glu Pro Ala Val Ser Leu
Ala Val Cys Ala Leu Leu Phe Leu Leu1 5 10 15Trp Val Arg Leu Lys Gly
Leu Glu Phe Val Leu Ile His Gln Arg Trp 20 25 30Val Phe Val Cys Leu
Phe Leu Leu Pro Leu Ser Leu Ile Phe Asp Ile 35 40 45Tyr Tyr Tyr Val
Arg Ala Trp Val Val Phe Lys Leu Ser Ser Ala Pro 50 55 60Arg Leu His
Glu Gln Arg Val Arg Asp Ile Gln Lys Gln Val Arg Glu65 70 75 80Trp
Lys Glu Gln Gly Ser Lys Thr Phe Met Cys Thr Gly Arg Pro Gly 85 90
95Trp Leu Thr Val Ser Leu Arg Val Gly Lys Tyr Lys Lys Thr His Lys
100 105 110Asn Ile Met Ile Asn Leu Met Asp Ile Leu Glu Val Asp Thr
Lys Lys 115 120 125Gln Ile Val Arg Val Glu Pro Leu Val Thr Met Gly
Gln Val Thr Ala 130 135 140Leu Leu Thr Ser Ile Gly Trp Thr Leu Pro
Val Leu Pro Glu Leu Asp145 150 155 160Asp Leu Thr Val Gly Gly Leu
Ile Met Gly Thr Gly Ile Glu Ser Ser 165 170 175Ser His Lys Tyr Gly
Leu Phe Gln His Ile Cys Thr Ala Tyr Glu Leu 180 185 190Val Leu Ala
Asp Gly Ser Phe Val Arg Cys Thr Pro Ser Glu Asn Ser 195 200 205Asp
Leu Phe Tyr Ala Val Pro Trp Ser Cys Gly Thr Leu Gly Phe Leu 210 215
220Val Ala Ala Glu Ile Arg Ile Ile Pro Ala Lys Lys Tyr Val Lys
Leu225 230 235 240Arg Phe Glu Pro Val Arg Gly Leu Glu Ala Ile Cys
Ala Lys Phe Thr 245 250 255His Glu Ser Gln Arg Gln Glu Asn His Phe
Val Glu Gly Leu Leu Tyr 260 265 270Ser Leu Asp Glu Ala Val Ile Met
Thr Gly Val Met Thr Asp Glu Ala 275 280 285Glu Pro Ser Lys Leu Asn
Ser Ile Gly Asn Tyr Tyr Lys Pro Trp Phe 290 295 300Phe Lys His Val
Glu Asn Tyr Leu Lys Thr Asn Arg Glu Gly Leu Glu305 310 315 320Tyr
Ile Pro Leu Arg His Tyr Tyr His Arg His Thr Arg Ser Ile Phe 325 330
335Trp Glu Leu Gln Asp Ile Ile Pro Phe Gly Asn Asn Pro Ile Phe Arg
340 345 350Tyr Leu Phe Gly Trp Met Val Pro Pro Lys Ile Ser Leu Leu
Lys Leu 355 360 365Thr Gln Gly Glu Thr Leu Arg Lys Leu Tyr Glu Gln
His His Val Val 370 375 380Gln Asp Met Leu Val Pro Met Lys Cys Leu
Gln Gln Ala Leu His Thr385 390 395 400Phe Gln Asn Asp Ile His Val
Tyr Pro Ile Trp Leu Cys Pro Phe Ile 405 410 415Leu Pro Ser Gln Pro
Gly Leu Val His Pro Lys Gly Asn Glu Ala Glu 420 425 430Leu Tyr Ile
Asp Ile Gly Ala Tyr Gly Glu Pro Arg Val Lys His Phe 435 440 445Glu
Ala Arg Ser Cys Met Arg Gln Leu Glu Lys Phe Val Arg Ser Val 450 455
460His Gly Phe Gln Met Leu Tyr Ala Asp Cys Tyr Met Asn Arg Glu
Glu465 470 475 480Phe Trp Glu Met Phe Asp Gly Ser Leu Tyr His Lys
Leu Arg Glu Lys 485 490 495Leu Gly Cys Gln Asp Ala Phe Pro Glu Val
Tyr Asp Lys Ile Cys Lys 500 505 510Ala Ala Arg His
515171422DNASaccharomyces cerevisiaeCDS(1)..(1422) 17atg gca aag
gat aat agt gag aag ctg cag gtg cag gga gag gag aaa 48Met Ala Lys
Asp Asn Ser Glu Lys Leu Gln Val Gln Gly Glu Glu Lys1 5 10 15aag tcc
aag caa ccg gtt aat ttc ctg cct cag ggt aaa tgg ctg aag 96Lys Ser
Lys Gln Pro Val Asn Phe Leu Pro Gln Gly Lys Trp Leu Lys 20 25 30cca
aat gaa atc gaa tat gag ttt ggt ggg act act ggt gtt att ggt 144Pro
Asn Glu Ile Glu Tyr Glu Phe Gly Gly Thr Thr Gly Val Ile Gly 35 40
45atg ctg atc ggg ttt cca ctg cta atg tac tat atg tgg att tgt gcg
192Met Leu Ile Gly Phe Pro Leu Leu Met Tyr Tyr Met Trp Ile Cys Ala
50 55 60gaa ttt tat cac ggt aag gtt gcc cta ccc aag gct ggt gaa tcg
tgg 240Glu Phe Tyr His Gly Lys Val Ala Leu Pro Lys Ala Gly Glu Ser
Trp65 70 75 80atg cac ttt atc aag cac cta tac cag tta gtc ttg gag
aac ggt atc 288Met His Phe Ile Lys His Leu Tyr Gln Leu Val Leu Glu
Asn Gly Ile 85 90 95cca gaa aag tat gac tgg act att ttc tta aca ttt
tgg gtg ttt cag 336Pro Glu Lys Tyr Asp Trp Thr Ile Phe Leu Thr Phe
Trp Val Phe Gln 100 105 110atc att ttc tac tat acg ttg ccc ggg att
tgg aca aaa ggt caa cca 384Ile Ile Phe Tyr Tyr Thr Leu Pro Gly Ile
Trp Thr Lys Gly Gln Pro 115 120 125ttg tct cat ttg aag gga aaa caa
ttg cct tac ttt tgt aat gcc atg 432Leu Ser His Leu Lys Gly Lys Gln
Leu Pro Tyr Phe Cys Asn Ala Met 130 135 140tgg acc ttg tat gta act
acc act ttg gtc ttg gtt ttg cac ttt acc 480Trp Thr Leu Tyr Val Thr
Thr Thr Leu Val Leu Val Leu His Phe Thr145 150 155 160aat ctt ttt
aga ttg tat gtc att att gac cgt ttt ggg agg atc atg 528Asn Leu Phe
Arg Leu Tyr Val Ile Ile Asp Arg Phe Gly Arg Ile Met 165 170 175aca
tgt gcc att att tca ggg ttt gcc ttc tcc atc ata ttg tac tta 576Thr
Cys Ala Ile Ile Ser Gly Phe Ala Phe Ser Ile Ile Leu Tyr Leu 180 185
190tgg act tta ttt atc tca cat gac tat cat aga atg aca gga aac cat
624Trp Thr Leu Phe Ile Ser His Asp Tyr His Arg Met Thr Gly Asn His
195 200 205cta tat gat ttc ttc atg gga
gct cca cta aac cct agg tgg ggg att 672Leu Tyr Asp Phe Phe Met Gly
Ala Pro Leu Asn Pro Arg Trp Gly Ile 210 215 220ttg gac ttg aag atg
ttt ttc gag gtt aga tta cct tgg ttc acc ctt 720Leu Asp Leu Lys Met
Phe Phe Glu Val Arg Leu Pro Trp Phe Thr Leu225 230 235 240tac ttt
atc act ttg ggt gcc tgt ttg aag cag tgg gag act tac ggc 768Tyr Phe
Ile Thr Leu Gly Ala Cys Leu Lys Gln Trp Glu Thr Tyr Gly 245 250
255tat gtg aca cca caa ttg ggg gtt gtc atg tta gct cat tgg ttg tac
816Tyr Val Thr Pro Gln Leu Gly Val Val Met Leu Ala His Trp Leu Tyr
260 265 270gcg aac gca tgt gct aaa ggt gaa gaa ttg att gtt cca acc
tgg gac 864Ala Asn Ala Cys Ala Lys Gly Glu Glu Leu Ile Val Pro Thr
Trp Asp 275 280 285atg gct tac gaa aag ttt gga ttt atg ctg atc ttc
tgg aat att gcc 912Met Ala Tyr Glu Lys Phe Gly Phe Met Leu Ile Phe
Trp Asn Ile Ala 290 295 300ggt gtc cca tac act tac tgt cat tgt acg
ttg tat ttg tac tac cat 960Gly Val Pro Tyr Thr Tyr Cys His Cys Thr
Leu Tyr Leu Tyr Tyr His305 310 315 320gac cca tct gaa tat cac tgg
tct aca ctg tac aat gtt tcg ctg tac 1008Asp Pro Ser Glu Tyr His Trp
Ser Thr Leu Tyr Asn Val Ser Leu Tyr 325 330 335gtt gtt cta tta tgc
gcc tac tac ttc ttt gac acg gca aat gct cag 1056Val Val Leu Leu Cys
Ala Tyr Tyr Phe Phe Asp Thr Ala Asn Ala Gln 340 345 350aaa aat gcc
ttc aga aag caa atg tct ggt gac aag aca ggt agg aag 1104Lys Asn Ala
Phe Arg Lys Gln Met Ser Gly Asp Lys Thr Gly Arg Lys 355 360 365act
ttc cca ttt ttg cca tac caa att ttg aag aat cca aag tat atg 1152Thr
Phe Pro Phe Leu Pro Tyr Gln Ile Leu Lys Asn Pro Lys Tyr Met 370 375
380gtt acc tcc aat gga tcg tac cta ttg att gat ggt tgg tac act ttg
1200Val Thr Ser Asn Gly Ser Tyr Leu Leu Ile Asp Gly Trp Tyr Thr
Leu385 390 395 400gct aga aaa att cac tac act gcc gat tgg act caa
tct ctc gtt tgg 1248Ala Arg Lys Ile His Tyr Thr Ala Asp Trp Thr Gln
Ser Leu Val Trp 405 410 415gcc ttg tct tgc ggg ttc aac tcg gtg ttc
cca tgg ttt ttc cca gta 1296Ala Leu Ser Cys Gly Phe Asn Ser Val Phe
Pro Trp Phe Phe Pro Val 420 425 430ttc ttc ctt gtt gtc ctg att cac
aga gcc ttc aga gac caa gca aaa 1344Phe Phe Leu Val Val Leu Ile His
Arg Ala Phe Arg Asp Gln Ala Lys 435 440 445tgt aag aga aag tac gga
aaa gat tgg gat gag tat tgt aaa cat tgc 1392Cys Lys Arg Lys Tyr Gly
Lys Asp Trp Asp Glu Tyr Cys Lys His Cys 450 455 460cct tac gtc ttt
att cct tat gtt ttc tag 1422Pro Tyr Val Phe Ile Pro Tyr Val Phe465
47018473PRTSaccharomyces cerevisiae 18Met Ala Lys Asp Asn Ser Glu
Lys Leu Gln Val Gln Gly Glu Glu Lys1 5 10 15Lys Ser Lys Gln Pro Val
Asn Phe Leu Pro Gln Gly Lys Trp Leu Lys 20 25 30Pro Asn Glu Ile Glu
Tyr Glu Phe Gly Gly Thr Thr Gly Val Ile Gly 35 40 45Met Leu Ile Gly
Phe Pro Leu Leu Met Tyr Tyr Met Trp Ile Cys Ala 50 55 60Glu Phe Tyr
His Gly Lys Val Ala Leu Pro Lys Ala Gly Glu Ser Trp65 70 75 80Met
His Phe Ile Lys His Leu Tyr Gln Leu Val Leu Glu Asn Gly Ile 85 90
95Pro Glu Lys Tyr Asp Trp Thr Ile Phe Leu Thr Phe Trp Val Phe Gln
100 105 110Ile Ile Phe Tyr Tyr Thr Leu Pro Gly Ile Trp Thr Lys Gly
Gln Pro 115 120 125Leu Ser His Leu Lys Gly Lys Gln Leu Pro Tyr Phe
Cys Asn Ala Met 130 135 140Trp Thr Leu Tyr Val Thr Thr Thr Leu Val
Leu Val Leu His Phe Thr145 150 155 160Asn Leu Phe Arg Leu Tyr Val
Ile Ile Asp Arg Phe Gly Arg Ile Met 165 170 175Thr Cys Ala Ile Ile
Ser Gly Phe Ala Phe Ser Ile Ile Leu Tyr Leu 180 185 190Trp Thr Leu
Phe Ile Ser His Asp Tyr His Arg Met Thr Gly Asn His 195 200 205Leu
Tyr Asp Phe Phe Met Gly Ala Pro Leu Asn Pro Arg Trp Gly Ile 210 215
220Leu Asp Leu Lys Met Phe Phe Glu Val Arg Leu Pro Trp Phe Thr
Leu225 230 235 240Tyr Phe Ile Thr Leu Gly Ala Cys Leu Lys Gln Trp
Glu Thr Tyr Gly 245 250 255Tyr Val Thr Pro Gln Leu Gly Val Val Met
Leu Ala His Trp Leu Tyr 260 265 270Ala Asn Ala Cys Ala Lys Gly Glu
Glu Leu Ile Val Pro Thr Trp Asp 275 280 285Met Ala Tyr Glu Lys Phe
Gly Phe Met Leu Ile Phe Trp Asn Ile Ala 290 295 300Gly Val Pro Tyr
Thr Tyr Cys His Cys Thr Leu Tyr Leu Tyr Tyr His305 310 315 320Asp
Pro Ser Glu Tyr His Trp Ser Thr Leu Tyr Asn Val Ser Leu Tyr 325 330
335Val Val Leu Leu Cys Ala Tyr Tyr Phe Phe Asp Thr Ala Asn Ala Gln
340 345 350Lys Asn Ala Phe Arg Lys Gln Met Ser Gly Asp Lys Thr Gly
Arg Lys 355 360 365Thr Phe Pro Phe Leu Pro Tyr Gln Ile Leu Lys Asn
Pro Lys Tyr Met 370 375 380Val Thr Ser Asn Gly Ser Tyr Leu Leu Ile
Asp Gly Trp Tyr Thr Leu385 390 395 400Ala Arg Lys Ile His Tyr Thr
Ala Asp Trp Thr Gln Ser Leu Val Trp 405 410 415Ala Leu Ser Cys Gly
Phe Asn Ser Val Phe Pro Trp Phe Phe Pro Val 420 425 430Phe Phe Leu
Val Val Leu Ile His Arg Ala Phe Arg Asp Gln Ala Lys 435 440 445Cys
Lys Arg Lys Tyr Gly Lys Asp Trp Asp Glu Tyr Cys Lys His Cys 450 455
460Pro Tyr Val Phe Ile Pro Tyr Val Phe465 470191152DNASaccharomyces
cerevisiaeCDS(1)..(1152) 19atg agt gaa aca gaa ttg aga aaa aga cag
gcc caa ttc act agg gag 48Met Ser Glu Thr Glu Leu Arg Lys Arg Gln
Ala Gln Phe Thr Arg Glu1 5 10 15tta cat ggt gat gat att ggt aaa aag
aca ggt ttg agt gca ttg atg 96Leu His Gly Asp Asp Ile Gly Lys Lys
Thr Gly Leu Ser Ala Leu Met 20 25 30tcg aag aac aac tct gcc caa aag
gaa gcc gtt cag aag tac ttg aga 144Ser Lys Asn Asn Ser Ala Gln Lys
Glu Ala Val Gln Lys Tyr Leu Arg 35 40 45aat tgg gat ggt aga acc gat
aaa gat gcc gaa gaa cgt cgt ctt gag 192Asn Trp Asp Gly Arg Thr Asp
Lys Asp Ala Glu Glu Arg Arg Leu Glu 50 55 60gat tat aat gaa gcc aca
cat tcc tac tat aac gtc gtt aca gat ttc 240Asp Tyr Asn Glu Ala Thr
His Ser Tyr Tyr Asn Val Val Thr Asp Phe65 70 75 80tat gaa tat ggt
tgg ggt tcc tct ttc cat ttc agc aga ttt tat aaa 288Tyr Glu Tyr Gly
Trp Gly Ser Ser Phe His Phe Ser Arg Phe Tyr Lys 85 90 95ggt gag agt
ttc gct gcc tcg ata gca aga cat gaa cat tat tta gct 336Gly Glu Ser
Phe Ala Ala Ser Ile Ala Arg His Glu His Tyr Leu Ala 100 105 110tac
aag gct ggt att caa aga ggc gat tta gtt ctc gac gtt ggt tgt 384Tyr
Lys Ala Gly Ile Gln Arg Gly Asp Leu Val Leu Asp Val Gly Cys 115 120
125ggt gtt ggg ggc cca gca aga gag att gca aga ttt acc ggt tgt aac
432Gly Val Gly Gly Pro Ala Arg Glu Ile Ala Arg Phe Thr Gly Cys Asn
130 135 140gtc atc ggt cta aac aat aac gat tac caa att gcc aag gca
aaa tat 480Val Ile Gly Leu Asn Asn Asn Asp Tyr Gln Ile Ala Lys Ala
Lys Tyr145 150 155 160tac gct aaa aaa tac aat ttg agt gac caa atg
gac ttt gta aag ggt 528Tyr Ala Lys Lys Tyr Asn Leu Ser Asp Gln Met
Asp Phe Val Lys Gly 165 170 175gat ttc atg aaa atg gat ttc gaa gaa
aac act ttc gac aaa gtt tat 576Asp Phe Met Lys Met Asp Phe Glu Glu
Asn Thr Phe Asp Lys Val Tyr 180 185 190gca att gag gcc aca tgt cac
gct cca aaa tta gaa ggt gta tac agc 624Ala Ile Glu Ala Thr Cys His
Ala Pro Lys Leu Glu Gly Val Tyr Ser 195 200 205gaa atc tac aag gtt
ttg aaa ccg ggt ggt acc ttt gct gtt tac gaa 672Glu Ile Tyr Lys Val
Leu Lys Pro Gly Gly Thr Phe Ala Val Tyr Glu 210 215 220tgg gta atg
act gat aaa tat gac gaa aac aat cct gaa cat aga aag 720Trp Val Met
Thr Asp Lys Tyr Asp Glu Asn Asn Pro Glu His Arg Lys225 230 235
240atc gct tat gaa att gaa cta ggt gat ggt atc cca aag atg ttc cat
768Ile Ala Tyr Glu Ile Glu Leu Gly Asp Gly Ile Pro Lys Met Phe His
245 250 255gtc gac gtg gct agg aaa gca ttg aag aac tgt ggt ttc gaa
gtc ctc 816Val Asp Val Ala Arg Lys Ala Leu Lys Asn Cys Gly Phe Glu
Val Leu 260 265 270gtt agc gaa gac ctg gcg gac aat gat gat gaa atc
cct tgg tat tac 864Val Ser Glu Asp Leu Ala Asp Asn Asp Asp Glu Ile
Pro Trp Tyr Tyr 275 280 285cca tta act ggt gag tgg aag tac gtt caa
aac tta gct aat ttg gcc 912Pro Leu Thr Gly Glu Trp Lys Tyr Val Gln
Asn Leu Ala Asn Leu Ala 290 295 300aca ttt ttc aga act tct tac ttg
ggt aga caa ttt act aca gca atg 960Thr Phe Phe Arg Thr Ser Tyr Leu
Gly Arg Gln Phe Thr Thr Ala Met305 310 315 320gtt act gta atg gaa
aaa tta ggt cta gcc cca gaa ggt tcc aag gaa 1008Val Thr Val Met Glu
Lys Leu Gly Leu Ala Pro Glu Gly Ser Lys Glu 325 330 335gtt act gct
gct cta gaa aat gct gcg gtt ggt tta gtt gcc ggt ggt 1056Val Thr Ala
Ala Leu Glu Asn Ala Ala Val Gly Leu Val Ala Gly Gly 340 345 350aag
tcc aag tta ttc act cca atg atg ctt ttc gtc gct agg aag cca 1104Lys
Ser Lys Leu Phe Thr Pro Met Met Leu Phe Val Ala Arg Lys Pro 355 360
365gaa aac gcc gaa acc ccc tcc caa act tcc caa gaa gca act caa taa
1152Glu Asn Ala Glu Thr Pro Ser Gln Thr Ser Gln Glu Ala Thr Gln 370
375 38020383PRTSaccharomyces cerevisiae 20Met Ser Glu Thr Glu Leu
Arg Lys Arg Gln Ala Gln Phe Thr Arg Glu1 5 10 15Leu His Gly Asp Asp
Ile Gly Lys Lys Thr Gly Leu Ser Ala Leu Met 20 25 30Ser Lys Asn Asn
Ser Ala Gln Lys Glu Ala Val Gln Lys Tyr Leu Arg 35 40 45Asn Trp Asp
Gly Arg Thr Asp Lys Asp Ala Glu Glu Arg Arg Leu Glu 50 55 60Asp Tyr
Asn Glu Ala Thr His Ser Tyr Tyr Asn Val Val Thr Asp Phe65 70 75
80Tyr Glu Tyr Gly Trp Gly Ser Ser Phe His Phe Ser Arg Phe Tyr Lys
85 90 95Gly Glu Ser Phe Ala Ala Ser Ile Ala Arg His Glu His Tyr Leu
Ala 100 105 110Tyr Lys Ala Gly Ile Gln Arg Gly Asp Leu Val Leu Asp
Val Gly Cys 115 120 125Gly Val Gly Gly Pro Ala Arg Glu Ile Ala Arg
Phe Thr Gly Cys Asn 130 135 140Val Ile Gly Leu Asn Asn Asn Asp Tyr
Gln Ile Ala Lys Ala Lys Tyr145 150 155 160Tyr Ala Lys Lys Tyr Asn
Leu Ser Asp Gln Met Asp Phe Val Lys Gly 165 170 175Asp Phe Met Lys
Met Asp Phe Glu Glu Asn Thr Phe Asp Lys Val Tyr 180 185 190Ala Ile
Glu Ala Thr Cys His Ala Pro Lys Leu Glu Gly Val Tyr Ser 195 200
205Glu Ile Tyr Lys Val Leu Lys Pro Gly Gly Thr Phe Ala Val Tyr Glu
210 215 220Trp Val Met Thr Asp Lys Tyr Asp Glu Asn Asn Pro Glu His
Arg Lys225 230 235 240Ile Ala Tyr Glu Ile Glu Leu Gly Asp Gly Ile
Pro Lys Met Phe His 245 250 255Val Asp Val Ala Arg Lys Ala Leu Lys
Asn Cys Gly Phe Glu Val Leu 260 265 270Val Ser Glu Asp Leu Ala Asp
Asn Asp Asp Glu Ile Pro Trp Tyr Tyr 275 280 285Pro Leu Thr Gly Glu
Trp Lys Tyr Val Gln Asn Leu Ala Asn Leu Ala 290 295 300Thr Phe Phe
Arg Thr Ser Tyr Leu Gly Arg Gln Phe Thr Thr Ala Met305 310 315
320Val Thr Val Met Glu Lys Leu Gly Leu Ala Pro Glu Gly Ser Lys Glu
325 330 335Val Thr Ala Ala Leu Glu Asn Ala Ala Val Gly Leu Val Ala
Gly Gly 340 345 350Lys Ser Lys Leu Phe Thr Pro Met Met Leu Phe Val
Ala Arg Lys Pro 355 360 365Glu Asn Ala Glu Thr Pro Ser Gln Thr Ser
Gln Glu Ala Thr Gln 370 375 380211617DNASaccharomyces
cerevisiaeCDS(1)..(1617) 21atg agt tct gtc gca gaa aat ata ata caa
cat gcc act cat aat tct 48Met Ser Ser Val Ala Glu Asn Ile Ile Gln
His Ala Thr His Asn Ser1 5 10 15acg cta cac caa ttg gct aaa gac cag
ccc tct gta ggc gtc act act 96Thr Leu His Gln Leu Ala Lys Asp Gln
Pro Ser Val Gly Val Thr Thr 20 25 30gcc ttc agt atc ctg gat aca ctt
aag tct atg tca tat ttg aaa ata 144Ala Phe Ser Ile Leu Asp Thr Leu
Lys Ser Met Ser Tyr Leu Lys Ile 35 40 45ttt gct act tta atc tgt att
ctt ttg gtt tgg gac caa gtt gca tat 192Phe Ala Thr Leu Ile Cys Ile
Leu Leu Val Trp Asp Gln Val Ala Tyr 50 55 60caa atc aag aaa ggt tcc
atc gca ggt cca aag ttt aag ttc tgg ccc 240Gln Ile Lys Lys Gly Ser
Ile Ala Gly Pro Lys Phe Lys Phe Trp Pro65 70 75 80atc atc ggt cca
ttt ttg gaa tcc tta gat cca aag ttt gaa gaa tat 288Ile Ile Gly Pro
Phe Leu Glu Ser Leu Asp Pro Lys Phe Glu Glu Tyr 85 90 95aag gct aag
tgg gca tcc ggt cca ctt tca tgt gtt tct att ttc cat 336Lys Ala Lys
Trp Ala Ser Gly Pro Leu Ser Cys Val Ser Ile Phe His 100 105 110aaa
ttt gtt gtt atc gca tct act aga gac ttg gca aga aag atc ttg 384Lys
Phe Val Val Ile Ala Ser Thr Arg Asp Leu Ala Arg Lys Ile Leu 115 120
125caa tct tcc aaa ttc gtc aaa cct tgc gtt gtc gat gtt gct gtg aag
432Gln Ser Ser Lys Phe Val Lys Pro Cys Val Val Asp Val Ala Val Lys
130 135 140atc tta aga cct tgc aat tgg gtt ttt ttg gac ggt aaa gct
cat act 480Ile Leu Arg Pro Cys Asn Trp Val Phe Leu Asp Gly Lys Ala
His Thr145 150 155 160gat tac aga aaa tca tta aac ggt ctt ttc act
aaa caa gct ttg gct 528Asp Tyr Arg Lys Ser Leu Asn Gly Leu Phe Thr
Lys Gln Ala Leu Ala 165 170 175caa tac tta cct tca ttg gaa caa atc
atg gat aag tac atg gat aag 576Gln Tyr Leu Pro Ser Leu Glu Gln Ile
Met Asp Lys Tyr Met Asp Lys 180 185 190ttt gtt cgt tta tct aag gag
aat aac tac gag ccc cag gtc ttt ttc 624Phe Val Arg Leu Ser Lys Glu
Asn Asn Tyr Glu Pro Gln Val Phe Phe 195 200 205cat gaa atg aga gaa
att ctt tgc gcc tta tca ttg aac tct ttc tgt 672His Glu Met Arg Glu
Ile Leu Cys Ala Leu Ser Leu Asn Ser Phe Cys 210 215 220ggt aac tat
att acc gaa gat caa gtc aga aag att gct gat gat tac 720Gly Asn Tyr
Ile Thr Glu Asp Gln Val Arg Lys Ile Ala Asp Asp Tyr225 230 235
240tat ttg gtt aca gca gca ttg gaa tta gtc aac ttc cca att att atc
768Tyr Leu Val Thr Ala Ala Leu Glu Leu Val Asn Phe Pro Ile Ile Ile
245 250 255cct tac act aaa aca tgg tat ggt aag aaa act gca gac atg
gcc atg 816Pro Tyr Thr Lys Thr Trp Tyr Gly Lys Lys Thr Ala Asp Met
Ala Met 260 265 270aag att ttc gaa aac tgt gct caa atg gct aag gat
cat att gct gca 864Lys Ile Phe Glu Asn Cys Ala Gln Met Ala Lys Asp
His Ile Ala Ala 275 280 285ggt ggt aag cca gtt tgt gtt atg gat gct
tgg tgt aag ttg atg cac 912Gly Gly Lys Pro Val Cys Val Met Asp Ala
Trp Cys Lys Leu Met His 290 295 300gat gca aag aat agt aac gat gat
gat tct aga atc tac cac aga gag 960Asp Ala Lys Asn Ser Asn Asp Asp
Asp Ser Arg Ile Tyr His Arg Glu305 310 315 320ttt act aac aag gaa
atc tcc gaa gct gtt ttc act ttc tta ttt gct 1008Phe Thr Asn Lys Glu
Ile Ser Glu Ala Val Phe Thr Phe Leu Phe Ala 325 330 335tct caa gat
gcc tct tct tct tta gct tgt tgg ttg ttc caa att gtt 1056Ser Gln Asp
Ala Ser Ser Ser Leu Ala Cys Trp Leu Phe Gln Ile Val 340
345 350gct gac cgt cca gat gtc tta gct aag atc aga gaa gaa caa ttg
gct 1104Ala Asp Arg Pro Asp Val Leu Ala Lys Ile Arg Glu Glu Gln Leu
Ala 355 360 365gtt cgt aac aat gac atg tct acc gaa ttg aac ttg gat
ttg att gag 1152Val Arg Asn Asn Asp Met Ser Thr Glu Leu Asn Leu Asp
Leu Ile Glu 370 375 380aaa atg aag tac acc aat atg gtc ata aaa gaa
act ttg cgt tac aga 1200Lys Met Lys Tyr Thr Asn Met Val Ile Lys Glu
Thr Leu Arg Tyr Arg385 390 395 400cct cct gtc ttg atg gtt cca tat
gtt gtt aag aag aat ttc cca gtt 1248Pro Pro Val Leu Met Val Pro Tyr
Val Val Lys Lys Asn Phe Pro Val 405 410 415tcc cct aac tat acc gca
cca aag ggc gct atg tta att cca acc tta 1296Ser Pro Asn Tyr Thr Ala
Pro Lys Gly Ala Met Leu Ile Pro Thr Leu 420 425 430tac cca gct tta
cat gat cct gaa gtt tac gaa aat cct gat gag ttc 1344Tyr Pro Ala Leu
His Asp Pro Glu Val Tyr Glu Asn Pro Asp Glu Phe 435 440 445atc cct
gaa aga tgg gta gaa ggc tct aag gct agt gaa gca aag aag 1392Ile Pro
Glu Arg Trp Val Glu Gly Ser Lys Ala Ser Glu Ala Lys Lys 450 455
460aat tgg ttg gtt ttt ggt tgt ggt cca cac gtt tgc tta ggt caa aca
1440Asn Trp Leu Val Phe Gly Cys Gly Pro His Val Cys Leu Gly Gln
Thr465 470 475 480tat gtc atg att acc ttc gcc gct ttg ttg ggt aaa
ttt gca cta tat 1488Tyr Val Met Ile Thr Phe Ala Ala Leu Leu Gly Lys
Phe Ala Leu Tyr 485 490 495act gat ttc cat cat aca gtg act cca tta
agt gaa aaa atc aag gtt 1536Thr Asp Phe His His Thr Val Thr Pro Leu
Ser Glu Lys Ile Lys Val 500 505 510ttc gct aca att ttc cca aaa gat
gat ttg tta ctg act ttc aaa aag 1584Phe Ala Thr Ile Phe Pro Lys Asp
Asp Leu Leu Leu Thr Phe Lys Lys 515 520 525aga gac cca att act gga
gaa gtc ttc gaa taa 1617Arg Asp Pro Ile Thr Gly Glu Val Phe Glu 530
53522538PRTSaccharomyces cerevisiae 22Met Ser Ser Val Ala Glu Asn
Ile Ile Gln His Ala Thr His Asn Ser1 5 10 15Thr Leu His Gln Leu Ala
Lys Asp Gln Pro Ser Val Gly Val Thr Thr 20 25 30Ala Phe Ser Ile Leu
Asp Thr Leu Lys Ser Met Ser Tyr Leu Lys Ile 35 40 45Phe Ala Thr Leu
Ile Cys Ile Leu Leu Val Trp Asp Gln Val Ala Tyr 50 55 60Gln Ile Lys
Lys Gly Ser Ile Ala Gly Pro Lys Phe Lys Phe Trp Pro65 70 75 80Ile
Ile Gly Pro Phe Leu Glu Ser Leu Asp Pro Lys Phe Glu Glu Tyr 85 90
95Lys Ala Lys Trp Ala Ser Gly Pro Leu Ser Cys Val Ser Ile Phe His
100 105 110Lys Phe Val Val Ile Ala Ser Thr Arg Asp Leu Ala Arg Lys
Ile Leu 115 120 125Gln Ser Ser Lys Phe Val Lys Pro Cys Val Val Asp
Val Ala Val Lys 130 135 140Ile Leu Arg Pro Cys Asn Trp Val Phe Leu
Asp Gly Lys Ala His Thr145 150 155 160Asp Tyr Arg Lys Ser Leu Asn
Gly Leu Phe Thr Lys Gln Ala Leu Ala 165 170 175Gln Tyr Leu Pro Ser
Leu Glu Gln Ile Met Asp Lys Tyr Met Asp Lys 180 185 190Phe Val Arg
Leu Ser Lys Glu Asn Asn Tyr Glu Pro Gln Val Phe Phe 195 200 205His
Glu Met Arg Glu Ile Leu Cys Ala Leu Ser Leu Asn Ser Phe Cys 210 215
220Gly Asn Tyr Ile Thr Glu Asp Gln Val Arg Lys Ile Ala Asp Asp
Tyr225 230 235 240Tyr Leu Val Thr Ala Ala Leu Glu Leu Val Asn Phe
Pro Ile Ile Ile 245 250 255Pro Tyr Thr Lys Thr Trp Tyr Gly Lys Lys
Thr Ala Asp Met Ala Met 260 265 270Lys Ile Phe Glu Asn Cys Ala Gln
Met Ala Lys Asp His Ile Ala Ala 275 280 285Gly Gly Lys Pro Val Cys
Val Met Asp Ala Trp Cys Lys Leu Met His 290 295 300Asp Ala Lys Asn
Ser Asn Asp Asp Asp Ser Arg Ile Tyr His Arg Glu305 310 315 320Phe
Thr Asn Lys Glu Ile Ser Glu Ala Val Phe Thr Phe Leu Phe Ala 325 330
335Ser Gln Asp Ala Ser Ser Ser Leu Ala Cys Trp Leu Phe Gln Ile Val
340 345 350Ala Asp Arg Pro Asp Val Leu Ala Lys Ile Arg Glu Glu Gln
Leu Ala 355 360 365Val Arg Asn Asn Asp Met Ser Thr Glu Leu Asn Leu
Asp Leu Ile Glu 370 375 380Lys Met Lys Tyr Thr Asn Met Val Ile Lys
Glu Thr Leu Arg Tyr Arg385 390 395 400Pro Pro Val Leu Met Val Pro
Tyr Val Val Lys Lys Asn Phe Pro Val 405 410 415Ser Pro Asn Tyr Thr
Ala Pro Lys Gly Ala Met Leu Ile Pro Thr Leu 420 425 430Tyr Pro Ala
Leu His Asp Pro Glu Val Tyr Glu Asn Pro Asp Glu Phe 435 440 445Ile
Pro Glu Arg Trp Val Glu Gly Ser Lys Ala Ser Glu Ala Lys Lys 450 455
460Asn Trp Leu Val Phe Gly Cys Gly Pro His Val Cys Leu Gly Gln
Thr465 470 475 480Tyr Val Met Ile Thr Phe Ala Ala Leu Leu Gly Lys
Phe Ala Leu Tyr 485 490 495Thr Asp Phe His His Thr Val Thr Pro Leu
Ser Glu Lys Ile Lys Val 500 505 510Phe Ala Thr Ile Phe Pro Lys Asp
Asp Leu Leu Leu Thr Phe Lys Lys 515 520 525Arg Asp Pro Ile Thr Gly
Glu Val Phe Glu 530 535231578DNAArtificial SequenceDescription of
Artificial Sequence Truncated HMG construct 23atg gac caa ttg gtg
aaa act gaa gtc acc aag aag tct ttt act gct 48Met Asp Gln Leu Val
Lys Thr Glu Val Thr Lys Lys Ser Phe Thr Ala1 5 10 15cct gta caa aag
gct tct aca cca gtt tta acc aat aaa aca gtc att 96Pro Val Gln Lys
Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val Ile 20 25 30tct gga tcg
aaa gtc aaa agt tta tca tct gcg caa tcg agc tca tca 144Ser Gly Ser
Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser Ser 35 40 45gga cct
tca tca tct agt gag gaa gat gat tcc cgc gat att gaa agc 192Gly Pro
Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu Ser 50 55 60ttg
gat aag aaa ata cgt cct tta gaa gaa tta gaa gca tta tta agt 240Leu
Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu Ser65 70 75
80agt gga aat aca aaa caa ttg aag aac aaa gag gtc gct gcc ttg gtt
288Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala Ala Leu Val
85 90 95att cac ggt aag tta cct ttg tac gct ttg gag aaa aaa tta ggt
gat 336Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys Lys Leu Gly
Asp 100 105 110act acg aga gcg gtt gcg gta cgt agg aag gct ctt tca
att ttg gca 384Thr Thr Arg Ala Val Ala Val Arg Arg Lys Ala Leu Ser
Ile Leu Ala 115 120 125gaa gct cct gta tta gca tct gat cgt tta cca
tat aaa aat tat gac 432Glu Ala Pro Val Leu Ala Ser Asp Arg Leu Pro
Tyr Lys Asn Tyr Asp 130 135 140tac gac cgc gta ttt ggc gct tgt tgt
gaa aat gtt ata ggt tac atg 480Tyr Asp Arg Val Phe Gly Ala Cys Cys
Glu Asn Val Ile Gly Tyr Met145 150 155 160cct ttg ccc gtt ggt gtt
ata ggc ccc ttg gtt atc gat ggt aca tct 528Pro Leu Pro Val Gly Val
Ile Gly Pro Leu Val Ile Asp Gly Thr Ser 165 170 175tat cat ata cca
atg gca act aca gag ggt tgt ttg gta gct tct gcc 576Tyr His Ile Pro
Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser Ala 180 185 190atg cgt
ggc tgt aag gca atc aat gct ggc ggt ggt gca aca act gtt 624Met Arg
Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr Val 195 200
205tta act aag gat ggt atg aca aga ggc cca gta gtc cgt ttc cca act
672Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe Pro Thr
210 215 220ttg aaa aga tct ggt gcc tgt aag ata tgg tta gac tca gaa
gag gga 720Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp Ser Glu
Glu Gly225 230 235 240caa aac gca att aaa aaa gct ttt aac tct aca
tca aga ttt gca cgt 768Gln Asn Ala Ile Lys Lys Ala Phe Asn Ser Thr
Ser Arg Phe Ala Arg 245 250 255ctg caa cat att caa act tgt cta gca
gga gat tta ctc ttc atg aga 816Leu Gln His Ile Gln Thr Cys Leu Ala
Gly Asp Leu Leu Phe Met Arg 260 265 270ttt aga aca act act ggt gac
gca atg ggt atg aat atg att tct aaa 864Phe Arg Thr Thr Thr Gly Asp
Ala Met Gly Met Asn Met Ile Ser Lys 275 280 285ggt gtc gaa tac tca
tta aag caa atg gta gaa gag tat ggc tgg gaa 912Gly Val Glu Tyr Ser
Leu Lys Gln Met Val Glu Glu Tyr Gly Trp Glu 290 295 300gat atg gag
gtt gtc tcc gtt tct ggt aac tac tgt acc gac aaa aaa 960Asp Met Glu
Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys Lys305 310 315
320cca gct gcc atc aac tgg atc gaa ggt cgt ggt aag agt gtc gtc gca
1008Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val Ala
325 330 335gaa gct act att cct ggt gat gtt gtc aga aaa gtg tta aaa
agt gat 1056Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu Lys
Ser Asp 340 345 350gtt tcc gca ttg gtt gag ttg aac att gct aag aat
ttg gtt gga tct 1104Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn
Leu Val Gly Ser 355 360 365gca atg gct ggg tct gtt ggt gga ttt aac
gca cat gca gct aat tta 1152Ala Met Ala Gly Ser Val Gly Gly Phe Asn
Ala His Ala Ala Asn Leu 370 375 380gtg aca gct gtt ttc ttg gca tta
gga caa gat cct gca caa aat gtt 1200Val Thr Ala Val Phe Leu Ala Leu
Gly Gln Asp Pro Ala Gln Asn Val385 390 395 400gaa agt tcc aac tgt
ata aca ttg atg aaa gaa gtg gac ggt gat ttg 1248Glu Ser Ser Asn Cys
Ile Thr Leu Met Lys Glu Val Asp Gly Asp Leu 405 410 415aga att tcc
gta tcc atg cca tcc atc gaa gta ggt acc atc ggt ggt 1296Arg Ile Ser
Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly Gly 420 425 430ggt
act gtt cta gaa cca caa ggt gcc atg ttg gac tta tta ggt gta 1344Gly
Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly Val 435 440
445aga ggc ccg cat gct acc gct cct ggt acc aac gca cgt caa tta gca
1392Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln Leu Ala
450 455 460aga ata gtt gcc tgt gcc gtc ttg gca ggt gaa tta tcc tta
tgt gct 1440Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu Ser Leu
Cys Ala465 470 475 480gcc cta gca gcc ggc cat ttg gtt caa agt cat
atg acc cac aac agg 1488Ala Leu Ala Ala Gly His Leu Val Gln Ser His
Met Thr His Asn Arg 485 490 495aaa cct gct gaa cca aca aaa cct aac
aat ttg gac gcc act gat ata 1536Lys Pro Ala Glu Pro Thr Lys Pro Asn
Asn Leu Asp Ala Thr Asp Ile 500 505 510aat cgt ttg aaa gat ggg tcc
gtc acc tgc att aaa tcc taa 1578Asn Arg Leu Lys Asp Gly Ser Val Thr
Cys Ile Lys Ser 515 520 52524525PRTArtificial SequenceDescription
of Artificial Sequence Synthetic protein construct 24Met Asp Gln
Leu Val Lys Thr Glu Val Thr Lys Lys Ser Phe Thr Ala1 5 10 15Pro Val
Gln Lys Ala Ser Thr Pro Val Leu Thr Asn Lys Thr Val Ile 20 25 30Ser
Gly Ser Lys Val Lys Ser Leu Ser Ser Ala Gln Ser Ser Ser Ser 35 40
45Gly Pro Ser Ser Ser Ser Glu Glu Asp Asp Ser Arg Asp Ile Glu Ser
50 55 60Leu Asp Lys Lys Ile Arg Pro Leu Glu Glu Leu Glu Ala Leu Leu
Ser65 70 75 80Ser Gly Asn Thr Lys Gln Leu Lys Asn Lys Glu Val Ala
Ala Leu Val 85 90 95Ile His Gly Lys Leu Pro Leu Tyr Ala Leu Glu Lys
Lys Leu Gly Asp 100 105 110Thr Thr Arg Ala Val Ala Val Arg Arg Lys
Ala Leu Ser Ile Leu Ala 115 120 125Glu Ala Pro Val Leu Ala Ser Asp
Arg Leu Pro Tyr Lys Asn Tyr Asp 130 135 140Tyr Asp Arg Val Phe Gly
Ala Cys Cys Glu Asn Val Ile Gly Tyr Met145 150 155 160Pro Leu Pro
Val Gly Val Ile Gly Pro Leu Val Ile Asp Gly Thr Ser 165 170 175Tyr
His Ile Pro Met Ala Thr Thr Glu Gly Cys Leu Val Ala Ser Ala 180 185
190Met Arg Gly Cys Lys Ala Ile Asn Ala Gly Gly Gly Ala Thr Thr Val
195 200 205Leu Thr Lys Asp Gly Met Thr Arg Gly Pro Val Val Arg Phe
Pro Thr 210 215 220Leu Lys Arg Ser Gly Ala Cys Lys Ile Trp Leu Asp
Ser Glu Glu Gly225 230 235 240Gln Asn Ala Ile Lys Lys Ala Phe Asn
Ser Thr Ser Arg Phe Ala Arg 245 250 255Leu Gln His Ile Gln Thr Cys
Leu Ala Gly Asp Leu Leu Phe Met Arg 260 265 270Phe Arg Thr Thr Thr
Gly Asp Ala Met Gly Met Asn Met Ile Ser Lys 275 280 285Gly Val Glu
Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr Gly Trp Glu 290 295 300Asp
Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr Asp Lys Lys305 310
315 320Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser Val Val
Ala 325 330 335Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu
Lys Ser Asp 340 345 350Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys
Asn Leu Val Gly Ser 355 360 365Ala Met Ala Gly Ser Val Gly Gly Phe
Asn Ala His Ala Ala Asn Leu 370 375 380Val Thr Ala Val Phe Leu Ala
Leu Gly Gln Asp Pro Ala Gln Asn Val385 390 395 400Glu Ser Ser Asn
Cys Ile Thr Leu Met Lys Glu Val Asp Gly Asp Leu 405 410 415Arg Ile
Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr Ile Gly Gly 420 425
430Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu Leu Gly Val
435 440 445Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg Gln
Leu Ala 450 455 460Arg Ile Val Ala Cys Ala Val Leu Ala Gly Glu Leu
Ser Leu Cys Ala465 470 475 480Ala Leu Ala Ala Gly His Leu Val Gln
Ser His Met Thr His Asn Arg 485 490 495Lys Pro Ala Glu Pro Thr Lys
Pro Asn Asn Leu Asp Ala Thr Asp Ile 500 505 510Asn Arg Leu Lys Asp
Gly Ser Val Thr Cys Ile Lys Ser 515 520 525251593DNASaccharomyces
cerevisiaeCDS(1)..(1593) 25atg tct gct acc aag tca atc gtt gga gag
gca ttg gaa tac gta aac 48Met Ser Ala Thr Lys Ser Ile Val Gly Glu
Ala Leu Glu Tyr Val Asn1 5 10 15att ggt tta agt cat ttc ttg gct tta
cca ttg gcc caa aga atc tct 96Ile Gly Leu Ser His Phe Leu Ala Leu
Pro Leu Ala Gln Arg Ile Ser 20 25 30ttg atc ata ata att cct ttc att
tac aat att gta tgg caa tta cta 144Leu Ile Ile Ile Ile Pro Phe Ile
Tyr Asn Ile Val Trp Gln Leu Leu 35 40 45tat tct ttg aga aag gac cgt
cca cct cta gtg ttt tac tgg att cca 192Tyr Ser Leu Arg Lys Asp Arg
Pro Pro Leu Val Phe Tyr Trp Ile Pro 50 55 60tgg gtc ggt agt gct gtt
gtg tac ggt atg aag cca tac gag ttt ttc 240Trp Val Gly Ser Ala Val
Val Tyr Gly Met Lys Pro Tyr Glu Phe Phe65 70 75 80gaa gaa tgt caa
aag aaa tac ggt gat att ttt tca ttc gtt ttg tta 288Glu Glu Cys Gln
Lys Lys Tyr Gly Asp Ile Phe Ser Phe Val Leu Leu 85 90 95gga aga gtc
atg act gtg tat tta gga cca aag ggt cac gaa ttt gtc 336Gly Arg Val
Met Thr Val Tyr Leu Gly Pro Lys Gly His Glu Phe Val 100 105 110ttc
aac gct aag ttg gca gat gtt tca gca gaa gct gct tac gct cat 384Phe
Asn Ala Lys Leu Ala Asp Val Ser Ala Glu Ala Ala Tyr Ala His 115 120
125ttg act act cca gtt ttc ggt aaa ggt gtt att tac gat tgt cca aat
432Leu Thr Thr Pro Val Phe Gly
Lys Gly Val Ile Tyr Asp Cys Pro Asn 130 135 140tct aga ttg atg gag
caa aag aag ttt gtt aag ggt gct cta acc aaa 480Ser Arg Leu Met Glu
Gln Lys Lys Phe Val Lys Gly Ala Leu Thr Lys145 150 155 160gaa gcc
ttc aag agc tac gtt cca ttg att gct gaa gaa gtg tac aag 528Glu Ala
Phe Lys Ser Tyr Val Pro Leu Ile Ala Glu Glu Val Tyr Lys 165 170
175tac ttc aga gac tcc aaa aac ttc cgt ttg aat gaa aga act act ggt
576Tyr Phe Arg Asp Ser Lys Asn Phe Arg Leu Asn Glu Arg Thr Thr Gly
180 185 190act att gac gtg atg gtt act caa cct gaa atg act att ttc
acc gct 624Thr Ile Asp Val Met Val Thr Gln Pro Glu Met Thr Ile Phe
Thr Ala 195 200 205tca aga tca tta ttg ggt aag gaa atg aga gca aaa
ttg gat acc gat 672Ser Arg Ser Leu Leu Gly Lys Glu Met Arg Ala Lys
Leu Asp Thr Asp 210 215 220ttt gct tac ttg tac agt gat ttg gat aag
ggt ttc act cca atc aac 720Phe Ala Tyr Leu Tyr Ser Asp Leu Asp Lys
Gly Phe Thr Pro Ile Asn225 230 235 240ttc gtc ttc cct aac tta cca
ttg gaa cac tat aga aag aga gat cac 768Phe Val Phe Pro Asn Leu Pro
Leu Glu His Tyr Arg Lys Arg Asp His 245 250 255gct caa aag gct atc
tcc ggt act tac atg tct ttg att aag gaa aga 816Ala Gln Lys Ala Ile
Ser Gly Thr Tyr Met Ser Leu Ile Lys Glu Arg 260 265 270aga aag aac
aac gac att caa gac aga gat ttg atc gat tcc ttg atg 864Arg Lys Asn
Asn Asp Ile Gln Asp Arg Asp Leu Ile Asp Ser Leu Met 275 280 285aag
aac tct acc tac aag gat ggt gtg aag atg act gat caa gaa atc 912Lys
Asn Ser Thr Tyr Lys Asp Gly Val Lys Met Thr Asp Gln Glu Ile 290 295
300gct aac ttg tta att ggt gtc tta atg ggt ggt caa cat act tct gct
960Ala Asn Leu Leu Ile Gly Val Leu Met Gly Gly Gln His Thr Ser
Ala305 310 315 320gcc act tct gct tgg att ttg ttg cac ttg gct gaa
aga cca gat gtc 1008Ala Thr Ser Ala Trp Ile Leu Leu His Leu Ala Glu
Arg Pro Asp Val 325 330 335caa caa gaa ttg tac gaa gaa caa atg cgt
gtt ttg gat ggt ggt aag 1056Gln Gln Glu Leu Tyr Glu Glu Gln Met Arg
Val Leu Asp Gly Gly Lys 340 345 350aag gaa ttg acc tac gat tta tta
caa gaa atg cca ttg ttg aac caa 1104Lys Glu Leu Thr Tyr Asp Leu Leu
Gln Glu Met Pro Leu Leu Asn Gln 355 360 365act att aag gaa act cta
aga atg cac cat cca ttg cac tct ttg ttc 1152Thr Ile Lys Glu Thr Leu
Arg Met His His Pro Leu His Ser Leu Phe 370 375 380cgt aag gtt atg
aaa gat atg cac gtt cca aac act tct tat gtc atc 1200Arg Lys Val Met
Lys Asp Met His Val Pro Asn Thr Ser Tyr Val Ile385 390 395 400cca
gca ggt tat cac gtt ttg gtt tct cca ggt tac act cat tta aga 1248Pro
Ala Gly Tyr His Val Leu Val Ser Pro Gly Tyr Thr His Leu Arg 405 410
415gac gaa tac ttc cct aat gct cac caa ttc aac att cac cgt tgg aac
1296Asp Glu Tyr Phe Pro Asn Ala His Gln Phe Asn Ile His Arg Trp Asn
420 425 430aaa gat tct gcc tcc tct tat tcc gtc ggt gaa gaa gtc gat
tac ggt 1344Lys Asp Ser Ala Ser Ser Tyr Ser Val Gly Glu Glu Val Asp
Tyr Gly 435 440 445ttc ggt gcc att tct aag ggt gtc agc tct cca tac
tta cct ttc ggt 1392Phe Gly Ala Ile Ser Lys Gly Val Ser Ser Pro Tyr
Leu Pro Phe Gly 450 455 460ggt ggt aga cac aga tgt atc ggt gaa cac
ttt gct tac tgt cag cta 1440Gly Gly Arg His Arg Cys Ile Gly Glu His
Phe Ala Tyr Cys Gln Leu465 470 475 480ggt gtt cta atg tcc att ttt
atc aga aca tta aaa tgg cat tac cca 1488Gly Val Leu Met Ser Ile Phe
Ile Arg Thr Leu Lys Trp His Tyr Pro 485 490 495gag ggt aag acc gtt
cca cct cct gac ttt aca tct atg gtt act ctt 1536Glu Gly Lys Thr Val
Pro Pro Pro Asp Phe Thr Ser Met Val Thr Leu 500 505 510cca acc ggt
cca gcc aag atc atc tgg gaa aag aga aat cca gaa caa 1584Pro Thr Gly
Pro Ala Lys Ile Ile Trp Glu Lys Arg Asn Pro Glu Gln 515 520 525aag
atc taa 1593Lys Ile 53026530PRTSaccharomyces cerevisiae 26Met Ser
Ala Thr Lys Ser Ile Val Gly Glu Ala Leu Glu Tyr Val Asn1 5 10 15Ile
Gly Leu Ser His Phe Leu Ala Leu Pro Leu Ala Gln Arg Ile Ser 20 25
30Leu Ile Ile Ile Ile Pro Phe Ile Tyr Asn Ile Val Trp Gln Leu Leu
35 40 45Tyr Ser Leu Arg Lys Asp Arg Pro Pro Leu Val Phe Tyr Trp Ile
Pro 50 55 60Trp Val Gly Ser Ala Val Val Tyr Gly Met Lys Pro Tyr Glu
Phe Phe65 70 75 80Glu Glu Cys Gln Lys Lys Tyr Gly Asp Ile Phe Ser
Phe Val Leu Leu 85 90 95Gly Arg Val Met Thr Val Tyr Leu Gly Pro Lys
Gly His Glu Phe Val 100 105 110Phe Asn Ala Lys Leu Ala Asp Val Ser
Ala Glu Ala Ala Tyr Ala His 115 120 125Leu Thr Thr Pro Val Phe Gly
Lys Gly Val Ile Tyr Asp Cys Pro Asn 130 135 140Ser Arg Leu Met Glu
Gln Lys Lys Phe Val Lys Gly Ala Leu Thr Lys145 150 155 160Glu Ala
Phe Lys Ser Tyr Val Pro Leu Ile Ala Glu Glu Val Tyr Lys 165 170
175Tyr Phe Arg Asp Ser Lys Asn Phe Arg Leu Asn Glu Arg Thr Thr Gly
180 185 190Thr Ile Asp Val Met Val Thr Gln Pro Glu Met Thr Ile Phe
Thr Ala 195 200 205Ser Arg Ser Leu Leu Gly Lys Glu Met Arg Ala Lys
Leu Asp Thr Asp 210 215 220Phe Ala Tyr Leu Tyr Ser Asp Leu Asp Lys
Gly Phe Thr Pro Ile Asn225 230 235 240Phe Val Phe Pro Asn Leu Pro
Leu Glu His Tyr Arg Lys Arg Asp His 245 250 255Ala Gln Lys Ala Ile
Ser Gly Thr Tyr Met Ser Leu Ile Lys Glu Arg 260 265 270Arg Lys Asn
Asn Asp Ile Gln Asp Arg Asp Leu Ile Asp Ser Leu Met 275 280 285Lys
Asn Ser Thr Tyr Lys Asp Gly Val Lys Met Thr Asp Gln Glu Ile 290 295
300Ala Asn Leu Leu Ile Gly Val Leu Met Gly Gly Gln His Thr Ser
Ala305 310 315 320Ala Thr Ser Ala Trp Ile Leu Leu His Leu Ala Glu
Arg Pro Asp Val 325 330 335Gln Gln Glu Leu Tyr Glu Glu Gln Met Arg
Val Leu Asp Gly Gly Lys 340 345 350Lys Glu Leu Thr Tyr Asp Leu Leu
Gln Glu Met Pro Leu Leu Asn Gln 355 360 365Thr Ile Lys Glu Thr Leu
Arg Met His His Pro Leu His Ser Leu Phe 370 375 380Arg Lys Val Met
Lys Asp Met His Val Pro Asn Thr Ser Tyr Val Ile385 390 395 400Pro
Ala Gly Tyr His Val Leu Val Ser Pro Gly Tyr Thr His Leu Arg 405 410
415Asp Glu Tyr Phe Pro Asn Ala His Gln Phe Asn Ile His Arg Trp Asn
420 425 430Lys Asp Ser Ala Ser Ser Tyr Ser Val Gly Glu Glu Val Asp
Tyr Gly 435 440 445Phe Gly Ala Ile Ser Lys Gly Val Ser Ser Pro Tyr
Leu Pro Phe Gly 450 455 460Gly Gly Arg His Arg Cys Ile Gly Glu His
Phe Ala Tyr Cys Gln Leu465 470 475 480Gly Val Leu Met Ser Ile Phe
Ile Arg Thr Leu Lys Trp His Tyr Pro 485 490 495Glu Gly Lys Thr Val
Pro Pro Pro Asp Phe Thr Ser Met Val Thr Leu 500 505 510Pro Thr Gly
Pro Ala Lys Ile Ile Trp Glu Lys Arg Asn Pro Glu Gln 515 520 525Lys
Ile 530271491DNASaccharomyces cerevisiaeCDS(1)..(1491) 27atg tct
gct gtt aac gtt gca cct gaa ttg att aat gcc gac aac aca 48Met Ser
Ala Val Asn Val Ala Pro Glu Leu Ile Asn Ala Asp Asn Thr1 5 10 15att
acc tac gat gcg att gtc atc ggt gct ggt gtt atc ggt cca tgt 96Ile
Thr Tyr Asp Ala Ile Val Ile Gly Ala Gly Val Ile Gly Pro Cys 20 25
30gtt gct act ggt cta gca aga aag ggt aag aaa gtt ctt atc gta gaa
144Val Ala Thr Gly Leu Ala Arg Lys Gly Lys Lys Val Leu Ile Val Glu
35 40 45cgt gac tgg gct atg cct gat aga att gtt ggt gaa ttg atg caa
cca 192Arg Asp Trp Ala Met Pro Asp Arg Ile Val Gly Glu Leu Met Gln
Pro 50 55 60ggt ggt gtt aga gca ttg aga agt ctg ggt atg att caa tct
atc aac 240Gly Gly Val Arg Ala Leu Arg Ser Leu Gly Met Ile Gln Ser
Ile Asn65 70 75 80aac atc gaa gca tat cct gtt acc ggt tat acc gtc
ttt ttc aac ggc 288Asn Ile Glu Ala Tyr Pro Val Thr Gly Tyr Thr Val
Phe Phe Asn Gly 85 90 95gaa caa gtt gat att cca tac cct tac aag gcc
gat atc cct aaa gtt 336Glu Gln Val Asp Ile Pro Tyr Pro Tyr Lys Ala
Asp Ile Pro Lys Val 100 105 110gaa aaa ttg aag gac ttg gtc aaa gat
ggt aat gac aag gtc ttg gaa 384Glu Lys Leu Lys Asp Leu Val Lys Asp
Gly Asn Asp Lys Val Leu Glu 115 120 125gac agc act att cac atc aag
gat tac gaa gat gat gaa aga gaa agg 432Asp Ser Thr Ile His Ile Lys
Asp Tyr Glu Asp Asp Glu Arg Glu Arg 130 135 140ggt gtt gct ttt gtt
cat ggt aga ttc ttg aac aac ttg aga aac att 480Gly Val Ala Phe Val
His Gly Arg Phe Leu Asn Asn Leu Arg Asn Ile145 150 155 160act gct
caa gag cca aat gtt act aga gtg caa ggt aac tgt att gag 528Thr Ala
Gln Glu Pro Asn Val Thr Arg Val Gln Gly Asn Cys Ile Glu 165 170
175ata ttg aag gat gaa aag aat gag gtt gtt ggt gcc aag gtt gac att
576Ile Leu Lys Asp Glu Lys Asn Glu Val Val Gly Ala Lys Val Asp Ile
180 185 190gat ggc cgt ggc aag gtg gaa ttc aaa gcc cac ttg aca ttt
atc tgt 624Asp Gly Arg Gly Lys Val Glu Phe Lys Ala His Leu Thr Phe
Ile Cys 195 200 205gac ggt atc ttt tca cgt ttc aga aag gaa ttg cac
cca gac cat gtt 672Asp Gly Ile Phe Ser Arg Phe Arg Lys Glu Leu His
Pro Asp His Val 210 215 220cca act gtc ggt tct tcg ttt gtc ggt atg
tct ttg ttc aat gct aag 720Pro Thr Val Gly Ser Ser Phe Val Gly Met
Ser Leu Phe Asn Ala Lys225 230 235 240aat cct gct cct atg cac ggt
cac gtt att ctt ggt agt gat cat atg 768Asn Pro Ala Pro Met His Gly
His Val Ile Leu Gly Ser Asp His Met 245 250 255cca atc ttg gtt tac
caa atc agt cca gaa gaa aca aga atc ctt tgt 816Pro Ile Leu Val Tyr
Gln Ile Ser Pro Glu Glu Thr Arg Ile Leu Cys 260 265 270gct tac aac
tct cca aag gtc cca gct gat atc aag agt tgg atg att 864Ala Tyr Asn
Ser Pro Lys Val Pro Ala Asp Ile Lys Ser Trp Met Ile 275 280 285aag
gat gtc caa cct ttc att cca aag agt cta cgt cct tca ttt gat 912Lys
Asp Val Gln Pro Phe Ile Pro Lys Ser Leu Arg Pro Ser Phe Asp 290 295
300gaa gcc gtc agc caa ggt aaa ttt aga gct atg cca aac tcc tac ttg
960Glu Ala Val Ser Gln Gly Lys Phe Arg Ala Met Pro Asn Ser Tyr
Leu305 310 315 320cca gct aga caa aac gac gtc act ggt atg tgt gtt
atc ggt gac gct 1008Pro Ala Arg Gln Asn Asp Val Thr Gly Met Cys Val
Ile Gly Asp Ala 325 330 335cta aat atg aga cat cca ttg act ggt ggt
ggt atg act gtc ggt ttg 1056Leu Asn Met Arg His Pro Leu Thr Gly Gly
Gly Met Thr Val Gly Leu 340 345 350cat gat gtt gtc ttg ttg att aag
aaa ata ggt gac cta gac ttc agc 1104His Asp Val Val Leu Leu Ile Lys
Lys Ile Gly Asp Leu Asp Phe Ser 355 360 365gac cgt gaa aag gtt ttg
gat gaa tta cta gac tac cat ttc gaa aga 1152Asp Arg Glu Lys Val Leu
Asp Glu Leu Leu Asp Tyr His Phe Glu Arg 370 375 380aag agt tac gat
tcc gtt att aac gtt ttg tca gtg gct ttg tat tct 1200Lys Ser Tyr Asp
Ser Val Ile Asn Val Leu Ser Val Ala Leu Tyr Ser385 390 395 400ttg
ttc gct gct gac agc gat aac ttg aag gca tta caa aaa ggt tgt 1248Leu
Phe Ala Ala Asp Ser Asp Asn Leu Lys Ala Leu Gln Lys Gly Cys 405 410
415ttc aaa tat ttc caa aga ggt ggc gat tgt gtc aac aaa ccc gtt gaa
1296Phe Lys Tyr Phe Gln Arg Gly Gly Asp Cys Val Asn Lys Pro Val Glu
420 425 430ttt ctg tct ggt gtc ttg cca aag cct ttg caa ttg acc agg
gtt ttc 1344Phe Leu Ser Gly Val Leu Pro Lys Pro Leu Gln Leu Thr Arg
Val Phe 435 440 445ttc gct gtc gct ttt tac acc att tac ttg aac atg
gaa gaa cgt ggt 1392Phe Ala Val Ala Phe Tyr Thr Ile Tyr Leu Asn Met
Glu Glu Arg Gly 450 455 460ttc ttg gga tta cca atg gct tta ttg gaa
ggt att atg att ttg atc 1440Phe Leu Gly Leu Pro Met Ala Leu Leu Glu
Gly Ile Met Ile Leu Ile465 470 475 480aca gct att aga gta ttc acc
cca ttt ttg ttt ggt gag ttg att ggt 1488Thr Ala Ile Arg Val Phe Thr
Pro Phe Leu Phe Gly Glu Leu Ile Gly 485 490 495taa
149128496PRTSaccharomyces cerevisiae 28Met Ser Ala Val Asn Val Ala
Pro Glu Leu Ile Asn Ala Asp Asn Thr1 5 10 15Ile Thr Tyr Asp Ala Ile
Val Ile Gly Ala Gly Val Ile Gly Pro Cys 20 25 30Val Ala Thr Gly Leu
Ala Arg Lys Gly Lys Lys Val Leu Ile Val Glu 35 40 45Arg Asp Trp Ala
Met Pro Asp Arg Ile Val Gly Glu Leu Met Gln Pro 50 55 60Gly Gly Val
Arg Ala Leu Arg Ser Leu Gly Met Ile Gln Ser Ile Asn65 70 75 80Asn
Ile Glu Ala Tyr Pro Val Thr Gly Tyr Thr Val Phe Phe Asn Gly 85 90
95Glu Gln Val Asp Ile Pro Tyr Pro Tyr Lys Ala Asp Ile Pro Lys Val
100 105 110Glu Lys Leu Lys Asp Leu Val Lys Asp Gly Asn Asp Lys Val
Leu Glu 115 120 125Asp Ser Thr Ile His Ile Lys Asp Tyr Glu Asp Asp
Glu Arg Glu Arg 130 135 140Gly Val Ala Phe Val His Gly Arg Phe Leu
Asn Asn Leu Arg Asn Ile145 150 155 160Thr Ala Gln Glu Pro Asn Val
Thr Arg Val Gln Gly Asn Cys Ile Glu 165 170 175Ile Leu Lys Asp Glu
Lys Asn Glu Val Val Gly Ala Lys Val Asp Ile 180 185 190Asp Gly Arg
Gly Lys Val Glu Phe Lys Ala His Leu Thr Phe Ile Cys 195 200 205Asp
Gly Ile Phe Ser Arg Phe Arg Lys Glu Leu His Pro Asp His Val 210 215
220Pro Thr Val Gly Ser Ser Phe Val Gly Met Ser Leu Phe Asn Ala
Lys225 230 235 240Asn Pro Ala Pro Met His Gly His Val Ile Leu Gly
Ser Asp His Met 245 250 255Pro Ile Leu Val Tyr Gln Ile Ser Pro Glu
Glu Thr Arg Ile Leu Cys 260 265 270Ala Tyr Asn Ser Pro Lys Val Pro
Ala Asp Ile Lys Ser Trp Met Ile 275 280 285Lys Asp Val Gln Pro Phe
Ile Pro Lys Ser Leu Arg Pro Ser Phe Asp 290 295 300Glu Ala Val Ser
Gln Gly Lys Phe Arg Ala Met Pro Asn Ser Tyr Leu305 310 315 320Pro
Ala Arg Gln Asn Asp Val Thr Gly Met Cys Val Ile Gly Asp Ala 325 330
335Leu Asn Met Arg His Pro Leu Thr Gly Gly Gly Met Thr Val Gly Leu
340 345 350His Asp Val Val Leu Leu Ile Lys Lys Ile Gly Asp Leu Asp
Phe Ser 355 360 365Asp Arg Glu Lys Val Leu Asp Glu Leu Leu Asp Tyr
His Phe Glu Arg 370 375 380Lys Ser Tyr Asp Ser Val Ile Asn Val Leu
Ser Val Ala Leu Tyr Ser385 390 395 400Leu Phe Ala Ala Asp Ser Asp
Asn Leu Lys Ala Leu Gln Lys Gly Cys 405 410 415Phe Lys Tyr Phe Gln
Arg Gly Gly Asp Cys Val Asn Lys Pro Val Glu 420 425 430Phe Leu Ser
Gly Val Leu Pro Lys Pro Leu Gln Leu Thr Arg Val Phe 435 440 445Phe
Ala Val Ala Phe Tyr Thr Ile Tyr Leu Asn Met Glu Glu Arg Gly 450 455
460Phe Leu Gly Leu Pro Met Ala Leu Leu Glu Gly Ile Met Ile Leu
Ile465 470 475 480Thr Ala Ile Arg Val Phe Thr Pro Phe Leu
Phe Gly Glu Leu Ile Gly 485 490 495291335DNASaccharomyces
cerevisiaeCDS(1)..(1335) 29atg gga aag cta tta caa ttg gca ttg cat
ccg gtc gag atg aag gca 48Met Gly Lys Leu Leu Gln Leu Ala Leu His
Pro Val Glu Met Lys Ala1 5 10 15gct ttg aag ctg aag ttt tgc aga aca
ccg cta ttc tcc atc tat gat 96Ala Leu Lys Leu Lys Phe Cys Arg Thr
Pro Leu Phe Ser Ile Tyr Asp 20 25 30cag tcc acg tct cca tat ctc ttg
cac tgt ttc gaa ctg ttg aac ttg 144Gln Ser Thr Ser Pro Tyr Leu Leu
His Cys Phe Glu Leu Leu Asn Leu 35 40 45acc tcc aga tcg ttt gct gct
gtg atc aga gag ctg cat cca gaa ttg 192Thr Ser Arg Ser Phe Ala Ala
Val Ile Arg Glu Leu His Pro Glu Leu 50 55 60aga aac tgt gtt act ctc
ttt tat ttg att tta agg gct ttg gat acc 240Arg Asn Cys Val Thr Leu
Phe Tyr Leu Ile Leu Arg Ala Leu Asp Thr65 70 75 80atc gaa gac gat
atg tcc atc gaa cac gat ttg aaa att gac ttg ttg 288Ile Glu Asp Asp
Met Ser Ile Glu His Asp Leu Lys Ile Asp Leu Leu 85 90 95cgt cac ttc
cac gag aaa ttg ttg tta act aaa tgg agt ttc gac gga 336Arg His Phe
His Glu Lys Leu Leu Leu Thr Lys Trp Ser Phe Asp Gly 100 105 110aat
gcc ccc gat gtg aag gac aga gcc gtt ttg aca gat ttc gaa tcg 384Asn
Ala Pro Asp Val Lys Asp Arg Ala Val Leu Thr Asp Phe Glu Ser 115 120
125att ctt att gaa ttc cac aaa ttg aaa cca gaa tat caa gaa gtc atc
432Ile Leu Ile Glu Phe His Lys Leu Lys Pro Glu Tyr Gln Glu Val Ile
130 135 140aag gag atc acc gag aaa atg ggt aat ggt atg gcc gac tac
atc tta 480Lys Glu Ile Thr Glu Lys Met Gly Asn Gly Met Ala Asp Tyr
Ile Leu145 150 155 160gat gaa aat tac aac ttg aat ggg ttg caa acc
gtc cac gac tac gac 528Asp Glu Asn Tyr Asn Leu Asn Gly Leu Gln Thr
Val His Asp Tyr Asp 165 170 175gtg tac tgt cac tac gta gct ggt ttg
gtc ggt gat ggt ttg acc cgt 576Val Tyr Cys His Tyr Val Ala Gly Leu
Val Gly Asp Gly Leu Thr Arg 180 185 190ttg att gtc att gcc aag ttt
gcc aac gaa tct ttg tat tct aat gag 624Leu Ile Val Ile Ala Lys Phe
Ala Asn Glu Ser Leu Tyr Ser Asn Glu 195 200 205caa ttg tat gaa agc
atg ggt ctt ttc cta caa aaa acc aac atc atc 672Gln Leu Tyr Glu Ser
Met Gly Leu Phe Leu Gln Lys Thr Asn Ile Ile 210 215 220aga gat tac
aat gaa gat ttg gtc gat ggt aga tcc ttc tgg ccc aag 720Arg Asp Tyr
Asn Glu Asp Leu Val Asp Gly Arg Ser Phe Trp Pro Lys225 230 235
240gaa atc tgg tca caa tac gct cct cag ttg aag gac ttc atg aaa cct
768Glu Ile Trp Ser Gln Tyr Ala Pro Gln Leu Lys Asp Phe Met Lys Pro
245 250 255gaa aac gaa caa ctg ggg ttg gac tgt ata aac cac ctc gtc
tta aac 816Glu Asn Glu Gln Leu Gly Leu Asp Cys Ile Asn His Leu Val
Leu Asn 260 265 270gca ttg agt cat gtt atc gat gtg ttg act tat ttg
gcc ggt atc cac 864Ala Leu Ser His Val Ile Asp Val Leu Thr Tyr Leu
Ala Gly Ile His 275 280 285gag caa tcc act ttc caa ttt tgt gcc att
ccc caa gtt atg gcc att 912Glu Gln Ser Thr Phe Gln Phe Cys Ala Ile
Pro Gln Val Met Ala Ile 290 295 300gca acc ttg gct ttg gta ttc aac
aac cgt gaa gtg cta cat ggc aat 960Ala Thr Leu Ala Leu Val Phe Asn
Asn Arg Glu Val Leu His Gly Asn305 310 315 320gta aag att cgt aag
ggt act acc tgc tat tta att ttg aaa tca agg 1008Val Lys Ile Arg Lys
Gly Thr Thr Cys Tyr Leu Ile Leu Lys Ser Arg 325 330 335act ttg cgt
ggc tgt gtc gag att ttt gac tat tac tta cgt gat atc 1056Thr Leu Arg
Gly Cys Val Glu Ile Phe Asp Tyr Tyr Leu Arg Asp Ile 340 345 350aaa
tct aaa ttg gct gtg caa gat cca aat ttc tta aaa ttg aac att 1104Lys
Ser Lys Leu Ala Val Gln Asp Pro Asn Phe Leu Lys Leu Asn Ile 355 360
365caa atc tcc aag atc gaa cag ttt atg gaa gaa atg tac cag gat aaa
1152Gln Ile Ser Lys Ile Glu Gln Phe Met Glu Glu Met Tyr Gln Asp Lys
370 375 380tta cct cct aac gtg aag cca aat gaa act cca att ttc ttg
aaa gtt 1200Leu Pro Pro Asn Val Lys Pro Asn Glu Thr Pro Ile Phe Leu
Lys Val385 390 395 400aaa gaa aga tcc aga tac gat gat gaa ttg gtt
cca acc caa caa gaa 1248Lys Glu Arg Ser Arg Tyr Asp Asp Glu Leu Val
Pro Thr Gln Gln Glu 405 410 415gaa gag tac aag ttc aat atg gtt tta
tct atc atc ttg tcc gtt ctt 1296Glu Glu Tyr Lys Phe Asn Met Val Leu
Ser Ile Ile Leu Ser Val Leu 420 425 430ctt ggg ttt tat tat ata tac
act tta cac aga gcg tga 1335Leu Gly Phe Tyr Tyr Ile Tyr Thr Leu His
Arg Ala 435 44030444PRTSaccharomyces cerevisiae 30Met Gly Lys Leu
Leu Gln Leu Ala Leu His Pro Val Glu Met Lys Ala1 5 10 15Ala Leu Lys
Leu Lys Phe Cys Arg Thr Pro Leu Phe Ser Ile Tyr Asp 20 25 30Gln Ser
Thr Ser Pro Tyr Leu Leu His Cys Phe Glu Leu Leu Asn Leu 35 40 45Thr
Ser Arg Ser Phe Ala Ala Val Ile Arg Glu Leu His Pro Glu Leu 50 55
60Arg Asn Cys Val Thr Leu Phe Tyr Leu Ile Leu Arg Ala Leu Asp Thr65
70 75 80Ile Glu Asp Asp Met Ser Ile Glu His Asp Leu Lys Ile Asp Leu
Leu 85 90 95Arg His Phe His Glu Lys Leu Leu Leu Thr Lys Trp Ser Phe
Asp Gly 100 105 110Asn Ala Pro Asp Val Lys Asp Arg Ala Val Leu Thr
Asp Phe Glu Ser 115 120 125Ile Leu Ile Glu Phe His Lys Leu Lys Pro
Glu Tyr Gln Glu Val Ile 130 135 140Lys Glu Ile Thr Glu Lys Met Gly
Asn Gly Met Ala Asp Tyr Ile Leu145 150 155 160Asp Glu Asn Tyr Asn
Leu Asn Gly Leu Gln Thr Val His Asp Tyr Asp 165 170 175Val Tyr Cys
His Tyr Val Ala Gly Leu Val Gly Asp Gly Leu Thr Arg 180 185 190Leu
Ile Val Ile Ala Lys Phe Ala Asn Glu Ser Leu Tyr Ser Asn Glu 195 200
205Gln Leu Tyr Glu Ser Met Gly Leu Phe Leu Gln Lys Thr Asn Ile Ile
210 215 220Arg Asp Tyr Asn Glu Asp Leu Val Asp Gly Arg Ser Phe Trp
Pro Lys225 230 235 240Glu Ile Trp Ser Gln Tyr Ala Pro Gln Leu Lys
Asp Phe Met Lys Pro 245 250 255Glu Asn Glu Gln Leu Gly Leu Asp Cys
Ile Asn His Leu Val Leu Asn 260 265 270Ala Leu Ser His Val Ile Asp
Val Leu Thr Tyr Leu Ala Gly Ile His 275 280 285Glu Gln Ser Thr Phe
Gln Phe Cys Ala Ile Pro Gln Val Met Ala Ile 290 295 300Ala Thr Leu
Ala Leu Val Phe Asn Asn Arg Glu Val Leu His Gly Asn305 310 315
320Val Lys Ile Arg Lys Gly Thr Thr Cys Tyr Leu Ile Leu Lys Ser Arg
325 330 335Thr Leu Arg Gly Cys Val Glu Ile Phe Asp Tyr Tyr Leu Arg
Asp Ile 340 345 350Lys Ser Lys Leu Ala Val Gln Asp Pro Asn Phe Leu
Lys Leu Asn Ile 355 360 365Gln Ile Ser Lys Ile Glu Gln Phe Met Glu
Glu Met Tyr Gln Asp Lys 370 375 380Leu Pro Pro Asn Val Lys Pro Asn
Glu Thr Pro Ile Phe Leu Lys Val385 390 395 400Lys Glu Arg Ser Arg
Tyr Asp Asp Glu Leu Val Pro Thr Gln Gln Glu 405 410 415Glu Glu Tyr
Lys Phe Asn Met Val Leu Ser Ile Ile Leu Ser Val Leu 420 425 430Leu
Gly Phe Tyr Tyr Ile Tyr Thr Leu His Arg Ala 435
440311929DNASaccharomyces cerevisiaeCDS(1)..(1929) 31atg gac aag
aag aag gat cta ctg gag aac gaa caa ttt ctc cgc atc 48Met Asp Lys
Lys Lys Asp Leu Leu Glu Asn Glu Gln Phe Leu Arg Ile1 5 10 15caa aag
ctc aac gct gcc gat gcg ggc aaa aga caa tct ata aca gtg 96Gln Lys
Leu Asn Ala Ala Asp Ala Gly Lys Arg Gln Ser Ile Thr Val 20 25 30gac
gac gag ggc gaa cta tat ggg tta gac acc tcc ggc aac tca cca 144Asp
Asp Glu Gly Glu Leu Tyr Gly Leu Asp Thr Ser Gly Asn Ser Pro 35 40
45gcc aat gaa cac aca gct acc aca att aca cag aat cac agc gtg gtg
192Ala Asn Glu His Thr Ala Thr Thr Ile Thr Gln Asn His Ser Val Val
50 55 60gcc tca aac gga gac gtc gca ttc atc cca gga act gct acc gaa
ggc 240Ala Ser Asn Gly Asp Val Ala Phe Ile Pro Gly Thr Ala Thr Glu
Gly65 70 75 80aat aca gag att gta act gaa gaa gtg att gag acc gat
gat aac atg 288Asn Thr Glu Ile Val Thr Glu Glu Val Ile Glu Thr Asp
Asp Asn Met 85 90 95ttc aag acc cat gtg aag act tta agc tcc aaa gag
aag gca cgg tat 336Phe Lys Thr His Val Lys Thr Leu Ser Ser Lys Glu
Lys Ala Arg Tyr 100 105 110agg caa ggg tcc tct aac ttt ata tcg tat
ttc gat gat atg tca ttt 384Arg Gln Gly Ser Ser Asn Phe Ile Ser Tyr
Phe Asp Asp Met Ser Phe 115 120 125gaa cac agg ccc agt ata tta gat
ggg tca gtt aac gag ccc ttc aag 432Glu His Arg Pro Ser Ile Leu Asp
Gly Ser Val Asn Glu Pro Phe Lys 130 135 140acc aaa ttc gtg gga cct
act tta gaa aag gag atc aga aga agg gag 480Thr Lys Phe Val Gly Pro
Thr Leu Glu Lys Glu Ile Arg Arg Arg Glu145 150 155 160aaa gag cta
atg gcc atg cgc aaa aat tta cac cac cgc aag tcc tcc 528Lys Glu Leu
Met Ala Met Arg Lys Asn Leu His His Arg Lys Ser Ser 165 170 175cca
gat gct gtc gac tca gta ggg aaa aat gat ggc gcc gcc cca act 576Pro
Asp Ala Val Asp Ser Val Gly Lys Asn Asp Gly Ala Ala Pro Thr 180 185
190act gtt cca act gcc gcc acc tca gaa acg gtg gtc acc gtt gaa acc
624Thr Val Pro Thr Ala Ala Thr Ser Glu Thr Val Val Thr Val Glu Thr
195 200 205acc ata att tca tcc aat ttc tcc ggg ttg tac gtg gcg ttt
tgg atg 672Thr Ile Ile Ser Ser Asn Phe Ser Gly Leu Tyr Val Ala Phe
Trp Met 210 215 220gct att gca ttt ggt gct gtc aag gct tta ata gac
tat tat tac cag 720Ala Ile Ala Phe Gly Ala Val Lys Ala Leu Ile Asp
Tyr Tyr Tyr Gln225 230 235 240cat aat ggt agc ttc aag gat tcg gag
atc ttg aaa ttt atg act acg 768His Asn Gly Ser Phe Lys Asp Ser Glu
Ile Leu Lys Phe Met Thr Thr 245 250 255aat ttg ttc act gtg gca tcc
gta gat ctt ttg atg tat ttg agc act 816Asn Leu Phe Thr Val Ala Ser
Val Asp Leu Leu Met Tyr Leu Ser Thr 260 265 270tat ttt gtc gtt gga
ata caa tac tta tgc aag tgg ggg gtc ttg aaa 864Tyr Phe Val Val Gly
Ile Gln Tyr Leu Cys Lys Trp Gly Val Leu Lys 275 280 285tgg ggc act
acc ggc tgg atc ttc acc tca att tac gag ttt ttg ttt 912Trp Gly Thr
Thr Gly Trp Ile Phe Thr Ser Ile Tyr Glu Phe Leu Phe 290 295 300gtt
atc ttc tac atg tat tta aca gaa aac atc cta aaa cta cac tgg 960Val
Ile Phe Tyr Met Tyr Leu Thr Glu Asn Ile Leu Lys Leu His Trp305 310
315 320ctg tcc aag atc ttc ctt ttt ttg cat tct tta gtt tta ttg atg
aaa 1008Leu Ser Lys Ile Phe Leu Phe Leu His Ser Leu Val Leu Leu Met
Lys 325 330 335atg cat tct ttc gcc ttc tac aat ggc tat cta tgg ggt
ata aag gaa 1056Met His Ser Phe Ala Phe Tyr Asn Gly Tyr Leu Trp Gly
Ile Lys Glu 340 345 350gaa cta caa ttt tcc aaa agc gct ctt gcc aaa
tac aag gat tct ata 1104Glu Leu Gln Phe Ser Lys Ser Ala Leu Ala Lys
Tyr Lys Asp Ser Ile 355 360 365aat gat cca aaa gtt att ggt gct ctt
gag aaa agc tgt gag ttt tgt 1152Asn Asp Pro Lys Val Ile Gly Ala Leu
Glu Lys Ser Cys Glu Phe Cys 370 375 380agt ttt gaa ttg agc tct cag
tct tta agc gac caa act caa aaa ttc 1200Ser Phe Glu Leu Ser Ser Gln
Ser Leu Ser Asp Gln Thr Gln Lys Phe385 390 395 400ccc aac aat atc
agt gca aaa agc ttt ttt tgg ttc acc atg ttt cca 1248Pro Asn Asn Ile
Ser Ala Lys Ser Phe Phe Trp Phe Thr Met Phe Pro 405 410 415acc cta
att tac caa att gaa tat cca aga act aag gaa atc aga tgg 1296Thr Leu
Ile Tyr Gln Ile Glu Tyr Pro Arg Thr Lys Glu Ile Arg Trp 420 425
430agc tac gta tta gaa aag atc tgc gcc atc ttc ggt acc att ttc tta
1344Ser Tyr Val Leu Glu Lys Ile Cys Ala Ile Phe Gly Thr Ile Phe Leu
435 440 445atg atg ata gat gct caa atc ttg atg tat cct gta gca atg
aga gca 1392Met Met Ile Asp Ala Gln Ile Leu Met Tyr Pro Val Ala Met
Arg Ala 450 455 460ttg gct gtg cgc aat tct gaa tgg act ggt ata ttg
gat aga tta ttg 1440Leu Ala Val Arg Asn Ser Glu Trp Thr Gly Ile Leu
Asp Arg Leu Leu465 470 475 480aaa tgg gtt gga ttg ctc gtt gat atc
gtc cca ggg ttt atc gtg atg 1488Lys Trp Val Gly Leu Leu Val Asp Ile
Val Pro Gly Phe Ile Val Met 485 490 495tac atc ttg gac ttc tat ttg
att tgg gat gcc att ttg aac tgt gtg 1536Tyr Ile Leu Asp Phe Tyr Leu
Ile Trp Asp Ala Ile Leu Asn Cys Val 500 505 510gct gaa ttg aca aga
ttt ggc gac aga tat ttc tac ggt gac tgg tgg 1584Ala Glu Leu Thr Arg
Phe Gly Asp Arg Tyr Phe Tyr Gly Asp Trp Trp 515 520 525aat tgt gtt
agt tgg gca gac ttc agt aga att tgg aac atc cca gtg 1632Asn Cys Val
Ser Trp Ala Asp Phe Ser Arg Ile Trp Asn Ile Pro Val 530 535 540cat
aag ttt ttg tta aga cat gtt tac cat agt tca atg agt tca ttc 1680His
Lys Phe Leu Leu Arg His Val Tyr His Ser Ser Met Ser Ser Phe545 550
555 560aaa ttg aac aag agt caa gca act ttg atg acc ttt ttc tta agt
tcc 1728Lys Leu Asn Lys Ser Gln Ala Thr Leu Met Thr Phe Phe Leu Ser
Ser 565 570 575gtc gtt cat gaa tta gca atg tac gtt atc ttc aag aaa
ttg agg ttt 1776Val Val His Glu Leu Ala Met Tyr Val Ile Phe Lys Lys
Leu Arg Phe 580 585 590tac ttg ttc ttc ttc caa atg ctg caa atg cca
tta gta gct tta aca 1824Tyr Leu Phe Phe Phe Gln Met Leu Gln Met Pro
Leu Val Ala Leu Thr 595 600 605aat act aaa ttc atg agg aac aga acc
ata atc gga aat gtt att ttc 1872Asn Thr Lys Phe Met Arg Asn Arg Thr
Ile Ile Gly Asn Val Ile Phe 610 615 620tgg ctc ggt atc tgc atg gga
cca agt gtc atg tgt acg ttg tac ttg 1920Trp Leu Gly Ile Cys Met Gly
Pro Ser Val Met Cys Thr Leu Tyr Leu625 630 635 640aca ttc taa
1929Thr Phe32642PRTSaccharomyces cerevisiae 32Met Asp Lys Lys Lys
Asp Leu Leu Glu Asn Glu Gln Phe Leu Arg Ile1 5 10 15Gln Lys Leu Asn
Ala Ala Asp Ala Gly Lys Arg Gln Ser Ile Thr Val 20 25 30Asp Asp Glu
Gly Glu Leu Tyr Gly Leu Asp Thr Ser Gly Asn Ser Pro 35 40 45Ala Asn
Glu His Thr Ala Thr Thr Ile Thr Gln Asn His Ser Val Val 50 55 60Ala
Ser Asn Gly Asp Val Ala Phe Ile Pro Gly Thr Ala Thr Glu Gly65 70 75
80Asn Thr Glu Ile Val Thr Glu Glu Val Ile Glu Thr Asp Asp Asn Met
85 90 95Phe Lys Thr His Val Lys Thr Leu Ser Ser Lys Glu Lys Ala Arg
Tyr 100 105 110Arg Gln Gly Ser Ser Asn Phe Ile Ser Tyr Phe Asp Asp
Met Ser Phe 115 120 125Glu His Arg Pro Ser Ile Leu Asp Gly Ser Val
Asn Glu Pro Phe Lys 130 135 140Thr Lys Phe Val Gly Pro Thr Leu Glu
Lys Glu Ile Arg Arg Arg Glu145 150 155 160Lys Glu Leu Met Ala Met
Arg Lys Asn Leu His His Arg Lys Ser Ser 165 170 175Pro Asp Ala Val
Asp Ser Val Gly Lys Asn Asp Gly Ala Ala Pro Thr 180 185 190Thr Val
Pro Thr Ala Ala Thr Ser Glu Thr Val Val Thr Val Glu Thr 195 200
205Thr Ile Ile Ser Ser Asn Phe Ser Gly Leu Tyr Val Ala Phe Trp Met
210 215 220Ala Ile Ala Phe Gly Ala Val Lys Ala Leu Ile Asp Tyr Tyr
Tyr Gln225 230 235 240His Asn Gly Ser Phe Lys Asp Ser Glu Ile Leu
Lys Phe Met Thr
Thr 245 250 255Asn Leu Phe Thr Val Ala Ser Val Asp Leu Leu Met Tyr
Leu Ser Thr 260 265 270Tyr Phe Val Val Gly Ile Gln Tyr Leu Cys Lys
Trp Gly Val Leu Lys 275 280 285Trp Gly Thr Thr Gly Trp Ile Phe Thr
Ser Ile Tyr Glu Phe Leu Phe 290 295 300Val Ile Phe Tyr Met Tyr Leu
Thr Glu Asn Ile Leu Lys Leu His Trp305 310 315 320Leu Ser Lys Ile
Phe Leu Phe Leu His Ser Leu Val Leu Leu Met Lys 325 330 335Met His
Ser Phe Ala Phe Tyr Asn Gly Tyr Leu Trp Gly Ile Lys Glu 340 345
350Glu Leu Gln Phe Ser Lys Ser Ala Leu Ala Lys Tyr Lys Asp Ser Ile
355 360 365Asn Asp Pro Lys Val Ile Gly Ala Leu Glu Lys Ser Cys Glu
Phe Cys 370 375 380Ser Phe Glu Leu Ser Ser Gln Ser Leu Ser Asp Gln
Thr Gln Lys Phe385 390 395 400Pro Asn Asn Ile Ser Ala Lys Ser Phe
Phe Trp Phe Thr Met Phe Pro 405 410 415Thr Leu Ile Tyr Gln Ile Glu
Tyr Pro Arg Thr Lys Glu Ile Arg Trp 420 425 430Ser Tyr Val Leu Glu
Lys Ile Cys Ala Ile Phe Gly Thr Ile Phe Leu 435 440 445Met Met Ile
Asp Ala Gln Ile Leu Met Tyr Pro Val Ala Met Arg Ala 450 455 460Leu
Ala Val Arg Asn Ser Glu Trp Thr Gly Ile Leu Asp Arg Leu Leu465 470
475 480Lys Trp Val Gly Leu Leu Val Asp Ile Val Pro Gly Phe Ile Val
Met 485 490 495Tyr Ile Leu Asp Phe Tyr Leu Ile Trp Asp Ala Ile Leu
Asn Cys Val 500 505 510Ala Glu Leu Thr Arg Phe Gly Asp Arg Tyr Phe
Tyr Gly Asp Trp Trp 515 520 525Asn Cys Val Ser Trp Ala Asp Phe Ser
Arg Ile Trp Asn Ile Pro Val 530 535 540His Lys Phe Leu Leu Arg His
Val Tyr His Ser Ser Met Ser Ser Phe545 550 555 560Lys Leu Asn Lys
Ser Gln Ala Thr Leu Met Thr Phe Phe Leu Ser Ser 565 570 575Val Val
His Glu Leu Ala Met Tyr Val Ile Phe Lys Lys Leu Arg Phe 580 585
590Tyr Leu Phe Phe Phe Gln Met Leu Gln Met Pro Leu Val Ala Leu Thr
595 600 605Asn Thr Lys Phe Met Arg Asn Arg Thr Ile Ile Gly Asn Val
Ile Phe 610 615 620Trp Leu Gly Ile Cys Met Gly Pro Ser Val Met Cys
Thr Leu Tyr Leu625 630 635 640Thr Phe3360DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
33atgtcgaaag ctacatataa ggaacgtgct gcatctcatc ccagctgaag cttcgtacgc
603462DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 34ttagttttgc tggccgcatc ttctcaaata tgcttcccag
gcataggcca ctagtggatc 60tg 623560DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 35gaatactcag gtatcgtaag
atgcaagagt tcgaatctct ccagctgaag cttcgtacgc 603662DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
36tctaccctat gaacatattc cattttgtaa tttcgtgtcg gcataggcca ctagtggatc
60tg 623760DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 37atgacagagc agaaagccct agtaaagcgt attacaaatg
ccagctgaag cttcgtacgc 603862DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 38ctacataaga acacctttgg
tggagggaac atcgttggta gcataggcca ctagtggatc 60tg
623960DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 39atgagtgaaa cagaattgag aaaaagacag gcccaattca
ccagctgaag cttcgtacgc 604062DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 40ttattgagtt gcttcttggg
aagtttggga gggggtttcg gcataggcca ctagtggatc 60tg
624160DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 41atgagttctg tcgcagaaaa tataatacaa catgccactc
ccagctgaag cttcgtacgc 604262DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 42ttattcgaag acttctccag
taattgggtc tctctttttg gcataggcca ctagtggatc 60tg
624333DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 43ctgcggccgc aacatgacca ccaatacggt ccc
334427DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 44ttctcgagtc tttagttatg cttgctc
274533DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 45ctgcggccgc aagatggacc tggttctcag tgc
334629DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 46ttctcgagct acttattctt tgtaaactc
294732DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 47ctgcggccgc aagatggagc ccgccgtgtc gc
324828DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 48aactcgagtc agtgccttgc cgccttgc
28491833DNASaccharomyces cerevisiaeCDS(1)..(1833) 49atg acg gag act
aag gat ttg ttg caa gac gaa gag ttt ctt aag atc 48Met Thr Glu Thr
Lys Asp Leu Leu Gln Asp Glu Glu Phe Leu Lys Ile1 5 10 15cgc aga ctc
aat tcc gca gaa gcc aac aaa cgg cat tcg gtc acg tac 96Arg Arg Leu
Asn Ser Ala Glu Ala Asn Lys Arg His Ser Val Thr Tyr 20 25 30gat aac
gtg atc ctg cca cag gag tcc atg gag gtt tcg cca cgg tcg 144Asp Asn
Val Ile Leu Pro Gln Glu Ser Met Glu Val Ser Pro Arg Ser 35 40 45tct
acc acg tcg ctg gtg gag cca gtg gag tcg act gaa gga gtg gag 192Ser
Thr Thr Ser Leu Val Glu Pro Val Glu Ser Thr Glu Gly Val Glu 50 55
60tcg act gag gcg gaa cgt gtg gca ggg aag cag gag cag gag gag gag
240Ser Thr Glu Ala Glu Arg Val Ala Gly Lys Gln Glu Gln Glu Glu
Glu65 70 75 80tac cct gtg gac gcc cac atg caa aag tac ctt tca cac
ctg aag agc 288Tyr Pro Val Asp Ala His Met Gln Lys Tyr Leu Ser His
Leu Lys Ser 85 90 95aag tct cgg tcg agg ttc cac cga aag gat gct agc
aag tat gtg tcg 336Lys Ser Arg Ser Arg Phe His Arg Lys Asp Ala Ser
Lys Tyr Val Ser 100 105 110ttt ttt ggg gac gtg agt ttt gat cct cgc
ccc acg ctc ctg gac agc 384Phe Phe Gly Asp Val Ser Phe Asp Pro Arg
Pro Thr Leu Leu Asp Ser 115 120 125gcc atc aac gtg ccc ttc cag acg
act ttc aaa ggt ccg gtg ctg gag 432Ala Ile Asn Val Pro Phe Gln Thr
Thr Phe Lys Gly Pro Val Leu Glu 130 135 140aaa cag ctc aaa aat tta
cag ttg aca aag acc aag acc aag gcc acg 480Lys Gln Leu Lys Asn Leu
Gln Leu Thr Lys Thr Lys Thr Lys Ala Thr145 150 155 160gtg aag act
acg gtg aag act acg gag aaa acg gac aag gca gat gcc 528Val Lys Thr
Thr Val Lys Thr Thr Glu Lys Thr Asp Lys Ala Asp Ala 165 170 175ccc
cca gga gaa aaa ctg gag tcg aac ttt tca ggg atc tac gtg ttc 576Pro
Pro Gly Glu Lys Leu Glu Ser Asn Phe Ser Gly Ile Tyr Val Phe 180 185
190gca tgg atg ttc ttg ggc tgg ata gcc atc agg tgc tgc aca gat tac
624Ala Trp Met Phe Leu Gly Trp Ile Ala Ile Arg Cys Cys Thr Asp Tyr
195 200 205tat gcg tcg tac ggc agt gca tgg aat aag ctg gaa atc gtg
cag tac 672Tyr Ala Ser Tyr Gly Ser Ala Trp Asn Lys Leu Glu Ile Val
Gln Tyr 210 215 220atg aca acg gac ttg ttc acg atc gca atg ttg gac
ttg gca atg ttc 720Met Thr Thr Asp Leu Phe Thr Ile Ala Met Leu Asp
Leu Ala Met Phe225 230 235 240ctg tgc act ttc ttc gtg gtt ttc gtg
cac tgg ctg gtg aaa aag cgg 768Leu Cys Thr Phe Phe Val Val Phe Val
His Trp Leu Val Lys Lys Arg 245 250 255atc atc aac tgg aag tgg act
ggg ttc gtt gca gtg agc atc ttc gag 816Ile Ile Asn Trp Lys Trp Thr
Gly Phe Val Ala Val Ser Ile Phe Glu 260 265 270ttg gct ttc atc ccc
gtg acg ttc ccc att tac gtc tac tac ttt gat 864Leu Ala Phe Ile Pro
Val Thr Phe Pro Ile Tyr Val Tyr Tyr Phe Asp 275 280 285ttc aac tgg
gtc acg aga atc ttc ctg ttc ctg cac tcc gtg gtg ttt 912Phe Asn Trp
Val Thr Arg Ile Phe Leu Phe Leu His Ser Val Val Phe 290 295 300gtt
atg aag agc cac tcg ttt gcc ttt tac aac ggg tat ctt tgg gac 960Val
Met Lys Ser His Ser Phe Ala Phe Tyr Asn Gly Tyr Leu Trp Asp305 310
315 320ata aag cag gaa ctc gag tac tct tcc aaa cag ttg caa aaa tac
aag 1008Ile Lys Gln Glu Leu Glu Tyr Ser Ser Lys Gln Leu Gln Lys Tyr
Lys 325 330 335gaa tct ttg tcc cca gag acc cgc gag att ctg caa aaa
agt tgc gac 1056Glu Ser Leu Ser Pro Glu Thr Arg Glu Ile Leu Gln Lys
Ser Cys Asp 340 345 350ttt tgc ctt ttc gaa ttg aac tac cag acc aag
gat aac gac ttc ccc 1104Phe Cys Leu Phe Glu Leu Asn Tyr Gln Thr Lys
Asp Asn Asp Phe Pro 355 360 365aac aac atc agt tgc agc aat ttc ttc
atg ttc tgt ttg ttc ccc gtc 1152Asn Asn Ile Ser Cys Ser Asn Phe Phe
Met Phe Cys Leu Phe Pro Val 370 375 380ctc gtg tac cag atc aac tac
cca aga acg tcg cgc atc aga tgg agg 1200Leu Val Tyr Gln Ile Asn Tyr
Pro Arg Thr Ser Arg Ile Arg Trp Arg385 390 395 400tat gtg ttg gag
aag gtg tgc gcc atc att ggc acc atc ttc ctc atg 1248Tyr Val Leu Glu
Lys Val Cys Ala Ile Ile Gly Thr Ile Phe Leu Met 405 410 415atg gtc
acg gca cag ttc ttc atg cac ccg gtg gcc atg cgc tgt atc 1296Met Val
Thr Ala Gln Phe Phe Met His Pro Val Ala Met Arg Cys Ile 420 425
430cag ttc cac aac acg ccc acc ttc ggc ggc tgg atc ccc gcc acg caa
1344Gln Phe His Asn Thr Pro Thr Phe Gly Gly Trp Ile Pro Ala Thr Gln
435 440 445gag tgg ttc cac ctg ctc ttc gac atg att ccg ggc ttc act
gtt ctg 1392Glu Trp Phe His Leu Leu Phe Asp Met Ile Pro Gly Phe Thr
Val Leu 450 455 460tac atg ctc acg ttt tac atg ata tgg gac gct tta
ttg aat tgc gtg 1440Tyr Met Leu Thr Phe Tyr Met Ile Trp Asp Ala Leu
Leu Asn Cys Val465 470 475 480gcg gag ttg acc agg ttt gcg gac aga
tat ttc tac ggc gac tgg tgg 1488Ala Glu Leu Thr Arg Phe Ala Asp Arg
Tyr Phe Tyr Gly Asp Trp Trp 485 490 495aat tgc gtt tcg ttt gaa gag
ttt agc aga atc tgg aac gtc ccc gtt 1536Asn Cys Val Ser Phe Glu Glu
Phe Ser Arg Ile Trp Asn Val Pro Val 500 505 510cac aaa ttt tta cta
aga cac gtg tac cac agc tcc atg ggc gca ttg 1584His Lys Phe Leu Leu
Arg His Val Tyr His Ser Ser Met Gly Ala Leu 515 520 525cat ttg agc
aag agc caa gct aca tta ttt act ttt ttc ttg agt gcc 1632His Leu Ser
Lys Ser Gln Ala Thr Leu Phe Thr Phe Phe Leu Ser Ala 530 535 540gtg
ttc cac gaa atg gcc atg ttc gcc att ttc aga agg gtt aga gga 1680Val
Phe His Glu Met Ala Met Phe Ala Ile Phe Arg Arg Val Arg Gly545 550
555 560tat ctg ttc atg ttc caa ctg tcg cag ttt gtg tgg act gct ttg
agc 1728Tyr Leu Phe Met Phe Gln Leu Ser Gln Phe Val Trp Thr Ala Leu
Ser 565 570 575aac acc aag ttt cta cgg gca aga ccg cag ttg tcc aac
gtt gtc ttt 1776Asn Thr Lys Phe Leu Arg Ala Arg Pro Gln Leu Ser Asn
Val Val Phe 580 585 590tcg ttt ggt gtc tgt tca ggg ccc agt atc att
atg acg ttg tac ctg 1824Ser Phe Gly Val Cys Ser Gly Pro Ser Ile Ile
Met Thr Leu Tyr Leu 595 600 605acc tta tga 1833Thr Leu
61050610PRTSaccharomyces cerevisiae 50Met Thr Glu Thr Lys Asp Leu
Leu Gln Asp Glu Glu Phe Leu Lys Ile1 5 10 15Arg Arg Leu Asn Ser Ala
Glu Ala Asn Lys Arg His Ser Val Thr Tyr 20 25 30Asp Asn Val Ile Leu
Pro Gln Glu Ser Met Glu Val Ser Pro Arg Ser 35 40 45Ser Thr Thr Ser
Leu Val Glu Pro Val Glu Ser Thr Glu Gly Val Glu 50 55 60Ser Thr Glu
Ala Glu Arg Val Ala Gly Lys Gln Glu Gln Glu Glu Glu65 70 75 80Tyr
Pro Val Asp Ala His Met Gln Lys Tyr Leu Ser His Leu Lys Ser 85 90
95Lys Ser Arg Ser Arg Phe His Arg Lys Asp Ala Ser Lys Tyr Val Ser
100 105 110Phe Phe Gly Asp Val Ser Phe Asp Pro Arg Pro Thr Leu Leu
Asp Ser 115 120 125Ala Ile Asn Val Pro Phe Gln Thr Thr Phe Lys Gly
Pro Val Leu Glu 130 135 140Lys Gln Leu Lys Asn Leu Gln Leu Thr Lys
Thr Lys Thr Lys Ala Thr145 150 155 160Val Lys Thr Thr Val Lys Thr
Thr Glu Lys Thr Asp Lys Ala Asp Ala 165 170 175Pro Pro Gly Glu Lys
Leu Glu Ser Asn Phe Ser Gly Ile Tyr Val Phe 180 185 190Ala Trp Met
Phe Leu Gly Trp Ile Ala Ile Arg Cys Cys Thr Asp Tyr 195 200 205Tyr
Ala Ser Tyr Gly Ser Ala Trp Asn Lys Leu Glu Ile Val Gln Tyr 210 215
220Met Thr Thr Asp Leu Phe Thr Ile Ala Met Leu Asp Leu Ala Met
Phe225 230 235 240Leu Cys Thr Phe Phe Val Val Phe Val His Trp Leu
Val Lys Lys Arg 245 250 255Ile Ile Asn Trp Lys Trp Thr Gly Phe Val
Ala Val Ser Ile Phe Glu 260 265 270Leu Ala Phe Ile Pro Val Thr Phe
Pro Ile Tyr Val Tyr Tyr Phe Asp 275 280 285Phe Asn Trp Val Thr Arg
Ile Phe Leu Phe Leu His Ser Val Val Phe 290 295 300Val Met Lys Ser
His Ser Phe Ala Phe Tyr Asn Gly Tyr Leu Trp Asp305 310 315 320Ile
Lys Gln Glu Leu Glu Tyr Ser Ser Lys Gln Leu Gln Lys Tyr Lys 325 330
335Glu Ser Leu Ser Pro Glu Thr Arg Glu Ile Leu Gln Lys Ser Cys Asp
340 345 350Phe Cys Leu Phe Glu Leu Asn Tyr Gln Thr Lys Asp Asn Asp
Phe Pro 355 360 365Asn Asn Ile Ser Cys Ser Asn Phe Phe Met Phe Cys
Leu Phe Pro Val 370 375 380Leu Val Tyr Gln Ile Asn Tyr Pro Arg Thr
Ser Arg Ile Arg Trp Arg385 390 395 400Tyr Val Leu Glu Lys Val Cys
Ala Ile Ile Gly Thr Ile Phe Leu Met 405 410 415Met Val Thr Ala Gln
Phe Phe Met His Pro Val Ala Met Arg Cys Ile 420 425 430Gln Phe His
Asn Thr Pro Thr Phe Gly Gly Trp Ile Pro Ala Thr Gln 435 440 445Glu
Trp Phe His Leu Leu Phe Asp Met Ile Pro Gly Phe Thr Val Leu 450 455
460Tyr Met Leu Thr Phe Tyr Met Ile Trp Asp Ala Leu Leu Asn Cys
Val465 470 475 480Ala Glu Leu Thr Arg Phe Ala Asp Arg Tyr Phe Tyr
Gly Asp Trp Trp 485 490 495Asn Cys Val Ser Phe Glu Glu Phe Ser Arg
Ile Trp Asn Val Pro Val 500 505 510His Lys Phe Leu Leu Arg His Val
Tyr His Ser Ser Met Gly Ala Leu 515 520 525His Leu Ser Lys Ser Gln
Ala Thr Leu Phe Thr Phe Phe Leu Ser Ala 530 535 540Val Phe His Glu
Met Ala Met Phe Ala Ile Phe Arg Arg Val Arg Gly545 550 555 560Tyr
Leu Phe Met Phe Gln Leu Ser Gln Phe Val Trp Thr Ala Leu Ser 565 570
575Asn Thr Lys Phe Leu Arg Ala Arg Pro Gln Leu Ser Asn Val Val Phe
580 585 590Ser Phe Gly Val Cys Ser Gly Pro Ser Ile Ile Met Thr Leu
Tyr Leu 595 600 605Thr Leu 6105133DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 51ctgcggccgc atcatgtctg
ctgttaacgt tgc 335230DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 52ttctcgagtt aaccaatcaa
ctcaccaaac 305335DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 53ctgcggccgc aggatgtctg ctaccaagtc
aatcg
355434DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 54atctcgagct tagatctttt gttctggatt tctc
345532DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 55ctgcggccgc accatgaagt ttttcccact cc
325633DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 56ttctcgagtt agaacttttt gttttgcaac aag
335735DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 57ctgcggccgc aatatggatt tggtcttaga agtcg
355831DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 58aactcgagtc agttgttctt cttggtattt g
315934DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 59ctgcggccgc actatggcaa aggataatag tgag
346032DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 60ttctcgagct agaaaacata aggaataaag ac 32
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