U.S. patent application number 10/346958 was filed with the patent office on 2004-01-08 for use of homologous amds genes as selectable markers.
Invention is credited to Bakhuis, Janna G., Bovenberg, Roelof A.L., Selten, Gerardus C.M., Swinkels, Bart W., Vollebregt, Adrianus W.H..
Application Number | 20040005692 10/346958 |
Document ID | / |
Family ID | 8220549 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040005692 |
Kind Code |
A1 |
Swinkels, Bart W. ; et
al. |
January 8, 2004 |
Use of homologous amdS genes as selectable markers
Abstract
The present invention discloses novel amdS genes from fungi
previously not known to contain and amdS gene, such as Aspergillus
niger and Penicillium chrysogenum. The novel amdS genes can be used
as homologous selectable marker genes in the transformation of
these fungi. Alternatively, the cloned amdS genes can be used to
inactivate the endogenous copy of the gene in order to reduce the
background in transformation experiments.
Inventors: |
Swinkels, Bart W.; (Delft,
NL) ; Selten, Gerardus C.M.; (Berkel En Rodenrijs,
NL) ; Bakhuis, Janna G.; (Delft, NL) ;
Bovenberg, Roelof A.L.; (Rotterdam, NL) ; Vollebregt,
Adrianus W.H.; (Naaldwijk, NL) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
3811 VALLEY CENTRE DRIVE
SUITE 500
SAN DIEGO
CA
92130-2332
US
|
Family ID: |
8220549 |
Appl. No.: |
10/346958 |
Filed: |
January 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10346958 |
Jan 17, 2003 |
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08809955 |
Apr 3, 1997 |
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6548285 |
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08809955 |
Apr 3, 1997 |
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PCT/EP96/03494 |
Aug 5, 1996 |
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Current U.S.
Class: |
435/228 ;
435/254.21; 435/254.3; 435/254.5; 435/320.1; 435/69.1;
536/23.2 |
Current CPC
Class: |
C12N 9/80 20130101 |
Class at
Publication: |
435/228 ;
435/69.1; 435/254.21; 435/254.3; 435/254.5; 536/23.2;
435/320.1 |
International
Class: |
C12N 009/80; C07H
021/04; C12N 001/16; C12N 001/18; C12N 015/74; C12P 021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 3, 1995 |
EP |
95202129.3 |
Claims
1. A DNA sequence encoding an acetamidase and characterized in that
the DNA sequence is derived from an organism other than Aspergillus
nidulans, Aspergillus oryzae and Saccharomyces cerevisiae.
2. A DNA sequence encoding an acetamidase which comprises an amino
acid sequence which is characterized in that: a) its amino acid
positional identity with one of the amino acid sequences of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 is
more than 30%, and b) its amino acid positional identity with each
of the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID
NO:3 is less than 100%.
3. A DNA sequence according to claims 1 or 2, and characterized in
that the DNA sequence is derived from an organism selected from the
group consisting of: fungi from the Aspergillus niger group, the
Aspergillus glaucus group, the Aspergillus terreus group, the
Aspergillus restrictus group, the Aspergillus fumigatus group, the
Aspergillus cervinus group, the Aspergillus ornatus group, the
Aspergillus clavatus group, the Aspergillus versicolor group, the
Aspergillus ustus group, the Aspergillus wentii group, the
Aspergillus ochraceus group, the Aspergillus candidus group, the
Aspergillus cremeus group, the Aspergillus sparsus group,
Penicillium species, Trichoderma species, Mucor species, Rhizopus
species, Phanerochaete species, Neurospora species, Humicola
species, Claviceps species, Sordaria species, Ustilago species,
Fusarium species, Schizophyllum species, Cephalosporium species,
Acremonium species, edible fungi of which preferably Agaricus
bisporus, Kluyveromyces species, Yarrowia species, Candida species,
Hansenula species, Pichia species and Phaffia species.
4. A DNA sequence according to claim 3 and characterized in that
the DNA sequence is derived from Aspergillus niger or Penicillium
chrysogenum.
5. A recombinant DNA construct which comprises a DNA sequence as
defined in any one of claims 1-4.
6. A recombinant DNA construct according to claim 5 and
characterized in that the DNA sequence is operably linked to a
promoter which is native to the DNA sequence.
7. A recombinant DNA construct according to claim 5 and
characterized in that the DNA sequence is operably linked to a
promoter which is foreign to the DNA sequence.
8. A recombinant DNA construct according to claim 7, wherein the
promoter foreign to the DNA sequence is a promoter derived from a
gene which is selected from the group consisting of the genes
encoding glycolytic enzymes and enzymes involved in alcohol
metabolism.
9. A recombinant DNA construct according to any one of claims 7-8,
wherein the DNA sequence and the promoter are derived from the same
species.
10. A recombinant DNA construct according to any one of claims 5-9,
further comprising a gene of interest.
11. A cell which comprises a recombinant DNA construct as defined
in any one of claims 5-10.
12. A cell characterized in that the cell does not contain an
active endogenous copy of an acetamidase gene comprising a DNA
sequence as defined in any one of claims 1-4.
13. A cell according to claim 12, characterized in that the
endogenous copy of the acetamidase gene has been inactivated by a
homologous recombination event.
14. A cell according to claim 13 and characterized in that the
homologous recombination event is a gene-replacement.
15. A cell according to any one of claims 11-14, further comprising
a recombinant gene of interest.
16. A cell according to any one of claims 11-15, further
characterized in that an endogenous gene of interest has been
inactivated.
17. A process for the production of a desired product, and
characterized in that the process comprises the steps of: a)
culturing a recombinant cell as defined in claims 15 or 16, under
conditions conducive to the production of the product, and b)
recovering the product.
18. A method for obtaining an DNA sequence as defined in any one of
claims 1-4, wherein the method comprises the steps of: a)
identification of conserved regions in the amino acid sequences of
known acetamidase genes, b) amplification of a DNA fragment in a
PCR, using as primers a set of degenerate oligonucleotides
corresponding to the conserved regions identified in a), on a
template nucleic acid containing the DNA sequence, c) isolation of
the DNA sequence using the DNA fragment amplified in b) as
hybridization probe to screen a DNA library containing the DNA
sequence.
19. A DNA sequence as defined in any one of claims 1-4, wherein the
DNA sequence is obtainable by the method of claim 18.
20. A method for inactivation of an endogenous copy of an
acetamidase gene comprising a DNA sequence as defined in any one of
claims 1-4, wherein the method comprises the steps of: a)
construction of an inactivation vector comprising a disrupted copy
of the acetamidase gene, b) transformation of a host cell with the
inactivation vector, c) selection of transformants with an
inactivated copy of the acetamidase gene.
21. A method according to claim 20, wherein the inactivation vector
is capable of replacing the endogenous copy of the acetamidase
gene.
22. A method of culturing cells at least a proportion of which
consists of cells according to claim 15 or 16 in a culture medium,
wherein the culture medium comprises acetamide as sole carbon
and/or nitrogen source.
23. The method according to claim 22, wherein said culturing
results in the enrichment of the proportion of cells according to
claim 15 or 16.
24. A living cell selected from the group consisting of fungi from
the Aspergillus niger group, the Aspergillus glaucus group, the
Aspergillus terreus group, the Aspergillus restrictus group, the
Aspergillus fumigatus group, the Aspergillus cervinus group, the
Aspergillus ornatus group, the Aspergillus clavatus group, the
Aspergillus versicolor group, the Aspergillus ustus group, the
Aspergillus wentii group, the Aspergillus ochraceus group, the
Aspergillus candidus group, the Aspergillus cremeus group, the
Aspergillus sparsus group, Penicillium species, Trichoderma
species, Mucor species, Rhizopus species, Phanerochaete species,
Neurospora species, Humicola species, Claviceps species, Sordaria
species, Ustilago species, Fusarium species, Schizophyllum species,
Cephalosporium species, Acremonium species, edible fungi of which
preferably Agaricus bisporus, Kluyveromyces species, Yarrowia
species, Candida species, Hansenula species, Pichia species and
Phaffia species, characterized by their ability to grow well on a
culture medium containing acetamide as sole carbon and/or nitrogen
source and wherein said ability is not caused by the expression of
a heterologous acetamidase gene.
Description
[0001] The use of homologous amdS genes as selectable markers
FIELD OF THE INVENTION
[0002] The present invention relates to the field of molecular
biology, in particular the invention is concerned with selectable
marker genes to be used in transformation of organisms.
BACKGROUND OF THE INVENTION
[0003] The Aspergillus nidulans amdS gene is probably the most
frequently used selectable marker for the transformation of
filamentous fungi and has been applied in most of the industrially
important filamentous fungi such as e.g. Aspergillus niger (Kelly
and Hynes 1985, EMBO J. 4: 475-479), Penicillium chrysogenum (Beri
and Turner 1987, Curr. Genet. 11: 639-641), Trichoderma reesei
(Pentill et al. 1987, Gene 61: 155-164), Aspergillus oryzae
(Christensen et al. 1988, Bio/technology 6: 1419-1422) and
Trichoderma harzianum (Pe'er et al. 1991, Soil Biol. Biochem. 23:
1043-1046).
[0004] The popularity of the amdS gene as a selectable marker is
most likely a result of the fact that it is the only available
non-antibiotic marker gene which can be used as a dominant
selectable marker in the transformation of fungi. Dominant
selectable markers provide the advantage that they can be used
directly in any strain without the requirement for mutant recipient
strains. The antibiotic-resistance genes are, however, not
preferred for use in industrial strains because the regulatory
authorities in most countries object to the use of antibiotic
markers in view of the potential risks of spread of
antibiotic-resistance genes in the biosphere upon large-scale use
of production strains carrying such genes.
[0005] The amdS gene has been used as a dominant marker even in
fungi known to contain an endogenous amdS gene, i.e. A. nidulans
(Tilburn et al. 1983, Gene 26: 205-221) and A. oryzae (Gomi et al.
1991, Gene 108: 91-98). In these cases the background of
non-transformants can be suppressed by the inclusion of CsCl in the
selection medium. In addition, high-copynumber transformants are
provided with a growth advantage over the non-transformants (when
acetamide is the sole nitrogen-source) because of the higher gene
dosage.
[0006] Apart from the A. nidulans and A. oryzae amdS genes, by
coincidence a sequence was found in the genome of the yeast
Saccharomyces cerevisiae, which shows homology to the A. nidulans
amdS gene (Chang and Abelson 1990, Nucleic Acids Res. 18:7180). The
yeast amdS-like sequence was shown not to be essential in yeast. It
is, however, not known whether the yeast amdS-like gene actually
encodes a protein with amidase activity which might allow to use
the gene as selectable marker. amdS genes have not been found in
other fungi, despite attempts to detect such genes with
heterologous hybridization using the A. nidulans amdS gene as probe
(see e.g. Kelly and Hynes 1985 EMBO J. 4: 475-479). This is also in
line with the observation that, in contrast to A. nidulans and A.
oryzae, most fungi grow very poor, if at all, on acetamide (see
e.g. Beri and Turner 1987, Curr. Genet. 11: 639-641; Pentill et al.
1987, Gene 61: 155-164). The cloning and sequencing of two
bacterial acetamidase genes has been reported, i.e. those of
Pseudomonas aeruginosa (Brammar et al. 1987, FEBS Lett. 215:
291-294) and of Mycobacterium smegatis (Mahenthiralingam et al.
1993, J. Gen. Microbiol. 139: 575-583). However, these bacterial
acetamidases appear to be unrelated to the above mentioned fungal
acetamidases since no sequence similarities can be detected and the
bacterial acetamidases are also much smaller than their fungal
counterparts. No reports of the use of these bacterial acetamidases
as selectable markers have appeared.
[0007] In addition to its dominant character, the amdS selectable
marker provides the advantage of being a bidirectional marker. This
means that, apart from the positive selection for the presence of
the amdS gene using acetamide as sole carbon- or nitrogen-source, a
counterselection can be applied using fluoracetamide to select
against the presence of the amdS gene (Hynes and Pateman 1970, Mol.
Gen. Genet. 108, 107-106). The fluoracetamide counterselection has
been applied to cure genetically engineered strains from
recombinant constructs carrying the amdS gene (e.g. Ward et al.
1993, Appl. Microbiol. Biotechnol. 39, 738-743).
[0008] A disadvantage of the amdS marker is the fact that the A.
nidulans amdS gene is a heterologous gene in industrial fungi such
as A. niger, A. oryzae, T. reesei and P. chrysogenum. Even though
this may seem trivial to most molecular biologists, regulatory
authorities often object that production strains containing the
heterologous A. nidulans amdS gene posses a new (the gene being
heterologous) and unnecessary (the marker gene not being necessary
once the transformant strain is obtained) property, the risks of
which cannot be foreseen. Unfortunately, the only industrial
filamentous fungus for which an homologous amdS gene is available
is A. oryzae.
[0009] We have previously addressed this problem by developing a
method to obtain recombinant fungal production strains that are
free of selectable markers (EP-A-0 635 574). In this method the
bidirectionality of the amdS marker is used to remove the marker
from specially constructed expression cassettes once they have been
introduced in the fungal genome. The method is, however, less
compatible with the high copy numbers which are often necessary in
industrial production strains. For these situations, a homologous
and dominant selectable marker would still be required.
SUMMARY OF THE INVENTION
[0010] The present invention discloses novel DNA sequences encoding
acetamidase genes from fungi other than Aspergillus nidulans,
Aspergillus oryzae and Saccharomyces cerevisiae.
[0011] Preferably, these DNA sequences encode acetamidases which
comprise an internal consensus fragment, the amino acid positional
identity of which is less than 100% when compared with each of the
amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3,
whereas this amino acid positional identity is more than 30% when
compared with one of the amino acid sequences of SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
[0012] The invention also discloses recombinant DNA constructs
comprising the DNA sequences encoding the acetamidases of the
invention, as well as recombinant cells containing these
constructs.
[0013] The invention further discloses recombinant cells in which
an endogenous copy of the gene encoding the acetamidase of the
invention has been inactivated.
[0014] In a further embodiment, the invention discloses a process
in which the recombinant cells of the invention are cultured in
order to obtain a product of interest.
[0015] Finally, the invention discloses methods for obtaining the
acetamidase genes of the invention, as well as methods for the
inactivation of endogenous copies of these acetamidase genes.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1A:
[0017] Amino acid comparison of amdS internal consensus fragments
of the amdS genes from A. nidulans, A. oryzae, S. cerevisiae, A.
niger and P. chrysogenum.
[0018] FIG. 1B:
[0019] Amino acid positional identities between each of the amdS
internal consensus fragments of FIG. 1A.
[0020] FIG. 2:
[0021] Partial restriction map of the phage clone .lambda.AMD-1 and
the subclones pGBAMD-2, pGBAMD-3 and pGEAMD-4.
[0022] Abbreviations used for the restriction enzymes:
[0023] B=BamHI, X=XbaI, P=PstI, S=SmaI, Sp=SpeI, K=KpnI,
E=EcoRI.
[0024] FIG. 3:
[0025] BamHI digests of two pGBAMD-4 transformants (lanes 1 and 2),
two pGBAMD-3 transformants (lanes 3 and 4) and the parental strain
A. niger CBS 513.88 (lane 5) probed with a .sup.32P labelled
EcoRI/BamHI fragment isolated from pGBAMD-1.
[0026] FIG. 4:
[0027] shows schematically the amdS gene replacement vector
pGBAMD-11.
[0028] Abbreviations used for the restriction enzymes:
[0029] B=BamHI, Sp=SpeI, K=KpnI, E=EcoRI, S=SmaI, N=NotI,
H=HindIII.
DETAILED DESCRIPTION OF THE INVENTION
[0030] Several terms used in the present description and claims are
defined as follows.
[0031] The term gene is herein defined as a DNA sequence encoding a
polypeptide, irrespective of whether the DNA sequence is a cDNA or
a genomic DNA sequence which may contain one or more introns.
[0032] The term selection marker gene (or selectable marker gene)
is herein defined as a gene which encodes a polypeptide that
provides a phenotype to the cell containing the gene such that the
phenotype allows either positive or negative, selection or
screening of cells containing the selection marker gene. The
selection marker gene may be used to distinguish between
transformed and non-transformed cells or may be used to identify
cells having undergone recombination or other kinds of genetic
modifications.
[0033] An acetamidase is herein defined as an enzyme which is
capable of catalysing the hydrolysis of acetamide into acetic acid
and ammonium, and/or which is capable of catalysing the hydrolysis
of related amide-compounds such as acrylamide or .omega.-amino
acids.
[0034] An amdS gene is herein defined as a gene, which is
preferably obtainable from a eukaryote, more preferably from a
fungus, and which encodes a polypeptide that is an acetamidase as
defined above. Preferably an amdS gene shows sequence similarity
with one or more of the three amdS genes known in the art, i.e: the
amdS genes from A. nidulans, A. oryzae or the amdS-like gene from
S. cerevisiae. A more accurate description of the sequence
similarity using the amino acid positional identity of an amdS
internal consensus fragment is provided below. An amdS gene
preferably encodes a protein of about 500 to 600 amino acids, more
preferably of about 520 to 570 amino acids and most preferably of
about 540 to 550 amino acids. An amdS gene is therefore usually
contained within a DNA fragment of about 2.0 kb. Of course the
presence of introns in a genomic amdS gene can increase the length
to e.g about 2.5 kb or more.
[0035] The terms homologous gene is herein defined as a gene which
is obtainable from a strain which belongs to the same species,
including variants thereof, as does the strain actually containing
the gene. Preferably, the donor and acceptor strain are the same.
It is to be understood that the same applies to polypeptides
encoded by homologous genes. Fragments and mutants of genes are
also considered homologous when the gene from which the mutants or
fragments are derived is a homologous gene. Also non-native
combinations of regulatory sequences and coding sequences are
considered homologous as long as the coding sequence is homologous.
It follows that the term heterologous herein refers to genes or
polypeptides for which donor and acceptor strains do not belong to
the same species or variants thereof.
[0036] The term endogenous gene is herein defined as a naturally
occurring copy of a gene in the genome of the organism in
question.
[0037] The term fungus herein refers to all members of the division
Eumycota of the kingdom Fungi and thus includes all filamentous
fungi and yeasts.
[0038] In view of recent changes in the nomenclature of black
Aspergilli, the term Aspergillus niger is herein defined as
including all (black) Aspergilli that can be found in the
Aspergillus niger Group as defined by Raper and Fennell (1965, In:
The Genus Aspergillus, The Williams & Wilkins Company,
Baltimore, pp 293-344). Similarly, also for the other Aspergillus
species we will refer to the Aspergillus groups as defined by Raper
and Fennell supra, thereby including all species and variants
included in a particular group by these authors.
[0039] The present application describes the cloning of amdS genes
from fungi not previously known to contain an amdS gene. A
comparison of the three available amdS sequences was used to
identify conserved regions in the amdS amino acid sequences. The
conserved regions are herein defeined as short peptide fragments,
e.g. 3-12 or more amino acids, which show a high degree of
conservation, i.e. more than 80% identity, in the amino acid
sequences of acetamidases from different organisms and which can be
used to identify novel acetamidase genes, thereby relying on the
fact that in the novel acetamidase these peptide fragments will
also be conserved. On the basis of these conserved regions
degenerate oligonucleotides were designed which were used as
primers in experiments using Polymerase Chain Reactions (PCR) on
genomic DNA isolated from A. niger and P. chrysogenum. Under
certain PCR conditions amplified fragments were obtained which were
subcloned and sequenced. The sequence analysis clearly identified
the amplified PCR fragment as derived from the amdS genes of A.
niger and P. chrysogenum by virtue of the homology of the encoded
amino acid sequences to the known (translated) amdS amino acid
sequences (see FIG. 1A).
[0040] The A. niger and P. chrysogenum amdS-PCR fragments, which
only contained a small part of the amdS genes (approximately 500
and 400 bp, respectively), were used as hybridization probes to
screen genomic libraries of A. niger and P. chrysogenum in order to
obtain cloned genomic DNA fragments containing the entire amdS gene
for these fungi (see e.g. FIG. 2 for A. niger). Restriction
fragments in the genomic clones that hybridized to the PCR probes
were subcloned into plasmids and subjected to sequence analysis.
The resulting nucleotide sequences of the genomic amdS genes of A.
niger and P. chrysogenum are presented in SEQ ID NO:18 and SEQ ID
NO:19, respectively. In the absence of the corresponding cDNA
sequences we cannot determine the exact positions of the introns in
these genomic sequences. We have therefore not deduced the
predicted amino acid sequences. Nevertheless, translation of all
three reading frames of the genomic sequences allows to identify
several areas (in addition to those corresponding to the above
mentioned PCR fragments which encode the internal consensus
fragments), which have significant amino acid positional identity
with the known amdS amino acid sequences.
[0041] The present disclosure of the presence of amdS genes in A.
niger and P. chrysogenum provides an incentive for the
identification of amdS genes in other organisms, preferably fungi,
which at present are not known to contain an amdS gene. The
preferred candidates in this respect are the industrially important
fungi such as the filamentous fungi belonging to the Aspergillus
niger group, the Aspergillus glaucus group, the Aspergillus terreus
group, the Aspergillus restrictus group, the Aspergillus fumigatus
group, the. Aspergillus cervinus group, the Aspergillus ornatus
group, the Aspergillus clavatus group, the Aspergillus versicolor
group, the Aspergillus ustus group, the Aspergillus wentii group,
the Aspergillus ochraceus group, the Aspergillus candidus group,
the Aspergillus cremeus group, the Aspergillus sparsus group,
Trichoderma species, such as T. reesei and T. harzianum, Mucor
species such as M. miehei, Rhizopus species, Phanerochaete species,
Neurospora species, Humicola species, Claviceps species, Sordaria
species, Ustilago species, Fusarium species, Schizophyllum species,
Penicillium species such as P. chrysogenum, Cephalosporium species,
Acremonium species and edible fungi such as Agaricus bisporus, and
yeasts such as Kluyveromyces species, Yarrowia species, Candida
species, Hansenula and Pichia species. As many of the above fungi
grow in their natural habitat by decomposing plant material, it is
not unlikely that also plants will express genes involved in the
metabolism of compounds like acetamide. Hence, plants may also
contain an acetamidase gene.
[0042] The amdS genes of the invention show sequence similarity
with other amdS (-like) genes. This sequence similarity is best
defined by the amino acid positional identity of an internal
consensus fragment within proteins encoded by amdS genes. The
internal consensus fragment is the DNA (or protein) fragment which
corresponds to a fragment in the A. nidulans amdS gene which
encodes amino acids 125 to 226 (or the corresponding protein
fragment), the amino acid sequence of which is provided in SEQ ID
NO:1. For the determination of the amino acid positional identity,
the (encoded) amino acid sequences of the internal consensus
fragments are lined up, introducing gaps if necessary for maximal
identity, as is shown in FIG. 1A. The amino acid positional
identity of two amdS sequences is subsequently expressed as the
percentage of identical amino acids in the sequence of the complete
internal consensus fragment of the shortest of the two amdS
sequences (FIG. 1B). Using the amino acid positional identity, the
amdS genes of the invention are defined as DNA sequences encoding
proteins which comprise an amino acid sequence of which the amino
acid positional identity with each of the amino acid sequences of
SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 is less than 80%,
preferably less than 90%, more preferably less than 95, and most
preferably less than 100%, and of which the amino acid positional
identity with one of the amino acid sequences of SEQ ID NO:1, SEQ
ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 is more than
30%, preferably more than 35, more preferably more than 40 and most
preferably more than 45%.
[0043] The novel amdS sequences of the present invention can be
used in conjunction with the already available amdS sequences to
more accurately define the conserved regions in the amdS amino acid
sequences and type of substitutions occurring therein. This will
facilitate the design of improved degenerate oligonucleotides which
will increase the chance of obtaining new amdS genes in PCRs or
hybridization experiments.
[0044] Even though the preferred method for cloning new amdS genes
is the method of the present invention, i.e. the use of degenerate
oligonucleotides in a PCR on genomic DNA (or cDNA) and subsequent
hybridization-screening of DNA (genomic- or cDNA) libraries to
obtain the full length amdS gene, other methods can also be used
for the cloning of new amdS genes. Such methods may include inverse
PCR, heterologous hybridization, hybridization with (degenerate)
oligonucleotides, (heterologous) complementation of amdS-negative
mutants, or even screening of expression-libraries with suitable
antibodies.
[0045] The novel amdS genes of the invention, e.g. those from A.
niger, P. chrysogenum, or one of the other fungi mentioned above,
can be used as a homologous selectable marker gene, which is herein
understood to mean that the amdS gene is used to select
transformants of the same species as the species from which the
amdS gene was originally derived. This offers the advantage that
the transformants obtained do not contain a foreign selectable
marker gene. In principle this allows to construct recombinant
strains which contain no foreign DNA other than absolutely
necessary, i.e. the (heterologous) gene of interest to be
expressed.
[0046] In a further embodiment of the invention, the native
promoter of the homologous amdS gene is replaced by a different
promoter. This replacement promoter, which is referred to as
foreign promoter herein, can either be stronger than the native
amdS promoter or it can be regulated in a different manner. Either
way, the replacement of the native amdS promoter is intended to
facilitate the selection of transformants, e.g. by increasing the
growth advantage of, transformants over non-transformants when
grown on acetamide or related amide-compounds as sole N- or
C-source. Preferably the foreign promoters are also homologous to
the host in which they are used. Suitable foreign promoters can be
derived from genes encoding glycolytic enzymes or enzymes involved
in alcohol metabolism, such as the promoters from genes encoding
phosphoglycerate kinases, glyceraldehyde-phosphate dehydrogenases,
triose-phosphate kinases, pyruvate kinase or alcohol
dehydrogenases.
[0047] In yet a further embodiment of the invention, the sequences
of the novel amdS gene are used to inactivate the endogenous copy
(or copies) of the amdS gene in the genome of the organism from
which the novel amdS gene is derived. To this extent an
inactivation vector can be constructed using the sequences of the
novel amdS gene to target the vector to an endogenous copy of the
gene by homologous recombination. The inactivation can then be
caused either by replacement of, or by insertion into the
endogenous amdS gene. Inactivation of the endogenous amdS gene
provides the advantage of reducing the background of
non-transformed cells in transformations using an amdS gene as
selectable marker for the introduction of a gene of interest.
Alternatively, the endogenous amdS locus can serve as a defined
site of integration for genes of interest to be expressed.
[0048] The homologous amdS genes of the invention can be used in
many different transformation procedures available to the skilled
person, including inter alia direct transformation of integrating
as well as autonomously replicating vectors, cotransformations in
which the DNA to be transformed and the selectable marker are not
physically linked, and transformation and subsequent curing of
transformants in order to obtain MARKER GENE FREE.TM. recombinant
strains as outlined in EP-A1-0 635 574, which is herein
incorporated by reference.
[0049] The invention also discloses a method of culturing cells, at
least a proportion of which consists of cells according to the
invention, in a culture medium, wherein the culture medium
comprises acetamide as sole carbon and/or nitrogen source, as well
as a method wherein said culturing results in the enrichment of the
proportion of cells according to invention.
[0050] The invention further discloses living cells according to
the invention, preferably fungal cells, with the ability to grow
well on a culture medium containing acetamide as sole carbon and/or
nitrogen source and wherein said ability is not caused by the
expression of a heterologous acetamidase gene but is rather caused
by the expression, preferably overexpression, of a homologous
acetamidase gene. The ability of a cell to grow well on a culture
medium containing acetamide as sole carbon and/or nitrogen source
is herein defined as the ability to grow faster than the
corresponding wild-type cell, wherein wild-type is understood to
mean wild-type with respect to its acetamidase genotype.
[0051] The present invention allows the preparation of recombinant
cells which contain a recombinant homologous amdS gene, and/or
which do not contain an active copy of an endogenous amdS gene.
Usually these recombinant cells will further comprise genes of
interest to be expressed and/or endogenous genes of interest which
have been inactivated. Any one of these recombinant cells can be
used in processes for the production of a product of interest. Such
a process will usually include the steps of culturing the
recombinant cells in a medium conducive to the production of the
product of interest and recovery of the product of interest from
the culture medium. The products of interest can be proteins, such
as an enzyme, and/or primary metabolites, such as CO.sub.2, alcohol
or organic acids, and/or secondary metabolites, such as antibiotics
or carotenoids. The product of interest can also be the recombinant
cells themselves, i.e. the biomass obtained in the process.
[0052] The following examples are given to illustrate the present
invention.
EXAMPLES
Experimental
[0053] General Molecular Cloning Techniques
[0054] In the examples described herein, standard molecular cloning
techniques such as isolation and purification of nucleic acids,
electrophoresis of nucleic acids, enzymatic modification, cleavage
and/or amplification of nucleic acids, transformation of E. coli,
etc., were performed as described in the literature (Sambrook et
al. (1989) "Molecular Cloning: a laboratory manual", Cold Spring
Harbour Laboratories, Cold Spring Harbour, N.Y.; Innis et al.
(eds.) (1990) "PCR protocols, a guide to methods and applications"
Academic Press, San Diego). Synthesis of oligo-deoxynucleotides and
DNA sequence analysis were performed on an Applied Biosystems 380B
DNA synthesizer and 373A DNA sequencer, respectively, according to
the user manuals supplied by the manufacturer.
[0055] Transformation of A. niger
[0056] Transformation of A. niger was performed according to the
method described by Tilburn, J. et.al. (1983) Gene 26, 205-221 and
Kelly, J. & Hynes, M. (1985) EMBO J., 4, 475-479 with the
following modifications:
[0057] spores were grown for 16 hours at 30.degree. C. in a rotary
shaker at 300 rpm in Aspergillus minimal medium. Aspergillus
minimal medium consists of the following components: Per liter: 6 g
NaNO.sub.3; 0.52 g KCl; 1.52 g KH.sub.2PO.sub.4; 1.12 ml 4M KOH;
0.52 g MgSO.sub.4.7H.sub.2O; 10 g glucose; 1 g casaminoacids; 22 mg
ZnSO.sub.4.7H.sub.2O; 11 mg H.sub.3BO.sub.3; 5 mg
FeSO.sub.4.7H.sub.2O; 1.7 mg CoCl.sub.2.6H.sub.2O; 1.6 mg
CuSO.sub.4.5H.sub.2O; 5 mg MnCl.sub.2.4H.sub.2O; 1.5 mg
Na.sub.2MoO.sub.4.2H.sub.2O; 50 mg EDTA; 2 mg riboflavin; 2 mg
thiamine.HCl; 2 mg nicotinamide; 1 mg pyridoxine.HCl; 0.2 mg
panthotenic acid; 4 .mu.g biotin; 10 ml Penicillin (5000
IU/ml)/Streptomycin (5000 UG/ml) solution (Gibco).
[0058] only Novozym 234 (Novo Industri), and no helicase, was used
for formation of protoplasts;
[0059] after protoplast formation (60-90 minutes), KC buffer (0.8 M
KCl, 9.5 mM citric acid, pH 6.2) was added to a volume of 45 ml.
and the protoplast suspension was centrifuged at 2500 g at
4.degree. C. for 10 minutes in a swinging-bucket rotor. The
protoplasts were resuspended in 20 ml. KC buffer. Then, 25 ml of
STC buffer (1.2 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM
CaCl.sub.2) was added and subsequently the protoplast suspension
was centrifuged at 2500 g at 4.degree. C. for 10 minutes in a
swinging-bucket rotor, washed in STC-buffer and resuspended in
STC-buffer at a concentration of 108 protoplasts/ml;
[0060] to 200 .mu.l of the protoplast suspension the DNA fragment,
in a volume of 10 .mu.l in TE buffer (10 mM Tris-HCl pH 7.5, 0.1 mM
EDTA), was added and subsequently 100 .mu.l of a PEG solution (20%
PEG 4000 (Merck), 0.8 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM
CaCl.sub.2);
[0061] after incubation of the DNA-protoplast suspension at room
temperature for 10 minutes, 1.5 ml PEG solution (60% PEG 4000
(Merck), 10 mM Tris-HCl pH 7.5, 50 mM CaCl.sub.2) was added slowly,
with repeated mixing of the tubes. After incubation at room
temperature for 20 minutes, the suspensions were diluted with 5 ml
STC buffer, mixed by inversion and centrifuged at 2000 g at room
temperature for 10 minutes. The protoplasts were resuspended gently
in 1 ml 1.2 M sorbitol and plated onto selective regeneration
medium consisting of Aspergillus minimal medium without riboflavin,
thiamine.HCL, nicotinamide, pyridoxine.HCl, panthotenic acid,
biotin, casaminoacids and glucose but with 10 mM acetamide as the
sole nitrogen source, 1 M sucrose, solidified with 2%
bacteriological agar #1 (Oxoid, England).
[0062] Following growth for 6-10 days at 30.degree. C., the plates
were replica plated onto selective acetamide plates consisting of
Aspergillus selective regeneration medium with 2% glucose instead
of sucrose and 1.5% agarose instead of agar. Single transformants
were isolated after 5-10 days of growth at 30.degree. C.
[0063] Isolation of Chromosomal DNA from Aspergillus.
[0064] The isolation of DNA from Aspergillus was performed
according to the procedure as described by Yelton, et al. (1984),
Proc. Natl. Acad. Sci. 81, 1470-1474.
[0065] Construction of a Genomic Library of Aspergillus niger CBS
513.88
[0066] Chromosomal DNA isolated from A. niger CBS 513.88 was
partially digested with Sau3AI, ligated to the BamHI sites of the
.lambda.EMBL3 arms (e.g. Promega), packaged and transfected to E.
coli according to Sambrook et al. (1989) "Molecular Cloning: a
laboratory manual", Cold Spring Harbour Laboratories, Cold Spring
Harbour.
[0067] Construction of a Genomic Library of Penicillium
chrysogenum
[0068] Chromosomal DNA isolated from P. chrysogenum Wisconsin
54-1255 was partially digested with Sau3AI. Fragments with length
varying between 7-12 Kb were ligated to the artificially created
BamHI sites of the .lambda.ZAPII arms (e.g. Stratagene), packaged
and transfected to E. coli according to Sambrook et al. (1989)
"Molecular Cloning: a laboratory manual", Cold Spring Harbour
Laboratories, Cold Spring Harbour.
[0069] Counter-Selection on Fluoracetamide
[0070] Removal of the A. nidulans amdS selection marker is achieved
by internal recombination between the 3'-A. niger amdS non coding
repeats that flank the A. nidulans amdS selection marker. Selection
of cells that have lost the amdS selection marker is achieved by
growth on plates containing fluoracetamide. Cells harbouring the
amdS gene metabolize fluoracetamide to ammonium and fluoracetate
which is toxic to the cell so only cells that have lost the amdS
gene are able to grow on plates containing fluoracetamide.
[0071] In case of removal of the amdS marker from Aspergillus
transformants, spores from these transformants were plated onto
selective regeneration medium (described above) containing 32 mM
fluoracetamide and 5 mM ureum instead of 10 mM acetamide, 1.1%
glucose instead of 1M sucrose and 1.1% instead of 2%
bacteriological agar #1 (Oxoid, England). After 7-10 days of growth
at 35.degree. C. single colonies were harvested and plated onto
0.4% potato dextrose agar (Oxoid, England).
Example 1
Cloning of the amdS of Aspergillus niger CBS 513.88
Example 1.1
[0072] Synthesis of an amdS Specific PCR Fragment
[0073] Oligonucleotide mixes corresponding to the coding and the
non-coding DNA strands were designed in well conserved amino acid
sequences of the amdS genes from Aspergillus nidulans (Corrick M.
C., Twomey A. P., Hynes M. J. (1987) Gene 53 63-71), Aspergillus
oryzae (Gomi K., Kitamoto K. Kumagai C. (1991) Gene 108 91-98) and
the amdY gene from Saccharomyces cerevisiae (Chang T. H., Abelson
J. (1990) Nucleic Acids Res. 18 7180. These oligonucleotide mixes
have the following sequences:
1 4073: 5' CGG GAT CCG CNT TTT GTA ANA GNG CNG C 3' C CC C 4079: 5'
CGG GAT CCN ATT AGN CTN AAG GAT CA 3' C TC T A C A 4080: 5' GGA ATT
CCC TCN CCN CCN CTN CTN CC 3' T GA GA 4081: 5' GGA ATT CTA ATN CTN
CCN CC 3' GT GA G 4082: 5' GGA ATT CCN CCA ATA TCN GTN CC 3' G G
T
[0074] The oligonucleotide mixes were used in PCR with chromosomal
DNA from A. niger CBS 513.88 as template. The combinations of two
oligonucleotide mixes 4078/4082; 4079/4080 and 4079/4082 (100 pmole
each) respectively, were used in reactions with a 50 .mu.l reaction
volume also containing 0.5 .mu.g chromosomal DNA from A. niger CBS
513.88, 100 nmole dNTP's, Amplitaq reaction buffer (Perkin Elmer)
and 1 U Amplitaq (Perkin-Elmer). The conditions for PCR were as
follows: After denaturation for 1 min at 94.degree. C., at
72.degree. C. the Amplitaq is added. Next, 30 cycles each 2 min.
94.degree. C.; 2 min. x.degree. C. (x=65.degree.-50.degree. C.;
every two cycles x decreases with 1.degree. C.) and 3 min.
72.degree. C. were carried out finally followed by 7 min.
72.degree. C.
[0075] The reaction products were analyzed by electrophoresis using
an 1% TBE-agarose gel. Only the combination of oligonucleotide
mixture 4078 and 4082 resulted in a reaction product, which was
approximately 500 bp in lenght. This PCR fragment was digested with
BamHI and EcoRI, purified by agarose electrophoresis and ethanol
precipitation and cloned into the BamHI and EcoRI sites of pTZ18R
(United States Biochemicals). The resulting plasmid was designated
pGBAMD-.
Example 1.2
[0076] Screening of the Aspergillus niger CBS 513.88 Genomic
Library for the amdS Gene
[0077] An A. niger CBS 513.88 genomic library, constructed in
.lambda.-EMBL3 as described in the experimental section, was
screened using the .sup.32P-labelled EcoRI/BamHI fragment isolated
from pGBAMD-1. Hybridization with the .sup.32P_labelled EcoRI/BamHI
fragment isolated from pGBAMD-1 took place overnight at 65.degree.
C. in hybridization buffer containing 4.times.SSC, 5.times.
Denhardt's solution, 0.1% SDS and 100 .mu.g/ml heat denatured calf
thymus DNA. After hybridization, the filters were washed in
4.times.SSC/0.1% SDS, 2.times.SSC/0.1% SDS and 1.times.SSC/0.1% SDS
at 65.degree. C.
[0078] Four plaques, hybridizing with the PCR fragment were
identified and isolated and purified. These phage clones were
designated .lambda.AMD1-.lambda.MD4.
Example 1.3
[0079] Restriction Analysis of amdS Containing Phage Clones
.lambda.AMD1-.lambda.AMD4
[0080] A partial restriction map was constructed for one of the
four phage clones, i.e. .lambda.AMD1. The isolated phage DNA was
digested with several restriction enzymes, run on a 0.7% agarose
gel, blotted onto nitrocellulose (0.2 .mu.m; Schleicher &
Schull) and hybridized with the .sup.32P-labelled EcoRI/BamHI
fragment isolated from pGBAMD-1. From the results obtained, a
partial restriction map was constructed (see FIG. 2).
Example 1.4
[0081] Subcloning Fragments of Phage Clone .lambda.AMD-1
[0082] Phage clone .lambda.AMD-1 contained an insert that was
supposed large enough to comprise the entire amdS gene. Several
fragments from this phage clone .lambda.AMD-1 were subcloned into
either pTZ18R or pTZ19R (United States Biochemicals). First, an
approximately 2.3 kb EcoRI fragment was isolated from .lambda.AMD-1
by digestion of the phage DNA by EcoRI, followed by agarose
electrophoresis. The fragment was cloned into the EcoRI site of
pTZ18R. The resulting plasmid was designated pGBAMD-2.
[0083] Next, the approximately 5 kb SpeI/KpnI fragment was isolated
by digesting the phage DNA with SpeI and KpnI followed by agarose
electrophoresis. The approximately 5 kb SpeI/KpnI fragment was
cloned into the XbaI and KpnI sites of pTZ19R. In this cloning step
both the SpeI and the XbaI sites are destroyed. The resulting
plasmid was designated pGBAMD-3.
[0084] Finally, the approximately 8 kb KpnI fragment was isolated
by digesting the phage DNA with KpnI followed by agarose
electrophoresis. The isolated fragment was cloned into the KpnI
site of pTZ19R. The resulting plasmid was designated pGBAMD-4. A
schematic overview of the different subclones is given in FIG.
2.
Example 1.5
[0085] Sequence Analysis of the A. niger amdS Gene
[0086] In order to determine whether the isolated PCR fragment was
a part of the A. niger amdS gene, the sequence of this fragment was
determined (presented in SEQ ID NO:16), translated to an amino acid
sequence and compared to the amino acid sequences of the A.
nidulans, A. oryzae amdS genes and the S. cerevisiae amdY gene (see
FIG. 1A). A considerable homology was found between the PCR
fragment and part of the amdS and amdY genes. Therefore it was
concluded that the PCR fragment is a part of the A. niger homologue
of the amdS gene. To obtain the entire genomic nucleotide sequence
of the A. niger amdS locus, the sequence of part (about 2.8 kb) of
the SpeI/KpnI DNA fragment of pGBAMD-3 was determined as well. This
sequence is presented in SEQ ID NO:18.
Example 2
[0087] Use of the A. niger amdS Gene as Selection Marker Gene in
Transformation of A. niger CBS 513.88
[0088] In order to determine whether the A. niger homologue of the
A. nidulans amdS gene could be used as a selection marker gene in
transformations of A. niger, DNA from subclones pGBAMD-2, pGBAMD-3
and pGBAMD-4 containing probably the entire coding region of the A.
niger, amdS gene with more or less of the regulatory sequences
(promoter and terminator sequences) was used to transform A. niger
CBS 513.88 according to the method described in the experimental
section.
Example 2.1
[0089] Transformation of A. niger CBS 513.88 with Subclones
pGBAMD-2, pGBAMD-3 and pGBAMD-4
[0090] From the plasmids pGBAMD-2, pGBAMD-3 and pGBAMD-410 .mu.g,
20 .mu.g and 50 .mu.g plasmid DNA was transformed to A. niger CBS
513.88 according to the method described in the experimental
section. Only with plasmids pGBAMD-3 and pGBAMD-4 transformants
could be generated that were able to grow on acetamide as sole
nitrogen source.
Example 2.2
[0091] Genetic Analysis of A. niger pGBAMD-3 resp. pGBAMD-4
Transformants
[0092] To verify that the generated transformants were genuine
transformants that had taken up the plasmid DNA two A.
niger/pGBAMD-3 transformants and two A. niger/pGBAMD-4
transformants were analysed using Southern analysis. From these
transformants and from the untransformed A. niger host strain, high
molecular weight DNA was isolated, digested with BamHI, separated
by agarose gelelectrophoresis and blotted onto nitrocellulose. The
blotted DNA was hybridized with the .sup.32P labelled EcoRI/BamHI
fragment isolated from pGBAMD-1. The results are presented in FIG.
3.
[0093] Characteristic for the endogenous amdS gene is an
approximately 9 kb hybridizing fragment (see FIG. 3, lane 5).
Characteristic for the presence of the pGBAMD-3 plasmid is an
approximately 5 kb hybridizing fragment and characteristic for the
presence of the pGBAMD-4 plasmid is an approximately 7.5 kb
hybridizing fragment. As can be seen in FIG. 3, lanes 3, 4, and
lanes 1, 2, hybridizing fragments characteristic for the presence
of pGBAMD-3 and pGBAMD-4, respectively, are detected in the
transformants. Therefore it can be concluded that the A. niger amdS
gene can be used as selection marker gene in transformations of A.
niger.
Example 3
Marker Gene Free.TM. Deletion of the A. niger amdS Gene
[0094] This example describes the deletion of the A. niger amdS
coding region and a (proximal) cart of the amdS promoter with a
replacement vector which integrates into the A. niger genome via a
double cross-over homologous recombination. The replacement vector
comprises a DNA region homologous to the target locus interrupted
by a selectable marker gene flanked by DNA repeats.
[0095] The replacement vector comprises a part of the A. niger amdS
genomic locus, wherein the amdS coding sequences as well as a part
of the amdS promoter sequences are replaced by the A. nidulans amdS
gene under control of the A. nidulans gpdA promoter as selection
marker flanked by 3'-untranslated A. niger amdS sequences as direct
repeats. Transformation of A. niger with this vector directs the
replacement of the A. niger amdS gene by the A. nidulans amdS gene.
By performing the fluoracetamide counter-selection on these
transformants as described in the experimental procedures, the A.
nidulans amdS gene is properly deleted by an internal recombination
event between the 3'-A. niger amdS repeats, resulting in a MARKER
GENE FREE.TM. .DELTA.amdS recombinant strain, containing no foreign
DNA sequences at all.
Example 3.1
[0096] Construction Pathway of the amdS Gene Replacement Vector
[0097] The first steps in the construction pathway of the A. niger
amdS gene replacement vector is the construction of a plasmid with
a suitable multiple cloning site. To achieve this, the plasmid
pTZ18R (United States Biochemicals) was digested with EcoRI and
HindIII and the approximately 2.8 kb fragment was purified by
agarose electrophoresis and ethanol precipitation and in this
fragment two different synthetic fragments of two oligonucleotides
were cloned. One synthetic fragment comprises the recognition sites
for the restriction enzymes NotI, EcoRI, KpnI, BglII, SmaI and
HindIII and has the following sequence:
2 5' AATTG GCGGCCGC GAATTC GGTACC AGATCT ATAG GGGCCC A 3' .linevert
split..linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split..linevert split. 3' C CGCCGGCG CTTAAG CCATGG TCTAGA TATC
CCCGGG TTCGA 5'
[0098] The resulting plasmid was designated pGBAMD5.
[0099] The other synthetic fragment comprises the recognition sites
for the restriction sites NotI, HindIII, KpnI, SpeI, BamHI, PstI,
and NotI and has the following sequence:
3 5' AATTG GCGGCCGC AAGCTT GGTACC ACTAGT GGATCC GCAA CTGCAG
GCGGCCGC T 3' .linevert split. .linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split. .linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split. .linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split. .linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split. .linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split. .linevert split..linevert
split..linevert split..linevert split. .linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split. .linevert split..linevert split..linevert split..linevert
split..linevert split..linevert split..linevert split..linevert
split. .linevert split. 3' C CGCCGGCG TTCGAA CCATGG TGATCA CCTAGG
CGTT GACGTC CGCCGGCG ATCGA 5'
[0100] The resulting plasmid was designated pGBAMD-6.
[0101] Next, the approximately 2.5 kb in size BamHI/SmaI fragment
from pGBAMD-4, comprising the supposed 3'non-coding region of the
A. niger amdS gene was cloned into the BglII and SmaI sites of
pGBAMD-5. In this cloning step both the BglII and the BamHI sites
were destroyed. The new plasmid was designated pGBAMD-7. This
plasmid was digested with EcoRI and KpnI and in these sites the
approximately 3.1 kb fragment comprising the A. nidulans amdS gene
under control of the A. nidulans gpdA promoter, isolated from
pGBGLA25 (EP 0 635 574 A1), was ligated. The new plasmid was named
pGBAMD-8.
[0102] Plasmid pGBAMD-6 was digested with BamHI and PstI and in
these sites was ligated the approximately 2 kb BamHI/PstI fragment
isolated from pGBAMD-4 and comprising part of the supposed 3'
non-coding region of the amdS gene. The resulting plasmid was named
pGBAMD-9.
[0103] Next, pGBAMD-9 was digested with KpnI and SpeI and in these
sites was ligated the approximately 2.7 kb KpnI/SpeI fragment
isolated from pGBAMD-4 amd comprising part of the 5' promoter
region of the amdS gene. The resulting plasmid was named
pGBAMD-10.
[0104] Finally, plasmid pGBAMD-8 was digested with NotI and in this
site was ligated the approximately 4.7 kb fragment isolated from
pGBAMD-10 and comprising a 5' part of the promoter region and part
of the 3' non-coding sequence both of the amdS gene. The resulting
plasmid with the cloned fragment in the correct orientation is
named pGBAMD-11 and is the replacement vector that is used to
delete the A. niger amdS gene using the MARKER GENE FREE.TM.
approach.
Example 3.2
[0105] Inactivation of the Endogenous A. niger amdS Gene
[0106] Prior to Transformation of A. niger with pGBAMD-11, the E.
coli sequences were removed by HindIII digestion and agarose gel
electrophoresis. The A. niger strain CBS 513.88 (deposited Oct. 10,
1988) was transformed with either 2.5, 5 or 10 .mu.g DNA fragment
by procedures as described in experimental procedures using
acetamide as sole N-source in selective plates. Single A. niger
transformants were purified several times onto selective acetamide
containing minimal plates. Spores of individual transformants were
collected by growing for about 5 days at 30.degree. C. on 0.4%
potato-dextrose (Oxoid, England) agar plates. Southern analyses
were performed to verify the presence of the truncated amdS
locus.
Example 3.3
[0107] Removal of the A. nidulans amdS Selection Marker Gene by
Counter-Selection on Fluoracetamide Containing Plates.
[0108] The A. nidulans amdS gene in the generated transformants was
removed again as described in the Experimental section. Correct
removal of the A. nidulans amdS selection marker gene was verified
by Southern analyses of chromosomal DNA of several fluoracetamide
resistant strains.
Example 4
Cloning of the Penicillium chrysogenum amdS Gene
Example 4.1
[0109] Amplification of an Internal Fragment of the Penicillium
chrysogenum amdS Gene Using Degenerate Oligonucleotides in a PCR on
Genomic DNA
[0110] At first, the same oligonucleotide combinations were used as
described in example 1.1. The oligonucleotide mixtures were used in
PCR with chromosomal DNA from P. chrysogenum Wisconsin 54-1255 as
template. The conditions for PCR were exactly the same as described
in example 1.1.
[0111] It was found that oligonucleotide mixtures with oligo AB4082
gave several reaction products. However, oligo AB 4082 itself also
could generate a PCR product of the expected molecular size.
Therefore it was decided to make a slightly different degenerate
oligonucleotide i.e. oligo AB 5224 (SEQ ID NO:15):
4 Oligo AB5224: 5' GGAATTCCAATNCTNCCNCCAATATC 3' TG GA G G T T
[0112] The new combination of oligonucleotides AB4079 and AB5424
gave a distinct PCR product: the other combinations of
oligonucleotides were not successful.
[0113] The PCR product obtained was cloned using the `InVitroGen`
TA cloning kit. Cloned PCR fragments were further characterized by
DNA sequence analysis (see SEQ ID NO:17). These DNA sequences were
analyzed for ORF's. The amino acid sequence as presented in SEQ ID
NO:5 was the result of this analysis. It was concluded that the
cloned PCR fragment was part of the P. chrysogenum amdS gene. The
plasmid with the cloned PCR fragment was called pPENAMDS2.
[0114] This PCR fragment was used to clone the entire P.
chrysogenum amdS gene, which can subsequently be used as homologous
selectable marker and/or to inactivate the endogenous P.
chrysogenum amdS gene as we have outlined above for the A. niger
amdS gene.
Example 4.2
[0115] Screening of the Penicillium chrysogenum Genomic Library for
the amdS Gene
[0116] A P. chrysogenum Wisconsin 54-1255 genomic library,
constructed in .lambda.-ZAPII (Stratagene, San Diego) as described
in the experimental section, was screened using the
.sup.32P-labelled EcoRI fragment isolated from pPENAMDS2.
Hybridization with the .sup.32P-labelled EcoRI fragment took place
overnight at 65.degree. C. in hybridization buffer containing
4.times.SSC, 5.times. Denhardt's solution, 0.1% SDS and 100
.mu.g/ml heat denatured calf thymus DNA. After hybridization, the
filters were washed in 4.times.SSC/0.1% SDS, 2.times.SSC/0.1% SDS
and 1.times.SSC/0.1% SDS at 65.degree. C.
[0117] One plaque, hybridizing with this probe was identified,
isolated and purified. This phage clone was designated
.lambda.PENAMD1.
Example 4.3
[0118] Sub-Cloning and Restriction Analysis of amdS Containing
Phage Clone .lambda.PENAMD1
[0119] Sub-cloning was done according the protocol of the
.lambda.-ZAPII system (Stratagene, San Diego). The result of the
sub-cloning experiment is a plasmid that exists of the pBluescript
SK vector and an insert of P. chrysogenum chromosomal DNA
(pPENAMDS101). A partial restriction map was constructed for clone
pPENAMDS101. The isolated plasmid DNA was digested with several
restriction enzymes, run on a 0.7% agarose gel, blotted onto
nitrocellulose (0.2 .mu.m; Schleicher & Schull) and hybridized
with the .sup.32P-labelled EcoRI fragment isolated from pPENAMDS2.
From the results obtained a partial restriction map was
constructed
Example 4.4
Subcloning and Sequencing of the Pc amdS Containing Fragment of
plasmid pPENAMDS101
[0120] Plasmid clone pPENAMDS101 contained an NruI-Sa/l insert of
3.3 Kb that was supposed large enough to comprise the entire, amdS
gene. The NruI/SalI fragment of pPENAMDS101 was isolated by
digesting the plasmid DNA with NruI and SalI followed by agarose
electrophoresis. The approximately 3 kb NruI/SalI fragment was
ligated in the pBluescript IIKS vector that was already digested
with SalI and SmaI. After ligation and transformation in E. coli
Inv.alpha.F, transformants were screened using restriction
analysis. The resulting plasmid was designated pPENAMDSFL. The
result of the DNA sequence analysis of clone pPENAMDSFL is given in
SEQ ID NO:19.
Sequence CWU 1
1
19 1 102 PRT A. nidulans 1 Pro Ile Ser Leu Lys Asp Gln Leu Arg Val
Lys Gly Tyr Glu Thr Ser 1 5 10 15 Met Gly Tyr Ile Ser Trp Leu Asn
Lys Tyr Asp Glu Gly Asp Ser Val 20 25 30 Leu Thr Thr Met Leu Arg
Lys Ala Gly Ala Val Phe Tyr Val Lys Thr 35 40 45 Ser Val Pro Gln
Thr Leu Met Val Cys Glu Thr Val Asn Asn Ile Ile 50 55 60 Gly Arg
Thr Val Asn Pro Arg Asn Lys Asn Trp Ser Cys Gly Gly Ser 65 70 75 80
Ser Gly Gly Glu Gly Ala Ile Val Gly Ile Arg Gly Gly Val Ile Gly 85
90 95 Val Gly Thr Asp Ile Gly 100 2 102 PRT A. oryzae 2 Pro Ile Ser
Leu Lys Asp Gln Leu Arg Val Lys Gly Thr Glu Thr Cys 1 5 10 15 Met
Ala Tyr Ile Ser Trp Leu Gly Lys Arg Asp Thr Ser Asp Ser Ile 20 25
30 Leu Thr Ala Leu Leu Arg Lys Ala Gly Ala Val Phe Leu Val Lys Thr
35 40 45 Ser Val Pro Gln Thr Leu Met Val Cys Glu Thr Val Asn Asn
Ile Ile 50 55 60 Gly Arg Thr Ser Asn Pro Arg Asn Leu Asn Leu Ser
Cys Gly Gly Ser 65 70 75 80 Ser Gly Gly Glu Gly Ala Met Ile Ala Met
Arg Gly Gly Ala Ile Gly 85 90 95 Ile Gly Thr Asp Ile Gly 100 3 104
PRT S. cerevisiae 3 Pro Ile Ser Leu Lys Asp Gln Cys Asn Val Glu Gly
Val Asp Thr Ser 1 5 10 15 Leu Gly Tyr Leu Cys Arg Thr Phe Lys Pro
Lys Thr Lys Asn Glu Glu 20 25 30 Ser Leu Ile Val Ser Phe Leu Arg
Asp Leu Gly Ala Ile Ile Phe Val 35 40 45 Lys Thr Thr Val Pro Ser
Ser Met Met Ala Thr Asp Thr Gln Ser Asn 50 55 60 Thr Phe Gly Tyr
Thr Tyr Asn Ser Ile Asn Leu Ser Phe Ser Ser Gly 65 70 75 80 Gly Ser
Ser Gly Gly Glu Gly Ser Leu Ile Gly Ala His Gly Ser Leu 85 90 95
Leu Gly Leu Gly Thr Asp Ile Gly 100 4 111 PRT A. niger 4 Pro Val
Ser Leu Lys Asp Gln Phe His Val Lys Gly Val Glu Thr Thr 1 5 10 15
Met Gly Tyr Val Gly Trp Ile Asn Thr Phe Gln Gly Lys Thr Asn Asp 20
25 30 Pro Arg Tyr Leu Thr His Glu Ser Glu Leu Val Lys Glu Leu Arg
Ala 35 40 45 Ala Gly Ala Val Leu Tyr Cys Lys Thr Ser Val Pro Met
Thr Leu Met 50 55 60 Ser Gly Glu Thr Met Asn Asn Ile Ile Thr Tyr
Thr His Asn Pro Lys 65 70 75 80 Asn Arg Leu Leu Ser Ser Gly Gly Ser
Ser Gly Gly Glu Gly Ala Leu 85 90 95 Ile Ala Leu Arg Gly Ser Pro
Ala Gly Phe Gly Thr Asp Ile Gly 100 105 110 5 102 PRT P.
chrysogenum 5 Pro Ile Trp Leu Lys Asp Gln Phe Asn Val Lys Gly Val
Asp Thr Thr 1 5 10 15 Leu Gly Tyr Val Gly Arg Ser Phe Ala Pro Ala
Gln Glu Asp Ala Val 20 25 30 Leu Val Gln Ile Leu Lys Asn Met Gly
Ala Ile Val Ile Ala Lys Thr 35 40 45 Asn Ile Pro Gln Ser Ile Met
Val Ala Glu Thr Glu Asn Pro Leu Trp 50 55 60 Gly Leu Thr Thr Asn
Pro Arg Asn Pro Ile Phe Ser Pro Gly Gly Ser 65 70 75 80 Thr Gly Gly
Glu Gly Ala Leu Leu Ala Leu His Gly Ser Leu Phe Gly 85 90 95 Phe
Gly Thr Asp Ile Gly 100 6 28 DNA Artificial sequence Synthetic
oligonucleotide designed in well conserved amino acid sequences of
the amdS genes from different fungi 6 cgggatccgc nttttgtaan
agngcngc 28 7 26 DNA Artificial sequence Synthetic oligonucleotide
designed in well conserved amino acid sequences of the amdS genes
from different fungi 7 cgggatccna ttagnctnaa ggatca 26 8 26 DNA
Artificial sequence Synthetic oligonucleotide designed in well
conserved amino acid sequences of the amdS genes from different
fungi 8 ggaattccct cnccnccnst nctncc 26 9 20 DNA Artificial
sequence Synthetic oligonucleotide designed in well conserved amino
acid sequences of the amdS genes from different fungi 9 ggaattctaa
tnstnccncc 20 10 23 DNA Artificial sequence Synthetic
oligonucleotide designed in well conserved amino acid sequences of
the amdS genes from different fungi 10 ggaattccnc caatatcngt ncc 23
11 42 DNA Artificial sequence Synthetic fragment that comprises the
recognition site for the different restriction enzymes 11
aattggcggc cgcgaattcg gtaccagatc tataggggcc ca 42 12 42 DNA
Artificial sequence Complementary chain for the synthetic
oligonucleotide disclosed in SEQ ID NO11 12 agcttgggcc cctatagatc
tggtaccgaa ttcgcggccg cc 42 13 56 DNA Artificial sequence Synthetic
fragment that comprises the recognition site for the different
restriction enzymes 13 aattggcggc cgcaagcttg gtaccactag tggatccgca
actgcaggcg gccgct 56 14 56 DNA Artificial sequence Complementary
chain for the synthetic oligonucleotide disclosed in SEQ ID NO13 14
agctagcggc cgcctgcagt tgcggatcca ctagtggtac caagcttgcg gccgcc 56 15
26 DNA Artificial sequence Synthetic oligonucleotide AB 5224 15
ggaattccaa tnstnccncc aatatc 26 16 542 DNA A. niger 16 cgggatccgc
gttttgccat agtgcagcat tggcgcatca actcgtacat tccccatcca 60
caaggagtgc tagtctgcgc tttactaatc gagaaaaagg taaactgctt gcatgaaatc
120 ttcttcgatg ccgcgcttga aaccgcccgc attctagacg accactacac
caagaccggc 180 aagccactcg gtccccttca cggcctccct gtcagtctga
aggatcaatt ccacgtcaag 240 ggcgtagaaa caaccatggg ttacgtcggc
tggataaaca ccttccaagg caagaccaat 300 gacccgcgct atcttacaca
cgaaagcgaa ctcgttaaag aactccgcgc cgcgggagcc 360 gtcctctact
gcaagactag cgtccccatg acgttgatgt caggtgaaac catgaacaat 420
atcataactt acacacataa cccgaagaac aggcttctca gttctggagg tagttccggg
480 ggcgaaggag cactgatcgc gttgcgggga tcaccagccg ggtttgggac
cgatatcggg 540 gg 542 17 384 DNA P. chrysogenum 17 cgggatccta
tctggcttaa gatcaattta acgtcaaagg cgtggacacg accctgggat 60
atgtgggtag atccttcgcc ccggcccagg aagacgcagt gcttgtgcag atcctgaaga
120 acatgggtgc catcgtcatt gcgaagacaa atatcccaca gagtatcatg
gttcgtccga 180 ggttgtcact ggcagtatct gattcggata ttgactctac
ctccagcggg ccgaaaccga 240 gaatcctctc tggggactga cgactaaccc
tcgcaatcct attttttcac cgggtgggtc 300 aactggcggc gaaggcgctt
tgctggcatt gcatggatca ctattcggat ttgggactga 360 cataggcggt
tcaataagaa ttcc 384 18 2869 DNA A. niger 18 tgtataacat agcggggtag
caagtgcctg tcagcttggc gcccatctat ccatccatct 60 accattcatt
catctccatc ttcatctcca tttcacccaa ataatgcaga attcccaatt 120
gtcgccgccc cgtatctcct ccctatctca tcgataactc aagtccgagc actatctgtc
180 tccgcgcatc aaacaagcta attctcccca ggatggatga taagcaagat
atattccgcg 240 ccattggctc cacttcctgc aatcccccgc cttcatatga
ctgacgaata gcaagaatag 300 gtaagacaac gggatgatca tcccaccgaa
cgcattgata agaaaggccc tatggtccac 360 cccctcttta tttaccatct
tatcccctca agacatccac ctccgcaaca gatactccta 420 caaccactgc
tttcaaaatg gccctcacat cctgggaaca aaccgcagcg gccaaacgcc 480
aatccgtcct caacgccatc cccgagaaat ggcgcatcaa gggtcctatc cccgcaccgt
540 cggagcagcg cgacgtaaca ggcccctaca tccagcagtt cctatcccca
cgcgaggttg 600 aaatcaccga aacagacgcc gtagggatca cagagcgaac
tacaacgggc cagtggacag 660 ctgtggaggt gaccgaggcg ttctgccatc
gcgcagcatt ggcgcatcaa ctcgtacatt 720 ccccatccac aaggagtgct
agtctgcgct ttactaatcg agaaaaaggt aaactgcttg 780 catgaaatct
tcttcgatgc cgcgcttgaa accgcccgca ttctagacga ccactacacc 840
aagaccggca agccactcgg tccccttcac ggcctccctg tcagtctgaa ggatcaattc
900 cacgtcaagg gcgtagaaac aaccatgggt tacgtcggct ggataaacac
cttccaaggc 960 aagaccaatg acccgcgcta tcttacacac gaaagcgaac
tcgttaaaga actccgcgcc 1020 gcgggagccg tcctctactg caagactagc
gtccccatga cgttgatgtc aggtgaaacc 1080 atgaacaata tcataactta
cacacataac ccgaagaaca ggcttctcag ttctggaggt 1140 agttccgggg
gcgaaggagc actgatcgcg ttgcggggat caccagccgg gtttggtacg 1200
gatatcgggg gtagtatccg tgttcctgcg tcgttcaatg gactgtatgg gatacggccg
1260 tctgtgggga gaatgccgta cgagggggcg gccaattcgg gcgatggaca
gaatactgtg 1320 ttgtcggttg tggggccgtt gtctccttcg gcgagagggt
tgatattgct gttcaagacg 1380 gtgttggggg caatgccgtg gttgggagat
cctggtgtgt tggagattcc ctggagggag 1440 gaaatcgtag aggagacgag
aaaattagtg cagggaaagc cagaggggct agcttttgga 1500 atattctacg
atgatggtca ggtaaagccg cagccaccgg tcgagagagc gatgcggatt 1560
gctgcagaga cgatcaagcg tctaggacat aaggtgagtg ccctccttct tcttgcgaca
1620 ctgctaacat tcatcccagc tcatcaattg ggaacccccc tctcacctaa
cagccgcctc 1680 cctcgcagta agtcccccat ccaacccact acaccacaac
cccctaacaa taaaccaacc 1740 cccagaaccg cgcctacaac atggacggcg
gcgccgacgt actccaaaac ttcgccctgt 1800 ccaacgaagc catccacacc
tccgtagtaa tcgacgcatc aggatccccc caaaagaccg 1860 cactagagat
cgccgcgcta aacgtcgaga agcgcgaata ccagaaacaa taccttgact 1920
actggaacag cacggcgcaa ttgacaggga ctggacgacc cgtcgacgcg gtcatttgtc
1980 cagtggcgcc gcatgcggcg tgcattccgg ggaagtatgc gacgatcggg
tatacggcgt 2040 ttattaatgt gttggattat acgagtgcgg ttgtgccggt
tacgagtgct gataggaggg 2100 tggatgttgt agggaaggaa ggaagggagt
attttgggga gttggatagg aagaccgagg 2160 gggagtgtaa gttcttccct
ttcttttctt ctttcttttc attgagctat ccaatttggt 2220 tggaggtctt
gtgtgtttgt ttgttcggag agtggtgatg gggttatgtg ctgactggat 2280
gtttctatct agacgatgcg gatgtgtttg atggggcgcc ggctgggatt cagctctttg
2340 gaagacggct tcaggaggag aagattctgg tactggctga gtatcttggt
gaggaattca 2400 agaaggctag tgcttgatca tagcgagtag tatgggaatc
gatcaaattg tctagtgata 2460 ttgagagaaa tgcagtgatg acacacattc
tgttgtgaga aacagacgaa tatacaacga 2520 agccgaaaaa tgtacagttg
taagtatcat agcatcatta tatctctacc atccctccag 2580 cggcgttact
ttcacacgga ccccgtcctt cggggtcact gtcgcggctt cacggagtat 2640
gagctctttc ttaggatcct cgagttcaaa ctggaatcgt ccaacaaatg cggccagcag
2700 acaagccagc tcggctttcg caaaaccctg cccaatgcaa ctacgcgggc
catgtatgaa 2760 ggtcaaaaag gcgtagttgc tggtggcacc gccagtgttg
gcttgccggg gccaaccacc 2820 gtcgggattg actgatcgca tcagggccca
aggactatcg tggttgtca 2869 19 3315 DNA P. chrysodenum 19 attgtaataa
tttttggata atttctaatg agagtttatt atcagattag caaaatatct 60
ctttccgaat ggtctaatat atgtagctat tgagtggtaa ctgattaaga tccaaaggtc
120 aagataaatc cccgttatat atagtggctc ccatactaaa ctcgcgatta
aaatcttgtc 180 tcactttgaa aattgacaac cctccatcca aaatacttac
tgatataatc tgtgatatat 240 attacattag gtttgtaatg ataaataatg
ataaaaaaaa aaaaaaaatg ttacaatata 300 aatctattga gagaataggg
ttcaagttgg tcagttttgg tgggaaaata gggagtggga 360 agatacttag
taacagggtc tgtatatgtc ccacactgta aacgaagcca ggcaggaatg 420
tcagccttct aatagacgag atttcacccc acaaagaccc tcgaaatcaa agatgtcatg
480 gttagagaga ggacatcaac aagatatccc aataacccct ggaccgaggc
tgtatgtgaa 540 gtatcgaggc gctaaaccta aaaaggaaaa actaaccgga
tacagttaca gtagttcact 600 ccgccgttta cagattcaaa atgctcaaga
actccaaaga ctccaaatta tttgggggga 660 acggcaaatc tcggttggat
aaagaaaaaa cgaaattact ccaaaaatga catttgacga 720 gagtgtgcca
cgtccccact tgataagagt ggccctcgga ctacagtccg agctgactat 780
attaggctag tcttgacctc caacagggct cttacaagtg caattcaaag taaaatgggc
840 agtcagacct gggaggagat tgtctcccag aaacgggcca tcagagacca
actcatcgca 900 ccgtacttag ccgatgtagc tcaacgtctg ccgcgagtac
agaatgccga ggagcgtact 960 cgactagaag atctgttgtt tcaaacgatt
acagacattg acaatgtcac ctctctgctg 1020 gaatgcatgg cgaaaggaga
gttccaggta gaacaggtga tcaaggcata tatccaacgg 1080 tatgtcttct
atcggggttg gaacaggccc tatactaatg ccatcggtag ggctgtgcta 1140
gcacatcaat tagtacgtgt cccacatctt ccttcctttc catttgcacc cttggccaag
1200 tcgcttatag aatctggcat gggtagacaa atagcctgac cgaggttctt
tttgaagatg 1260 ccctaggaca ggcaaagcag ctagacgccg aatttgcaga
aactggaaag ctcagaggtc 1320 ccctgcatgg aattccaatc acggtgaaag
accaatttaa cgtcaaaggc gtggacacga 1380 ccctgggata tgtgggtaga
tccttcgccc cggcccagga agacgcagtg cttgtgcaga 1440 tcctgaagaa
catgggtgcc atcgtcattg cgaagacaaa tatcccacag agtatcatgg 1500
ttcgtccgag gttgtcactg gcagtatctg attcggatat tgactctacc tccagtgggc
1560 cgaaaccgag aatcctctct ggggactgac gactaaccct cgcaatccta
ttttttcacc 1620 gggtgggtca actggcggcg aaggcgcttt gctggcattg
catggatcac tattcggatt 1680 tgggactgat attggcggaa gtgtaaggat
cccacaggct acagtgggct tgtacggatt 1740 caaaccaagc gtaagtacca
accgccatga acaaactgtc ctttcttttc cattttttta 1800 atatgtgtcg
atgattcctg aaagcagagc gcccgacttc cttaccaggg cgtacccgtc 1860
tccactgagg gtcaagaaca tgtcccgtct tcaatcggcc cgatggcccg ggatctctcg
1920 tctatctgcc acatgagccg tctgatagcg aacagccagc cgtgggatgt
tgatccgcgg 1980 tgcgctcctc ttccttggaa tgacactgca ttccaagaac
ttcaagtccg acctatggta 2040 atcggcttga tcctggatga cggtgtagta
aaggtccacc cgcctattgc gcgtgccctg 2100 ctagaactct cagcagtact
tagagcacat ggccacgaag ttgtggtctg ggatacattt 2160 gatcatgcgg
agtgcattga gattatggat atcttctaca cggtcgatgg gggtgaggat 2220
attcgtcggg atgtagccgc tgccggcgag ccgtttattc ctcatgttga agggctggtt
2280 aaccgcggca aggctatatc ggtttatgag tattggcagc tgaacaagcg
gaaaactgca 2340 gtgcagaaga aatatctgga caaatggaac gcggtgcgat
ctccgtcggg tcgggctgtc 2400 gatgttctgc tgagtcctac cttgccgcat
acgactgtgc ctcatcggaa attccgttgg 2460 gttggctata ctaagatttg
gaatttgttg gactacccgg ctttgacgtt cccagtggat 2520 agagtgaggg
ctgaggtgga tgtgttgcca tcggagcctt atatcccgag aaacagcctc 2580
gacgagtgga attggaatat tttcgatgcc aaacaagcgg atggatgtcc agtgaatctg
2640 cagatcatcg gaaaaaaact ccacgaagag aaggtactgg gggctgctac
agttattgag 2700 aggctctgga aaagtcatat cgacgaatcc aattgaacca
tctgggatgt atgggtagaa 2760 aatgaagttg ggttcactcg cagactgaac
gacgtgtatc gcagttgact gaactgaatt 2820 tggaataaat atggtagaca
taactcatta tgcagcttgg tgggatatct gcctccagag 2880 tcatataaat
cacaaacgcc gtgggaatat gcaacaagac aagcactctt gagtctcaag 2940
ccttgggagg ctcgaacaga tcgggtttca tgttaatttt gcaaagcttc gccacacagc
3000 cgctcagttg aagcagtgac tgcacgccgt ctagtatacg catatgcgtg
aatccaatct 3060 cccggataaa ctccaacttg gaatgttcag acaatgtggg
aatggtcttg gtgactcgga 3120 acatggtact gatgatatca tgcgcagaat
aacccagcgt cctgtggata caaaatgatt 3180 aggacctcct gcagttgaac
caaacagatg tgtaacatac catagctcgt tcaacccctc 3240 cagtgccaca
tccaccttgc cttcccagca agccttgatc atggcctgga ctttgaccgg 3300
gtgcgggctg tcgac 3315
* * * * *