U.S. patent number 5,190,931 [Application Number 07/436,598] was granted by the patent office on 1993-03-02 for regulation of gene expression by employing translational inhibition of mrna utilizing interfering complementary mrna.
This patent grant is currently assigned to The Research Foundation of State University of New York. Invention is credited to Masayori Inouye.
United States Patent |
5,190,931 |
Inouye |
March 2, 1993 |
Regulation of gene expression by employing translational inhibition
of MRNA utilizing interfering complementary MRNA
Abstract
Gene expression in a cell can be regulated or inhibited by
incorporating into or associating with the genetic material of the
cell a non-native nucleic acid sequence which is transcribed to
produce an mRNA which is complementary to and capable of binding to
the mRNA produced by the genetic material of said cell.
Inventors: |
Inouye; Masayori (Bridgewater,
NJ) |
Assignee: |
The Research Foundation of State
University of New York (Albany, NY)
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Family
ID: |
27569389 |
Appl.
No.: |
07/436,598 |
Filed: |
November 15, 1989 |
Related U.S. Patent Documents
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Application
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300741 |
Jan 23, 1989 |
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228852 |
Aug 3, 1988 |
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300741 |
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585282 |
Mar 1, 1984 |
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543528 |
Oct 20, 1983 |
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228852 |
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Current U.S.
Class: |
435/91.32;
435/252.3; 435/252.33; 435/254.11; 435/320.1; 435/455; 435/471;
435/91.33; 514/44A; 536/24.1 |
Current CPC
Class: |
C07H
21/00 (20130101); C12N 15/113 (20130101); A61K
38/00 (20130101); C12N 2310/111 (20130101) |
Current International
Class: |
C07H
21/00 (20060101); C12N 15/11 (20060101); A61K
38/00 (20060101); C12N 005/10 (); C12N 001/11 ();
C12N 015/09 (); C07H 015/12 () |
Field of
Search: |
;435/91,172.3,252.3,252.33,948,235,240.2 ;536/27 ;514/43,44
;935/33,34,35,36,37 ;424/93 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Paterson et al. "Structural gene identification and mapping by
DNA:mRNA hybrid-arrested cell-free translation" Chemical Abstract
87:196,773t (1977). .
Paterson et al. "Structural gene identification and mapping by
DNA:mRNA hybrid-arrested cell-free translation" Proc. Natl. Acid.
Sci. U.S.A. 74:4370-4374 (1977). .
Hastie et al. "Analysis of mRNA populations by cDNA-mRNA
hybrid-mediated inhibition of cell-free protein synthesis" Proc.
Natl. Acad. Sci. U.S.A. 75:1217-1221. .
Inglis et al. "Polypeptides Specified by the Influenza Virus
Genome, 2. Assignment of Protein Coding Functions to Individual
Genome Segments by in vitro Translation", Virology 78:522-536
(1977). .
Meril, et al. "E. coli gal Operaon Proteins Made After Prophage
Lambda Induction", J. Bact. 147:875-887 (1981). .
Metzler, D. E, 1977, in: Biochemistry. The Chemical Reactions of
Living Cells. Academic Press. New York. p. 103. .
Eisenbert et al 1979. in: Physical Chemistry with Applications to
the Life Sciences. Benjamin/Cummings. Publ. Co. CA. pp. 155-158, SI
units table. .
Wong et al. 1982. Biochem. Biophys. Res. Commun. 107:584-587. .
Chilton, M. D. 1983. Sci. Am. 248, 50-59. .
Weinstein et al. (eds.) 1983. in: Genes and Proteins in Orcogenes.
Academic Press. New York pp. 253-265. .
Biol. Abs. 73, 3743. abstract No. 36372 (Naora et al.). .
Herskowitz, I. H. 1977. in: Principles of Genetics. Second Edition,
MacMillan Publishing Co. New York. pp. 56, 492-493. .
Kolter et al. 1982. Ann. Rev. Genet. 16, 113-114. .
Simons et al. 1983 Cell 34, 683-691. .
Tomizawa et al. 1982 Cell 31, 575-583. .
Light et al 1983. EMBO. J. 2, 93-98. .
Saito et al. 1981. Cell 27, 533-542. .
Light et al. 1982. Molec. Cell. Genet. 187, 486-493. .
Tomizawa et al. 1981. Proc. Nat'l. Acad. Sci. U.S.A. 78,
6096-6108..
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Primary Examiner: Low; Christopher S. F.
Attorney, Agent or Firm: Moroz; Eugene Feiler; William S.
Morry; Mary J.
Government Interests
This invention was made with Government support under Grant No.
R01-GM-19043 awarded by National Science Foundation. The Government
has certain rights in the invention.
Parent Case Text
REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of copending coassigned
U.S. patent application Ser. No. 07/300,741, filed Jan. 23, 1989,
now abandoned, and U.S. patent application Ser. No. 07/228,852,
filed Aug. 3, 1988, now abandoned. Application Ser. No. 07/300,741,
now abandoned, is in turn a continuation application of U.S. patent
application Ser. No. 06/585,282, filed Mar. 1, 1984, now abandoned,
which is in turn a continuation-in-part application of U.S. patent
application Ser. No. 06/543,528, filed Oct. 20, 1983, now
abandoned. Application Ser. No. 07/288,852, now abandoned, is a
continuation application of application Ser. No. 06/543,528, filed
Oct. 20, 1983, now abandoned.
Claims
What is claimed is:
1. A prokaryotic or eukaryotic cell containing a non-native DNA
construct, which construct produces an RNA which regulates the
function of a gene, said DNA construct containing the following
operably linked DNA segments:
a. a transcriptional promoter segment;
b. a transcription termination segment; and therebetween
c. a DNA segment;
whereby transcription of the DNA segment produces a ribonucleotide
sequence which does not naturally occur in the cell, is
complementary to a ribonucleotide sequence transcribed from said
gene, and said non-naturally occurring ribonucleotide sequence
regulates the function of said gene.
2. A prokaryotic or eukaryotic cell containing a non-native DNA
construct, which construct produces an RNA which regulates the
function of a gene, said DNA construct containing the following
operably linked DNA segments:
a. a transcriptional promoter segment;
b. a transcription termination segment; and
c. a DNA segment comprising a segment of said gene, said gene
segment located between said promoter segment and said termination
segment and being inverted with respect to said promoter segment
and said termination segment, whereby the RNA produced by
transcription of the inverted gene segment regulates the function
of said gene.
3. A method of regulating the function of a gene in a prokaryotic
or eukaryotic cell which comprises introducing into said cell the
DNA construct of claim 1.
4. A method of regulating the function of a gene in a prokaryotic
or eukaryotic cell which comprises:
introducing into said cell the DNA construct of claim 1 whereby a
transformed cell is obtained; and
growing said transformed cell whereby the RNA produced by
transcription of said DNA segment regulates the functioning of said
gene.
5. A non-native DNA construct which, when present in a prokaryotic
or eukaryotic cell containing a gene, produces an RNA which
regulates the function of said gene, said DNA construct containing
the following operably linked DNA segments:
a. a transcriptional promoter segment;
b. a transcription termination segment; and
c. a DNA segment comprising a segment of said gene, said gene
segment located between said promoter segment and said termination
segment and being inverted with respect to said promoter segment
and said termination segment, whereby the RNA produced by
transcription of the inverted gene segment regulates the function
of said gene.
6. A non-native DNA construct which, when present in a prokaryotic
or eukaryotic cell containing a gene, produces an RNA which
regulates the function of said gene, said DNA construct containing
the following operably linked DNA segments:
a. a transcriptional promoter segment;
b. a transcription termination segment; and therebetween
c. a DNA segment;
whereby transcription of the DNA segment produces a ribonucleotide
sequence which does not naturally occur in the cell, is
complementary to a ribonucleotide sequence transcribed from said
gene, and said non-naturally occurring ribonucleotide sequence
regulates the function of said gene.
7. A method of regulating the function of a gene in a prokaryotic
or eukaryotic cell which comprises introducing into said cell the
DNA construct of claim 5.
8. A method of regulating the function of a gene in a prokaryotic
or eukaryotic cell which comprises:
introducing into said cell the DNA construct of claim 5 whereby a
transformed cell is obtained; and
growing said transformed cell whereby the RNA produced by
transcription of the inverted gene segment regulates the
functioning of said gene.
9. A non-native polynucleotide construct which, when present in a
cell containing a gene, produces an RNA which regulates the
function of said gene, said polynucleotide construct containing the
following operably linked polynucleotide segments:
a. a transcriptional promoter segment;
b. a transcription termination segment; and
c. a polynucleotide segment comprising a segment of said gene, said
gene segment located between said promoter segment and said
termination segment and being inverted with respect to said
promoter segment and said termination segment, whereby the RNA
produced by transcription of the inverted gene segment regulates
the function of said gene.
10. The polynucleotide construct of claim 9 wherein said cell is
prokaryotic.
11. The polynucleotide construct of claim 9 wherein said cell is
eukaryotic.
12. A polynucleotide construct of claim 9 wherein said
transcriptional promoter segment comprises an inducible
promoter.
13. A polynucleotide construct of claim 9 wherein said gene is an
oncogene.
14. A polynucleotide construct of claim 9 wherein said gene is a
viral gene.
15. A polynucleotide construct of claim 9 wherein said gene encodes
a protein.
16. A polynucleotide construct of claim 15 wherein said
transcriptional promoter segment comprises an inducible
promoter.
17. A polynucleotide construct of claim 15 wherein said gene
segment includes the 5' non-coding region of said gene.
18. A polynucleotide construct of claim 15 wherein said gene
segment includes the ribosome binding portion of said gene.
19. A polynucleotide construct of claim 15 wherein said gene
segment includes the translation initiation portion of said
gene.
20. A vector having incorporated therein a polynucleotide construct
according to any one of claims 9-19.
21. A vector according to claim 20 wherein said vector is a
plasmid.
22. A vector according to claim 20 wherein said vector is a viral
vector.
23. A pharmaceutical composition which comprises the polynucleotide
construct of any one of claims 9-19.
24. A pharmaceutical composition which comprises the vector of
claim 20.
25. A pharmaceutical composition which comprises the vector of
claim 21.
26. A pharmaceutical composition which comprises the vector of
claim 22.
27. A method of regulating the function of a gene in a cell which
comprises introducing into said cell the polynucleotide construct
of any one of claims 9 or 12-19.
28. A method of regulating the function of a gene in a cell which
comprises introducing into said cell the vector of claim 20.
29. The method of claim 27 wherein said cell is prokaryotic.
30. The method of claim 27 wherein said cell is eukaryotic.
31. The method of claim 28 wherein said cell is prokaryotic.
32. The method of claim 28 wherein said cell is eukaryotic.
33. A method of regulating the function of a gene in a cell which
comprises:
introducing into said cell the polynucleotide construct of any one
of claims 9-19 whereby a transformed cell is obtained; and
growing said transformed cell whereby the RNA produced by
transcription of the inverted gene segment regulates the
functioning of said gene.
34. A non-native polynucleotide construct which, when present in a
cell containing a gene, produces an RNA which regulates the
function of said gene, said polynucleotide construct containing the
following operably linked polynucleotide segments:
a. a transcriptional promoter segment;
b. a transcription termination segment; and therebetween
c. a polynucleotide segment;
whereby transcription of the polynucleotide segment produces a
ribonucleotide sequence which does not naturally occur in the cell,
is complementary to a ribonucleotide sequence transcribed from said
gene, and said non-naturally occurring ribonucleotide sequence
regulates the function of said gene.
35. The polynucleotide construct of claim 34 wherein said cell is
prokaryotic.
36. The polynucleotide construct of claim 34 wherein said cell is
eukaryotic.
37. A polynucleotide construct of claim 34 wherein said
transcriptional promoter segment comprises an inducible
promoter.
38. A polynucleotide construct of claim 34 wherein said gene is an
oncogene.
39. A polynucleotide construct of claim 34 wherein said gene is a
viral gene.
40. A polynucleotide construct of claim 34 wherein said gene
encodes a protein.
41. A polynucleotide construct of claim 40 wherein said
transcriptional promoter segment comprises an inducible
promoter.
42. A polynucleotide construct of claim 40 wherein said
polynucleotide segment encodes a ribonucleotide sequence
complementary to a 5' end non-coding portion of said ribonucleotide
sequence transcribed from said gene.
43. A polynucleotide construct of claim 40 wherein said
polynucleotide segment encodes a ribonucleotide sequence
complementary to a ribosome binding portion of said ribonucleotide
sequence transcribed from said gene.
44. A polynucleotide construct of claim 40 wherein said
polynucleotide segment encodes a ribonucleotide sequence
complementary to the translation initiation portion of said
ribonucleotide sequence transcribed from said gene.
45. A vector having incorporated therein a polynucleotide construct
according to any one of claims 34-44.
46. A vector according to claim 45 wherein said vector is a
plasmid.
47. A vector according to claim 45 wherein said vector is a viral
vector.
48. A pharmaceutical composition which comprises the polynucleotide
construct of any one of claims 34-44.
49. A pharmaceutical composition which comprises the vector of
claim 45.
50. A pharmaceutical composition which comprises the vector of
claim 46.
51. A pharmaceutical composition which comprises the vector of
claim 47.
52. A cell containing a non-native polynucleotide construct, which
construct produces an RNA which regulates the function of a gene,
said polynucleotide construct containing the following operably
linked polynucleotide segments:
a. a transcriptional promoter segment;
b. a transcription termination segment; and therebetween
c. a polynucleotide segment;
whereby transcription of the polynucleotide segment produces a
ribonucleotide sequence which does not naturally occur in the cell,
is complementary to a ribonucleotide sequence transcribed from said
gene, and said non-naturally occurring ribonucleotide sequence
regulates the function of said gene.
53. The cell of claim 52 wherein said cell is prokaryotic.
54. The cell of claim 52 wherein said cell is eukaryotic.
55. The cell of claim 52 wherein said transcriptional promoter
segment comprises an inducible promoter.
56. The cell of claim 52 wherein said gene is an oncogene.
57. The cell of claim 52 wherein said gene is a viral gene.
58. The cell of claim 52 wherein said gene encodes a protein.
59. The cell of claim 58 wherein said transcriptional promoter
segment comprises an inducible promoter.
60. The cell of claim wherein said polynucleotide segment encodes a
ribonucleotide sequence complementary to a 5' end non-coding
portion of said ribonucleotide sequence transcribed from said
gene.
61. The cell of claim 58 wherein said polynucleotide segment
encodes a ribonucleotide sequence complementary to a ribosome
binding portion of said ribonucleotide sequence transcribed from
said gene.
62. The cell of claim 58 wherein said polynucleotide segment
encodes a ribonucleotide sequence complementary to the translation
initiation portion of said ribonucleotide sequence transcribed from
said gene.
63. The cell according to any one of claims 52-62 wherein said
polynucleotide construct is incorporated into a vector.
64. The cell according to claim 63 wherein said vector is a
plasmid.
65. The cell according to claim 63 wherein said vector is a viral
vector.
66. A method of regulating the function of a gene in a cell which
comprises introducing into said cell the polynucleotide construct
of any one of claims 34 or 37-44.
67. A method of regulating the function of a gene in a cell which
comprises introducing into said cell the vector of claim 45.
68. The method of claim 66 wherein said cell is prokaryotic.
69. The method of claim 66 wherein said cell is eukaryotic.
70. The method of claim 67 wherein said cell is prokaryotic.
71. The method of claim 67 wherein said cell is eukaryotic.
72. A method of regulating the function of a gene in a cell which
comprises:
introducing into said cell the polynucleotide construct of any one
of claims 34-44 whereby a transformed cell is obtained; and
growing said transformed cell whereby the RNA produced by
transcription of said polynucleotide segment regulates the
functioning of said gene.
73. A cell containing a non-native polynucleotide construct, which
construct produces an RNA which regulates the function of a gene,
said polynucleotide construct containing the following operably
linked polynucleotide segments:
a. a transcriptional promoter segment;
b. a transcription termination segment; and
c. a polynucleotide segment comprising a segment of said gene, said
gene segment located between said promoter segment and said
termination segment and being inverted with respect to said
promoter segment and said termination segment, whereby the RNA
produced by transcription of the inverted gene segment regulates
the function of said gene.
74. The cell of claim 73 wherein said cell is prokaryotic.
75. The cell of claim 73 wherein said cell is eukaryotic.
76. The cell of claim 73 wherein said transcriptional promoter
segment comprises an inducible promoter.
77. The cell of claim 73 wherein said gene is an oncogene.
78. The cell of claim 73 wherein said gene is a viral gene.
79. The cell of claim 73 wherein said gene encodes a protein.
80. The cell of claim 79 wherein said transcriptional promoter
segment comprises an inducible promoter.
81. The cell of claim 79 wherein said gene segment includes the 5'
non-coding region of said gene.
82. The cell of claim 79 wherein said gene segment includes the
ribosome binding portion of said gene.
83. The cell of claim 79 wherein said gene segment includes the
translation initiation portion of said gene.
84. The cell of any one of claims 79-83 wherein the polynucleotide
construct is incorporated into a vector.
85. The cell of claim 84 wherein said vector is a plasmid.
86. The cell of claim 84 wherein said vector is a viral vector.
Description
BACKGROUND OF THE INVENTION
Regulatory control of gene expression has received special
attention by scientists. In special circumstances, gene expression
has been achieved by employing recombinant DNA as well as other
techniques.
For example, in the PCT Patent Application WO 83/01451, published
Apr. 23, 1983, there is disclosed a technique employing an
oligonucleotide, preferably in phosphotriester form having a base
sequence substantially complementary to a Portion of messenger
ribonucleic acid (mRNA) coding for a biological component of an
organism. This oligonucleotide is introduced into the organism and,
due to the complementary relationship between the oligonucleotide
and the messenger ribonucleotide, the two components hybridize
under appropriate conditions to control or inhibit synthesis of the
organism's biological component coded for by the messenger
ribonucleotide. If the biological component is vital to the
organism's viability, then the oligonucleotide could act as an
antibiotic. A related technique for the regulation of gene
expression in an organism is described in Simons, et al.,
"Translational Control of IS10 Transposition", Cell 34, 683-691
(1983). The disclosures of the above-identified publications are
herein incorporated and made part of this disclosure.
In U.S. patent application Ser. No. 543,528 filed Oct. 20, 1983 of
which this application is in turn a continuation-in-part, gene
expression is regulated, inhibited and/or controlled by
incorporating in or along with the genetic material of the
organism, DNA which is transcribed to produce an mRNA having at
least a portion complementary to or capable of hybridizing with an
mRNA of said organism, such that upon binding or hybridizing to the
mRNA, the translation of the mRNA is inhibited and/or prevented.
Consequently, production of the protein coded for by the mRNA is
precluded. In the instance here, because the mRNA codes for a
protein vital to the growth of the organism, the organism becomes
disabled. It is also disclosed that this technique for regulating
or inhibiting gene expression is applicable to both prokaryotic and
eukaryotic organisms, including yeast.
As indicated hereinabove, it is known that the expression of
certain genes can be regulated at the level of transcription.
Transcriptional regulation is carried out either negatively
(repressors) or positively (activators) by a protein factor.
It is also known that certain specific protein factors regulate
translation of specific mRNAs. As indicated hereinabove, it has
become evident that RNAs are involved in regulating the expression
of specific genes and it has been reported that a small mRNA
transcript of 174 bases is produced, upon growing Escherichia coli
in a medium of high osmolarity, which inhibits the expression of a
gene coding for a protein called Omp F. See Mizuno et al.
"Regulation of Gene Expression by a Small RNA Transcript (micRNA)
in Escherichia coli: K-12", Proc. Jap. Acad., 59, 335-338 (1983).
The inhibition of OmpF protein production by the small mRNA
transcript (mic-RNA, i.e. mRNA interfering complementary RNA) is
likely due to the formation of a hybrid between the micRNA and the
ompF mRNA over a region of approximately 80 bases including the
Shine-Dalgarno sequence and the initiation codon.
A similar regulation by a small complementary mRNA has also been
described for the Tn10 transposase gene, see Simons et al.
"Translational Control of IS10 Transposition", Cell, 34, 683-691
(1983). In this case, however, the gene for the transposase protein
and the gene for the micRNA are transcribed in opposite directions
off the same segment of DNA such that the 5'-ends of the
transcripts can form a complementary hybrid. The hybrid is thought
to inhibit translation of the transposase mRNA. However, the
transposase situation is in contrast to the ompF situation in which
the ompF gene and the micRNA gene (micF) are completely unlinked
and map at 21 and 47 minutes, respectively, on the E. coli
chromosomes.
It is an object of this invention to provide a technique useful for
the regulation of gene expression of a cell and/or an organism.
It is another object of this invention to provide transformed cells
and/or organisms having special properties with respect to the gene
expression of the genetic material making up said organisms.
It is yet another object of this invention to provide DNA and viral
or plasmid vectors containing the DNA, wherein said DNA is
transcribed to produce mRNA which is complementary to and capable
of binding or hybridizing to the mRNA produced by said gene to be
regulated.
It is a further object of this invention to provide an improved
technique and materials useful in connection therewith for the
regulation or inhibition of gene expression.
It is also an object of this invention to provide transformed
organisms, having been transformed with plasmids or viral vectors
containing a gene that produces a micRNA which regulates and/or
inhibits the gene expression of a gene located within the host
organism.
Another object of this invention is to provide DNA, or vectors
including plasmids and viral vectors containing said DNA which is
transcribed to produce an mRNA (micRNA) which is complementary to
and capable of binding or hybridizing with the mRNA transcribed by
the gene to be regulated.
How these and other objects of this invention are achieved will
become apparent in the light of the accompanying disclosure and
with reference to the accompanying drawings wherein:
FIG. 1 describes the construction of a subclone or a gene and
various plasmids carrying the promoter region therefor;
FIG. 2 sets forth the nucleotide sequence of the promoter region
and upstream region of an ompC gene of E. coli;
FIG. 3 illustrates the hybrid formation between certain RNA in
accordance with the practices of this invention;
FIG. 4 illustrates the homologous sequences between the micF and
the ompC genes of E. coli;
FIG. 5 illustrates a possible model for the role of micF RNA useful
in and in accordance with the practices of this invention;
FIG. 6 illustrates the construction of mic vector pJDC402 and
mic(lpp);
FIG. 7 illustrates the homology between the ompC mRNA and the lpp
mRNA; and
FIG. 8 illustrates fragments used to construct mic(ompA) genes.
SUMMARY OF THE INVENTION
Gene expression in an organism in accordance with the practices of
this invention is regulated, inhibited and/or controlled by
incorporating in or along with the genetic material of the organism
non-native DNA which transcribes to produce an RNA which is
complementary to and capable of binding or hybridizing to a mRNA
produced by a gene located within said organism. Upon binding to or
hybridization with the mRNA, the translation of the mRNA is
prevented. Consequently, the protein coded for by the mRNA is not
produced. In the instance where the mRNA translated product, e.g.
protein, is vital to the growth of the organism or cellular
material, the organism is so transformed or altered such that it
becomes, at least, disabled.
In accordance with the practices of this invention there has been
constructed a mic system designed to regulate the expression of a
gene. More particularly, one can construct in accordance with the
practices of this invention an artificial mic system to regulate
the expression of any specific gene in E. coli.
Further, in accordance with the practices of this invention, a
micRNA system for a gene is constructed by inserting a small DNA
fragment from the gene, in the opposite orientation, after a
promoter. Such a system provides a way, heretofore unknown, for
specifically regulating the expression of any gene. More
particularly, by inserting the micDNA fragments under the control
of an inducible promoter, particularly as embodied in E. coli, the
expression of essential E. coli genes can be regulated. It would
appear, therefore, that in accordance with the practices of this
invention, the inducible lethality thus-created may be an effective
tool in the study of essential genes.
Hereinafter, in accordance with the practices of this invention,
there is described the construction of an artificial mic system and
the demonstration of its function utilizing several E. coli genes.
The mic system in accordance with this invention is an effective
way to regulate the expression of specific prokaryotic genes. This
invention accordingly provides the basis for accomplishing similar
regulation of biologically important genes in eukaryotes. For
example, the mic system can be used to block the expression of
harmful genes, such as oncogenes and viral genes, and to influence
the expression substantially of any other gene, harmful or
otherwise.
The practices of this invention are thus applicable to both
procaryotic and eukaryotic cellular materials or microorganisms,
including yeast, and are generally applicable to organisms which
contain expressed genetic material.
Accordingly, in the practices of this invention from a genetic
point of view as evidenced by gene expression, new organisms are
readily produced. Further, the practices of this invention provide
a powerful tool or technique for altering gene expression of
organisms. The practices of this invention may cause the organisms
to be disabled or incapable of functioning normally or may impart
special properties thereto. The DNA employed in the practices of
this invention can be incorporated into the treated or effected
organisms by direct introduction into the nucleus of a eukaryotic
organism or by way of a plasmid or suitable vector containing the
special DNA of this invention in the case of a procaryotic
organism.
DETAILED DESCRIPTION OF THE INVENTION
By way of further background of the practices of this invention, it
has been found that gene expression of the major outer membrane
proteins, OmpF and OmpC, of Escherichia coli are osmoregulated. The
ompC locus was found to be transcribed bidirectionally under
conditions of high osmolarity. The upstream stretch of mRNA of
approximately 170 bases was found to inhibit the production of OmpF
protein. This mRNA (micRNA) has a long sequence which is
complementary to the 5'-end region of the ompF mRNA that includes
the ribosome-binding site and the coding region of the first nine
amino acid residues of pro-OmpF protein. Thus, it is proposed that
the micRNA inhibits the translation of ompF mRNA by hybridizing
with it. This novel mechanism can account for the observation that
the total amount of the OmpF and of the OmpC proteins is always
constant in E. coli.
The major outer membrane proteins of Escherichia coli, OmpF and
OmpC, are essential proteins which function as passive diffusion
pores for small, hydrophilic molecules. These matrix porin proteins
are encoded by the structural genes ompF and ompC, which map at 21
and 47 min on the E. coli chromosome, respectively, see Reeves, P.
in Bactrial Outer Membranes: Biogenesis and Function (ed. Inouye,
M.) 255-291 (John Wiley and Sons, New York, 1979). The expression
of these genes is regulated by the osmolarity of the culture
medium. There is a strict compensatory production of both proteins:
as the osmolarity of the culture medium increases, the production
of OmpF protein decreases while the production of OmpC protein
increases so that the total amount of the OmpF plus OmpC proteins
is constant. This osmoregulation of the ompF and ompC genes is
controlled by another unlinked locus, ompB, which maps at 74 min,
see Hall, M. N. & Silhavy, T. J., J. Mol. Biol. 146, 23-43
(1981) and Hall, M. N. & Silhavy, T. J., J. Mol. Biol. 151,
1-15 (1981). The ompB locus contains two genes called ompR and
envZ. The DNA sequences of both genes have been determined and
their gene products have been characterized, see Wurtzel, E. T. et
al., J. Biol. Chem 257, 13685-13691 (1982) and Mizuno, T., et al.,
J. Biol. Chem. 257, 13692-13698 (1982). The EnvZ protein, assumed
to be a membrane receptor protein, serves as an osmosensor
transmitting the signal from the culture medium to the OmpR
protein. The OmpR protein then serves as a positive regulator for
the expression of the ompF and ompC genes. The ompF and ompC genes
were sequenced, and extensive homology was found in their coding
regions, however, there was very little homology in their promoter
regions.
During the characterization of the ompC gene, the novel regulatory
mechanism of gene expression mediated by a new species of RNA
called mRNA interfering complementary RNA (micRNA) in accordance
with this invention was discovered and/or elicited. MicRNA is
produced from an independent transcriptional unit (the micF gene).
This gene is located immediately upstream of the ompC gene but is
transcribed in the opposite direction. The 174-base micRNA blocks
the translation of the ompF mRNA by hybridizing to it. Since the
production of micRNA is assumed to be proportional to the
production of ompC mRNA, this regulatory mechanism appears to be a
very efficient way to maintain a constant total amount of OmpF and
OmpC proteins.
A DNA Fragment Suppressing ompF Expression
While characterizing the ompC promoter, it was found that a DNA
fragment of approximately 300 bp, located upstream of the ompC
promoter, completely blocked the production of OmpF protein when
OmpF.sup.+ cells were transformed with a multi-copy plasmid
harboring this DNA fragment. For this experiment, plasmid pMY150
was constructed from the original ompC clone, pMY111, see Mizuno,
T. et al., J.Biol. Chem. 258, 6932-6940 (1982), by changing the
HpaI sites of pMY111 to XbaI sites followed by removal of the 1.1
kb SalI fragment as described in FIG. 1a of FIG. 1.
FIG. 1 shows the construction of the subclone of the ompC gene and
various plasmids carrying the ompC promoter region.
(a) Schematic presentation of the subcloning of the ompC gene.
Plasmid pMY111 carrying a 2.7 Kb E. coli chromosomal DNA in pBR322
was described previously. The plasmid (1 .mu.g of DNA) was digested
with HpaI and religated in the presence of an XbaI linker
(CTCTAGAG, 150 p mole). Thus, a 400 bp HapI fragment was removed
and a unique XbaI site was newly created (pMY100). Plasmid pMY100
(1 .mu.g of DNA) was further digested with SalI and religated to
remove a 1.1 kb SalI fragment (pMY150). In order to obtain an ompC
promoter fragment of different sizes, plasmid pMY150 was digested
by Bal31 nuclease after cleavage of the unique BglII site (see FIG
1b). Subsequently, the plasmid was religated in the presence of an
XbaI linker. Plasmid pCX28, thus constructed, is one of clones
carrying approximately a 300-bp XbaI-XbaI fragment as shown in FIG.
1b.
(b) Simplified restriction map of the plasmid pMY150 carrying the
entire ompC gene. The 1.8 Kb HindIII-SalI fragment (boxed region)
in pBR322 contains the entire ompC coding region as well as the 5'-
and 3'-non-coding region. Transcription of the ompC gene proceeds
in the direction shown by an arrow. A bidirectional arrow indicates
an approximate deleted region (ca. 600 bp) for plasmid pCX28.
(c) Various .beta.-galactosidase (lacZ) gene fusions to the DNA
fragments derived from the ampC promoter and its upstream region
Plasmid I, 507-bp XbaI-RsaI fragment was isolated from pMY150 (an
RsaI site is present just downstream of the ATG codon), and
inserted between XbaI-SmaI sites of plasmid pICIII which is derived
from plasmid pINIII carrying the lacz gene. During the ligation, a
HindIII linker was inserted between the RsaI and SmaI ligation
site. The XbaI-HindIII fragment was isolated from the plasmid thus
constructed and reinserted into plasmid pKM005 to create a lacZ
gene fusion in the right reading frame. Characteristic features of
plasmids pICIII and pKM005 were described previously. Plasmids II
and IV carrying approximately 430-bp MspI-BamHI fragment was
isolated from clone I (a BamHI site is present just downstream of
the ATG codon for the .beta.-galactosidase coding sequence in
plasmid I), and treated with S1 nuclease to create blunt ends.
After adding XbaI linkers at both ends, the XbaI-XbaI fragment thus
obtained was inserted into plasmid pKM005 at its XbaI site in the
possible two orientations. Plasmids III and V, an approximately 300
bp XbaI-XbaI fragment was isolated from plasmid pCX28 (FIG. 1a) and
inserted into plasmid pKM005 at its XbaI site in the two possible
orientations. These plasmids (I-V) were transformed into a lacZ
deletion strain SB4288 (F.sup.- recA thi-1 relA ma124 spc12 supE-50
proB lac), and those .beta.-galactosidase activities were tested on
MacConkey plates (Difco). Results are shown as LacZ.sup.+ or
LacZ.sup.-. Ability of these clones to inhibit the expression of
OmpF protein are also shown as MicF.sup.+ or MicF.sup.-.
The resulting plasmid, pMY150 (FIG. 1b) contains the entire coding
region of the ompC gene and approximately 500 bp of upstream
sequences including the ompC promoter and the DNA encoding the
5'-end untranslated region of ompC mRNA. In order to obtain an ompC
promoter fragment of different sizes, pMY150 was digested by Bal31
nuclease at the unique BglII site, followed by the addition of XbaI
linkers. The plasmid constructed in this manner carry XbaI
fragments that vary in size due to the position of the XbaI site
furthest from the SalI site (see FIG. 1b). The different XbaI
fragments were subsequently transferred to a promoter-cloning
vector, pKM005 which can express the lacZ gene only when a promoter
fragment is inserted in the right orientation into its unique XbaI
site. These experiments revealed that transcription of the ompC
gene initiates at a site located between 390 and 440 bp downstream
from the upstream XbaI site (originally HpaI site). Surprisingly,
E. coli transformed with these pKM005 derivatives, including the
clone of the shortest XbaI fragment of only 300 bp, CX28 (subcloned
from pCX28; FIG. 1a and b) lost the ability to produce OmpF
protein. OmpF protein was clearly produced in the host cells
(ompB.sup.+ ompF.sup.+ ompC.sup.+), while the same cells carrying
the clone of the CX28 fragment were not able to produce OmpF
protein. The same effect could be observed with cells harboring a
clone of a longer fragment such as plasmid I in FIG. 1c. In this
clone the lacZ gene was fused immediately after the initiation
codon of the ompC gene resulting in the LacZ.sup.+ phenotype of the
cells carrying this plasmid. However, when the XbaI-MspI fragment
of 87 bp was removed from plasmid I, the cells carrying the
resulting plasmid (plasmid II in FIG. 1c) were able to produce OmpF
protein. It should be mentioned that a similar DNA fragment of 430
bp in length containing the upstream region of the ompF gene did
not block the production of both OmpF and OmpC proteins.
DNA Sequence Homology Between CX28 and the ompF Gene
The results described above demonstrate that the stretch of DNA
approximately 300 bp long, located upstream of the ompC promoter,
is able to block ompF expression. In order to elucidate the
function of this DNA fragment (CX28), the DNA sequence of this
region was determined.
Reference is now made to FIG. 2 which shows the nucleotide sequence
of the promoter region and upstream of the ompC gene. Restriction
DNA fragments prepared from pMY111 or pMY150 were labeled at their
3'-end by the method of Sakano et al., Nature, 280, 288-294 (1979),
using [.alpha.-.sup.32 P]dNTP's and DNA polymerase I large fragment
(Klenow fragment). Singly end-labeled DNA fragment was obtained by
digestion with a second restriction enzyme. DNA sequence were
determined by the method of Maxam and Gilbert, Methods in
Enzymology 65, 499-560 (1981), using 20%, 10% and 6% polyacrylamide
gels in 7M urea. The RNA polymerase recognition site (-35 region)
and the Pribnow box (-10 region) for the ompC and micF promoter, as
well as the initiation codon of the ompC gene are boxed. The
transcriptional initiation sites are determined by S1 nuclease
mapping for the ompC and micF genes.
FIG. 2 shows the DNA sequence of 500 bp from the XbaI site
(originally HpaI) to the initiation codon, ATG, of the ompC gene.
The DNA sequence downstream of residue 88 was determined
previously. It was found that the sequence from residue 99 to 180
(FIG. 2) has 70% homology with the 5'-end region of the ompF mRNA
which includes the Shine-Dalgarno sequence, the initiation codon,
and the codons for the first nine amino acid residues of pro-OmpF
protein (bases marked by + are homologous to the ompF sequence). A
plausible model to explain the above result is that the 300-bp CX28
fragment (FIG. 1c) contains a transcription unit which is directed
towards the region upstream of the ompC gene so that the RNA
transcript from this region has a sequence complementary to the
ompF mRNA. The hybridization between the two RNAs thus blocks the
translation of ompF mRNA to OmpF protein.
Existence of a New Transcription Unit
To determine whether the CX28 fragment contained an independent
transcription unit oriented in a direction opposite from the ompC
gene, the lacZ gene was fused at two different sites within the
CX28 fragment. In plasmid V, the CX28 fragment was inserted in the
opposite orientation with respect to plasmid III (FIG. 1c). This
clone was still fully active in suppressing the production of OmpF
protein, although it did not produce .beta.-galactosidase
(LacZ.sup.-) (see FIG. 1c). When the fusion junction was shifted to
the MspI site at nucleotide 88 (FIG. 2, also see FIG. 1c), the
newly constructed clone (plasmid IV) was capable of producing
.beta.-galactosidase. However, this plasmid was no longer able to
suppress the production of OmpF protein. Although this plasmid
contains additional DNA (approximately 200 bp) upstream from the
lacZ and the CX28 sequences (from residue 300 to 500; FIG. 2), this
should not affect the functions of the CX28 fragment since plasmid
V is fully active in the suppression of OmpF protein production.
These results demonstrate that there is a transcription unit in the
CX28 fragment which is independent from the ompC gene promoter and
that the CX28 fragment and the ompC gene are transcribed in
divergent directions. The fact that plasmid IV can produce
.beta.-galactosidase and plasmid IV does not, indicates that the
CX28 transcription unit terminates between residue 1 and 88 (FIG.
1c). In fact, a very stable stem-and-loop structure can form
between nucleotides 70 and 92 (arrows with letter a in FIG. 2)
which is followed by oligo-[T]. This structure is characteristic of
.rho.-factor independent transcription termination sites in
prokaryotes. The .DELTA.G value for this structure was calculated
to be -12.5 Kcal according to Salser, W., Cold Spring Harbor Symp.
Quant. Biol. 12, 985-1002 (1977).
The initiation site for the CX28 transcript was positioned at
nucleotide 237 (FIG. 2) by S1-nuclease mapping. This result
indicates that the CX28 DNA fragment is transcribed to produce a
transcript of 174 nucleotides. This was further proven by Northern
blot hybridization. In the RNA preparation extracted from cells
carrying plasmid III (FIG. 1c), an RNA species is clearly observed
to hybridize with the CX28 fragment, which migrates a little slower
than 5S RNA. In the control cells, only a small amount of the same
RNA was detected. The size of the RNA (CX28 RNA) was estimated on
gel to be approximately 6S which is in very strong agreement with
the size estimated from the sequence (174 bases).
Function of the CX28 RNA
As pointed out earlier, the CX28 DNA fragment has extensive
homologies with a portion of the ompF gene. Thus, part of CX28 RNA
is complementary to the ompF mRNA and can form an extremely stable
hybrid with the ompF mRNA as shown in FIG. 3. The .DELTA.G value
for this hybrid formation was calculated to be -55.5 Kcal.
Forty-four bases of the 5'-end untranslated region of ompF mRNA,
including the Shine-Delgarno sequence for ribosome-binding and 28
bases from the coding region, are involved in the hybrid formation.
This hybrid structure is sandwiched by the two stable stem-and-loop
structures of the CX28 RNA; one for the 3'-end p-independent
transcription termination signal (loop a) and the other at the
5'-end (loop b). The .DELTA.G values for loops a and b were
calculated to be -12 5 and -4.5 Kcal, respectively.
Referring now to FIG. 3 of the drawings, there is illustrated
therein hybrid formation between micF and ompf mRNA. The sequence
of micF RNA corresponds to the sequence from residue 237 to 64 in
FIG. 2. The ompF mRNA sequence was cited from Inokuchi, K. et al.,
Nucleic Acids Res. 10, 6957-6968 (1982). The .DELTA.G values for
the secondary structures a, b and c were calculated to be -12.5,
-4.5 and +2.9 Kcal, respectively.
In FIG. 3 another loop (loop c) is shown. This loop, however, is
unlikely to be formed because of its .DELTA.G value (+2.9 Kcal). It
appears that the formation of the hybrid blocks the translation of
ompF mRNA. This would explain why clones carrying the CX28 DNA
fragment suppress the production of OmpF protein. Thus, CX28 RNA is
designated as the mRNA-interferring complementary RNA for ompF
(micRNA for ompF) and the gene is designated micF. It should be
noted that when loop a was eliminated by fusing the micF gene with
the lacZ gene, the MicF function was abolished (plasmid IV, FIG.
1c). This may be due to the stability of the micF RNA or
alternatively due to the requirement of loop a for the micF
function.
It seemed of interest to examine whether the micF gene is under the
control of the ompB locus as is the ompC gene. Various lacZ clones
were therefore put into four different ompB mutants Reference is
now made to Table I.
TABLE I
__________________________________________________________________________
.beta.-Galactosidase Activities of Various Promoter-lacZ Gene
Fusion Clones in ompB Mutant Strains .beta.-Galactosidase Activity
(U) Plasmids pKM004 Plasmid I Plasmid IV pOmpF.sup.p -Al Strains
(1pp.sup.p -lacZ) (ompC.sup.p -lacZ) (mic.sup.p -lacZ) (ompF.sup.p
-lacZ)
__________________________________________________________________________
Mc4100 (wild type) 1360 1808 796 2071 OmpC.sup.+ OmpF.sup.+ MH1160
(ompR1) 1415 102 133 43 OmpC.sup.- OmpF MH760 (ompR2) 1219 21 102
1521 OmpC.sup.- OmpF.sup.+ MH1461 (envZ) 905 1500 616 1063
OmpC.sup.+ OmpF.sup.-
__________________________________________________________________________
Various ompB mutant strains, MC4100 (F.sup.- lacV169 araD139 rspL
thiA tibB relA; wild type), MH1160 [ompB101 (ampR1) mutant from
MC4100]MH760 [ampB427 (ompR2) mutant from MC4100], MH1461 [tpoll
(envZ) mutant from MC4100] were transformed by various
promoter-lacZ gene fusion clones. Cells were grown in 10 ml of
nutrient broth at 37.degree. C. to Klett unit of 1.2. 100 ul of the
cultures were used for .beta.-galactosidase activity measurement
according to the method of Miller, H. J., in Experiments of
Molecular Genetics (ed. Miller, H. J.) 352-355 (Cold Spring Harbor
Laboratory, New York (1972)). Plasmid pK004 was derived from pKM005
and pKM004 contains the lpp (the gene for outer membrane
lipoprotein) promoter fused to the lacZ gene. Plasmid I and IV are
described in FIG. 1c. Plasmid pOmpF.sup.p -A1 contains the lacZ
gene under the control of the ompF promoter.
As shown in Table I, the lacZ gene under micF control (plasmid IV
in FIG. 1C) produces .beta.-galactosidase in the same manner as the
lacZ gene under ompC promoter control (plasmid I in FIG. 1C): high
.beta.-galactosidase activity was found in both the wild type and
envZ.sup.- strains but low activity was observed in ompR1.sup.- and
ompR2.sup.- mutants. On the other hand, the lacZ gene under the
control of the ompF promoter was not expressed in the ompR1.sup.-
cells. In addition, lacZ gene under the control of the lipoprotein
promoter, used as a control, was expressed in all strains. These
results indicate that the micF gene is regulated by the ompB locus
in the same fashion as the ompC gene. It is interesting to note
that the lacZ gene under the control of the ompF promoter is
constitutively expressed in the envZ.sup.- (Ompc.sup.+ GmpF.sup.-)
strain. This suggests that the OmpF.sup.- phenotype of this
envZ.sup.- strain is due to the inhibition of translation of the
ompF mRNA by micRNA.
Promoters of the micF and ompC Genes
Since both the micF and ompC genes appear to be regulated by the
ompB locus, the promoters of these genes should have sequence
homologies. In order to search for the homologies, the
transcription initiation site for the ompC gene was first
determined by S1-nuclease mapping. Major transcription initiation
takes place at the T residues at position 410 and 411 (FIG. 2; also
see FIG. 4).
In FIG. 4 the homologous sequences between the micF and the ompC
genes are shown. Nucleotide numbers correspond to those in FIG. 2.
The sequences in the box show the homologous sequences between the
two genes. Bars between the two sequences indicate the identical
bases. The arrows indicate the transcription initiation sites. The
-10 and -35 regions are underlined.
Thus, -10 regions for the micF and ompC genes are assigned as
AATAAT (nucleotides 250 to 245 in FIG. 2) and GAGAAT (nucleotides
400 to 405 in FIG. 2), respectively (FIG. 4), both of which show
good homology to the consensus sequence, TATAAT. RNA polymerase
recognition sites, (-35 regions), for the micF and ompC genes are
also assigned as TAAGCA and TTGGAT, respectively (FIG. 4), both of
which show 50% homology to the consensus sequence, TTGACA. However,
no significant sequence homologies are found between the micF
promoter of 63 bp (nucleotides 300 to 238) and the ompC promoter
(nucleotides 301 to 409 in FIG. 2). On the other hand, homologous
sequences are found in the 5'-end regions of both the transcripts
as shown in FIG. 4. Twenty-eight out of 44 bases are homologous
(64% homology), and these regions are probably the sites recognized
by OmpR protein. It is interesting to note that a homologous
sequence to these sequences has also been found in the 5 '-end
untranslated region of ompF mRNA. Binding experiments of the micF
gene and the ompC gene with purified OmpR protein are now in
progress.
As indicated hereinabove, regulation of gene expression in E. coli
is generally controlled at the level of transcription. It has been
well established that expression of some genes are suppressed by
their specific repressors or activated by their specific inducers.
Positive protein factors such as cAMP receptor protein and OmpR
protein are also known to regulate gene expression at the level of
transcription. Another transcriptional regulatory mechanism is
attenuation which plays an important role in controlling expression
of operations involved in the biosynthesis of various amino acids
of other compounds, see Kolter, R. & Yanofsky, C. Ann. Rev.
Genet. 16, 113-134 (1982).
In addition, some proteins have been shown to regulate gene
expression at the level of translation. The results herein
demonstrate the regulation of bacterial gene expression at the
level of translation by means of a complementary RNA factor to the
translational start region. This novel regulatory mechanism
mediated by micRNA is illustrated in FIG. 5.
FIG. 5 illustrates a possible model for the role of micF RNA. OmpR
protein binds to the ompF gene under the low osmolarity and
promotes the production of OmpF protein. Under the high osmolarity,
OmpR protein binds to both the micF and the ompC genes. The micF
RNA thus produced hybridizes with the ompF mRNA to arrest its
translation.
The possibility that micRNA blocks the expression of the ompF gene
at the level of transcription has not been ruled out. However, this
is highly unlikely since the lacZ gene fused with the ompF promoter
was expressed in the envZ.sup.- cells (OmpC.sup.+ OmpF.sup.- ;
Table 1. In this case lacZ expression is probably due to the
inability of lacZ mRNA transcribed from the clone to form a stable
hybrid with micRNA. Furthermore, if micRNA is able to bind the
nonsense strand of the ompF gene, it would more likely stimulate
gene expression rather than block it, since the binding would make
the ompF gene more accessible to RNA polymerase.
Regulation by micRNA appears to be an extremely efficient way to
block production of a specific protein without hampering other
protein production. At present, the relative ratio between micRNA
and ompC production is not known (.beta.-galactosidase activities
in Table I do not necessarily reflect their accurate promoter
activities, since the promoter regions were not inserted in the
same fashion, see FIG. 1c). However, it is reasonable to assume
that the micRNA and the ompC are produced coordinately. Therefore,
when OmpC protein is produced, micRNA is produced in the same
manner. micRNA then blocks the production of OmpF protein
proportionally, so that the total amount of OmpC plus OmpF protein
is constant.
The binding of micRNA to the ribosome-binding site and the
initiation codon is a very effective way to block the translation
of the particular mRNA. A similar mechanism has been proposed to
explain a translational block in a mutant of bacteriophage T7. It
was suggested that the sequence of the 3'-end of a mutant mRNA
hybridizes with its own ribosome-binding site to block translation,
see Saito, H. & Richardson, C. C., Cell, 27, 533-542 (1981). It
seems reasonable that the micRNA regulatory system may be a general
regulatory phenomenon in E. coli and in other organisms including
eukaryotes. It is a particularly attractive and rapid mechanism to
very rapidly stop the formation of a protein or to control the
ratio of one protein with another. RNA species may have additional
roles in the regulation of various cellular activities. In fact,
small RNA species have been shown to be involved in the regulation
of DNA replication of some plasmids.
Construction of an Artificial Mic Gene
The micF gene produces a 174-base RNA that blocks production of the
OmpF protein. This small RNA has two stem-and-loop structures, one
at the 3'-end and the other at the 5'-end. Since these structures
are considered to play an important role for the function of the
micRNA, it was attempted to use these features in the construction
of an artificial mic system using the gene for the major outer
membrane lipoprotein (lpp) cloned in an inducible expression
vector, pIN-II, see Nakamura et al., "Construction of Versatile
Expression Cloning Vehicles Using the Lipoprotein Gene of
Escherichia coli", EMBO J., 1,771-775 (1982).
pIN-II vectors are high expression vectors that have the lac.sup.po
downstream of the lipoprotein promoter, thus allowing high level
inducible expression of an inserted gene. The pIN-II promoter was
fused to the lpp gene at a unique XbaI site immediately upstream of
the Shine-Dalgarno sequence of the lpp mRNA. The resulting plasmid
was designated as pYM140. When the expression of the lpp gene, in
pYM140, is induced by isopropyl-.beta.-D-thiogalactoside (IPTG), a
lac inducer, the RNA transcript derived from the lpp gene has a
possible stem-and-loop structure (at the 5' end). Immediately
upstream of the unique XbaI site, see FIG. 6-A, is another stable
stem-and-loop structure at its 3' end. The latter loop is derived
from the .rho.-independent transcription termination signal of the
lpp gene. The construction of a general mic cloning vector, pJDC402
was achieved by removing the DNA fragment in pMH044 between the two
loops as shown in FIG. 6-A. An RsaI site immediately upstream of
the termination site was changed to an EcoRI site by partial
digestion of pYM140 followed by insertion of an EcoRI linker. The
resulting plasmid, pMHO44 was partially digested with EcoRI,
followed by a complete digestion with XbaI. The single stranded
portions of the linear DNA fragment were filled in with DNA
polymerase I (large fragment), and then treated with T4 DNA ligase,
resulting in the formation of the plasmid, pJDC402, which lost the
fragment between the XbaI and the RsaI sites.
As a result of this procedure, both an EcoRI and an XbaI site were
recreated at the junction. Thus the unique XBaI site can serve as
the insertion site for any DNA fragment, and the RNA transcript
from the artificial mic gene produces an RNA which has a similar
structure to the micF RNA; the portion derived from the inserted
DNA is sandwiched by two loop structures, one at the 5' and one at
the 3'-end.
The following is a more detailed description of FIG. 6-A and FIG.
6-B. As illustrated in FIG. 6-A for the construction of pJDC402,
restriction sites are indicated as follows: X, XbaI; P, PvuII; E,
EcoRI. lpp.sup.p and lac.sup.po are the lipoprotein promoter and
the lactose promoter operator, respectively. Amp.sup.r is the
Ampicillin resistance gene. Cross hatches represent the lipoprotein
promoter. Solid dots represent the lactose promoter operator.
Slashes indicate the lipoprotein signal sequence, and the solid bar
represents the coding region for the mature portion of the
lipoprotein. The open dots represent the transcription termination
region derived from the lpp gene. The open bar represents the 5'
nontranslated region of the lipoprotein mRNA.
In FIG. 6-B for the construction of mic (lpp) pJDC412, open arrows
represent promoters. The PvuII site was converted to an XbaI site
by inserting an XbaI linker (TCTAGAG). This fragment was inserted
into the unique XbaI site of pJDC402 in the reverse orientation
forming pJDC412. a and b show the mic(lpp) RNAs initiating at the
lpp and lac promoters, respectively.
Construction of the mic(lpp) Gene
Using the mic cloning vector pJDC402, it was first attempted to
create a mic system for the lpp gene of E. coli, in order to block
the synthesis of the lipoprotein upon induction of the mic(lpp)
gene. For this purpose it is necessary to first isolate the DNA
fragment containing the Shine-Dalgarno sequence for ribosome
binding, and the coding region for the first few amino acid
residues of prolipoprotein. To do this the PvuII site immediately
after the coding region of prolipoprotein signal peptide was
changed to an XbaI site by inserting an XbaI linker at this
position. The resulting plasmid was then digested with XbaI, and
the 112-bp XbaI-XbaI (originally PvuII-XbaI) fragment was purified.
This fragment encompassing the Shine-Dalgarno sequence and the
coding region for the first 29 amino residues from the amino
terminus of prolipoprotein was purified. This fragment was then
inserted into the unique XbaI site of pJDC402 in the opposite
orientation from the normal lpp gene. The resulting plasmid,
designated as pJDC412, is able to produce mic(lpp) RNA, an RNA
transcript complementary to the lpp mRNA, upon induction with
IPTG.
The inclusion of a HinfI site immediately upstream of the lpp
promoter and another one immediately downstream of the
transcription termination site in the mic expression vector pJDC402
is important. These two HinfI sites can be used to remove a DNA
fragment containing the entire mic transcription unit which can
then be inserted back into the unique pvuII site of the vector. In
this manner, the entire mic gene can be duplicated in a single
plasmid. One would expect a plasmid containing two identical mic
genes to produce twice as much micRNA as a plasmid containing a
single mic gene. Such a plasmid was constructed containing two
mic(lpp) genes and designated as pJDC422.
Expression of the mic(lpp) Gene
In order to examine the effect of the artificial mic(lpp) RNA,
cells were pulse-labeled for one minute, with [.sup.35
S]-methionine, one hour after induction of the mic(lpp) with 2 mM
IPTG. The cells harboring the vector, pJDC402, produce the same
amount of lipoprotein either in the absence or the presence of the
inducer, IPTG, as quantitated by densitometric scanning of the
autoradiogram and normalizing. Lipoprotein production was reduced
approximately two-fold in the case of cells carrying pJDC412 in the
absence of IPTG and approximately 16-fold in the presence of IPTG.
The reduction in lipoprotein synthesis in the absence of IPTG is
attributed to incomplete repression of the mic(lpp) gene. In the
case of cells carrying pJDC422, where the mic(lpp) gene was
duplicated, lipoprotein production is now reduced 4-fold in the
absence of IPTG, and 31-fold in the presence of IPTG. These results
clearly demonstrate that the production of the artificial mic(lpp)
RNA inhibits lipoprotein production, and that the inhibition is
proportional to the amount of the mic(lpp) RNA produced. It should
be noticed that the mic(lpp) RNA is specifically blocking the
production of lipoprotein, and that it does not block the
production of any other proteins except for OmpC protein. The fact
that the induction of the mic(lpp) gene reduces the production of
the OmpC plus OmpF proteins was found to be due to unusual homology
between the lpp and the ompC gene as discussed hereinafter.
There are several mechanisms by which the mic inhibition may occur.
One mechanism is that the micRNA binds to the mRNA preventing the
ribosome from binding the mRNA. Other possible mechanisms include:
destabilization of the mRNA, attenuation of the mRNA due to
premature termination of transcription, or inhibition of
transcription initiation. If the inhibitory effect of the micRNA is
solely at the level of attenuation or transcription initiation one
would expect the mic effect to be somewhat delayed due to the fact
that the functional half-life of the lipoprotein mRNA is 12
minutes. Therefore, it was examined how rapidly lipoprotein
production is inhibited upon induction of the mic(lpp) RNA by
pulse-labeling E. coli JA221/F'lacI.sup.q harboring pJDC412, with
[.sup.35 S]-methionine at various time points after induction with
IPTG. It was determined that lipoprotein production was maximally
inhibited by 16-fold within 5 minutes after the addition of IPTG.
This result indicates that inhibition of lipoprotein production is
primarily due to the binding of the mic(lpp) RNA to the lpp mRNA,
resulting in the inhibition of translation of the lpp mRNA and/or
destabilization of the mRNA.
lpp mRNA Production in the Presence of mic(lpp) RNA
It appeared interesting to examine whether the mic(lpp) RNA also
affects the level of the lpp mRNA, since the expression of the micF
gene substantially reduced the amount of the ompF mRNA. For this
purpose, total cellular RNA one hour after the induction of the
mic(lpp) gene with IPTG was isolated. The RNA preparation was
analyzed after electrophoresis in a formaldehyde agarose gel and
subsequently transferred onto nitrocellulose paper. The paper was
then hybridized with a probe specific to the mic(lpp) RNA, or to
the lpp mRNA. A probe specific for the ompA mRNA was used as an
internal control. Again pJDC402 shows no difference in the
production of the lpp mRNA in the absence or presence of IPTG. Due
to the fact that the double stranded primer used to make the probe
for these experiments contains a portion of the lac operon, the
probes hybridize to any transcript containing the lac promoter such
as the mic(lpp) RNA from JDC412 and the short nonsense transcript
from pJDC402.
Cells harboring pJDC412 contain a reduced amount of the lpp mRNA in
the absence of IPTG and a greatly reduced amount of the lpp mRNA in
the presence of IPTG. The production of the mic(lpp) RNA in the
absence and the presence of IPTG in cells harboring pJDC412 was
shown. Therefore, even in the absence of IPTG, a significant amount
of the mic(lpp) RNA is produced. This is consistent with the
results of the lipoprotein production observed earlier. The fact
that the lpp mRNA disappears upon induction of the mic(lpp) RNA
indicates that the mechanism of action of the micRNA is not solely
at the level of translation. Tests demonstrated there are two
mic(lpp) RNAs of different sizes. The sizes of these transcripts
were estimated to be 281 and 197 bases, which correspond to
transcripts initiating at the lipoprotein promoter (the larger RNA)
and initiating at the lac promoter (the smaller RNA).
Inhibition of OmpC Production with the mic(ompC) Gene
An almost complete inhibition of OmpC synthesis by artificially
constructing mic(ompC) genes was achieved. The first construct,
pAM320, carrying two mic(ompC) genes gives rise to an RNA molecule
complementary to 20 nucleotides of the leader region and 100
nucleotides of the coding region of the ompC mRNA. This was done by
changing the unique BglII site in the ompC structural gene and the
MnII site, 20 nucleotides upstream of the ATG initiation codon to
XbaI sites. The resulting 128-bp XbaI fragment was then inserted
into pJDC402 in the opposite orientation from the OmpC gene and a
second copy of the mic(ompC) gene was introduced in a manner
similar to that described for the pJDC422 construction. The
resulting plasmid, pAM320, has the second mic(ompC) gene inserted
in the orientation opposite to the first one. Reversing the
orientation of the second mic gene did not change the expression or
stability of the plasmid. A second construct, pAM321, was designed
to extend the complementarity between the micRNA and the ompC mRNA
to include a longer leader sequence than in the case of pAM320, 72
nucleotides of the leader region instead of 20. This plasmid was
constructed as described for pAM320, except that the MnlI site
changed to an XbaI site was located 72 nucleotides bp upstream of
the ompC initiation codon.
Commassie Brilliant Blue stained gel patterns of the outer membrane
proteins isolated from E. coli JA221/F'lacI.sup.q harboring the mic
cloning vector pJDC402, pAM320 and pAM321 were obtained. The effect
of the addition of IPTG was clearly seen by the appearance of
.beta.-galactosidase. The induction of the mic(ompC) RNA from
pAM320 caused a substantial decrease (approximately 5-fold) in OmpC
production, compared to pJDC402. Induction of the longer mic(ompC)
RNA from pAM321 decreased OmpC synthesis more dramatically
(approximately 20-fold compared to pJDC402).
OmpC production could hardly be detected in the cells harboring
pAM321 when they were pulse-labeled for one minute after a one-hour
induction with IPTG. In the same experiment, OmpC synthesis
decreased approximately 7-fold when the mic(ompC) gene in cells
harboring pAM320 was induced with IPTG. Marked decreases in OmpC
expression were also observed when plasmids containing single
copies of the mic(ompC) genes were induced. Again, the longer
mic(ompC) gene had a greater effect. The increased efficiency of
mic-mediated inhibition with pAM320 may indicate that the
effectiveness of the micRNA function is related to the extent of
complementarity to the 5'-end of the mRNA.
It was interesting to note that the synthesis of either of the
mic(ompC) RNAs described above caused a decrease not only in OmpC
synthesis but also in lipoprotein synthesis. This inhibitory effect
of the mic(ompC) RNA on lipoprotein production appears to be due to
the unexpected homology between the lpp mRNA sequence and the ompC
mRNA as illustrated in FIG. 2. This feature explains why pAM320 and
pAM321 are exerting a mic effect on lipoprotein production. Such an
explanation would predict that induction of the mic(lpp) RNA from
pJDC412 and pJDC422 should decrease the synthesis of the OmpC
protein, and this was found to be the case.
In FIG. 7, a region of homology between the lpp mRNA (top line) and
the ompC mRNA (bottom line) is illustrated. Bars connect identical
bases. Both mic(ompC) RNAs have the potential to hybridize across
this homologous region. The Shine-Dalgarno Sequences (S.D.) and AUG
initiation codons are boxed.
Inhibition of OmpA Production with mic(ompA) RNA
In an effort to determine what components contribute to the
effectiveness of a micRNA, several mic genes were constructed from
the ompA gene. The ompA gene was selected for this because the
leader and the coding regions of the ompA mRNA have been
characterized extensively. Five DNA fragments (see I through V of
FIG. 8) were individually cloned into the XbaI site of pJDC402 in
the orientation promoting the production of mic(ompA) RNAs. The
resulting mic(ompA) plasmids containing fragments I-V were
designated as pAM301, pAM307, pAM313, pAM314, and pAM318,
respectively. Each plasmid contains only one copy of the described
mic(ompA) gene.
In FIG. 8, the top line shows the structure of the E. coli ompA
gene. The arrow represents the promoter and the open bar represents
the region encoding the 5'-leader region of the ompA mRNA. The
slashed bar and shaded bar represent the portions of the ompA gene
encoding the signal sequence and the mature OmpA protein,
respectively. Restriction fragment I (HphI-HpaI) was inserted into
the XbaI site of pJDC402, see FIG. 6-A, in the orientation opposite
from that depicted here, as outlined in FIG. 6-B for mic(lpp), to
create the plasmid, pAM301. The other mic(ompA) plasmids were
similarly constructed from: fragment II, pAM307; fragment III,
pAM313; fragment IV, pAM314; fragment V, pAM318. The positions of
the Shine-Dalgarno sequence (SD), ATG initiation codon (ATG), and
relevant restriction sites are shown.
E. coli JA221/F'lacI.sup.q containing each of the mic(ompA)
plasmids was pulse-labeled with [.sup.35 S]-methionine for one
minute with and without a one-hour prior preincubation with IPTG.
Electrophoretic patterns of the outer membrane proteins isolated
from these cultures were obtained. The autoradiographs revealed
that each of the five mic(ompA) genes is capable of inhibiting OmpA
synthesis. The mic(ompA) genes appear to be less effective than the
mic(lpp) and mic(ompC) genes described earlier. However, this
problem was circumvented by increasing the mic(ompA) gene
dosage.
The plasmid pAM301, encoding an mRNA complementary to a 258 base
region of the ompA mRNA encompassing the translation initiation
site (fragment I in FIG. 3) was found to inhibit OmpA synthesis by
approximately 45 percent. A similar inhibition was obtained with
pAM307, by approximately 51 percent. This plasmid contains fragment
II (see FIG. 3) which does not include any DNA sequences
corresponding to the ompA structural gene. The inhibition by pAM307
was not surprising because the mic(ompC) experiments described
earlier showed that increased complementarity to the 5'-leader
region of the mRNA was more effective in micRNA-mediated
inhibition. On the other hand, pAM313, which produces a micRNA that
is complementary only to the portion of the ompA structural gene
covered by fragment III (See FIG. 8) which spans the coding region
for amino acid residues 4 through 45 of pro-OmpA, was also
effectively able to inhibit OmpA synthesis by approximately 54
percent, indicating that the micRNA does not need to hybridize to
the initiation site for protein synthesis and/or to the 5'-leader
region of the target mRNA in order to function. This was also
confirmed using mic(lpp) genes. Two mic(lpp) RNAs which were
complementary to only the coding region of the lpp mRNA have also
been found to inhibit lipoprotein production. The effect of the
mic(lpp) genes in pJDC413 and pJDC414 which were constructed from
the lpp structural gene fragments coding for amino acid residues 3
to 29, and 43 to 63 of prolipoprotein, respectively, were observed.
Both pJDC413 and pJDC414, however, exhibit only a 2-fold inhibition
of lipoprotein synthesis indicating that a DNA fragment covering
the translation initiation site (which caused a 16-fold inhibition)
is more effective in the case of the mic(lpp) genes. Fragment IV
(see FIG. 8) was chosen to test the effectiveness of a micRNA
complementary only to the 5' leader region of the ompA mRNA. The
resulting construct pAM314, synthesizes a micRNA complementary to a
68-base stretch of the ompA mRNA leader located 60 bases upstream
of the AUG initiation codon. pAM314 exhibits a very weak mic
effect, inhibiting OmpA synthesis by only about 18 percent. The
significant differences in the mic effect between fragments II and
IV (see FIG. 8) clearly demonstrates that the complementary
interaction within the region of the mRNA that interacts with the
ribosome i.e., the Shine-Dalgarno sequence and/or the coding
region, is very important for the effective mic function, although
it is not absolutely required. It is also interesting to note that
shortening the mic(ompA) gene from fragment I to V had little
effect on its efficiency, a 45 percent compared to a 48 percent
decrease, respectively.
In order to construct a plasmid capable of inhibiting OmpA
synthesis more effectively than those discussed above, plasmids
were constructed containing more than one mic(ompA) gene. The
plasmid, pAM307 and its derivatives pAM319 and pAM315 were
compared. The latter two plasmids contain two and three copies of
the mic(ompA) gene in pAM307, respectively. While pAM307 inhibited
OmpA synthesis by approximately 47 percent, pAM315 and pAM319
inhibited OmpA synthesis by 69 percent and 73 percent,
respectively.
The results presented hereinabove clearly demonstrate that the
artificial mic system and techniques of this invention can be used
for specifically regulating the expression of a gene of interest.
In particular, the inducible mic system for a specific gene is a
novel and very effective way to study the function of a gene. If
the gene is essential, conditional lethality may be achieved upon
the induction of the mic system, somewhat similar to a
temperature-sensitive mutation. It should be noted, however, that
the mic system blocks the synthesis of the specific protein itself
while temperature sensitive mutations block only the function of
the protein without blocking its synthesis.
From this invention, the following has become evident:
(a) The production of an RNA transcript (micRNA) that is
complementary to a specific mRNA inhibits the expression of that
mRNA.
(b) The production of a micRNA specifically blocks the expression
of only those genes which share complementarity to the micRNA.
(c) The induction of micRNA production blocks the expression of the
specific gene very rapidly in less than the half-life of the
mRNA.
(d) The micRNA also reduces the amount of the specific mRNA in the
cell, as was found when the natural micF gene was expressed, as
well as when the artificially constructed mic(lpp) gene was
expressed in the present invention.
(e) There is a clear effect of gene dosage; the more a micRNA is
produced, the more effectively the expression of the target gene is
blocked.
In the practices of this invention, it appears that regions of the
micRNAs that are complementary to regions of the mRNA known to
interact with ribosomes are the most effective. Using the lpp gene
as an example, it appears that a mic(lpp) RNA that can hybridize to
the Shine-Dalgarno sequence and the translation-initiation site of
the lpp mRNA inhibits lipoprotein synthesis more efficiently than
one which cannot. However, for the ompA gene, micRNAs complementary
to both the Shine-Dalgarno sequence and the translation-initiation
site, just the Shine-Dalgarno sequence, or the structural gene
alone were equally effective.
For some genes, such as ompC and lpp, the region of the gene
encompassing the translation-initiation site may not contain a
unique sequence, and micRNA induction results in the inhibition of
the production of more than one protein. In these cases, another
region of the gene may be used to construct the mic gene. The
length of the micRNA is another variable to be considered. The
longer mic(ompC) RNA was 4-fold more effective at inhibiting OmpC
production than the shorter mic(ompC) RNA. It should be noted that
the inhibition of lipoprotein expression by the mic(ompC) RNA was
less effective with the longer mic(ompC) RNA, in spite of the fact
that the region of the two mic(ompC) RNAs complementary to the
lipoprotein mRNA is the same. This indicates that higher
specificity may be achieved by using longer micRNAs. In contrast to
the mic(ompC) genes, length did not appear to be a significant
factor for the mic(ompA) RNA-mediated inhibition of OmpA
production. In addition, the secondary structure of the micRNA most
likely plays an important role in micRNA function.
There are several mechanisms by which the micRNA may function to
inhibit expression of the specific gene. It is most likely that the
micRNA primarily acts by binding to the mRNA, thereby preventing
the interaction with ribosomes as proposed earlier. This hypothesis
is supported by the fact that the mic(lpp) RNA inhibited
lipoprotein production much faster than the time expected if only
transcription was affected based on the half-life of the lpp mRNA.
Concerning how micRNA causes a reduction in the amount of
lipoprotein mRNA, a plausible model to explain this reduction is
that the mRNA is less stable when ribosomes are not traversing the
entire mRNA.
Another possible model to explain this reduction in mRNA level is
that complementary hybrid formation between the micRNA and the mRNA
causes premature termination of transcription or destabilization of
the mRNA. Alternatively, the micRNA may directly inhibit the
initiation of transcription, or cause pausing of mRNA elongation in
a manner similar to that described for the function of a small
complementary RNA species in ColEl replication, see Tomizawa et
al., "The importance of RNA secondary structure in ColEl primer
formation." Cell, 31, 575-583 (1982).
In accordance with the practices of this invention the accompanying
disclosure presents a powerful tool and technique for regulating
gene expression. Gene expression in accordance with the practices
of this invention is regulated by incorporating foreign DNA that
associates with the genetic material of an organism (i.e.
transformation). The organism may possess only its native genetic
material or may have been genetically altered by the deletion of
native genetic material or the addition of foreign genetic
material. Upon transcription of the DNA of said organism, an
oligoribonucleotide or polyribonucleotide RNA is produced. This
mRNA is complementary to and/or capable of hybridizing with an mRNA
produced by the DNA of the organism so that expression or
translation of said mRNA is inhibited or prevented.
Gene expression regulation of an organism in accordance with the
practices of this invention is carried out in a transformed
organism. Along with the genetic material of said organism there is
incorporated non-native DNA which is transcribed along with the DNA
of the said organism. Through transcription, the non-native DNA
produces mRNA that is complementary to and capable of hybridizing
to the mRNA that is produced from the native DNA. Hybridization,
thus, inhibits or prevents translation of the mRNA into
protein.
In the practices of this invention, the non-native DNA that is
transcribed along with the native DNA into mRNA that is
complementary to the mRNA produced by the native DNA may be
incorporated into the native DNA directly or indirectly. Direct
incorporation of the DNA necessitates inserting the DNA directly
into the nucleus that contains the organism's DNA. This may be
accomplished through microinjection. Indirect incorporation is done
through incorporating the non-native DNA into a plasmid or viral
vector and then transforming the said organism with the plasmid or
viral vector. The plasmid or viral vector may be inserted into the
organism through the membrane thereof into the cytoplasm and travel
to the nucleus and associate with the DNA that characterizes the
organism. Where desired, convenient, or practical, microinjection
may be employed to insert the DNA or the plasmid or viral vector
containing the DNA insert into the organism into the nucleus or
cytoplasm of the organism. It is usually convenient to transform
the organism with the DNA or the plasmid or viral vector containing
the DNA insert through the membrane that encompasses the organism
by known methods, such as, electroporation, coprecipitation or
microinjection.
The practices of this invention are generally applicable with
respect to any organism containing genetic material which is
capable of being expressed. Suitable organisms include the
prokaryotic and eukaryotic organisms, such as bacteria, yeast and
other cellular organisms. The practices of this invention are also
applicable to viruses, particularly where the viruses are
incorporated in the organisms.
In its application, the mic system of this invention has great
potential to block the expression of various toxic or harmful genes
permanently or upon induction. These genes include drug resistance
genes, oncogenes, and phage or virus genes among others.
In the development and demonstration of the practices of this
invention as described herein, the following materials and
procedures were employed.
Strain and Medium
E. coli JA221 (hsdr leuB6 lacY thi recA trpE5)F'(lacI.sup.q proAB
lacZYA) was used in all experiments. This strain was grown in M9
medium (J. H. Miller, Experiments in Molecular Genetics. Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1972))
supplemented with 0.4 percent glucose, 2 .mu.g/ml thiamine, 40
.mu.g/ml each of leucine and tryptophan, and 50 .mu.g/ml
ampicillin, unless otherwise indicated.
Materials
Restriction enzymes were purchased from either Bethesda Research
Laboratories or New England BioLabs. T4 DNA ligase and E. coli DNA
polymerase I(large fragment) were purchased from Bethesda Research
Laboratories. All enzymes were used in accordance with the
instructions provided by the manufacturer. XbaI linkers (CTCTAGAG)
were purchased from New England BioLabs.
DNA Manipulation
Plasmids pJDC402, pJDC412, and pJDC422 were constructed as
described herein and FIG. 1. Plasmids pJDC413 and pJDC414 were
constructed by isolating the 80-bp AluI fragment from the lpp gene
encoding amino acid residues 3 through 29 of prolipoprotein for
pJDC413 and the 58-bp AluI fragment encoding amino acid residues 43
through 63 of prolipoprotein for pJDC414. The fragments were blunt
end ligated into pJDC402 which was first digested with XbaI
followed by treatment with DNA polymerase I (large fragment).
The isolation of the appropriate ompC fragments for mic(ompC)
construction involved a subcloning step due to the absence of
suitable unique restriction sites between the ompC promoter and
structural gene. Two derivatives of the ompC containing plasmid,
pMY150, lacking either the 471-bp XbaI-MnI ompC promoter containing
fragment (pDR001 and pDR002, respectively), but containing an XbaI
site in its place, were isolated. The unique BglII sites in each of
these plasmids were changed to XbaI sites by treatment with DNA
polymerase I (large fragment) and ligation with synthetic XbaI
linkers. Following XbaI digestion, the 123-bp XbaI fragment from
pDR001 and the 175-bp XbaI fragment from pDR002 were individually
isolated and cloned into the XbaI site of pJDC402 to create pAM308
and pAM309, respectively. pAM320 contains the HinfI fragment
covering the mic(ompC) gene isolated from pAM308 cloned into the
PvuII site of pAM308. pAM321 was similarly constructed from pAM309
to also contain two mic(ompC) genes.
The mic(ompA) plasmids pAM301, pAM307, pAM313, pAM314, and pAM318
were constructed as described in a manner similar to the
construction of the mic(lpp) and the mic(ompC) genes. To construct
pAM319, the HinfI fragment containing the mic(ompA) gene was
isolated from pAM307 and inserted back into the PvuII site of
pAM307. pAM315 was constructed in the same manner as pAM319 except
that it contains two HinfI fragments inserted into the PvuII site
of pAM307.
Analysis of outer membrane protein production
E. coli JA221/F'lacI.sup.q carrying the appropriate plasmid were
grown to a Klett-Summerson colorimeter reading of 30, at which time
IPTG was added to a final concentration of 2 mM. After one
additional hour of growth (approximately one doubling), 50 uCi of
[.sup.35 S]-Methionine (Amersham, 1000 Ci/mMole) was added to 1 ml
of the culture. The mixture was then incubated with shaking for one
minute, at which time the labeling was terminated by addition of 1
ml ice cold stop solution (20 mM sodium phosphate [pH 7.1],
containing 1 percent formaldehyde, and 1 mg/ml methionine). Cells
were washed once with 10 mM sodium phosphate [pH 7.1], suspended in
1 ml of the same buffer, and sonicated with a Heat Systems
Ultrasonics sonicator model W-220E with a cup horn adapter for 3
minutes (in 30 second pulses). Unbroken cells were removed by low
speed centrifugation prior to collecting the outer membrane.
Cytoplasmic membranes were solubilized during a 30 minute
incubation at room temperature in the presence of 0.5 percent
sodium lauroyl sarcosinate and the outer membrane fraction was
precipitated by centrifugation at 105,000.times.g for 2 hours.
Lipoprotein and OmpA were analyzed by Tris-SDS polyacrylamide gel
electrophoresis (SDS-PAGE). To analyze OmpC production, urea-SDS
polyacrylamide gel electrophoresis (urea-SDS-PAGE) was used.
Proteins were dissolved in the sample buffer and the solution was
incubated in a boiling water bath for 8 minutes prior to gel
application. The autoradiographs of dried gels were directly
scanned by a Shimadzu densitometer. To determine relative amounts
of the band of interest, the ratio of the area of the peak of
interest to the area of an unaffected protein peak, was determined
for each sample.
RNA Analysis
Cells were grown and labeled with [3H]-uridine, then cell growth
was stopped by rapidly chilling the culture on ice for less than 5
minutes. The cells were collected by centrifugation at 8000 rpm for
5 minutes. RNA was isolated using the following procedure. The
cells were quickly resuspended in hot lysis solution (10 mM
Tris-HCl [pH 8.0], 1 mM EDTA, 350 mM NaCl, 2 percent SDS and 7M
urea) with vigorous vortexing for 1 minute. The mixture was
immediately extracted, twice with phenol:chloroform (1:1) and twice
with chloroform alone. One tenth volume of 3M sodium acetate (pH
5.2) was added to the mixture and 3 volumes of ethanol was added to
precipitate the RNA. The precipitate was then dissolved in TE
buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA). For gel
electrophoresis, equal counts were loaded in each lane. The RNA was
separated on a 1.5 percent agarose gel containing 6 percent
formaldehyde. The running buffer was 20 mM MOPS
(3-[N-morpholino]propanesulfonic acid [Sigma]), 5 mM sodium acetate
and 1 mM EDTA, pH 7.0.
RNA was transferred to nitrocellulose paper. M13 hybridization
probes specific for the mic(lpp) RNA and lpp mRNA were individually
constructed by cloning the 112-bp XbaI fragment shown in FIG. 1-B
into M13 mp9 in the appropriate orientation. A probe specific for
the ompA mRNA was constructed by inserting a 1245-bp XbaI-EcoRI
fragment (originally an EcoRV-PSTI fragment) into M13 mp10 and the
probes were labeled.
As will be apparent to those skilled in the art in the light of the
foregoing disclosure, many modifications, alterations and
substitutions are possible in the practices of this invention
without departing from the spirit or scope thereof.
* * * * *