U.S. patent application number 11/180928 was filed with the patent office on 2005-11-10 for control of gene expression.
This patent application is currently assigned to Benitec Australia Limited. Invention is credited to Graham, Michael Wayne, Rice, Robert Norman.
Application Number | 20050250208 11/180928 |
Document ID | / |
Family ID | 3806754 |
Filed Date | 2005-11-10 |
United States Patent
Application |
20050250208 |
Kind Code |
A1 |
Graham, Michael Wayne ; et
al. |
November 10, 2005 |
Control of gene expression
Abstract
The present invention relates generally to synthetic genes for
modifying endogenous gene expression in a cell, tissue or organ of
a transgenic organism, in particular a transgenic animal or plant.
More particularly, the present invention provides novel synthetic
genes and generic constructs which are capable of repressing
delaying or otherwise reducing the expression of an endogenous gene
or a target gene in an organism when introduced thereto.
Inventors: |
Graham, Michael Wayne;
(Jindalee, AU) ; Rice, Robert Norman; (Sinnamon
Park, AU) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
|
Assignee: |
Benitec Australia Limited
Commonwealth Scientific Industrial Research Org
|
Family ID: |
3806754 |
Appl. No.: |
11/180928 |
Filed: |
July 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11180928 |
Jul 13, 2005 |
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10759841 |
Jan 15, 2004 |
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10759841 |
Jan 15, 2004 |
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10346853 |
Jan 17, 2003 |
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10346853 |
Jan 17, 2003 |
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09100812 |
Jun 19, 1998 |
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6573099 |
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Current U.S.
Class: |
435/468 ;
536/23.1 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2840/20 20130101; C12N 15/8218 20130101; C12N 15/8216
20130101; C12N 2830/38 20130101; C12N 2840/102 20130101; C12N
2800/108 20130101; C12N 2830/55 20130101; C12N 9/503 20130101; C12N
15/85 20130101; C12N 2830/00 20130101; C12N 2830/002 20130101; C12N
15/69 20130101; C12N 2830/42 20130101; C12N 9/0071 20130101; C12N
9/1051 20130101; C12N 15/63 20130101; C12N 9/127 20130101; C12N
2830/60 20130101; C12N 15/8283 20130101; C12N 2830/15 20130101 |
Class at
Publication: |
435/468 ;
536/023.1 |
International
Class: |
A01H 001/00; C12N
015/82; C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 20, 1998 |
AU |
PP 2492 |
Claims
1-33. (canceled)
34. An isolated nucleic acid molecule for use in repressing,
delaying or otherwise reducing the expression of a target gene in a
non-human eukaryotic cell, tissue or organ, the isolated nucleic
acid molecule comprising multiple copies of a nucleotide sequence
which is at least 95% identical to a target gene or a region of
said target gene or complementary thereto, wherein at least two
copies of said multiple copies are in different orientations
relative to each other and wherein the nucleotide sequence
comprises at least 20 nucleotides.
35. The isolated nucleic acid molecule according to claim 34
wherein the isolated nucleic acid molecule is non-endogenous to
said non-human eukaryotic cell.
36. The isolated nucleic acid molecule according to claim 34
wherein the target gene is non-endogenous to said non-human
eukaryotic cell.
37. The isolated nucleic acid molecule according to claim 36
wherein the target gene is a viral gene.
38. The isolated nucleic acid molecule according to claim 34 which
comprises RNA.
39. The isolated nucleic acid molecule according to claim 34 which
comprises DNA.
40. The isolated nucleic acid molecule according to claim 34
wherein the two copies are separated by a sequence of
nucleotides.
41. The isolated nucleic acid molecule according to claim 40
wherein the sequence of nucleotides separating said two copies of
said multiple copies is an intron.
42. The isolated nucleic acid molecule according to claim 34
wherein the non-human eukaryotic cell is a mammalian cell.
43. The isolated nucleic acid molecule according to claim 34
wherein the non-human eukaryotic cell is an insect cell.
44. The isolated nucleic acid molecule according to claim 34
wherein the non-human eukaryotic cell is a aquatic animal cell.
45. A genetic construct comprising a promoter that functions in a
non-human eukaryotic cell which is operably linked to the isolated
nucleic acid molecule of claim 34 or a nucleotide sequence encoding
the isolated nucleic acid molecule.
46. A non-human eukaryotic cell comprising the isolated nucleic
acid molecule according to claim 45 or the genetic construct of
claim 45.
47. The cell of claim 46 which is a mammalian cell.
48. The cell of claim 46 which is an insect cell.
49. The cell of claim 46 which is an aquatic animal cell.
50. A composition comprising the isolated nucleic acid molecule
according to claim 34 or the genetic construct of claim 45 and a
veterinarially acceptable carrier.
51. A method of repressing, delaying or otherwise reducing the
expression of a target gene in a non-human eukaryotic cell, tissue
or organ, comprising introducing the isolated nucleic acid molecule
according to claim 34 or the genetic construct according to claim
45 into said non-human eukaryotic cell, tissue or organ.
52. The method of claim 51 wherein the non-human eukaryotic cell,
tissue or organ is from a mammal.
53. The method of claim 51 wherein the non-human eukaryotic cell,
tissue or organ is from an insect.
54. The method of claim 51 wherein the non-human eukaryotic cell,
tissue or organ is from an aquatic animal.
55. A method of modulating the expression of a target gene in a
non-human eukaryotic cell, tissue or organ, comprising the steps of
selecting an isolated nucleic acid molecule according to claim 34,
and introducing said isolated nucleic acid molecule into said
non-human eukaryotic cell, tissue or organ for a time and under
conditions sufficient for the mRNA product of said target to be
modified.
56. The method of claim 55 wherein the non-human eukaryotic cell,
tissue or organ is from a mammal.
57. The method of claim 55 wherein the non-human eukaryotic cell,
tissue or organ is from an insect.
58. The method of claim 55 wherein the non-human eukaryotic cell,
tissue or organ is from an aquatic animal.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to synthetic genes
for modifying endogenous gene expression in a cell, tissue or organ
of a transgenic organism, in particular a transgenic animal or
plant. More particularly, the present invention provides novel
synthetic genes and genetic constructs which are capable of
repressing delaying or otherwise reducing the expression of an
endogenous gene or a target gene in an organism when introduced
thereto.
BACKGROUND TO THE INVENTION
[0002] Controlling metabolic pathways in eukaryotic organisms is
desirable for the purposes of producing novel traits therein or
introducing novel traits into a particular cell, tissue or organ of
said organism. Whilst recombinant DNA technology has provided
significant progress in an understanding of the mechanisms
regulating eukaryotic gene expression, much less progress has been
made in the actual manipulation of gene expression to produce novel
traits. Moreover, there are only limited means by which human
intervention may lead to a repression, delay or reduction in
eukaryotic gene expression.
[0003] Current methods for down-regulating gene expression using
recombinant DNA technology comprise the introduction of a transgene
to the cell which is capable of repressing expression of an
endogenous target gene, either transcriptionally or
post-transcriptionally. However, the precise mechanism is not
known. Moreover, the efficiency of current approaches is low and
the results are variable and unpredictable.
[0004] Attempts to improve the accuracy and predictability of
methods for regulating gene expression in cells, in particular the
repression, delay or reduction in expression of viral target genes
in eukaryotic cells, foreign transgenes or other foreign genes
introduced into cells, tissues or organs by natural means, or
endogenous genes which are expressed to produce undesirable traits
for a particular purpose, have been largely unsuccessful possibly
due to a lack of knowledge of the precise mechanisms involved. As a
consequence, the efficiency of methods currently available remains
low and highly variable.
[0005] In work leading up to the present invention, the inventors
sought elucidate the mechanisms involved in down-regulating gene
expression in an attempt to provide improved methods therefor. In
so doing the inventors have developed a wide range of synthetic
genes capable of modulating gene expression in both prokaryotic and
eukaryotic cells and genetic constructs comprising same.
[0006] Bibliographic details of the publications referred to by
author in this specification are collected at the end of the
description. Sequence identity numbers (SEQ ID NOs.) for the
nucleotide and amino acid sequences referred to in the
specification are defined after the bibliography.
[0007] Throughout this specification and the claims that follow,
unless the context requires otherwise, the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element or integer or group of
elements or integers but not the exclusion of any other element or
integer or group of elements or integers.
[0008] As used herein, the term "derived from" shall be taken to
indicate that a particular integer or group of integers has
originated from the species specified, but has not necessarily been
obtained directly from the specified source.
SUMMARY OF THE INVENTION
[0009] The, present invention provides novel synthetic genes and
improved genetic constructs comprising same for modifying
endogenous or target gene expression in cells, tissues and/or
organs which are either transfected or stably transformed
therewith.
[0010] Accordingly, one aspect of the present invention provides a
synthetic gene which is capable of modifying target gene expression
in a cell, tissue or organ of a prokaryotic or eukaryotic organism
which is transfected or transformed therewith, wherein said
synthetic gene at least comprises a structural gene sequence
comprising a nucleotide sequence which is substantially identical
to the nucleotide sequence of said target gene or a derivative
thereof or a complementary sequence thereto placed operably under
the control of a promoter sequence which is operable in said cell,
tissue or organ.
[0011] A further aspect of the invention provides a synthetic gene
which is capable of modifying the expression of a target gene in a
cell, tissue or organ of a prokaryotic or eukaryotic organism which
is transfected or transformed therewith, wherein said synthetic
gene at least comprises multiple structural gene sequences, wherein
each of said structural gene sequences comprises a nucleotide
sequence which is substantially identical to the nucleotide
sequence of said target gene or a derivative thereof or a
complementary sequence thereto and wherein said multiple structural
gene sequences arc placed operably under the control of a single
promoter sequence which is operable in said cell, tissue or
organ.
[0012] A third aspect of the present invention provides a synthetic
gene which is capable of modifying the expression of a target gene
in a cell, tissue or organ of a prokaryote or eukaryote which is
transfected or transformed therewith wherein said synthetic gene at
least comprises multiple structural gene sequences wherein each of
said structural gene sequences is placed operably under the control
of a promoter sequence which is operable in said cell, tissue or
organ and *herein each of said structural gene sequences comprises
a nucleotide sequence which is substantially identical to the
nucleotide sequence of said target gene or a derivative thereof or
a complementary sequence thereto.
[0013] A further aspect of the present invention provides a genetic
construct which is capable of modifying the expression of an
endogenous gene or target gene in a transformed or transfected
cell, tissue or organ wherein said genetic construct at least
comprises the synthetic gene of the invention and one or more
origins of replication and/or selectable marker gene sequences.
[0014] A still further aspect of the invention provides a cell,
tissue, organ or organism comprising the synthetic genes and
genetic constructs described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a copy of a diagrammatic representation of the
plasmid pEGFP-N1 MCS.
[0016] FIG. 2 is a copy of a diagrammatic representation of the
plasmid pCMV.cass.
[0017] FIG. 3 is a copy of a diagrammatic representation of the
plasmid pCR.Bgl-GFP-Bam.
[0018] FIG. 4 is a copy of a diagrammatic representation of the
plasmid pCR.SV40L.
[0019] FIG. 5 is a copy of a diagrammatic representation of the
plasmid pCMV.SV40L.cass.
[0020] FIG. 6 is a copy of a diagrammatic representation of the
plasmid pCR.BEV.1.
[0021] FIG. 7 is a copy of a diagrammatic representation of the
plasmid pCR.BEV.2.
[0022] FIG. 8 is a copy of a diagrammatic representation of the
plasmid pCR.BEV.3.
[0023] FIG. 9 is a copy of a diagrammatic representation of the
plasmid pEGFP.BEV.1.
[0024] FIG. 10 is a copy of a diagrammatic representation of the
plasmid pCMV.BEV.2.
[0025] FIG. 11 is a copy of a diagrammatic representation of the
plasmid pCMV.VEB.
[0026] FIG. 12 is a copy of a diagrammatic representation of the
plasmid pCMV.BEVnt.
[0027] FIG. 13 is a copy of a diagrammatic representation of the
plasmid pCMV.BEVx2.
[0028] FIG. 14 is a copy of a diagrammatic representation of the
plasmid pCMV.BEV.VEB.
[0029] FIG. 15 is a copy of a diagrammatic representation of the
plasmid pCMV.BEV.GFP.VEB.
[0030] FIG. 16 is a copy of a diagrammatic representation of the
plasmid pCMV.BEV.SV40L-0.
[0031] FIG. 17 is a copy of a diagrammatic representation of the
plasmid pCMV.0.SV40L.BEV.
[0032] FIG. 18 is a copy of a diagrammatic representation of the
plasmid pCMV.0.SV40L.VEB.
[0033] FIG. 19 is a copy of a diagrammatic representation of the
plasmid pCMV.BEV.SV40L.BEV.
[0034] FIG. 20 is a copy of a diagrammatic representation of the
plasmid pCMV.BEV.SV40L.VEB.
[0035] FIG. 21 is a copy of a diagrammatic representation of the
plasmid pCMV.SV40LR.cass.
[0036] FIG. 22 is a copy of a diagrammatic representation of the
plasmid pCMV.BEV.SV40LR.
[0037] FIG. 23 is a copy of a diagrammatic representation of the
plasmid pCMV.TYR.
[0038] FIG. 24 is a copy of a diagrammatic representation of t
plasmid pCMV.TYR.
[0039] FIG. 25 is a copy of a diagrammatic representation of the
plasmid pCMV.Lac.
[0040] FIG. 26 is a copy of a diagrammatic representation of the
plasmid pCMVLacI.OPRSV1.cass.
[0041] FIG. 27 is a copy of a diagrammatic representation of the
plasmid pCMVLacI.OPRSV1.GFP.cass.
[0042] FIG. 28 is a copy of a diagrammatic representation of the
plasmid pCMVLacI.TYR.OPRSV1.GFP.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0043] One aspect of the present invention provides a synthetic
gene which is capable of modifying the expression of a target gene
in a cell, tissue or organ wherein said synthetic gene at least
comprises a structural gene comprising a nucleotide sequence which
is substantially identical to the nucleotide sequence of said
target gene or a derivative thereof or a complementary sequence
thereto placed operably under the control of a promoter which is
operable in said cell, tissue or organ.
[0044] Reference herein to a "gene" is to be taken in its broadest
context and includes:
[0045] (i) a classical genomic gene consisting of transcriptional
and/or translational regulatory sequences and/or a coding region
and/or non-translated sequences (i.e. introns, 5'- and
3'-untranslated sequences);
[0046] (ii) mRNA or cDNA corresponding to the coding regions (i.e.
exons) optionally comprising 5'- or 3'-untranslated sequences
linked thereto; or
[0047] (iii) an amplified DNA fragment or other recombinant nucleic
acid molecule produced in vitro and comprising all or a part of the
coding region and/or 5'- or 3'-untranslated sequences linked
thereto.
[0048] The term "gene" is also used to describe synthetic or fusion
molecules encoding all or part of a functional product, in
particular a sense or antisense mRNA product or a peptide,
oligopeptide or polypeptide or a biologically-active protein,
[0049] The term "synthetic gene" refers to a non-naturally
occurring gene as hereinbefore defied which preferably comprises at
least one or more transcriptional and/or translational regulatory
sequences operably linked to a structural gene sequence.
[0050] The term "structural gene" shall be taken to refer to a
nucleotide sequence which is capable of being transmitted to
produce mRNA and optionally, encodes a peptide, oligopeptide,
polypeptide or biologically active protein molecule. Those skilled
in the an will be aware that not all mRNA is capable of being
translated into a peptide, oligopeptide, polypeptide or protein,
for example if the mRNA lacks a functional translation start signal
or alternatively, if the mRNA is antisense mRNA. The present
invention clearly encompasses synthetic genes comprising nucleotide
sequences which are not capable of encoding peptides,
oligopeptides, polypeptides or biologically-active proteins. In
particular, the present inventors have found that such synthetic
genes may be advantageous in modifying target gene expression in
cells, tissues or organs of a prokaryotic or eukaryotic
organism.
[0051] The term "target gene" shall be taken to refer to any gene,
the expression of which is to be modified using the synthetic gene
of the invention. Preferred target genes include, but are not
limited to viral genes and foreign genes which have been introduced
into the cell, tissue or organ or alternatively, genes which are
endogenous to the cell, tissue or organ.
[0052] Wherein the target gene is a viral gene, it is particularly
preferred that the viral gene encodes a function which is essential
for replication or reproduction of the virus, such as but not
limited to a DNA polymerase or RNA polymerase gene or a viral coat
protein gene, amongst others. In a particularly preferred
embodiment, the target gene comprises an RNA polymerase gene
derived from a single-stranded (+) RNA virus such as bovine
enterovirus (BEV). Sinbis alphavirus or a lentivirus such as, but
not limited to, an immunodeficiency virus (eg. HIV-1) or
alternatively, a DNA polymerase derived from a double-stranded DNA
virus such as bovine herpesvirus or herpes simplex virus I (HSVI),
amongst others.
[0053] Wherein the target gene is a foreign gene, those skilled in
the art will be aware that it will have been introduced to the
cell, tissue or organ using transformation technology or
alternatively, comprise a gene derived from a pathogen which has
been introduced to said cell, tissue or organ by
naturally-occurring gene transfer processes.
[0054] Particularly preferred foreign target genes include any
transgene which has been introduced to the cell, tissue or
organ.
[0055] Wherein the target gene is a gene which is endogenous to the
cell, tissue or organ, it is particular preferred that its
expression is capable of being monitored by a visual assay, enzyme
assay or immunoassay. Particularly preferred endogenous target
genes are those detected by visual assay means.
[0056] The synthetic genes of the present invention may be derived
from naturally-occurring genes by standard recombinant techniques,
the only requirement being that the synthetic gene is substantially
identical at the nucleotide sequence level to at least a part of
the target gene, the expression of which is to be modified. By
"substantially identical" is meant that the structural gene
sequence of the synthetic gene is at least about 80%-90% identical
to 30 or more contiguous nucleotides of the target gene, more
preferably at least about 90-95% identical to 30 or more contiguous
nucleotides of the target gene and even more preferably at least
about 95-99% identical or absolutely identical to 30 or ore
contiguous nucleotides of the target gene.
[0057] Generally, a gene of the invention may be subjected to
mutagenesis to produce single or multiple nucleotide substitutions,
deletions and/or additions without affecting its ability to modify
target gene expression. Nucleotide insertional derivatives of the
synthetic gene of the present invention include 5' and 3' terminal
fusions as well as intra-sequence insertions of single or multiple
nucleotides. Insertional nucleotide sequence variants are those in
which one or more nucleotides arc introduced into a predetermined
site in the nucleotide sequence although random insertion is also
possible with suitable screening of the resulting product.
[0058] Deletional variants are characterised by the removal of one
or more nucleotides from the sequence. Substitutional nucleotide
variants are those in which at least one nucleotide in the sequence
has been removed and a different nucleotide Inserted in its place.
Such a substitution may be "silent" in that the substitution does
not change the amino acid defined by the codon. Alternatively,
substituents are designed to alter one amino acid for another
similar acting amino acid or amino acid of like charge, polarity,
or hydrophobicity.
[0059] Accordingly, the present invention extends to homologues,
analogues and derivatives of the synthetic genes described
herein.
[0060] For the present purpose, "homologues" of a gene as
hereinbefore defined or of a nucleotide sequence shall be taken to
refer to an isolated nucleic acid molecule which is substantially
the same as the nucleic acid molecule of the present invention or
its complementary nucleotide sequence, notwithstanding the
occurrence within said sequence, of one or more nucleotide
substitutions, insertions, deletions, or rearrangements.
[0061] "Analogues" of a gene as hereinbefore defined or of a
nucleotide sequence set forth herein shall be take to refer to an
isolated nucleic acid molecule which is substantially the same as a
nucleic acid molecule of the present invention or its complementary
nucleotide sequence, notwithstanding the occurrence of any
non-nucleotide constituents not normally present in said isolated
nucleic acid molecule, for example carbohydrates, radiochemicals
including radionucleotides, reporter molecules such as, but not
limited to DIG, alkaline phosphatase or horseradish peroxidase,
amongst others.
[0062] "Derivatives" of a gene as hereinbefore defined or of a
nucleotide sequence set forth herein shall be taken to refer to any
isolated nucleic acid molecule which contains significant sequence
similarity to said sequence or a part thereof. Generally, the
nucleotide sequence of the present invention may be subjected to
mutagenesis to produce single or multiple nucleotide substitutions,
deletions and/or insertions. Nucleotide insertional derivatives of
the nucleotide sequence of the present invention include 5' and 3'
terminal fusions as well as intra-sequence insertions of single or
multiple nucleotides or nucleotide analogues. Insertional
nucleotide sequence variants are those in which one or more
nucleotides or nucleotide analogues are introduced into a
predetermined site in the nucleotide sequence of said sequence,
although random insertion is also possible with suitable screening
of the resulting product being performed. Deletional variants are
characterised by the removal of one or more nucleotides from the
nucleotide sequence. Substitutional nucleotide variants are those
in which at least one nucleotide in the sequence has been removed
and a different nucleotide or nucleotide analogue inserted in its
place.
[0063] Accordingly, the structural gene component of the synthetic
gene may comprise a nucleotide sequence which is at least about 80%
identical to at least about 30 contiguous nucleotides of an
endogenous target gene, a foreign target gene or a viral target
gene present in a cell, tissue or organ or a homologue, analogue,
derivative thereof or a complementary sequence thereto.
[0064] Preferred structural gene components of the synthetic gene
of the invention comprise at least about 20-30 nucleotides in
length derived from a viral DNA polymerase, viral RNA polymerase,
viral coat protein or visually-detectable gene, more particularly
an RNA polymerase gene derived from a virus selected from the list
comprising BEV, Sindbis alphavirus, HIV-1, bovine herpes virus and
HSV1 or a visually-detectable gene which is involved in determining
pigmentation, cell death or other external phenotype on a cell,
tissue, organ or organism, amongst others.
[0065] In a particularly preferred embodiment, the structural gene
component of the synthetic gene comprises at least about 20-30
nucleotides in length derived from the BEV RNA-dependent RNA
polymerase gene or the murine tyrosinase gene or the Escherichia
coli lac repressor gene lacI or a complementary sequence
thereto.
[0066] The structural gene component may comprise a nucleotide
sequence which encodes an amino acid sequence, with or without a
translation start signal (ATG) or a nucleotide sequence which is
complementary thereto. Those skilled in the art will be aware that,
in the absence of the translation start signal in an appropriate
reading frame, the mRNA encoded by the structural gene will not be
translated in most eukaryotic and prokaryotic cells.
[0067] Alternatively, the structural gene may comprise a nucleotide
sequence which does not encode an amino acid sequence or more
commonly, comprises one or more open reading frames which encode
one or more peptides. oligopeptides or polypeptides which are
unrelated at the amino acid sequence level to the amino acid
sequence encoded by the target gene. For example, the mRNA product
of the structural gene may be inserted into the synthetic gene of
the invention so as to alter or disrupt the reading frame of the
structural gene and produce one or more frame shift mutations in
the translation product thereof relative to the translation product
encoded by the target gene, notwithstanding a substantial identity
between the structural gene and the target gene on the one hand and
the corresponding mRNA products of the structural gene and the
target gene on the other hand. Such effects may be produced by
introducing one or two nucleotide substitutions or deletions into
the structural gene, relative to the target gene sequence or
alternatively, by introducing a translation start codon 5'-ATG-3'
upstream of any nucleotide in the structural germ which occurs at a
particular position in a codon of the corresponding target gene
such that the position of said nucleotide in the codon of the
structural gene is altered.
[0068] Alternatively, the structural gene may encode no amino acid
sequence or one or more amino acid sequences which are unrelated to
the amino acid sequence encoded by the target gene wherein said
structural gene is transcribed in the antisense orientation from
the synthetic gene promoter, relative to the direction of
transcription of the corresponding target gene. In such
circumstances, the mRNA product of the structural gene will
comprise a nucleotide sequence which is complementary to the
nucleotide sequence in the corresponding region of them mRNA
encoded by the target gene.
[0069] The present invention clearly encompasses synthetic genes
wherein the structural gene component is operably connected in the
sense or antisense orientation to a promoter sequence and
irrespective of the capacity of said structural gene to encode an
amino acid sequence which is encoded by the target gene.
Accordingly, the structural gene component may further comprise
5'-untranslated region and/or 3'-untranslated region and/or intron
(eg. SV40 intron) and/or a coding region derived from the target
gene or a complementary nucleotide sequence thereto.
[0070] Reference herein to a "promoter" is to be taken in its
broadest context and includes the transcriptional regulatory
sequences of a classical genomic gene, including the TATA box which
is required for accurate transcription initiation in eukaryotic
cells, with or without a CCAAT box sequence and additional
regulatory elements (i.e. upstream activating sequences, enhancers
and silencers). For expression in prokaryotic cells, such as
bacteria, the promoter should at least contain the -35 box and -10
box sequences.
[0071] A promoter is usually, but not necessarily,
positioned-upstream or 5', of the structural gene component of the
synthetic gene of the invention, the expression of which it
regulates. Furthermore, the regulatory elements comprising a
promoter are usually positioned within 2 kb of the start Site of
transcription of the structural gene.
[0072] In the present context, the term "promoter" is also used to
describe a synthetic or fusion molecule, or derivative which
confers, activates or enhances expression of an isolated nucleic
acid molecule, in a cell, such as a plant, animal, insect, fungal,
yeast or bacterial cell. Preferred promoters may contain additional
copies of one or more specific regulatory elements, to further
enhance expression of a structural gene which expression it
regulates and/or to alter the spatial expression and/or temporal
expression of same. For example, regulatory elements which confer
inducibility on the expression of the structural gene may be placed
adjacent to a heterologous promoter sequence driving expression of
a nucleic acid molecule.
[0073] Placing a structural gene under the regulatory control of a
promoter sequence means positioning said molecule such that
expression is controlled by the promoter sequence. Promoters are
generally positioned 5' (upstream) to the genes that they control.
In the construction of heterologous promoter/structural gene
combinations it Is generally preferred to position the promoter at
a distance from the gene transcription start site that is
approximately the same as the distance between that promoter and
the gene it controls in its natural setting, i.e. the gene from
which the promoter is derived. As is known in the art, some
variation in this distance can be accommodated without loss of
promoter function. Similarly, the preferred positioning of a
regulatory sequence clement with respect to a heterologous gene to
be placed under its control is defined by the positioning of the
element in its natural setting, i.e., the genes from which it is
derived. Again, as is known in the art, some variation in this
distance can also occur.
[0074] Examples of promoters suitable for use in the synthetic
genes of the present invention include viral, fungal, bacterial,
animal and plant derived promoters capable of functioning in plant,
animal, insect, fungal, yeast or bacterial cells. The promoter may
regulate the expression of the structural gene component
constitutively, or differentially with respect to cell, the tissue
or organ in which expression occurs, or with respect to the
developmental stage at which expression occurs, or in response to
external stimuli such as physiological stresses, or pathogens, or
metal ions, amongst others.
[0075] Preferably, the promoter is capable of regulating expression
of a nucleic acid molecule in a eukaryotic cell, tissue or organ,
at least during the period of time over which the target gene is
expressed therein and more preferably also immediately preceding
the commencement of detectable expression of the target gene in
said cell, tissue or organ.
[0076] Accordingly, strong constitutive promoters are particularly
preferred for the purposes of the present inventions or promoters
which may be induced by virus infection or the commencement of
target gene expression.
[0077] Examples of preferred promoters include the bacteriophage T7
promoter, bacteriophage T3 promoter, SP6 promoter, lac
operator-promoter, lac promoter, SV40 late promoter, SV40 early
promoter, RSV-LTR promoter, CMV IE promoter and the like.
[0078] Particularly preferred promoters contemplated herein include
promoters operable in eukaryotic cells, for example the SV40 early
promoter, SV40 late promoter or the CMV IE promoter sequence. Those
skilled in the art will readily be aware of additional promoter
sequences other than those specifically described.
[0079] In the present context, the terms "in operable connection
with" or "operably under the control" or similar shall be taken to
indicate that expression of the structural gene is under the
control of the promoter sequence with which it is spatially
connected; in a cell, tissue, organ or whole organism.
[0080] In a more particularly preferred embodiment of the
invention, the synthetic gene according to this aspect of the
invention comprises the coding region of the BEV polymerase gene
placed in the sense orientation operably under the control of the
CMV IE promoter or SV40 late promoter. In an alternative
embodiment, the synthetic gene comprises a nucleotide sequence
derived from the coding region of the BEV polymerase gene but
lacking a translation--start site, placed in the sense orientation
in operable connection with the CMV IE promoter or SV40 late
promoter. In a further alternative embodiment, the synthetic gene
comprises a nucleotide sequence derived from the BEV polymerase
gene placed in the antisense orientation relative to the BEV
polymerase gene and in operable connection with the CMV IE promoter
or the SV40 late promoter sequence.
[0081] For the present purposes, the term "BEV polymerase" as used
herein shall be taken to refer to a structural gene, cDNA molecule,
genomic gene or nucleotide sequence at least about 30-50
nucleotides in length which is derived from the nucleotide sequence
of the bovine enterovirus(BEV) RNA-dependent RNA polymerase gene,
including both translatable and non-translatable nucleotide
sequences and nucleotide sequences which arc complementary to a
part of the nucleotide sequence of the full-length BEV
RNA-dependent RNA polymerase gene.
[0082] In a further alternative embodiment, the synthetic gene
according to this aspect of the invention comprises the coding
region of a tyrosinase gene, in particular the murine tyrosinase
gene, placed in the sense orientation operably under the control of
the CMV IE promoter or SV40 late promoter. As with other
embodiments described herein, the synthetic gene (i.e. tyrosinase
gene) may lack a functional translation start site or be introduced
in the antisense orientation. The present invention clearly
encompasses all such embodiments.
[0083] As used herein, the tern "tyrosinase gene" shall be taken to
refer to a structural gene, cDNA molecule, genomic gene or
nucleotide sequence which is capable of encoding the tyrosinase
enzyme or a polypeptide fragment thereof or alternatively, a
nucleotide sequence which is complementary to said structural gene,
cDNA molecule, genomic gene or nucleotide sequence. Particularly
preferred tyrosinase genes for use in the performance of the
present invention include, but are not limited to, those described
by Kwon et at (1988) and homologues, analogues and derivatives
thereof and complementary nucleotide sequences thereto.
[0084] In still a further alternative embodiment, the synthetic
gene according to this aspect of the invention comprises the coding
region of the lacI gene, placed in the sense orientation operably
under the control of the CMV IE promoter or SV40 late promoter. As
with other embodiments described herein, the synthetic gene (i.e.
E. coli lacI gene) may lack a functional translation start site or
be introduced in the antisense orientation. The present invention
clearly encompasses all such embodiments.
[0085] As used herein, he term "lacI gene" shall be taken to refer
to a structural gene, cDNA molecule, genomic gene or nucleotide
sequence which is capable of encoding a polypeptide repressor of
the lacZ gene which encodes the enzyme .beta.-galactosidase or
alternatively, a nucleotide sequence which is complementary to said
structural gene, cDNA molecule, genomic gene or nucleotide
sequence. Those skilled in the art will be aware that the lac
repressor is a DNA-binding protein which acts on the lac
operator-promoter sequence. In the presence of one of a variety of
.beta.-galactosides, the affinity of the lac repressor for the lac
operator-promoter sequence is lowered, thereby allowing RNA
polymerase to bind the lac operator-promoter region to activate
transcription of the lac operon.
[0086] Standard methods may be used to produce the structural genes
of the present invention, in particular the BEV polymerase and
tyrosinase genes which are derived from publicly available
material. For example, the BEV polymerase and tyrosinase genes may
be amplified using the polymerase chain reaction or alternatively,
isolated using standard hybridisation techniques known to those
skilled in the art.
[0087] For the purposes of nomenclature, the nucleotide sequence of
the cDNA encoding murine tyrosinase is publicly available under
GenBank Accession No. M20234.
[0088] A second aspect of the present invention provides a
synthetic gene which is capable of modifying the expression of a
target gene in a cell, tissue or organ, wherein said synthetic gene
at least comprises multiple structural gene sequences wherein each
of said structural gene sequences comprises a nucleotide sequence
which is substantially identical to the nucleotide sequence of the
target gene or a derivative thereof or a complementary sequence
thereto and wherein said multiple structural gene sequences are
placed operably under the control of a single promoter sequence
which is operable in said cell, tissue or organ
[0089] As used herein, the term "multiple structural gene
sequences" or similar term shall be taken to refer to any number of
structural genes as defined herein which is greater than or equal
to two. Accordingly, a multiple structural gene sequence may
comprise a tandem repeat or concatemer of two or more identical
nucleotide sequences or alternatively, a tandem array or concatemer
of non-identical nucleotide sequences, the only requirement being
that each of the structural gene sequences contained therein is
substantially identical to the target gene sequence or a
complementary sequence thereto. In this regard, those skilled in
the art will be aware that a cDNA molecule may also be regarded as
a multiple structural gene sequence in the context of the present
invention, in so far as it comprises a tandem array or concatemer
of exon sequences derived from a genomic target gene. Accordingly,
cDNA molecules and any tandem array, tandem repeat or concatemer of
exon sequences and/or intron sequences and/or 5'-untranslated
and/or 3'-untranslated sequences are clearly encompassed by this
embodiment of the invention.
[0090] Preferably, the multiple structural gene comprises at least
2-4 individual structural gene sequences, more preferably at least
about 4-6 individual structural gene sequences and more preferably
at least about 6-8 individual structural gene sequences.
[0091] The optimum number of structural gene sequences which may be
involved in the synthetic gene of the present invention will vary
considerably, depending upon the length of each of said structural
gene sequences, their orientation and degree of identity to each
other. For example, those skilled in the art will be aware of the
inherent instability of palindromie nucleotide sequences in vivo
and the difficulties associated with constructing long synthetic
genes comprising inverted repeated nucleotide sequences, because of
the tendency for such sequences to form hairpin loops and to
recombine in vivo. Notwithstanding such difficulties, the optimum
number of structural gene sequences to be included in the synthetic
genes of the present invention may be determined empirically by
those skilled in the art, without any undue experimentation and by
following standard procedures such as the construction of the
synthetic gene of the invention using recombinase-deficient cell
lines, reducing the number of repeated sequences to a level which
eliminates or minimises recombination events and by keeping the
total length of the multiple structural gene sequence to an
acceptable limit, preferably no more than 5-10 kb, more preferably
no more than 2-5 kb and even more preferably no more than 0.5-2.0
kb in length.
[0092] In an alternative embodiment, each structural gene contained
within the multiple structural gene unit of the subject synthetic
gene may comprise a nucleotide sequence which is substantially
identical to a different target gene in the same organism. Such an
arrangement may be of particular utility when the synthetic gene is
intended to provide protection against a pathogen in a cell, tissue
or organ, in particular a viral pathogen, by modifying the
expression of viral target genes. For example, the multiple
structural gene may comprise nucleotide sequences which are
substantially identical to two or more target genes selected from
the list comprising DNA polymerase, RNA polymerase and coat protein
or other target gene which is essential for viral infectivity,
replication or reproduction. However, it is preferred with this
arrangement that the structural germ units are selected such that
the target genes to which they are substantially identical are
normally expressed at approximately the same time (or later) in an
infected cell, tissue or organ as (than) the multiple structural
gene of the subject synthetic gene is expressed under control of
the promoter sequence. This means that the promoter controlling
expression of the multiple structural gene will usually be selected
to confer expression in the cell, tissue or organ over the entire
life cycle of the virus when the viral target genes are expressed
at different stages of infection.
[0093] The individual structural gene units of the multiple
structural gene according to the embodiments described herein may
be spatially connected in any orientation relative to each other,
for example head-to-head, head-to-tail or tail-to-tail and all such
configurations are within the scope of the invention.
[0094] Preferably, the multiple structural gene unit comprises two
structural genes in a head-to-tail or head-to-head configuration
More preferably, the multiple structural gene unit comprises two
identical or substantially identical structural genes or a
homologue, analogue or derivative thereof in a head-to-tail
configuration as a direct repeat or alternatively, in a
head-to-head configuration as an inverted repeat or palindrome.
[0095] In a particularly preferred embodiment, the multiple
structural gene unit comprises two identical or substantially
identical structural genes comprising nucleotide sequences derived
from the BEV polymerase or tyrosinase gene or a homologue, analogue
or derivative thereof, placed in a head-to-head or head-to-tail
configuration.
[0096] According to this aspect of the invention, wherein the
multiple structural gene or any individual structural gene unit
thereof is intended to be both transcribed and translated, a
translation start signal may be included at the 5' end of that open
reading frame. In a particularly preferred embodiment, the
structural gene unit which is positioned nearer the 5' end of the
synthetic gene comprises an in-frame translation start signal of
facilitate translation of the first open reading frame of the
multiple structural gene in a cell, tissue or organ into which the
synthetic gene is introduced. Those skilled in the an will be aware
that it is also possible to produce a fusion polypeptide from such
an arrangement provided that the individual structural gene units
are positioned such that their open reading frames are in-frame
with respect to each other or alternatively, the individual
structural gene units are separated by intron/exon splice boundary
sequences such that splicing of the mRNA product of the synthetic
gene produces a translatable mRNA wherein the said open reading
frames are in-frame with respect to each other. Such embodiments
are clearly contemplated by the present invention. Intron/exon
splice junction sequences are well-known in the art and the skilled
person would readily be able to introduce such sequences to the 5'-
and 3'-ends of a structural gene unit of the synthetic genes
described herein.
[0097] The individual structural genes comprising the multiple
structural gene unit may be further spatially separated by the
addition of a linker molecule or "stuffer fragment" there between.
The stuffer fragment may comprise any combination of nucleotide or
amino acid residues, carbohydrate molecules or oligosaccharide
molecules or carbon atoms or a homologue, analogue or derivative
thereof which is capable of being linked covalently to a nucleic
acid molecule.
[0098] Preferably, embodiment, the stuffer fragment comprises a
sequence of nucleotides or a homologue, analogue or derivative
thereof.
[0099] More preferably, the stuffer fragment comprises a sequence
of nucleotides of at least about 10-50 nucleotides in length, even
more preferably at least about 50-100 nucleotides in length and
still more preferably at least about 100-500 nucleotides in
length.
[0100] Wherein the multiple structural gene unit comprises
intron/exon splice junction sequences, the stuffer fragment may
serve as an intron sequence placed between the 3'-splice site of
the structural gene nearer the 5'-end of the gene and the 5'-splice
site of the next downstream structural gene. Alternatively, wherein
it is desirable for more than two adjacent structural genes to be
translated, the stuffer fragment placed there between should not
include an in-frame translation stop codon, absent intron/exon
splice junction sequences at both ends of the stuffer fragment or
the addition of a translation start codon at the 5' end of each
structural gene unit, as will be obvious to those skilled in the
art.
[0101] Preferred stuffer fragments are those which encode
detectable marker proteins or biologically-active analogues and
derivatives thereof, for example luciferase, .beta.-galacturonase,
.beta.-galactosidase, chloramphenicol acetyltansferase or green
fluorescent protein, amongst others.
[0102] According to this embodiment, the detectable marker or an
analogue or derivative thereof serves to indicate the expression of
the synthetic gene of the invention in a cell, tissue or organ by
virtue of its ability to confer a specific detectable phenotype
thereon, preferably a visually-detectable phenotype.
[0103] In a more particularly preferred embodiment of the
invention, the multiple structural gene comprises an interrupted
direct repeat or interrupted palindrome comprising two identical or
substantially-identical BEV polymerase structural gene sequences or
alternatively, two identical or substantially-identical tyrosinase
structural gene sequences or a homologue, analogue or derivative
thereof separated by a stuffer fragment comprising a nucleotide
sequence which encodes green-fluorescent protein or a
biologically-active analogue or derivative thereof.
[0104] As used herein, the term "green fluorescent protein" or
"GFP" shall be taken to refer to a protein, polypeptide or peptide
which is capable of producing a strong green fluorescence when
excited with near ultraviolet radiation or blue light or a
homologue, analogue or derivative thereof. Accordingly, the term
"GFP gene" shall be taken to refer to a nucleotide sequence which
is capable of encoding GFP or a complementary nucleotide sequence
thereto. Particularly preferred GFPs and GFP genes according to the
present invention are derived from the jellyfish Aequoria victoria
as described by Prasher et al (1992) or in International Patent
Publication No. WO 95/07463, amongst others.
[0105] A further aspect of the invention provides for each
structural gene of the multiple structural gene unit to be placed
operably under the control of a separate promoter sequence.
[0106] According to this embodiment, the promoters controlling
expression of the structural gene unit are preferably different
promoter sequences, to reduce competition there between for
cellular transcription factors and RNA polymerases. Preferred
promoters are selected from those referred to supra.
[0107] Those skilled in the art wilt know how to modify the
arrangement or configuration of the individual structural genes as
described supra to regulate their expression from separate promoter
sequences.
[0108] In a particularly preferred embodiment, the multiple
structural gene unit comprises two or more BEV polymerase
structural genes or two or more tyrosinase structural genes wherein
each of said structural genes is placed operably in connection with
a different promoter sequence. More particularly preferred, the
multiple structural gene unit comprises two BEV polymerase
structural genes or two tyrosinase structural genes positioned as
inverted repeats or direct repeats wherein one of said structural
genes is placed operably in connection with the CMV IE promoter.
Even more preferably, at least one of the BEV polymerase structural
genes or tyrosinase genes comprising the multiple structural gene
is presented it the sense orientation and comprises a translation
start signal to facilitate translation of mRNA encoded
therefrom.
[0109] Those skilled in the art will be aware that the structural
genes comprising the multiple structural gene unit according to
this aspect of the invention are expressed as physically-distinct
mRNA species and, as a consequence, wherein said mRNA species are
translated, no fusion polypeptide will be produced there between.
However, the present invention clearly extends to synthetic gene
which comprises two or more structural genes operably connected to
a first promoter sequence and one or more structural genes operably
connected to one or more additional promoter sequences.
[0110] The synthetic genes described supra are capable of being
modified further, for example by the inclusion of marker nucleotide
sequences encoding a detectable marker enzyme or a functional
analogue or derivative thereof, to facilitate detection of the
synthetic gene in a cell, tissue or organ in which it is expressed.
According to this embodiment, the marker nucleotide sequences will
be present in a translatable format and expressed, for example as a
fusion polypeptide with the translation product(s) of any one or
more of the structural genes or alternatively as a non-fusion
polypeptide.
[0111] Alternatively or in addition, the synthetic genes described
supra may further comprise one or more transcription termination
sequences placed at the 3'-end of the transoriptional unit of the
synthetic gene sequence.
[0112] The term "terminator" refers to a DNA sequence at the end of
a transcriptional unit which signals termination of transcription.
Terminators are 3'-non-translated DNA sequences containing a
polyadenylation signal, which facilitates the addition of
polyadenylate sequences to the 3'-end of a primary transcript.
Terminators active in cells derived from viruses, yeasts, moulds,
bacteria, insects, birds, mammals and plants are known and
described in the literature. They may be isolated from bacteria,
fungi, viruses, animals and/or plants.
[0113] Examples of terminators particularly suitable for use in the
synthetic genes of the present invention include the SV40
polyadenylation signal, the HSV TK polyadenylation signal, the CYC1
terminator, ADH terminator, SPA terminator, nopaline synthase (NOS)
gene terminator of Agrobacterium tumefaciens, the terminator of the
Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator
from Zea mays, the Rubisco small subunit gene (SSU) gene terminator
sequences, subclover stunt virus (SCSV) gene sequence terminators,
any rho-independent E. coli terminator, or the lacZ alpha
terminator, amongst others.
[0114] In a particularly preferred embodiment, the terminator is
the SV40 polyadenylation signal or the HSV TK polyadenylation
signal which are operable in animal cells, tissues and organs or
the lacZ alpha terminator which is active in prokaryotic cells.
[0115] Those skilled in the art will be aware of additional
promoter sequences and terminator sequences which may be suitable
for use in performing the invention. Such sequences may readily be
used without any undue experimentation.
[0116] The synthetic genes of the present invention may be
introduced to a suitable cell, tissue or organ without modification
as linear DNA in the form of a genetic construct, optionally
contained within a suitable carrier, such as a cell, virus particle
or liposome, amongst others. To produce a genetic construct, the
synthetic gene of the invention is inserted into a suitable vector
or episome molecule, such as a bacteriophage vector, viral vector
or a plasmid, cosmid or artificial chromosome vector which is
capable of being maintained and/or replicated and/or expressed in
the host cell, tissue or organ into which it is subsequently
introduced.
[0117] Accordingly a further aspect of the invention provides a
genetic construct which at least comprises the synthetic gene
according to any one or more of the embodiments described herein
and one or more origins of replication and/or selectable marker
gene sequences.
[0118] Usually, an origin of replication or a selectable marker
gene suitable for use in bacteria is physically-separated from
those genetic sequences contained in the genetic construct which
are intended to be expressed or transferred to a eukaryotic cell,
or integrated into the genome of a eukaryotic cell.
[0119] In a particularly preferred embodiment, the origin of
replication is functional in a bacterial cell and comprises the pUC
or the ColE1 origin or alternatively the origin of replication is
operable in a eukaryotic cell, tissue and more preferably comprises
the 2 micron (2 .mu.M) origin of replication or the SV40 origin of
replication.
[0120] As used herein, the term "selectable marker gene" includes
any gene which confers a phenotype on a cell in which it is
expressed to facilitate the identification and/or selection of
cells which are transfected or transformed with a genetic construct
of the invention or a derivative thereof.
[0121] Suitable selectable marker genes contemplated herein include
the ampicillin-resistance gene (Amp.sup.r), tetracycline-resistance
gene (Tc.sup.r), bacterial kanamycin-resistance gene (Kan.sup.r),
is the zeocin resistance gene (Zeocin is a drug of bleomycin family
which is trademark of InVitrogen Corporation), the AURI-C gene
which confers resistance to the antibiotic aureobasidin A,
phosphinothricin-resistance gene, neomycin phosphotransferase gene
(nptII), hygromycin-resistance gene, .beta.-glucuronidase (GUS)
gene, chloramphenicol acetyltransferase (CAT) gene, green
fluorescent protein-encoding gene or the luciferase gene, amongst
others.
[0122] Preferably, the selectable marker gene is the nptII gene or
Kan.sup.r gene or green fluorescent protein (GFP)-encoding
gene.
[0123] Those skilled in the art will be aware of other selectable
marker genes useful in the performance of the present invention and
the subject invention is not limited by the nature of the
selectable marker gene.
[0124] The present invention extends to all genetic constructs
essentially as defined herein, which include further genetic
sequences intended for the maintenance and/or replication of said
genetic construct in prokaryotes and/or the integration of said
genetic construct or a part thereof into the genome of a eukaryotic
cell or organism.
[0125] The present invention further extends to an isolated cell,
tissue or organ comprising the synthetic gene described herein or a
genetic construct comprising same. Any standard means may be used
for their introduction including cell mating, transformation or
transfection procedures known to those skilled in the art or
described by Ausubel et al (1992),
[0126] The present invention is further described by reference to
the following non-limiting Examples.
EXAMPLE 1
Base Plasmids
[0127] Plasmid pEGFP-N1 MCS
[0128] Plasmid pEGFP-N1 MCS (FIG. 1; Clontech) contains the CMV IE
promoter operably connected to an open reading fame encoding a
red-shifted variant of wild-type green fluorescent protein (GFP;
Prasher et al., 1992; Chalfie et al.,. 1994; Inouye and Tsuji,
1994), which has been optimised for brighter fluorescence. The
specific GFP variant encoded by pEGFP-N1 MCS has been disclosed by
Cormack et al. (1996). Plasmid pEGFP-N1 MCS contains a multiple
cloning site comprising BglII and BamHI sites and many other
restriction endonuclease cleavage sites, located between the CMV IE
promoter and the GFP open reading frame. Structural genes cloned
into the multiple cloning site will be expressed at the
transcriptional level if they lack a functional translation start
site, however such structural gene sequences will not be expressed
at the protein level (i.e. translated). Structural gene sequences
inserted into the multiple cloning site which comprise a functional
translation start site will be expressed as GFP fusion polypeptides
if they are cloned in-frame with the GFP-encoding sequence. The
plasmid further comprises an SV40 polyadenylation signal downstream
of thee GFP open reading frame to direct proper processing of the
3'-end of mRNA transcribed from the CMV-IE promoter sequence. The
plasmid Her comprises the SV40 origin of replication functional in
animal cells; the neomycin-resistance gene comprising SV40 early
promoter (SV40 EP in FIG. 1) operably connected to the
neomycin/kanamycin-resistance gene derived from Tn5 (Kan/neo in
FIG. 1) and the HSV thymidine kinase polyadenylation signal (HSV TK
poly (A) in FIG. 1), for selection of transformed cells on
kamanycin, neomycin or G418; the pUC19 origin of replication which
is functional in bacterial cells (pUC Ori in FIG. 1); and the f1
origin of replication for single-stranded DNA production (f1 Ori in
FIG. 1).
[0129] pCMVLacI
[0130] Plasmid pCMVLacI is a commercially-obtainable mammalian
expression vector (Stratagene) comprising the lacI gene encoding
the lac repressor and a gene coding for hygromycin resistance
(Hyg.sup.r).
[0131] Plasmid pOPRSVI/MCS
[0132] Plasmid pOPRSVI/MCS is a commercially-obtainable mammalian
expression vector (Stratagene), comprising the OPRSV1 promoter
sequence (a modified RSV-LTR promoter), SV40 intron sequence, lac
operator sequence, multiple cloning site and thymidine kinase (TK)
gene transcription terminator sequence [i.e. TK poly(A)
signal].
[0133] Plasmid pSVL
[0134] Plasmid pSVL is commercially-obtainable from Pharmacia and
serves as a source of the SV40 late promoter sequence. The
nucleotide sequence of pSVL is also publicly available as GenBank
Accession Number U13868.
[0135] Plasmid pCMV.cass
[0136] Plasmid pCMV.cass (FIG. 2) is an expression cassette for
driving expression of a structural gene sequence under control of
the CMV-IE promoter sequence. Plasmid pCMV.cass was derived from
pEGFP-N1 MCS by deletion of the GFP open reading frame as follows:
Plasmid pEGFP-N1 MCS was digested with PinAI and Not I, blunt-ended
using PfuI polymerase and then re-ligated. Structural gene
sequences are cloned into pCMV.cass using the multiple cloning
site, which is identical to the multiple cloning site of pEGFP-N1
MCS, except it lacks the PinAI site.
[0137] Plasmid pCR2.1
[0138] Plasmid pCR2.1 is commercially available from Stratagene and
comprises the lacZ promoter sequence and lacZ-.alpha. transcription
terminator, with a cloning site for the insertion of structural
gene sequences there between. Plasmid pCR2.1 is designed to clone
nucleic acid fragments by virtue of the A-overhang frequently
synthesized by Taq polymerase during the polymerase chain reaction.
The plasmid further comprises the ColE1 and f1 origins of
replication and kanamycin-resistance and ampicillin-resistance
genes.
[0139] Plasmid pCR.Bgl-GFP-Bam
[0140] Plasmid pCR.Bgl-GFP-Bam (FIG. 3) comprises an internal
region of the GFP open reading frame derived from plasmid pEGFP-N1
MCS (FIG. 1) placed operably under the control of the lacZ
promoter. To produce this plasmid, a region of the GFP open reading
frame was amplified from pEGFP-N1 MCS using the amplification
primers Bgl-GFP (SEQ ID NO:5) and GFP-Bam (SEQ ID NO:6) and cloned
into plasmid pCR2.1. The internal GFP-encoding region in plasmid
pCR.Bgl-GFP-Bam lacks functional translational start and stop
codons.
[0141] Plasmid pCR.SV40L
[0142] Plasmid pCR.SV40L (FIG. 4) comprises the SV40 late promoter
derived from plasmid pSVL. (GenBank Accession No. U13868;
Pharmacia), cloned into pCR2.1 (Stratagene). To produce this
plasmid, the SV40 late promoter was amplified using the primers
SV40-1 (SEQ ID NO:7) and SV40-2 (SEQ ID NO:8) which comprise Sal I
cloning sites to facilitate sub-cloning of the amplified DNA
fragment into pCMV.cass. SEQ ID No. 7 also contains a synthetic
poly (A) site at the 5' end, such that the amplicification product
comprises the synthetic poly(A) site at the 5' end of the SV40
promoter sequence.
[0143] Plasmid pCMV.SV40L.cass
[0144] Plasmid pCMV.SV40L.cass (FIG. 5) comprises the synthetic
poly A site and the SV40 late promoter sequence from plasmid
pCR.SV40L FIG. 4), sub-cloned as a Sal I fragment, into the Sal I
site of plasmid pCMV.cass (FIG. 2), such that the CMV-IE promoter
and SV40 late promoter sequences are capable of directing
transcription in the same direction. Accordingly, the synthetic
poly(A) site at the 5' end of the SV40 promoter sequence is used as
a transcription terminator for structural genes expressed from the
CMV IE promoter in this plasmid, which also provides for the
insertion of said structural gene via the multiple cloning site
present between the SV40 late promoter and the synthetic poly(A)
site (FIG. 5). The multiple cloning sites are located behind the
CMV-IE and SV40 late promoters, including BamHI and BglII
sites.
EXAMPLE 2
BEV Polymerase-Containing Genes
[0145] Plasmid pCR.BEV.1
[0146] The BEV RNA-dependent RNA polymerase coding region was
amplified as a 1.385 bp DNA fragment from a full-length cDNA clone
encoding same, using the primers designated BEV-1 (SEQ ID NO: 1)
and BEV-2 (SEQ ID NO :2), under standard amplification conditions.
The amplified DNA contained a 5'-BglII restriction enzyme site,
derived from the BEV-1 primer sequence and a 3'BamHI restriction
enzyme site, derived from the BEV-2 primer sequence. Additionally,
as the BEV-1 primer sequence contains a translation start signal
5'-ATG-3' engineered at positions 15-17 of SEQ ID NO:1, the
amplified BEV polymerase structural gene comprises the start site
in-frame with BEV polymerase-encoding nucleotide sequences, Thus,
the amplified BEV polymerase structural gene comprises the ATG
start codon immediately upstream (ie. juxtaposed) to the BEV
polymerase-encoding sequence. There is no translation stop codon in
the amplified DNA.
[0147] The amplified BEV polymerase structural gene was cloned into
plasmid pCR2.1 to produce pCR.BEV.1 (FIG. 6).
[0148] Plasmid pCR.BEV.2
[0149] The complete BEV polymerase coding region was amplified from
a full-length cDNA clone encoding same, using primers BEV-1 (SEQ ID
NO:1) and BEV-3 (SEQ ID NO:3). Primer BEV-3 comprises a BamHI
restriction enzyme site at positions 5 to 10 inclusive of SEQ ID
NO:3 and the complement of a translation stop signal at positions
11 to 13 of SEQ ID NO:3. As a consequence, an open reading frame
comprising a translation start signal and translation stop signal,
contained between the BglII and BamHI restriction sites. The
amplified fragment was cloned into pCR2.1 to produce plasmid
pCR2.BEV.2 (FIG. 7).
[0150] Plasmid pCR.BEV.3
[0151] A non-translatable BEV polymerase structural gene was
amplified from a fall-length BEV polymerase cDNA clone using the
amplification primers BEV-3 (SEQ ID NO:3) and BEV4 (SEQ ID NO:4).
Primer BEV-4 comprises a BglII cloning site at positions 5-10 of
SEQ ID NO:4 and sequences downstream of this BglII site are
homologous to nucleotide sequences of the BEV polymerase gene.
There is no functional ATG start codon in the amplified DNA product
of primers BEV-3 and BEV-4. The BEV polymerase is expressed as part
of a polyprotein and, as a consequence, there is no ATG translation
start site in this gene. The amplified DNA was cloned into plasmid
pCR2.1 to yield plasmid pCR.BEV.3.(FIG. 8).
EXAMPLE 3
Synthetic Genes Comprising a BEV Polymerase Structural Gene
Operably Connected to the CMV-IE Promoter Sequence
[0152] Plasmid pEGFP.BEV.1
[0153] Plasmid pEGFP.BEV.1 (FIG. 9) is capable of expressing the
BEV polymerase structural gene as a GFP fusion polypeptide under
the control of the CMV-IE promoter sequence. To produce plasmid
pEGFP.BEV.1, the BEV polymerase sequence from pCR.BEV.1 (FIG. 6)
was cloned as a BglII-to-BamHI fragment into BglII/BamHI-digested
pEGFP.N1 MCS (FIG. 1).
[0154] Plasmid pCMV.BEV.2
[0155] Plasmid pCMV.BEV.2 (FIG. 10) is capable of expressing the
entire BEV polymerase open reading frame under the control of
CMV-IE promoter sequence. To produce pCMV.BEV.2, the BEV polymerase
sequence from pCR.BEV.2 (FIG. 7) was sub-cloned in the sense
orientation as a BglII-to-BamHI fragment into BglII/BamHI-digested
pCMV.cass (FIG. 2).
[0156] Plasmid pCMV.VEB
[0157] Plasmid pCMV.VEB (FIG. 11) expresses an antisense BEV
polymerase mRNA under the control of the CMV-IE promoter sequence.
To produce plasmid pCMV.VEB, the BEV polymerase sequence from
pCR.BEV.2 (FIG. 7) was sub-cloned in the antisense orientation as a
BglII-to-BamHI fragment into BglII/BamHI-digested pCMV.cass (FIG.
2).
[0158] Plasmid pCMV.BEVnt
[0159] Plasmid pCMV.BEVnt (FIG. 12) expresses a non-translatable
BEV polymerase structural gene in the sense orientation under the
control of the CMV-IE promoter sequence. To produce pCMV.BEVnt, the
BEV polymerase sequence from pCR.BEV.3 (FIG. 8) was sub-cloned in
the sense orientation as a BglII-to-BamHI fragment into
BglII/BamHI-digested pCMV.cass (FIG. 2).
[0160] Plasmid pCMV.BEVx2
[0161] Plasmid pCMV.BEVx2 (FIG. 13) comprises a direct repeat of a
complete BEV polymerase open reading frame under the control of the
CMV-IE promoter sequence. In eukaryotic cells at least, the open
reading frame located nearer the CMV-IE promoter is translatable.
To produce pCMV.BEVx2, the BEV polymerase structural gene from
plasmid pCR.BEV.2 (FIG. 7) was sub-cloned in the sense orientation
as a BglII-to-BamHI fragment into BamHI-digested pCMV.BEV.2 (FIG.
10), immediately downstream of the BEV polymerase structural gene
already present therein.
[0162] Plasmid pCMV.BEV.VEB
[0163] Plasmid pCMV.BEV.VEB (FIG. 14) comprises an inverted repeat
or palindrome of a complete BEV polymerase open reading frame under
the control of the CMV-IE promoter sequence. In eukaryotic cells at
least, the open reading frame located nearer the CMV-IE promoter is
translatable. To produce pCMV.BEV.VEB, the BEV polymerase
structural gene from plasmid pCR.BEV.2 (FIG. 7) was sub-cloned in
the antisense orientation as a BglII-to-BamHI fragment into
BamHI-digested pCMV.BEV.2 (FIG. 10), immediately downstream of the
BEV polymerase structural gene already present therein.
[0164] Plasmid pCMV.BEV.GFP.VEB
[0165] Plasmid pCMV.BEV.GFP.VEB (FIG. 15) is similar to plasmid
pCMV.BEV.VEB except that the BEV structural gene inverted repeat or
palindrome is interrupted by the insertion of a GFP open reading
fame (stuffer fragment) therein. To produce plasmid
pCMV.BEV.GFP.VEB, the GFP stuffer fragment from pCR.Bgl-GFP-Bam
(FIG. 3) was first sub-cloned in the sense orientation as a
BglII-to-BamHI fragment into BamHI-digested pCMV.BEV.2 (FIG. 7) to
produce an intermediate plasmid pCMV.BEV.GFP wherein the BEV
polymerase-encoding and GFP-encoding sequences are contained within
the same 5'-BglII-to-BamHI-3' fragment. The BEV polymerase
structural gene from pCMV.BEV.2 (FIG. 7) was then cloned in the
antisense orientation as a BglII-to-BamHI fragment into
BamHI-digested pCMV.BEV.GFP. The BEV polymerase structural gene
nearer the CMV-IE promoter sequence in plasmid pCMV.BEV.GFP.VEB is
capable of being translated, at least in eukaryotic cells.
EXAMPLE 4
Synthetic Genes Comprising BEV Polymerase Structural Genes Operably
Connected to Multiple Promoter Sequences
[0166] Plasmid pCMV.BEV.SV40L-O
[0167] Plasmid pCMV.BEV.SV40L-O (FIG. 16) comprises a translatable
BEV polymerase structural gene derived from plasmid pCR.BEV,2 (FIG.
7) inserted in the sense orientation between the CMV-IE promoter
and the SV40 late promoter sequences of plasmid pCMV.SV40L.cass
(FIG. 5). To produce plasmid pCMV.BEV.SV40L-O, the BEV polymerase
structural gene was sub-cloned as a BglII-to-BamHI fragment into
BglII-digested pCMV.SV40L.cass DNA.
[0168] Plasmid pCMV.O.SV40L.BEV
[0169] Plasmid pCMV.O.SV40L.BEV (FIG. 17) comprises a translatable
BEV polymerase structural gene derived from plasmid pCR.BEV.2 (FIG.
7) cloned downstream of tandem CMV-IE promoter and SV40 late
promoter sequences present in plasmid pCMV.SV40L.cass (FIG. 5). To
produce plasmid pCMV.O.SV40L.BEV, the BEV polymerase structural
gene was sub-cloned in the sense orientation as a BglII-to-BamHI
fragment into BamHI-digested pCMV.SV40L.cass DNA.
[0170] Plasmid pCMV.O.SV40L.VEB
[0171] Plasmid pCMV.O.SV40L.VEB (FIG. 18) comprises an antisense
BEV polymerase structural gene derived from plasmid pCR.BEV.2 (FIG.
7) cloned downstream of tandem CMV-IE promoter and SV40 late
promoter sequences present in plasmid pCMV.SV40L.cass (FIG. 5). To
produce plasmid pCMV.O.SV40L.VEB, the BEV polymerase structural
gene was sub-cloned in the antisense orientation as a
BglII-to-BamHI fragment into BamHI-digested pCMV.SV40L.cass
DNA.
[0172] Plasmid pCMV.BEV.SV40L.BEV
[0173] Plasmid pCMV.BEV.SV40L.BEV(FIG. 19) comprises a multiple
structural gene unit comprising two BEV polymerase structural genes
placed operably and separately under control of the CMV-IE promoter
and SV40 late promoter sequences. To produce plasmid
pCMV.BEV.SV40L.BEV, the translatable BEV polymerase structural gene
present in pCR.BEV.2 (FIG. 7) was sub-cloned in the sense
orientation as a BglII-to-BamHI fragment behind the SV40 late
promoter sequence present in BamHI-digested pCMV.BEV.SV40L-O (FIG.
16).
[0174] Plasmid pCMV.BEV.SV40L.VEB
[0175] Plasmid pCMV.BEV.SV40L.VEB (FIG. 20) comprises a multiple
structural gene unit comprising two BEV polymerase structural genes
placed operably and separately under control of the CMV-IE promoter
and SV40 late promoter sequences. To produce plasmid
pCMV.BEV.SV40L.VEB, the translatable BEV polymerase structural gene
present in pCR.BEV.2 (FIG. 7), was sub-cloned in the antisense
orientation as a BglII-to-BamHI fragment behind the SV40 late
promoter sequence present in BamHI-digested pCMV.BEV.SV40L-O (FIG.
16). In this plasmid, the BEV polymerase structural gene is
expressed in the sense orientation under control of the CMV-IE
promoter to produce a translatable mRNA, whilst the BEV polymerase
structural gene is also expressed under control of the SV40
promoter to produce an antisense mRNA species.
[0176] Plasmid pCMV.SV40LR.cass
[0177] Plasmid pCMV.SV40LR.cass (FIG. 21) comprises the SV40 late
promoter sequence derived from plasmid pCR.SV40L (FIG. 4),
sub-cloned as a SalI fragment into the SalI site of the plasmid
pCMV.cass (FIG. 2), such that the CMV-IE or the SV40 late promoter
may drive transcription of a structural gene or a multiple
structural gene unit, in the sense or antisense orientation, as
desired. A multiple cloning site is positioned between the opposing
CMV-IE and SV40 late promoter sequences sis plasmid to facilitate
the introduction of a structural gene sequence. In order for
expression of a structural gene sequence to occur from this
plasmid, it must be introduced with its own transcription
termination sequence located at the 3' end, because there are no
transcription termination sequences located between the opposing
CMV-IE and SV40 late promoter sequences in this plasmid.
Preferably, the structural gene sequence or multiple structural
gene unit which is to be introduced into pCMV.SV40LR.cass will
comprise both a 5' and a 3' polyadenylation signal as follows:
[0178] (i) where the structural gene sequence or multiple
structural gene unit is to be expressed in the sense orientation
from the CMV IE promoter sequence and/or in the antisense
orientation from the SV40 late promoter, the 5' polyadenylation
signal will be in the antisense orientation and the 3'
polyadenylation signal will be in the sense orientation; and
[0179] (ii) where the structural gene sequence or multiple
structural gene unit is to be expressed in the antisense
orientation the CMV IE promoter sequence and/or in the sense
orientation from t SV40 late promoter, the 5' polyadenylation
signal will be in the sense orientation and the 3' polyadenylation
signal will be in the antisense orientation.
[0180] Alternatively or in addition, suitably-oriented terminator
sequences may be placed at the 5'-end of the CMV and SV40L
promoters, as shown in FIG. 21.
[0181] Alternatively, plasmid pCMV.SV40LR.cass is further modified
to produce a derivative plasmid which comprises two polyadenylation
signals located between the CMV IE and SV40 late promoter
sequences, in appropriate orientations to facilitate expression of
any structural gene located therebetween in the sense or antisense
orientation from either the CMV IE promoter or the SV40 promoter
sequence. The present invention clearly encompasses such
derivatives.
[0182] Plasmid pCMV.BEV.SV40LR
[0183] Plasmid pCMV.BEV.SV40LR (FIG. 22) comprises a structural
gene comprising the entire BEV polymerase open reading frame placed
operably and separately under control of opposing CMV-IE promoter
and SV40 late promoter sequences, thereby potentially producing BEV
polymerase scripts at least from both strands of the full-length
BEV polymerase structural gene. To produce plasmid pCMV.BEV.SV40LR,
the translatable BEV polymerase structural gene present in
pCR.BEV.2 (FIG. 7) was sub-cloned, as a BglII-to-BamHI fragment,
into the unique BglII site of plasmid pCMV-SV40LR.cass (FIG. 21),
such that the BEV open reading frame is present in the sense
orientation relative to the CMV-IE promoter sequence.
[0184] Those skilled in the art will recognise that it is possible
to generate a plasmid wherein the BEV polymerase fragment from
pCR.BEV.2 is inserted in the antisense orientation, relative to the
CMV IE promoter sequence, using this cloning strategy. The present
invention further encompasses such a genetic construct.
EXAMPLE 5
Synthetic Genes and Genetic Constructs Comprising the Tyrosinase
Open Reading Frame
[0185] Isolation of the Tyrosinase Open Reading Frame
[0186] The tyrosinase structural gene is isolated by polymerase
chain reaction, from mRNA derived from murine cells, using the
following oligonucleotide primers under standard polymerase chian
reaction conditions:
1 Tyr 5' (forward primer; SEQ ID NO:9):
5'-CCCGGGGCTTAGTGTAAAACAGGCTGAGAG-3'; and Tyr 3' (reverse primer;
SEQ ID NO:10): 5'-CCCGGGCAAATCCCAGTCATTTCTTAG- AAAC-3'.
[0187] Nucleotide residues 1 to 6 in each primer represent a SmaI
cloning site. Nucleotides 7 to 30 of primer Tyr 5' correspond to
the 5'-end of the murine tyrosinase cDNA sequence disclosed in
GenBank Accession No. M20234 (Kwon et al, 1988). Nucleotides 7 to
31 of primer Tyr 3' correspond to the complement of the nucleotide
sequence of the 3'-end of the murine tyrosinase cDNA sequence.
[0188] Plasmid pCR.tyr
[0189] Plasmid pCR.tyr is produced by sub-cloning the amplified
tyrosinase structural gene into plasmid pCR2.1 (Example 1),
substantially according to the manufacturer's protocol. Plasmid
pCR.tyr can be used as a base plasmid to produce a range of genetic
constructs designed to express the tyrosinase structural gene or a
multiple structural gene unit comprising same, under the control of
one or more promoter sequences.
[0190] Plasmid pCMV.TYR
[0191] Plasmid pCMV.TYR (FIG. 23) comprises the complete mouse
tyrosinase cDNA sequence placed operably in connection, in the
sense orientation, with the CMV-IE promoter sequence and upstream
of the SV40 polyadenylation sequence. To produce pCMV.TYR, the
full-length mouse tyrosinase cDNA sequence was excised from plasmid
pCR.tyr by digestion with SmaI and then ligated, into the SmaI
cloning site of pCMV.cass (FIG. 2). Clones possessing the
tyrosinase structural gene in the sense orientation relative to the
CMV-IE promoter were then selected.
[0192] Plasmid pCMV.TYRLIB
[0193] Plasmid pCMV.TYRLIB (FIG. 24) comprises a structural gene or
multiple structural gene unit which comprises one or more
tyrosinase gene fragments of 100 to 200 base pairs in length each,
placed operably in connection with the CMV-IE promoter sequence and
upsteam of the SV40 polyadenylation signal. To produce pCMV.TYRLIB,
blunt-ended fragments of the tyrosinase gene are ligated into
SmaI-digested, dephosphorylated plasmid pCMV.cass DNA (FIG. 2). The
tyrosinase gene fragments are produced, for example, by sonication
or mechanical shearing and end-repair using T4 DNA polymerase.
Accordingly, the structural gene insert in plasmid pCMV.TYRLIB is
variable and an representative library of pCMV.TYRLIB plasmids,
covering the complete tyrosinase gene sequence, may be produced
using such procedures. The present invention clearly encompasses
such representative libraries. Those skilled in the art will
recognise that such procedures are also useful for structural genes
other than tyrosinase and, as a consequence, the present invention
clearly extends to synthetic genes and genetic constructs wherein
the structural gene present in pCMV.TYRLIB is a structural gene
other than a tyrosinase gene fragment.
EXAMPLE 6
Synthetic Genes and Genetic Constructs Comprising the lacI Open
Reading Frame
[0194] Plasmid pCMV.Lac
[0195] Plasmid pCMV.Lac (FIG. 25) contains a CMV IE promoter
driving expression of the lac repressor protein encoded by the
Escherichia coli lacI gene. Accordingly, the open reading frame of
the LacI gene is cloned in the sense orientation with respect to
the CMV IE promoter sequence in this plasmid. This construct also
contains the selectable marker for neomycin resistance.
[0196] To produce plasmid pCMV.Lac, the lacI gene was excised from
plasmid pCMV.LacI (Stratagene) by digestion with HindIII and BsaBI
and then ligated, in the sense orientation, into the multiple
cloning site (MCS) of plasmid pCMV.cass (FIG. 2) which had been
digested with HindIII and SmaI.
[0197] Plasmid pCMVLacI.OPRSV1.cass
[0198] Plasmid pCMVLacI.OPRSV1.cass (FIG. 26) is a dual expression
construct in which the CMV-IE promoter drives expression of the
LacI structural gene to produce the lac repressor protein and the
OPRSVI promoter drives the expression of a second structwal gene or
multiple structural gene unit placed operably under control of lac
repressor protein.
[0199] To produce plasmid pCMVLac.OPRSV1.cass, a DNA fragment
comprising the OPRSVI promoter, SV40 intron, lac operator sequence,
multiple cloning site (MCS) and TK poly(A) sequence was excised
from plasmid pOPRSVI/MCS (Stratagene), by digestion with SnaBl and
Asel restriction enzymes, then end-filled using PfuI polymerase and
ligated into the end-filled BglII cloning site of plasmid pCMVLacI
(Stratagene).
EXAMPLE 7
Synthetic Genes and Genetic Constructs Comprising the lacI and
Green Fluorescent Protein (GFP)Open Reading Frames
[0200] Plasmid pCMVLacI.OPRSV1.GFP.cass
[0201] Plasmid pCMVLacI.OPRSV1.GFP.cass (FIG. 27) is designed such
that a structural gene or multiple structural gene unit can be
fused to the 3' untranslated region of the lacI gene, by cloning
directly into the unique BsaB1 cloning site which is located after
the lacI stop codon and before an SV40 polyadenylation signal.
Alternatively, the BSAB1 site may be modified to facilitate
cloning, for example by the addition of linkers or adaptors. This
construct also contains the antibiotic selectable marker for
hygromycin resistance.
[0202] To produce plasmid pCMVLacI.OPRSVI.GFP.cass, the enhanced
GFP coding sequence was excised from plasmid pEGFP-N1 MCS (FIG. 1)
by digestion with Xhol and Notl and the DNA fragment thus produced
was ligated into the Xhol and Notl cloning sites of the multiple
cloning site present in plasmid pCMVLacLOPRSVI.cass (FIG. 26).
EXAMPLE 8
Synthetic Genes and Genetic Constructs Comprising the lacI and
Green Fluorescent Protein (GFP) and Tyrosinase Open Reading
Frames
[0203] Plasmid pCMVLacI.TYR.OPRSV1.GFP
[0204] Plasmid pCMVLacI.TYR.OPRSV1.GFP (FIG. 28) is a dual
construct in which the CMV IE promoter drives expression of the
lacI gene and the mRNA of the mouse tyrosinase cDNA or a fragment
thereof, whilst the OPRSVI promoter drives expression of GFP
operably under control of the lacI gene. The construct is designed
such that the mouse tyrosinase gene is fused to the 3 '
untranslated region of the lacI gene via a unique BsaB1 cloning
site. This cloning site is located after the stop codon of the lacI
coding sequence, but before the SV40 polyadenylation signal The
construct also contains the hygromycin-resistance gene as a
selection marker.
[0205] To produce plasmid pCMVLacI.TYR.OPRSV1.GFP, the complete
tyrosinase gene present in plasmid pCR tyr (Stratagene; Example 1)
is isolated from host cells, digested with SmaI and ligated into
BsaB1-digested and dephosphotylated plasmid
pCMVLacI.OPRSVI.GFP.cass DNA (FIG. 27).
Sequence CWU 1
1
10 1 38 DNA Artificial Sequence Primer Bev-1 1 cggcagatct
aacaatggca ggacaaatcg agtacatc 38 2 31 DNA Artificial Sequence
Primer Bev-2 2 cccgggatcc tcgaaagaat cgtaccactt c 31 3 29 DNA
Artificial Sequence Primer Bev-3 3 gggcggatcc ttagaaagaa tcgtaccac
29 4 28 DNA Artificial Sequence Primer Bev-4 4 cggcagatct
ggacaaatcg agtacatc 28 5 26 DNA Artificial Sequence Primer Bgl-GFP
5 agatctgtaa acggccacaa gttcag 26 6 26 DNA Artificial Sequence
Primer GFP-Bam 6 ggatccttgt acagctcgtc catgcc 26 7 74 DNA
Artificial Sequence Primer SV40-1 7 gtcgacaata aaatatcttt
attttcatta catctgtgtg ttggtttttt gtgtgatttt 60 tgcaaaagcc tagg 74 8
31 DNA Artificial Sequence Primer SV40-2 8 gtcgacgttt agagcagaag
taacacttcc g 31 9 30 DNA Artificial Sequence Forward Primer Tyr 5'
9 cccggggctt agtgtaaaac aggctgagag 30 10 31 DNA Artificial Sequence
Reverse Primer Tyr 3' 10 cccgggcaaa tcccagtcat ttcttagaaa c 31
* * * * *