U.S. patent application number 13/458704 was filed with the patent office on 2012-11-01 for control of gene expression.
This patent application is currently assigned to Commonwealth Scientific and Industrial Research Organisation. Invention is credited to Michael Wayne Graham, Robert Norman Rice.
Application Number | 20120277285 13/458704 |
Document ID | / |
Family ID | 25645735 |
Filed Date | 2012-11-01 |
United States Patent
Application |
20120277285 |
Kind Code |
A1 |
Graham; Michael Wayne ; et
al. |
November 1, 2012 |
CONTROL OF GENE EXPRESSION
Abstract
The present invention relates generally to a method of modifying
gene expression and 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 utilizes recombinant DNA
technology to post-transcriptionally modify or modulate the
expression of a target gene in a cell, tissue organ or whole
organism, thereby producing novel phenotypes. Novel synthetic genes
and genetic constructs which are capable of repressing delaying or
otherwise reducing the expression of an endogenous gene or target
gene in an organism when introduced thereto are also provided.
Inventors: |
Graham; Michael Wayne;
(Chapel Hill, AU) ; Rice; Robert Norman;
(Queensland, AU) |
Assignee: |
Commonwealth Scientific and
Industrial Research Organisation
|
Family ID: |
25645735 |
Appl. No.: |
13/458704 |
Filed: |
April 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11218999 |
Sep 2, 2005 |
8168774 |
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13458704 |
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09646807 |
Dec 5, 2000 |
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PCT/AU99/00195 |
Mar 19, 1999 |
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11218999 |
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09100812 |
Jun 19, 1998 |
6573099 |
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09646807 |
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09100813 |
Jun 19, 1998 |
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09100812 |
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Current U.S.
Class: |
514/44A ;
435/320.1; 435/375; 536/23.5 |
Current CPC
Class: |
C12N 2830/00 20130101;
C12N 9/127 20130101; C12N 15/113 20130101; A61P 31/04 20180101;
A01K 2217/05 20130101; C12N 2310/111 20130101; C12N 15/69 20130101;
C12N 2310/14 20130101; A61P 31/12 20180101; C12N 9/503 20130101;
C12N 2330/30 20130101; C12N 2830/002 20130101; A61K 48/00 20130101;
C12N 2840/20 20130101; C12N 15/8218 20130101; C12N 2310/531
20130101; C12N 15/8216 20130101; C12N 2330/50 20130101; C12N 15/111
20130101; C12N 2800/108 20130101; C12N 2330/51 20130101; C12N
2320/30 20130101; C12N 15/1131 20130101; C12N 2830/42 20130101;
C12N 2830/55 20130101; C12N 2830/60 20130101; C12N 2830/15
20130101; A61P 43/00 20180101; C12N 15/8283 20130101; C12N 9/1051
20130101; C12N 15/63 20130101; C12N 2320/10 20130101; C12N 15/85
20130101; C12N 2830/38 20130101 |
Class at
Publication: |
514/44.A ;
435/375; 435/320.1; 536/23.5 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; C12N 15/85 20060101 C12N015/85; C07H 21/04 20060101
C07H021/04; C12N 5/07 20100101 C12N005/07 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 20, 1998 |
AU |
PP2492 |
Mar 20, 1998 |
AU |
PP2499 |
Claims
1-43. (canceled)
44. A method of repressing, delaying or otherwise reducing the
expression of a target gene in a cell, tissue or organ, said method
comprising introducing to said cell, tissue or organ one or more
dispersed nucleic acid molecules or foreign nucleic acid molecules
comprising multiple copies of a nucleotide sequence which is
substantially identical to the nucleotide sequence of said target
gene or a region thereof or complementary thereto for a time and
under conditions sufficient for translation of the mRNA product of
said target gene to be modified, subject to the proviso that the
transcription of said mRNA product is not exclusively repressed or
reduced.
45. The method according to claim 44, wherein the dispersed nucleic
acid molecules or foreign nucleic acid molecules comprise inverted
repeats of the target gene sequence or a region thereof or
complementary thereto.
46. The method according to claim 44, wherein the number of copies
of the target gene sequence or region thereof or complementary
thereto in the dispersed nucleic acid molecule or foreign nucleic
acid molecule is two.
47. The method according to claim 44, wherein the ce tissue or
organ is an animal cell, tissue or organ.
48. The method according to claim 44, wherein the target gene is a
gene which is contained within the genome of the cell, tissue or
organ.
49. The method according to claim 44, wherein the target gene is
derived from the genome of a pathogen of the cell, tissue or organ
or an organism comprising said cell, tissue or organ.
50. The method according to claim 49, wherein the pathogen is a
virus.
51. The method according to claim 50, wherein the virus is an
animal pathogen.
52. The method according to claim 44, further comprising selecting
the dispersed nucleic acid molecule(s) or foreign nucleic acid
molecule(s) according to their ability to effectively modulate
expression of the target gene.
53. A method of conferring resistance or immunity to a viral
pathogen upon a cell, tissue, organ or whole organism, comprising
introducing one or more dispersed nucleic acid molecules or foreign
nucleic acid molecules which comprise inverted repeats of a
nucleotide sequence derived from the viral pathogen or a
complementary sequence thereto for a time and under conditions
sufficient for translation of the mRNA product of a virus gene to
be delayed or otherwise reduced, subject to the proviso that the
transcription of said mRNA product is not exclusively repressed or
reduced.
54. The method according to claim 53, wherein the virus is an
animal pathogen.
55. The method according to claim 53, further comprising selecting
the dispersed nucleic acid molecule(s) or foreign nucleic acid
molecule(s) according to their ability to confer resistance or
immunity on the cell, tissue, organ or organism.
56. The method according to claim 53, wherein the dispersed nucleic
acid molecules or foreign nucleic acid molecules comprise multiple
copies of nucleotide sequence encoding a viral replicase,
polymerase, coat protein or uncoating gene.
57. The method according to claim 53, wherein the dispersed nucleic
acid molecules or foreign nucleic acid molecules comprise multiple
copies of nucleotide sequence encoding a viral polymerase.
58. The method according to claim 53, wherein the dispersed nucleic
acid molecules or foreign nucleic acid molecules comprise multiple
copies of nucleotide sequence encoding a viral coat protein.
59. A synthetic gene which is capable of repressing, delaying or
otherwise reducing the expression of a target gene in a cell,
tissue, organ or whole organism, wherein said synthetic gene
comprises a dispersed nucleic acid molecule or a foreign nucleic
acid molecule comprising multiple copies of 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, organ or whole
organism.
60. The synthetic gene according to claim 59, wherein the dispersed
nucleic acid molecule or a foreign nucleic acid molecule comprises
inverted repeats of a genetic sequence that is endogenous to the
genome of the cell, tissue, organ or organism or which is derived
from a non-endogenous gene of the cell, tissue, organ or
organism.
61. The synthetic gene according to claim 60, wherein the
non-endogenous gene is derived from a viral pathogen of the cell,
tissue, organ or organism.
62. The synthetic gene according to claim 61, wherein the
non-endogenous gene is derived from an animal virus.
63. A genetic construct comprising the synthetic gene according to
claim 59.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to a method of
modifying gene expression and 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 utilises recombinant DNA
technology to post-transcriptionally modify or modulate the
expression of a target gene in a cell, tissue, organ or whole
organism, thereby producing novel phenotypes. 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 are also provided.
GENERAL
[0002] Bibliographic details of the publications referred to in
this specification are collected at the end of the description.
[0003] As used herein the term "derived from" shall be taken to
indicate that a specified integer may be obtained from a particular
specified source or species, albeit not necessarily directly from
that specified source or species.
[0004] Throughout this specification, 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 step or element or integer or group of steps or elements or
integers but not the exclusion of any other step or element or
integer or group of elements or integers.
[0005] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations or any two or more of said steps or features.
[0006] The present invention is not to be limited in scope by the
specific embodiments described herein, which are intended for the
purposes of exemplification only. Functionally-equivalent products,
compositions and methods are clearly within the scope of the
invention, as described herein.
[0007] Sequence identity numbers (SEQ ID NOS.) containing
nucleotide and amino acid sequence information included in this
specification are collected after the Abstract and have been
prepared using the programme PatentIn Version 2.0. Each nucleotide
or amino acid sequence is identified in the sequence listing by the
numeric indicator <210> followed by the sequence identifier
(e.g. <210>1, <210>2, etc). The length, type of
sequence (DNA, protein (PRT), etc) and source organism for each
nucleotide or amino acid sequence are indicated by information
provided in the numeric indicator fields <211>, <212>
and <213>, respectively. Nucleotide and amino acid sequences
referred to in the specification are defined by the information
provided in numeric indicator field <400> followed by the
sequence identifier (eg. <400>1, <400>2, etc).
[0008] The designation of nucleotide residues referred to herein
are those recommended by the IUPAC-IUB Biochemical Nomenclature
Commission, wherein A represents Adenine, C represents Cytosine, G
represents Guanine, T represents thymine, Y represents a pyrimidine
residue, R represents a purine residue, M represents Adenine or
Cytosine, K represents Guanine or Thymine, S represents Guanine or
Cytosine, W represents Adenine or Thymine, H represents a
nucleotide other than Guanine, B represents a nucleotide other than
Adenine, V represents a nucleotide other than Thymine, D represents
a nucleotide other than Cytosine and N represents any nucleotide
residue.
[0009] The designation of amino acid residues referred to herein,
as recommended by the IUPAC-IUB Biochemical Nomenclature
Commission, are listed in Table 1.
TABLE-US-00001 TABLE 1 Amino Acid Three-letter code One-letter code
Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D
Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G
Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K
Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S
Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
Aspartate/Asparagine Baa B Glutamate/Glutamine Zaa Z Any amino acid
Xaa X
BACKGROUND TO THE INVENTION
[0010] 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 modulation of the level of eukaryotic
gene expression.
[0011] One approach to repressing, delaying or otherwise reducing
gene expression utilise a mRNA molecule which is transcribed from
the complementary strand of a nuclear gene to that which is
normally transcribed and capable of being translated into a
polypeptide. Although the precise mechanism invoked in this
approach is not established, it has been postulated that a
double-stranded mRNA may form by base pairing between the
complementary nucleotide sequences, to produce a complex which is
translated at low efficiency and/or degraded by intracellular
ribonuclease enzymes prior to being translated.
[0012] Alternatively, the expression of an endogenous gene in a
cell, tissue or organ may be suppressed when one or more copies of
said gene, or one or more copies of a substantially similar gene
are introduced into the cell. Whilst the mechanism involved in this
phenomenon has not been established and appears to be involve
mechanistically heterogeneous processes. For example, this approach
has been postulated to involve transcriptional repression, in which
case somatically-heritable repressed states of chromatin are formed
or alternatively, a post-transcriptional silencing wherein
transcription initiation occurs normally but the RNA products of
the co-suppressed genes are subsequently eliminated.
[0013] The efficiency of both of these approaches in targeting the
expression of specific genes is very low and highly variable
results are usually obtained. Inconsistent results are obtained
using different regions of genes, for example 5'-untranslated
regions, 3'-untranslated regions, coding regions or intron
sequences to target gene expression. Accordingly, there currently
exists no consensus as to the nature of genetic sequences which
provide the most efficient means for repressing, delaying or
otherwise reducing gene expression using existing technologies.
Moreover, such a high degree of variation exists between
generations such that it is not possible to predict the level of
repression of a specific gene in the progeny of an organism in
which gene expression was markedly modified.
[0014] Recently, Dorer and Henikoff (1994) demonstrated the
silencing of tandemly repeated gene copies in the Drosophila genome
and the transcriptional repression of dispersed Drosophila Adh
genes by Polycomb genes (i.e. the Pc-G system; Pal-Bhadra et al,
1997). However, such silencing of tandemly repeated gene copies is
of little utility in an attempt to manipulate gene expression in an
animal cell by recombinant means, wherein the sequences capable of
targeting the expression of a particular gene are introduced at
dispersed locations in the genome, absent the combination of this
approach with gene-targeting technology. Whilst theoretically
possible, such combinations would be expected to work at only
low-efficiency, based upon the low efficiency of gene-targeting
approaches used in isolation and further, would require complicated
vector systems. Additionally, the utilisation of transcriptional
repression, such as the Drosophila Pc-G system, would appear to
require some knowledge of the regulatory mechanisms capable of
modulating the expression of any specific target gene and, as a
consequence, would be difficult to implement in practice as a
general technology for repressing, delaying or reducing gene
expression in animal cells.
[0015] The poor understanding of the mechanisms involved in these
phenomena has meant that there have been few improvements in
technologies for modulating the level of gene expression, in
particular technologies for delaying, repressing or otherwise
reducing the expression of specific genes using recombinant DNA
technology.
[0016] Furthermore, as a consequence of the unpredictability of
these approaches, there is currently no commercially-viable means
for modulating the level of expression of a specific gene in a
eukaryotic or prokaryotic organism.
[0017] Thus, there exists a need for improved methods of modulating
gene expression, in particular repressing, delaying or otherwise
reducing gene expression in animal cells for the purpose of
introducing novel phenotypic traits thereto. In particular, these
methods should provide general means for phenotypic modification,
without the necessity for performing concomitant gene-targeting
approaches.
SUMMARY OF THE INVENTION
[0018] The invention is based in part on the surprising discovery
by the inventors that cells which exhibit one or more desired
traits can be produced and selected from transformed cells
comprising a nucleic acid molecule operably linked to a promoter,
wherein the transcription product of the nucleic acid molecule
comprises a nucleotide sequence which is substantially identical to
the nucleotide sequence of a transcript of an endogenous or
non-endogenous target gene, the expression of which is intended to
be modulated. The transformed cells are regenerated into whole
tissues, organs or organisms capable of exhibiting novel traits, in
particular virus resistance and modified expression of endogenous
genes.
[0019] Accordingly, one aspect of the present invention provides a
method of modulating the expression of a target gene in an animal
cell, tissue or organ, said method at least comprising the step of
introducing to said cell, tissue or organ one or more dispersed
nucleic acid molecules or foreign nucleic acid molecules comprising
multiple copies of a nucleotide sequence which is substantially
identical to the nucleotide sequence of said target gene or a
region thereof or complementary thereto for a time and under
conditions sufficient for translation of the mRNA product of said
target gene to be modified, subject to the proviso that the
transcription of said mRNA product is not exclusively repressed or
reduced.
[0020] In a particularly preferred embodiment, the dispersed
nucleic acid molecules or foreign nucleic acid molecules comprises
a nucleotide sequence which encodes multiple copies of an mRNA
molecule which is substantially identical to the nucleotide
sequence of the mRNA product of the target gene. More preferably,
the multiple copies of the target molecule are tandem direct repeat
sequences.
[0021] In a more particularly preferred embodiment, the dispersed
nucleic acid molecule or foreign nucleic acid molecule is in an
expressible form such that it is at least capable of being
transcribed to produce mRNA.
[0022] The target gene may be a gene which is endogenous to the
animal cell or alternatively, a foreign gene such as a viral or
foreign genetic sequence, amongst others. Preferably, the target
gene is a viral genetic sequence.
[0023] The invention is particutarty useful in the modulation of
eukaryotic gene expression, in particular the modulation of human
or animal gene expression and even more particularly in the
modulation of expression of genes derived from vertebrate and
invertebrate animals, such as insects, aquatic animals (eg. fish,
shellfish, molluscs, crustaceans such as crabs, lobsters and
prawns, avian animals and mammals, amongst others).
[0024] A variety of traits are selectable with appropriate
procedures and sufficient numbers of transformed cells. Such traits
include, but are not limited to, visible traits, disease-resistance
traits, and pathogen-resistance traits. The modulatory effect is
applicable to a variety of genes expressed in plants and animals
including, for example, endogenous genes responsible for cellular
metabolism or cellular transformation, including oncogenes,
transcription factors and other genes which encode polypeptides
involved in cellular metabolism.
[0025] For example, an alteration in the pigment production in mice
can be engineered by targeting the expression of the tyrosinase
gene therein. This provides a novel phenotype of albinism in black
mice. By targeting genes required for virus replication in a plant
cell or an animal cell, a genetic construct which comprises
multiple copies of nucleotide sequence encoding a viral replicase,
polymerase, coat protein or uncoating gene, or protease protein,
may be introduced into a cell where it is expressed, to confer
immunity against the virus upon the cell.
[0026] In performance of the present invention, the dispersed
nucleic acid molecule or foreign nucleic acid molecule will
generally comprise a nucleotide sequence having greater than about
85% identity to the target gene sequence, however, a higher
homology might produce a more effective modulation of expression of
the target gene sequence. Substantially greater-homology, or more
than about 90% is preferred, and even more preferably about 95% to
absolute identity is desirable.
[0027] The introduced dispersed nucleic acid molecule or foreign
nucleic acid molecule sequence, needing less than absolute
homology, also need not be full length, relative to either the
primary transcription product or fully processed mRNA of the target
gene. A higher homology in a shorter than full length sequence
compensates for a longer less homologous sequence. Furthermore, the
introduced sequence need not have the same intron or exon pattern,
and homology of non-coding segments will be equally effective.
Normally, a sequence of greater than 20-100 nucleotides should be
used, though a sequence of greater than about 200-300 nucleotides
would be preferred, and a sequence of greater than 500-1000
nucleotides would be especially preferred depending on the size of
the target gene.
[0028] A second 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 dispersed nucleic acid
molecular foreign nucleic acid molecule comprising multiple copies
of 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.
[0029] A third 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 are placed operably under the control of a single
promoter sequence which is operable in said cell, tissue or
organ.
[0030] A fourth 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 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.
[0031] A fifth 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.
[0032] In order to observe many novel traits in multicellular
organisms such as plants and animals, in particular those which are
tissue-specific or organ-specific or developmentally-regulated,
regeneration of a transformed cell carrying the synthetic genes and
genetic constructs described herein into a whole organism will be
required. Those skilled in the art will be aware that this means
growing a whole organism from a transformed plant cell or animal
cell, a group of such cells, a tissue or organ. Standard methods
for the regeneration of certain plants and animals from isolated
cells and tissues are known to those skilled in the art.
[0033] Accordingly, a sixth 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
[0034] FIG. 1 is a diagrammatic representation of the plasmid
pEGFP-N1 MCS.
[0035] FIG. 2 is a diagrammatic representation of the plasmid
pCMV.cass.
[0036] FIG. 3 is a diagrammatic representation of the plasmid
pCMV.SV40L.cass.
[0037] FIG. 4 is a diagrammatic representation of the plasmid
pCMV.SV40LR.cass.
[0038] FIG. 5 is a diagrammatic representation of the plasmid
pCR.Bgl-GFP-Bam.
[0039] FIG. 6 is a diagrammatic representation of the plasmid
pBSII(SK+).EGFP.
[0040] FIG. 7 is a diagrammatic representation of the plasmid
pCMV.EGFP.
[0041] FIG. 8 is a diagrammatic representation of the plasmid
pCR.SV40L.
[0042] FIG. 9 is a diagrammatic representation of the plasmid
pCR.BEV.1.
[0043] FIG. 10 is a diagrammatic represenation of the plasmid
pCR.BEV.2.
[0044] FIG. 11 is a diagrammatic representation of the plasmid
pCR.BEV.3.
[0045] FIG. 12 is a diagrammatic representation of the plasmid
pCMV.EGFP.BEV2.
[0046] FIG. 13 is a diagrammatic representation of the plasmid
pCMV.BEV.2.
[0047] FIG. 14 is a diagrammatic representation of the plasmid
pCMV.BEV.3.
[0048] FIG. 15 is a diagrammatic representation of the plasmid
pCMV.VEB.
[0049] FIG. 16 is a diagrammatic representation of the plasmid
pCMV.BEV.GFP.
[0050] FIG. 17 is a diagrammatic representation of the plasmid
pCMV.BEV.SV40L-0.
[0051] FIG. 18 is a diagrammatic representation of the plasmid
pCMV.0.SV40L.BEV.
[0052] FIG. 19 is a diagrammatic representation of the plasmid
pCMV.0.SV40L.VEB.
[0053] FIG. 20 is a diagrammatic representation of the plasmid
pCMV.BEVx2.
[0054] FIG. 21 is a diagrammatic representation of the plasmid
pCMV.BEVx3.
[0055] FIG. 22 is a diagrammatic representation of the plasmid
pCMV.BEVx4.
[0056] FIG. 23 is a diagrammatic representation of the plasmid
pCMV.BEV.SV40L.BEV.
[0057] FIG. 24 is a diagrammatic representation of the plasmid
pCMV.BEV.SV40L.VEB.
[0058] FIG. 25 is a diagrammatic representation of the plasmid
pCMV.BEV.GFP.VEB.
[0059] FIG. 26 is a diagrammatic representation of the plasmid
pCMV.EGFP.BEV2.PFG.
[0060] FIG. 27 is a diagrammatic representation of the plasmid
pCMV.BEVSV40LR.
[0061] FIG. 28 is a diagrammatic representation of the plasmid
pCDNA3.Galt.
[0062] FIG. 29 is a diagrammatic representation of the plasmid
pCMV.Galt.
[0063] FIG. 30 is a diagrammatic representation of the plasmid
pCMV.EGFP.Galt.
[0064] FIG. 31 is a diagrammatic representation of the plasmid
pCMV.Galt.GFP.
[0065] FIG. 32 is a diagrammatic representation of the plasmid
pCMV.Galt.SV40L.O.
[0066] FIG. 33 is a diagrammatic representation of the plasmid
pCMV.GaILSV40L.tlaG.
[0067] FIG. 34 is a diagrammatic representation of the plasmid
pCMV.0.SV40L.Galt.
[0068] FIG. 35 is a diagrammatic representation of the plasmid
pCMV.Galtx2.
[0069] FIG. 36 is a diagrammatic representation of the plasmid
pCMV.Galtx4.
[0070] FIG. 37 is a diagrammatic representation of the plasmid
pCMV.Galt.SV40L.Galt.
[0071] FIG. 38 is a diagrammatic representation of the plasmid
pCMV.Galt.SV40L.tlaG.
[0072] FIG. 39 is a diagrammatic representation of the plasmid
pCMV.Galt.GFP.tlaG.
[0073] FIG. 40 is a diagrammatic representation of the plasmid
pCMVEGFP.Galt.PFG.
[0074] FIG. 41 is a diagrammatic representation of the plasmid
pCMV.Galt.SV40LR.
[0075] FIG. 42 is a diagrammatic representation of the plasmid
pART7.
[0076] FIG. 43 is a diagrammatic representation of the plasmid
pART7.35S.SCBV.cass.
[0077] FIG. 44 is a diagrammatic representation of the plasmid
pBC.PVY.
[0078] FIG. 45 is a diagrammatic representation of the plasmid
pSP72.PVY.
[0079] FIG. 46 is a diagrammatic representation of the plasmid
pClapBC.PVY.
[0080] FIG. 47 is a diagrammatic representation of the plasmid
pBC.PVYx2.
[0081] FIG. 48 is a diagrammatic representation of the plasmid
pSP72.PVYx2.
[0082] FIG. 49 is a diagrammatic representation of the plasmid
pBC.PVYx3.
[0083] FIG. 50 is a diagrammatic representation of the plasmid
pBC.PVYx4.
[0084] FIG. 51 is a diagrammatic representation of the plasmid
pBC.PVY.LNYV.
[0085] FIG. 52 is a diagrammatic representation of the plasmid
pBC.PVY.LNYV.PVY.
[0086] FIG. 53 is a diagrammatic representation of the plasmid
pBC.PVY.LNYV.YVP4.
[0087] FIG. 54 is a diagrammatic representation of the plasmid
pBC.PVY.LNYVYVP.
[0088] FIG. 55 is a diagrammatic representation of the ptasmid
pART27.PVY
[0089] FIG. 56 is a diagrammatic representation of the plasmid
pART27.35S.PVY.SCBV.O.
[0090] FIG. 57 is a diagrammatic representation of the plasmid
pART27.35S.O.SCBV.P.VY.
[0091] FIG. 58 is a diagrammatic representation of the plasmid
pART27.35S.O.SCBV.YVP.
[0092] FIG. 59 is a diagrammatic representation of the plasmid
pART7.PVYx2.
[0093] FIG. 60 is a diagrammatic representation of the plasmid
pART7.PVYx3.
[0094] FIG. 61 is a diagrammatic representation of the plasmid
pART7.PVYx4.
[0095] FIG. 62 is a diagrammatic representation of the plasmid
pART7.PVY.LNYV.PVY.
[0096] FIG. 63 is a diagrammatic representation of the plasmid
pART7.PVY.LNYV.YVP6.
[0097] FIG. 64 is a diagrammatic representation of the plasmid
pART7.PVY.LNYV.YVP.
[0098] FIG. 65 is a diagrammatic representation of
pART7.35S.PVY.SCBV.YVP.
[0099] FIG. 66 is a diagrammatic representation of
pART7.35S.PVYx3.SCBV.YVPx3.
[0100] FIG. 67 is a diagrammatic representation of
pART7.PVYx3.LNYVYVPx3.
[0101] FIG. 68 is a diagrammatic representation of the plasmid
pART7.PVYMULTI.
DETAILED DESCRIPTION OF THE INVENTION
[0102] The present invention provides a method of modulating the
expression of a target gene in a cell, tissue or organ, said method
at least comprising the step of introducing to said cell, tissue or
organ one or more dispersed nucleic acid molecules or foreign
nucleic acid molecules comprising multiple copies of a nucleotide
sequence which is substantially identical to the nucleotide
sequence of said target gene or a region thereof or complementary
thereto for a time and under conditions sufficient for translation
of the mRNA product of said target gene to be modified, subject to
the proviso that the transcription of said mRNA product is not
exclusively repressed or reduced.
[0103] By "multiple copies" is meant that two or more copies of the
target gene are presented in close physical connection or
juxtaposed, in the same or different orientation, on the same
nucleic acid molecule, optionally separated by a stuffer fragment
or intergenic region to facilitate secondary structure formation
between each repeat where this is required. 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.
[0104] Preferably, embodiment, the stuffer fragment comprises a
sequence of nucleotides or a homologue, analogue or derivative
thereof.
[0105] 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.
[0106] Wherein the dispersed or foreign nucleic acid molecule
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 unit thereof.
Alternatively, wherein it is desirable for more than two adjacent
nucleotide sequence units of the dispersed foreign nucleic 5 acid
molecule 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 unit, as will be obvious to those skilled in the
art.
[0107] 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 acetyltransferase or green
fluorescent protein, amongst others. Additional stuffer fragments
are not excluded.
[0108] 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.
[0109] As used herein, the term "modulating" shall be taken to mean
that expression of the target gene is reduced in amplitude and/or
the timing of gene expression is delayed and/or the developmental
or tissue-specific or cell-specific pattern of target gene
expression is altered, compared to the expression of said gene in
the absence of the inventive method described herein.
[0110] Whilst not limiting the scope of the invention described
herein, the present invention is directed to a modulation of gene
expression which comprises the repression, delay or reduction in
amplitude of target gene expression in a specified cell, tissue or
organ of a eukaryotic organism, in particular a plant such as a
monocotyledonous or dicotyledonous plant, or a human or other
animal and even more particularly a vertebrate and invertebrate
animal, such as an insect, aquatic animal (eg. fish, shellfish,
mollusc, crustacean such as a crab, lobster or prawn, an avian
animal or a mammal, amongst others).
[0111] More preferably, target gene expression is completely
inactivated by the dispersed nucleic acid molecules or foreign
nucleic acid molecules which has been introduced to the cell,
tissue or organ.
[0112] Whilst not being bound by any theory or mode of action, the
reduced or eliminated expression of the target gene which results
from the performance of the invention may be attributed to reduced
or delayed translation of the mRNA transcription product of the
target gene or alternatively, the prevention of translation of said
mRNA, as a consequence of sequence-specific degradation of the mRNA
transcript of the target gene by an endogenous host cell
system.
[0113] It is particularly preferred that, for optimum results,
sequence-specific degradation of the mRNA transcript of the target
gene occurs either prior to the time or stage when the mRNA
transcript of the target gene would normally be translated or
alternatively, at the same time as the mRNA transcript of the
target gene would normally be translated. Accordingly, the
selection of an appropriate promoter sequence to regulate
expression of the introduced dispersed nucleic acid molecule or
foreign nucleic acid molecule is an important consideration to
optimum performance of the invention. For this reason, strong
constitutive promoters or inducible promoter systems are especially
preferred for use in regulating expression of the introduced
dispersed nucleic acid molecules or foreign nucleic acid
molecules.
[0114] The present invention clearly encompasses reduced expression
wherein reduced expression of the target gene is effected by
lowered transcription, subject to the proviso that a reduction in
transcription is not the sole mechanism by which this occurs and
said reduction in transcription is at least accompanied by reduced
translation of the steady-state mRNA pool.
[0115] The target gene may be a genetic sequence which is
endogenous to the animal cell or alternatively, a non-endogenous
genetic sequence, such as a genetic sequence which is derived from
a virus or other foreign pathogenic organism and is capable of
entering a cell and using the cell's machinery following
infection.
[0116] Wherein the target gene is a non-endogenous genetic sequence
to the animal cell, it is desirable that the target gene encodes a
function which is essential for replication or reproduction of the
viral or other pathogen. In such embodiments, the present invention
is particularly useful in the prophylactic and therapeutic
treatment of viral infection of an animal cell or for conferring or
stimulating resistance against said pathogen.
[0117] Preferably, the target gene comprises one or more nucleotide
sequences of a viral pathogen of a plant or an animal cell, tissue
or organ.
[0118] For example, in the case of animals and humans, the viral
pathogen may be a retrovirus, for example a lentivirus such as the
immunodeficiency viruses, a single-stranded (+) RNA virus such as
bovine enterovirus (BEV) or Sinbis alphavirus. Alternatively, the
target gene can comprise one or more nucleotide sequences of a
viral pathogen of an animal cell, tissue or organ, such as but not
limited to a double-stranded DNA virus such as bovine herpes virus
or herpes simplex virus I (HSV I), amongst others.
[0119] In the case of plants, the virus pathogen is preferably a
potyvirus, caulimovirus, badnavirus, geminivirus, reovirus,
rhabdovirus, bunyavirus, tospovirus, tenuivirus, tombusvirus,
luteovirus, sobemovirus, bromovirus, cucomovirus, ilavirus,
alfamovirus, tobamovirus, tobravirus, potexvirus and clostrovirus,
such as but not limited to CaMV, SCSV, PVX, PVY, PLRV, and TMV,
amongst others.
[0120] With particular regard to viral pathogens, those skilled in
the art are aware that virus-encoded functions may be complemented
in trans by polypeptides encoded by the host cell. For example, the
replication of the bovine herpes virus genome in the host cell may
be facilitated by host cell DNA polymerases which are capable of
complementing an inactivated viral DNA polymerase gene.
[0121] Accordingly, wherein the target gene is a non-endogenous
genetic sequence to the animal cell, a further alternative
embodiment of the invention provides for the target gene to encode
a viral or foreign polypeptide which is not capable of being
complemented by a host cell function, such as a virus-specific
genetic sequence. Exemplary target genes according to this
embodiment of the invention include, but are not limited to genes
which encode virus coat proteins, uncoating proteins and
RNA-dependent DNA polymerases and RNA-dependent RNA polymerases,
amongst others.
[0122] In a particularly preferred embodiment of the present
invention, the target gene is the BEV RNA-dependent RNA polymerase
gene or a homologue, analogue or derivative thereof or PVY Nia
protease-encoding sequences.
[0123] The cell in which expression of the target gene is modified
may be any cell which is derived from a multicellular plant or
animal, including cell and tissue cultures thereof. Preferably, the
animal cell is derived from an insect, reptile, amphibian, bird,
human or other mammal. Exemplary animal cells include embryonic
stem cells, cultured skin fibroblasts, neuronal cells, somatic
cells, haematopoietic stem cells, T-cells and immortalised cell
lines such as COS, VERO, HeLa, mouse C127, Chinese hamster ovary
(CHO), WI-38, baby hamster kidney (BHK) or MDBK cell lines, amongst
others. Such cells and cell lines are readily available to those
skilled in the art. Accordingly, the tissue or organ in which
expression of the target gene is modified may be any tissue or
organ comprising such animal cells.
[0124] Preferably the plant cell is derived from a monocotyledonous
or dicotyledonous plant species or a cell line derived
therefrom.
[0125] As used herein, the term "dispersed nucleic acid molecule
shall be taken to refer to a nucleic acid molecule which comprises
one or more multiple copies, preferably tandem direct repeats, of a
nucleotide sequence which is substantially identical or
complementary to the nucleotide sequence of a gene which originates
from the cell, tissue or organ into which said nucleic acid
molecule is introduced, wherein said nucleic acid molecule is
non-endogenous in the sense that it is introduced to the cell,
tissue or organ of an animal via recombinant means and will
generally be present as extrachromosomal nucleic acid or
alternatively, as integrated chromosomal nucleic acid which is
genetically-unlinked to said gene. More particularly, the
"dispersed nucleic acid molecule" will comprise chromosomal or
extrachromosomal nucleic acid which is unlinked to the target gene
against which it is directed in a physical map, by virtue of their
not being tandemly-linked or alternatively, occupying a different
chromosomal position on the same chromosome or being localised on a
different chromosome or present in the cell as an episome, plasmid,
cosmid or virus particle,
[0126] By "foreign nucleic acid molecule" is meant an isolated
nucleic acid molecule which has one or more multiple copies,
preferably tandem direct repeats, of a nucleotide sequence which
originates from the genetic sequence of an organism which is
different from the organism to which the foreign nucleic acid
molecule is introduced. This definition encompasses a nucleic acid
molecule which originates from a different individual of the same
lowest taxonomic grouping (i.e. the same population) as the
taxonomic grouping to which said nucleic acid molecule is
introduced, as well as a nucleic acid molecule which originates
from a different individual of a different taxonomic grouping as
the taxonomic grouping to which said nucleic acid molecule is
introduced, such as a gene derived from a viral pathogen.
[0127] Accordingly, a target gene against which a foreign nucleic
acid molecule acts in the performance of the invention may be a
nucleic acid molecule which has been introduced from one organism
to another organism using transformation or introgression
technologies. Exemplary target genes according to this embodiment
of the invention include the green fluorescent protein encoding
gene derived from the jellyfish Aequoria victoria (Prasher et al.,
1992; International Patent Publication No. WO 95/07463), tyrosinase
genes and in particular the murine tyrosinase gene (Kwon et al.,
1988), the Escherichia coli lacI gene which is capable of encoding
a polypeptide repressor of the lacZ gene, the porcine
.alpha.-1,3-galactosyltransferase gene (NCBI Accession No. L36535)
exemplified herein, and the PW and BEV structural genes exemplified
herein , or a homologue, analogue or derivative of said genes or a
complementary nucleotide sequence thereto.
[0128] The present invention is further useful for simultaneously
targeting the expression of several target genes which are
co-expressed in a particular cell, for example by using a dispersed
nucleic acid molecule or foreign nucleic acid molecule which
comprises nucleotide sequences which are substantially identical to
each of said co-expressed target genes.
[0129] By "substantially identical" is meant that the introduced
dispersed or foreign nucleic acid molecule of the invention and the
target gene sequence are sufficiently identical at the nucleotide
sequence level to permit base-pairing there between under standard
intracellular conditions.
[0130] Preferably, the nucleotide sequence of each repeat in the
dispersed or foreign nucleic acid molecule of the invention and the
nucleotide sequence of a part of the target gene sequence are at
least about 80-85% identical at the nucleotide sequence level, more
preferably at least about 85-90% identical, even more preferably at
least about 90-95% identical and still even more preferably at
least about 95-99% or 100% identical at the nucleotide sequence
level.
[0131] Notwithstanding that the present invention is not limited by
the precise number of repeated sequences in the dispersed nucleic
acid molecule or the foreign nucleic acid molecule of the
invention, it is to be understood that the present invention
requires at least two copies of the target gene sequence to be
expressed in the cell.
[0132] Preferably, the multiple copies of the target gene sequence
are presented in the dispersed nucleic acid molecule or the foreign
nucleic acid molecule as tandem inverted repeat sequences and/or
tandem direct repeat sequences. Such configurations are exemplified
by the "test plasmids" described herein that comprise Galt, BEV or
PVY gene regions.
[0133] Preferably, the dispersed or foreign nucleic acid molecule
which is introduced to the cell, tissue or organ comprises RNA or
DNA.
[0134] Preferably, the dispersed or foreign nucleic acid molecule
further comprises a nucleotide sequence or is complementary to a
nucleotide sequence which is capable of encoding an amino acid
sequence encoded by the target gene. Even more preferably, the
nucleic acid molecule includes one or more ATG or AUG translational
start codons.
[0135] Standard methods may be used to introduce the dispersed
nucleic acid molecule or foreign nucleic acid molecule into the
cell, tissue or organ for the purposes of modulating the expression
of the target gene. For.example, the nucleic acid molecule may be
introduced as naked DNA or RNA, optionally encapsulated in a
liposome, in a virus particle as attenuated virus or associated
with a virus coat or a transport protein or inert carrier such as
gold or as a recombinant viral vector or bacterial vector or as a
genetic construct, amongst others.
[0136] Administration means include injection and oral ingestion
(e.g. in medicated food material), amongst others.
[0137] The subject nucleic acid molecules may also be delivered by
a live delivery system such as using a bacterial expression system
optimised for their expression in bacteria which can be
incorporated into gut flora. Alternatively, a viral expression
system can be employed. In this regard, one form of viral
expression is the administration of a live vector generally by
spray, teed or water where an infecting effective amount of the
live vector (e.g. virus or bacterium) is provided to the animal.
Another form of viral expression system is a non-replicating virus
vector which is capable of infecting a cell but not replicating
therein. The non-replicating viral vector provides a means of
introducing to the human or animal subject genetic material for
transient expression therein. The mode of administering such a
vector is the same as a live viral vector.
[0138] The carriers, excipients and/or diluents utilised in
delivering the subject nucleic acid molecules to a host cell should
be acceptable for human or veterinary applications. Such carriers,
excipients and/or diluents are well-known to those skilled in the
art. Carriers and/or diluents suitable for veterinary use include
any and all solvents, dispersion media, aqueous solutions,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, use thereof in the composition is contemplated.
Supplementary active ingredients can also be incorporated into the
compositions.
[0139] In an alternative embodiment, the invention provides a
method of modulating the expression of a target gene in a cell,
tissue or organ, said method at least comprising the steps of:
[0140] (i) selecting one or more dispersed nucleic acid molecules
or foreign nucleic acid molecules which comprise multiple copies of
a nucleotide sequence which is substantially identical to the
nucleotide sequence of said target gene or a region thereof or
which is complementary thereto; and [0141] (ii) introducing said
dispersed nucleic acid molecules or foreign nucleic acid molecules
to said cell, tissue or organ for a time and under conditions
sufficient for translation of the mRNA product of said target gene
to be modified, subject to the proviso that the transcription of
said mRNA product is not exclusively repressed or reduced.
[0142] To select appropriate nucleotide sequences for targeting
expression of the target gene, several approaches may be employed.
In one embodiment, multiple copies of specific regions of
characterised genes may be cloned in operable connection with a
suitable promoter and assayed for efficacy in reducing target gene
expression. Alternatively, shotgun libraries comprising multiple
copies of genetic sequences may be produced and assayed for their
efficacy in reducing target gene expression. The advantage
associated with the latter approach is that it is not necessary to
have any prior knowledge of the significance of any particular
target gene in specifying an undesirable phenotype in the cell. For
example, shotgun libraries comprising virus sub-genomic fragments
may be employed and tested directly for their ability to confer
virus immunity on the animal host cell, without prior knowledge of
the role which any virus genes play in pathogenesis of the host
cell.
[0143] As used herein, the term "shotgun library" is a set of
diverse nucleotide sequences wherein each member of said set is
preferably contained within a suitable plasmid, cosmid,
bacteriophage or virus vector molecule which is suitable for
maintenance and/or replication in a cellular host. The term
"shotgun library" includes a representative library, in which the
extent of diversity between the nucleotide sequences is numerous
such that all sequences in the genome of the organism from which
said nucleotide sequences is derived are present in the "set" or
alternatively, a limited library in which there is a lesser degree
of diversity between said sequences. The term "shotgun library"
further encompasses random nucleotide sequences, wherein the
nucleotide sequence comprises viral or cellular genome fragments,
amongst others obtained for example by shearing or partial
digestion of genomic DMA using restriction endonucleases, amongst
other approaches. A "shotgun library" further includes cells, virus
particles and bacteriophage particles comprising the individual
nucleotide sequences of the diverse set,
[0144] Preferred shotgun libraries according to this embodiment of
the invention are "representative libraries", comprising a set of
tandem repeated nucleotide sequences derived from a viral pathogen
of a plant or an animal.
[0145] In a particularly preferred embodiment of the invention, the
shotgun library comprises cells, virus particles or bacteriophage
particles comprising a diverse set of tandem-repeated nucleotide
sequences which encode a diverse set of amino acid sequences,
wherein the member of said diverse set of nucleotide sequences are
placed operably under the control of a promoter sequence which is
capable of directing the expression of said tandem-repeated
nucleotide sequence in the cell.
[0146] Accordingly, the nucleotide sequence of each unit in the
tandem-repeated sequence may comprise at least about 1 to 200
nucleotides in length. The use of larger fragments, particularly
employing randomly sheared nucleic acid derived from viral, plant
or animal genomes, is not excluded,
[0147] The introduced nucleic acid molecule is preferably in an
expressible form.
[0148] By "expressible fore is meant that the subject nucleic acid
molecule is presented in an arrangement such that it may be
expressed in the cell, tissue, organ or whole organism, at least at
the transcriptional level (i.e. it is expressed in the animal cell
to yield at least an mRNA product which is optionally translatable
or translated to produce a recombinant peptide, oligopeptide or
polypeptide molecule).
[0149] By way of exemplification, in order to obtain expression of
the dispersed nucleic acid molecule or foreign nucleic acid
molecule in the cell, tissue or organ of interest, a synthetic gene
or a genetic construct comprising said synthetic gene is produced,
wherein said synthetic gene comprises a nucleotide sequence as
described supra in operable connection with a promoter sequence
which is capable of regulating expression therein. Thus, the
subject nucleic acid molecule will be operably connected to one or
more regulatory elements sufficient for eukaryotic transcription to
occur.
[0150] Accordingly, a further alternative embodiment of the
invention provides a method of modulating the expression of a
target gene in an animal cell, tissue or organ, said method at
least comprising the steps of: [0151] (i) selecting one or more
dispersed nucleic acid molecules or foreign nucleic acid molecules
which comprise multiple copies, preferably tandem repeats, of a
nucleotide sequence which is substantially identical to the
nucleotide sequence of said target gene or a region thereof or
which is complementary thereto; [0152] (ii) producing a synthetic
gene comprising said dispersed nucleic acid molecules or foreign
nucleic acid molecules; [0153] (iii) introducing said synthetic
gene to said cell, tissue or organ; and [0154] (iv) expressing said
synthetic gene in said cell, tissue or organ for a time and under
conditions sufficient for translation of the mRNA product of said
target gene to be modified, subject to the proviso that the
transcription of said mRNA product is not exclusively repressed or
reduced.
[0155] Reference herein to a "gene" or "genes" is to be taken in
its broadest ntext and includes:
[0156] (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); and/or
[0157] (ii) mRNA or cDNA corresponding to the coding regions (i.e.
exons) and 5'- and 3'-untranslated sequences of the gene;
and/or
[0158] (iii) a structural region corresponding to the coding
regions (i.e. exons) optionally further comprising untranslated
sequences and/or a heterologous promoter sequence which consists of
transcriptional and/or translational regulatory regions capable of
conferring expression characteristics on said structural
region.
[0159] 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.
[0160] The term "synthetic gene" refers to a non-naturally
occurring gene as hereinbefore defined which preferably comprises
at least one or more transcriptional and/or translational
regulatory sequences operably linked to a structural gene
sequence.
[0161] 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 art 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.
[0162] The term "structural gene region" refers to that part of a
synthetic gene which comprises a dispersed nucleic acid molecule or
foreign nucleic acid molecule as described herein which is
expressed in a cell, tissue or organ under the control of a
promoter sequence to which it is operably connected. A structural
gene region may comprise one or more dispersed nucleic acid
molecules and/or foreign nucleic acid molecules operably under the
control of a single promoter sequence or multiple promoter
sequences. Accordingly, the structural gene region of a synthetic
gene may comprise a nucleotide sequence which is capable of
encoding an amino acid sequence or is complementary thereto. In
this regard, a structural gene region which is used in the
performance of the instant invention may also comprise a nucleotide
sequence which encodes an animo acid sequence yet lacks a
functional translation initiation codon and/or a functional
translation slop codon and, as a consequence, does not comprise a
complete open reading frame. In the present context, the term
"structural gene region" also extends to a non-coding nucleotide
sequences, such as 5'-upstream or 3'-downstream sequences of a gene
which would not normally be translated in a eukaryotic cell which
expresses said gene,
[0163] Accordingly, in the context of the present invention, a
structural gene region may also comprise a fusion between two or
more open reading frames of the same or different genes. In such
embodiments, the invention may be used to modulate the expression
of one gene, by targeting different non-contiguous regions thereof
or alternatively, to simultaneously modulate the expression of
several different genes, including different genes of a multigene
family. In the case of a fusion nucleic acid molecule which is
non-endogenous to the animal cell and in particular comprises two
or more nucleotide sequences derived from a viral pathogen, the
fusion may provide the added advantage of conferring simultaneous
immunity or protection against several pathogens, by targeting the
expression of genes in said several pathogens. Alternatively or in
addition, the fusion may provide more effective immunity against
any pathogen by targeting the expression of more than one gene of
that pathogen.
[0164] Particularly preferred structural gene regions according to
this aspect of the invention are those which include at least one
translatable open reading frame, more preferably further including
a translational start codon located at the 5'-end of said open
reading frame, albeit not necessarily at the 5'-terminus of said
structural gene region. In this regard, notwithstanding that the
structural gene region may comprise at least one translatable open
reading frame and/or AUG or ATG translational start codon, the
inclusion of such sequences in no way suggests that the present
invention requires, translation of the introduced nucleic acid
molecule to occur in order to modulate the expression of the target
gene. Whilst not being bound by any theory or mode of action, the
inclusion of at least one translatable open reading frame and/or
translational start codon in the subject nucleic acid molecule may
serve to increase stability of the mRNA transcription product
thereof, thereby improving the efficiency of the invention.
[0165] 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 palindromic 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 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.
[0166] Wherein the structural gene region comprises more than one
dispersed nucleic acid molecule or foreign nucleic acid molecule it
shall be referred to herein as a "multiple structural gene region"
or similar term. The present invention clearly extends to the use
of multiple structural gene regions which preferably comprise a
direct repeat sequence, inverted repeat sequence or interrupted
palindrome sequence of a particular structural gene, dispersed
nucleic acid molecule or foreign nucleic acid molecule, or a
fragment thereof.
[0167] Each dispersed or foreign nucleic acid molecule 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 (i.e. two or more
dispersed or foreign nucleic acid molecules) which are
substantially identical to two or more target genes selected from
the list comprising DNA polymerase, RNA polymerase, Nia protease,
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 gene 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.
[0168] As with the individual sequence units of a dispersed or
foreign nucleic acid molecule, the individual units of the multiple
structural gene 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.
[0169] For expression in eukaryotic cells, the synthetic gene
generally comprises, in addition to the nucleic acid molecule of
the invention, a promoter and optionally other regulatory sequences
designed to facilitate expression of the dispersed nucleic acid
molecule or foreign nucleic acid molecule.
[0170] 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, with or without
a CCAAT box sequence and additional regulatory elements (i.e.
upstream activating sequences, enhancers and silencers) which alter
gene expression in response to developmental and/or external
stimuli, or in a tissue-specific manner. A promoter is usually, but
not necessarily, positioned upstream or 5', of a structural gene
region; 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 gene.
[0171] 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 a nucleic acid
molecule in a cell.
[0172] Preferred promoters may contain additional copies of one or
more specific regulatory elements, to further enhance expression of
the sense molecule and/or to alter the spatial expression and/or
temporal expression of said sense molecule. For example, regulatory
elements which confer copper inducibility may be placed adjacent to
a heterologous promoter sequence driving expression of a sense
molecule, thereby conferring copper inducibility on the expression
of said molecule.
[0173] Placing a dispersed or foreign nucleic acid molecule under
the regulatory control of a promoter sequence means positioning the
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 element 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.
[0174] 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.
[0175] 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.
[0176] Accordingly, strong constitutive promoters are particularly
preferred for the purposes of the present invention or promoters
which may be induced by virus infection or the commencement of
target gene expression.
[0177] Plant-operable and animal-operable promoters are
particularly preferred for use in the synthetic genes of the
present invention. Examples of preferred promoters include the
bacteriophage 17 promoter, bacteriophage T3 promoter, SP6 promoter,
lac operator-promoter, tac promoter, SV40 late promoter, SV40 early
promoter, RSV-LTR promoter, CMV IE promoter, CaMV 35S promoter,
SCSV promoter, SCBV promoter and the like.
[0178] In consideration of the preferred requirement for high-level
expression which coincides with expression of the target gene or
precedes expression of the target gene, it is highly desirable that
the promoter sequence is a constitutive strong promoter such as the
CMV-IE promoter or the SV40 early promoter sequence, the SV40 late
promoter sequence, the CaMV 35S promoter, or the SCBV promoter,
amongst others. Those skilled in the art will readily be aware of
additional promoter sequences other than those specifically
described.
[0179] 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 region or multiple
structural gene region is under the control of the promoter
sequence with which it is spatially connected; in a cell, tissue,
organ or whole organism.
[0180] In a preferred embodiment of the invention, a structural
gene region (i.e. dispersed nucleic acid molecule or foreign
nucleic acid molecule) or multiple structural gene region is placed
operably in connection with a promoter orientation relative to the
promoter sequence, such that when it is transcribed an mRNA product
is synthesized which, if translated, is capable of encoding a
polypeptide product of the target gene or a fragment thereof.
[0181] However, the present invention is not to be limited to the
use of such an arrangement and the invention clearly extends to the
use of synthetic genes and genetic constructs wherein the a
structural gene region or multiple structural gene region is placed
in the "antisense" orientation relative to the promoter sequence,
such that at least a part of the mRNA transcription product thereof
is complementary to the mRNA encoded by the target gene or a
fragment thereof.
[0182] Clearly, as the dispersed nucleic acid molecule, foreign
nucleic acid molecule or multiple structural gene region comprises
tandem direct and/or inverted repeat sequences of the target gene,
all combinations of the above-mentioned configurations are
encompassed by the invention.
[0183] In an alternative embodiment of the invention, the
structural gene region or multiple structural gene region is
operably connected to both a first promoter sequence and a second
promoter sequence, wherein said promoters are located at the distal
and proximal ends thereof such that at least one unit of said a
structural gene region or multiple structural gene region is placed
in the "sense" orientation relative to the first promoter sequence
and in the "antisense" orientation relative to the second promoter
sequence. According to this embodiment, it is also preferred that
the first and second promoters be different, to prevent competition
there between for cellular transcription factors which bind
thereto. The advantage of this arrangement is that the effects of
transcription from the first and second promoters in reducing
target gene expression in the cell may be compared to determine the
optimum orientation for each nucleotide sequence tested.
[0184] The synthetic gene preferably contains additional regulatory
elements for efficient transcription, for example a transcription
termination sequence.
[0185] 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 plant cells are known and described in the
literature. They may be isolated from bacteria, fungi, viruses,
animals and/or plants or synthesized de novo.
[0186] As with promoter sequences, the terminator may be any
terminator sequence which is operable in the cells, tissues or
organs in which it is intended to be used.
[0187] 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 CYCI
terminator, ADH terminator, SPA terminator, nopaline synthase (NOS)
gene terminator of Agrobacterium tumefaciens, the terminator of the
Cauliflower mosaic virus (CaMV) 355 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.
[0188] 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,
octopine synthase (OCS) or nopaline synthase (NOS) terminator
active in plant cells, tissues or organs, or the lacZ alpha
terminator which is active in prokaryotic cells.
[0189] Those skilled in the art will be aware of additional
terminator sequences which may be suitable for use in performing
the invention. Such sequences may readily be used without any undue
experimentation.
[0190] Means for introducing (i.e. transfecting or transforming)
cells with the synthetic genes described herein or a genetic
construct comprising same are well-known to those skilled in the
art.
[0191] In a further alternative embodiment, a genetic construct is
used which comprises two or more structural gene regions or
multiple structural gene regions wherein each of said structural
gene regions is placed operably under the control of its own
promoter sequence. As with other embodiments described herein, the
orientation of each structural gene region may be varied to
maximise its modulatory effect on target gene expression.
[0192] 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.
[0193] Those skilled in the art will know how to modify the
arrangement or configuration of the individual structural genes as
described supra to regulate their expression from separate promoter
sequences.
[0194] 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.
[0195] Those skilled in the art will be aware of how to produce the
synthetic genes described herein and of the requirements for
obtaining the expression thereof, when so desired, in a specific
cell or cell-type under the conditions desired. In particular, it
will be known to those skilled in the art that the genetic
manipulations required to perform the present invention may require
the propagation of a genetic construct described herein or a
derivative thereof in a prokaryotic cell such as an E. coil cell or
a plant cell or an animal cell.
[0196] 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.
[0197] 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.
[0198] Genetic constructs are particularly suitable for the
transformation of a eukaryotic cell to introduce novel genetic
traits thereto, in addition to the provision of resistance
characteristics to viral pathogens. Such additional novel traits
may be introduced in a separate genetic construct or, alternatively
on the same genetic construct which comprises the synthetic genes
described herein. Those skilled in the art will recognise the
significant advantages, in particular in terms of reduced genetic
manipulations and tissue culture requirements and increased
cost-effectiveness, of including genetic sequences which encode
such additional traits and the synthetic genes described herein in
a single genetic construct.
[0199] 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 eukaryolic cell.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] Preferably, the selectable marker gene is the nptII gene or
Kan.sup.r gene or green fluorescent protein (GFP)-encoding
gene.
[0204] 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.
[0205] The present invention extends to all genetic constructs
essentially as described herein, which include further genetic
sequences intended for the maintenance and/or replication of said
genetic construct in prokaryotes or eukaryotes and/or the
integration of said genetic construct or a part thereof into the
genome of a eukaryotic cell or organism.
[0206] As with dispersed or foreign nucleic acid molecules,
standard methods described supra may be used to introduce synthetic
genes and genetic constructs into the cell, tissue or organ for the
purposes of modulating the expression of the target gene, for
example liposome-mediated transfection or transformation,
transformation of cells with attenuated virus particles or
bacterial cells, cell mating, transformation or transfection
procedures known to those skilled in the art or described by
Ausubel et al. (1992).
[0207] Additional means for introducing recombinant DNA into plant
tissue or cells include, but are not limited to, transformation
using CaCl.sub.2 and variations thereof, in particular the method
described by Hanahan (1983), direct DNA uptake into protoplasts
(Krens et al, 1982; Paszkowski et al, 1984), PEG-mediated uptake to
protoplasts (Armstrong et al, 1990) microparticle bombardment,
electroporation (Fromm et al., 1985), microinjection of DNA
(Crossway at al., 1986), microparticle bombardment of tissue
explants or cells (Christou et al, 1988; Sanford, 1988),
vacuum-infiltration of tissue with nucleic acid, or in the case of
plants, T-DNA-mediated transfer from Agrobacterium to the plant
tissue as described essentially by An et al. (1985),
Herrera-Estrella et al. (1983a, 1983b, 1985).
[0208] For microparticle bombardment of cells, a microparticle is
propelled into a cell to produce a transformed cell. Any suitable
ballistic cell transformation methodology and apparatus can be used
in performing the present invention. Exemplary apparatus and
procedures are disclosed by Stomp etal. (U.S. Pat. No. 5,122,466)
and Sanford and Wolf (U.S. Pat. No. 4,945,050). When using
ballistic transformation procedures, the genetic construct may
incorporate a plasmid capable of replicating in the cell to be
transformed.
[0209] Examples of microparticles suitable for use in such systems
include 1 to 5 .mu.m gold spheres. The DNA construct may be
deposited on the microparticle by any suitable technique, such as
by precipitation.
[0210] In a further embodiment of the present invention, the
synthetic genes and genetic constructs described herein are adapted
for integration into the genome of a cell in which it is expressed.
Those skilled in the art will be aware that, in order to achieve
integration of a genetic sequence or genetic construct into the
genome of a host cell, certain additional genetic sequences may be
required. In the case of plants, left and right border sequences
from the T-DNA of the Agrobacterium tumefaciens Ti plasmid will
generally be required.
[0211] The present invention further extends to an isolated cell,
tissue or organ comprising the synthetic gene described herein or a
genetic construct comprising same. The present invention extends
further to regenerated tissues, organs and whole organisms derived
from said cells, tissues and organs and to propagules and progeny
thereof.
[0212] For example, plants may be regenerated from transformed
plant cells or tissues or organs on hormone-containing media and
the regenerated plants may take a variety of forms, such as
chimeras of transformed cells and non-transformed cells; clonal
transformants (e.g., all cells transformed to contain the
expression cassette); grafts of transformed and untransformed
tissues (e.g., a transformed root stock grafted to an untransformed
scion in citrus species). Transformed plants may be propagated by a
variety of means, such as by clonal propagation or classical
breeding techniques. For example, a first generation (or T1)
transformed plants may be selfed to give homozygous second
generation (or T2) transformed plants, and the T2 plants further
propagated through classical breeding techniques.
[0213] The present invention is further described with reference to
the following non-limiting Examples.
EXAMPLE 1
Genetic Constructs Comprising BEV Polymerase Gene Sequences Linked
to the CMV Promoter Sequence and/or the SV40L Promoter Sequence
[0214] 1. Commercial Plasmids
[0215] Plasmid pBluescript II (SK+)
[0216] Plasmid pOluescript II (SK+) is commercially available from
Stratagene and comprises the LacZ promoter sequence and lacZ-alpha
transcription terminator, with a multiple cloning site for the
insertion of structural gene sequences therein. The plasmid further
comprises the ColE1 and fl origins of replication and
ampicillin-resistance gene.
[0217] Plasmid pSVL
[0218] 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.
[0219] Plasmid pCR2.1
[0220] Plasmid pCR2.1 is commercially available from Invitrogen 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.
PCR fragments cloned in this fashion are flanked by two EcoRI
sites. The plasmid further comprises the ColE1 and f1 origins of
replication and kanamycin-resistance and ampicillin-resistance
genes.
[0221] Plasmid pEGFP-N1 MCS
[0222] Plasmid pEGFP-N1 MCS (FIG. 1; Clontech) contains the CMV IE
promoter operably connected to an open reading frame 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 Bg/II 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 the GFP open reading frame to direct proper processing of the
3'-end of mRNA transcribed from the CMV-IE promoter sequence. The
plasmid further 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 6418; the pUC19 origin of replication which
is functional in bacterial cells (pUC On in FIG. 1); and the f1
origin of replication for single-stranded DNA production (f1 Ori in
FIG. 1).
[0223] 2. Expression Cassettes
[0224] Plasmid pCMV.cass
[0225] 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.
[0226] Plasmid pCMV.SV40L.cass
[0227] Plasmid pCMV.SV40L.cass (FIG. 3) 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.
[0228] Plasmid pCMV.SV40LR.cass
[0229] Plasmid pCMV.SV40LR cass (FIG. 4) comprises the SV40 late
promoter sequence derived from plasmid pCR.SV40L, sub-cloned as a
Sall fragment into the Sall 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 in this 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: [0230] (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
[0231] (ii) where the structural gene sequence or multiple
structural gene unit is to be expressed in the antisense
orientation from the CMV IE promoter sequence and/or in the sense
orientation from the SV40 late promoter, the 5' polyadenylation
signal will be in the sense orientation and the 3' polyadenylation
signal will be in the antisense orientation.
[0232] 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. 4.
[0233] 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 approriate 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.
[0234] Alternatively appropriately oriented terminators could be
placed upstream of the CMV and SV40L promoters such that
transcriptional termination could occur after readthrough of each
of the two promoters in the antisense orientation.
[0235] 3. Intermediate Constructs
[0236] Plasmid pCR.Bgl-GFP-Bam
[0237] Plasmid pCR.Bgl-GFP-Bam (FIG. 5) 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 and GFP-Bam and cloned into plasmid pCR2.1. The
internal GFP-encoding region in plasmid pCR.Bgl-GFP-Barn lacks
functional translational start and stop codons.
[0238] Plasmid pBSII(SK+).EGFP
[0239] Plasmid pBSII(SK+).EGFP (FIG. 6) comprises the EGFP open
reading frame derived from plasmid pEGFP-N1 MCS (FIG. 1) placed
operably under the control of the lacZ promoter. To produce this
plasmid, the EGFP encoding region of pEGFP-N1 MCS was excised as a
Not1/Xho1 fragment and cloned into the Not1/Xho1 cloning sites of
plasmid pBluescript II (SK+).
[0240] Plasmid pCMV.EGFP
[0241] Plasmid pCMV.EGFP (FIG. 7) is capable of expressing the EGFP
structural gene under the control of the CMV-IE promoter sequence,
To produce this plasmid the EGFP sequence from plasmid
pBSII(SK+).EGFP was excised as BamHI/SacI fragment and cloned into
the Bg/II/SacI sites of plasmid pCMV.cass (FIG. 2).
[0242] Plasmid pCR.SV40L
[0243] Plasmid pCR.SV40L (FIG. 8) comprises the SV40 late promoter
derived from plasmid pSVL (GenBank Accession No. 1113868;
Pharmacia), cloned into pCR2.1 (Stratagene). To produce this
plasmid, the SV40 late promoter was amplified using the primers
SV40-1 and SV40-2 which comprise Sal I cloning sites to facilitate
subcloning of the amplified DNA fragment into pCMV.cass. The primer
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.
[0244] Plasmid pCR.BEV.1
[0245] The BEV RNA-dependent RNA polymerase coding region was
amplified as a 1,385 by DNA fragment from a full-length cDNA clone
encoding same, using primers designated BEV-1 and BEV-2, under
standard amplification conditions. The amplified DNA contained a
5'-Bgl II 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, the amplified BEV polymerase structural gene
comprises the start site in-frame with BEV polymerase-encoding
nucleotide sequences, Thus, the ampfified 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. This plasmid is
present as FIG. 9.
[0246] Plasmid pCR.BEV.2
[0247] The complete BEV polymerase coding region was amplified from
a full-length cDNA clone encoding same, using primers BEV-1 and
BEV-3. Primer BEV-3 comprises a BamHI restriction enzyme site at
positions 5 to 10 inclusive and the complement of a translation
stop signal at positions 11 to 13. As a consequence, an open
reading frame comprising a translation start signal and translation
stop signal, contained between the Bgl II and BamHI restriction
sites. The amplified fragment was cloned into pCR2.1 (Stratagene)
to produce plasmid pCR2.BEV.2 (FIG. 10).
[0248] Plasmid pCR.BEV.3
[0249] A non-translatable BEV polymerase structural gene was
amplified from a full-length BEV polymerase cDNA clone using the
amplification primers BEV-3 and BEV-4. Primer BEV-4 comprises a
BglII cloning site at positions 5-10 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 (Stratagene) to
yield plasmid pCR.BEV.3 (FIG. 11).
[0250] Plasmid pCMV.EGFP.BEV2
[0251] Plasmid pCMV.EGFP.BEV2 (FIG. 12) was produced by cloning the
BEV polymerase sequence from pCR.BEV.2 as a BglII/BamHI fragment
into the BamHI site of pCMV.EGFP.
[0252] 4. Control Plasmids
[0253] Plasmid pCMV.BEV.2
[0254] Plasmid pCMV.BEV.2 (FIG. 13) 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 was sub-cloned in the sense orientation as
a Bgfl-to-BamHI fragment into BglII/BamHI-digested pCMV.cass (FIG.
2).
[0255] Plasmid pCMV.BEV.3
[0256] Plasmid pCMV.BEV.3 (FIG. 14) 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 was sub-cloned in the sense
orientation as a BglII-to-BamHI fragment into BglII/BamHI-digested
pCMV.cass (FIG. 2).
[0257] Plasmid pCMV.VEB
[0258] Plasmid pCMV.VEB (FIG. 15) 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 was sub-cloned in the antisense orientation as a
BglII-to-BamHI fragment into BglII/BamHI-digested pCMV.cass (FIG.
2).
[0259] Plasmid pCMV.BEV.GFP
[0260] Plasmid pCMV.BEV.GFP (FIG. 16) was constructed by cloning
the GFP fragment from pCR.Bgl-GFP-Bam as a BglII/BamHI fragment
into BamHI-digested pCMV.BEV.2. This plasmid serves as a control in
some experiments and also as an intermediate construct.
[0261] Plasmid pCMV.BEV.SV40-L
[0262] Plasmid pCMV.BEV.SV40-L (FIG. 17) comprises a translatable
BEV polymerase structural gene derived from plasmid pCR.BEV.2
inserted in the sense orientation between the CMV-IE promoter and
the SV40 late promoter sequences of plasmid pCMV.SV40L.cass. To
produce plasmid pCMV.BEV.SV40L-O, the BEV polymerase structural
gene was sub-cloned as a BglII-to-BamHI fragment into EMI-digested
pCMV.SV40L.cass DNA.
[0263] Plasmid pCMV.O.SV40L.BEV
[0264] Plasmid pCMV.O.SV40L.BEV (FIG. 18) comprises a translatable
BEV polymerase structural gene derived from plasmid pCR.BEV.2
cloned downstream of tandem CMV-IE promoter and SV40 late promoter
sequences present in plasmid pCMV.SV40Lcass. 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.
[0265] Plasmid pCMV.O.SV40LVEB
[0266] Plasmid pCMV.O.SV40L.VEB (FIG. 19) comprises an antisense
BEV polymerase structural gene derived from plasmid pCR.BEV.2
cloned downstream of tandem CMV-IE promoter and SV40 late promoter
sequences present in plasmid pCMV.SV40L.cass. 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.
[0267] 5. Test Plasmids
[0268] Plasmid pCMV.BEVx2
[0269] Plasmid pCMV.BEVx2 (FIG. 20) 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 was sub-cloned in the sense orientation as a
BglII-to-BamHI fragment into BamHI-digested pCMV.BEV.2, immediately
downstream of the BEV polymerase structural gene already present
therein.
[0270] Plasmid pCMV.BEVx3
[0271] Plasmid pCMV.BEVx3 (FIG. 21) comprises a direct repeat of
three complete BEV polymerase open reading frames under the control
of the CMV-1E promoter. To produce pCMV.BEVx3, the BEV polymerase
fragment from pCR.BEV.2 was cloned in the sense orientation as a
BglIIIBamHI fragment into the BamHI site of pCMV.BEVx2, immediately
downstream of the BEV polymerase sequences already present
therein.
[0272] Plasmid pCMV.BEVx4
[0273] Plasmid pCMV.BEVx4 (FIG. 22) comprises a direct repeal of
four complete BEV polymerase open reading frames under the control
of the CMV-1E promoter. To produce pCMV.BEVx4, the BEV polymerase
fragment from pCR.BEV.2 was cloned in the sense orientation as a
BglII/BamHI fragment into the BamHI site of pCMV.BEVx3, immediately
downstream of the BEV polymerase sequences already present
therein.
[0274] Plasmid pCMV.BEV.SV40LBEV
[0275] Plasmid pCMV.BEV.SV40L.BEV (FIG. 23) 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 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.
[0276] Plasmid pCMV.BEV.SV40L.VEB
[0277] Plasmid pCMV.BEV.SV40L.VEB (FIG. 24) 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 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. 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.
[0278] Plasmid pCMV.BEV.GFP.VEB
[0279] Plasmid pCMV.BEV.GFP.VEB (FIG. 25) comprises a BEV
structural gene inverted repeat or palindrome, interrupted by the
insertion of a GFP open reading frame (stuffer fragment) between
each BEV structural gene sequence in the inverted repeat. To
produce plasmid pCMV.BEV.GFP.VEB, the GFP stuffer fragment from
pCR.Bgl-GFP-Bam was first sub-cloned in the sense orientation as a
BglII-to-BamHI fragment into BamHI-digested pCMV.BEV.2 to produce
an intermediate plasmid pCMV.BEV.GFP wherein the BEV
polymerase-encoding and GFP-encoding sequences are contained within
thesame 5'-BglII-to-BamH1-3' fragment. The BEV polymerase
structural gene from pCMV.BEV.2 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.
[0280] Plasmid pCMV.EGFP.BEV2.PFG
[0281] Plasmid pCMV.EGFP.BEV2.PFG (FIG. 26) comprise a GFP
palindrome, interrupted by the insertion of a BEV polymerase
sequence between each GFP structural gene in the inverted repeat.
To produce this plasmid the GFP fragment from pCR.Bgl-GFP-Bam was
cloned as a BglII/BamHI fragment into the BamHI site of
pCMV.EGFP.BEV2 in the antisense orientation relative to the CMV
promoter.
[0282] Plasmid pCMV.BEV.SV40LR
[0283] Plasmid pCMV.BEV.SV40LR (FIG. 27) composes 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 transcripts 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 was sub-cloned, as a BglII-to-BamHI fragment,
into the unique BglII site of plasmid pCMV.SV40L.R.cass, such that
the BEV open reading frame is present in the sense orientation
relative to the CMV-IE promoter sequence.
[0284] 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 2
Genetic Constructs Comprising the Porcine
.alpha.-1,3-galactosyltransferase (Galt) Structural Gene Sequence
or Sequences Operably Connected to the CMV Promoter Sequence and/or
the SV40L Promoter Sequence
[0285] 1. Commercial Plasmids
[0286] Plasmid pcDNA3
[0287] Plasmid pcDNA3 is commercially available from Invitrogen and
comprises the CMV-IE promoter and BGHpA transcription terminator,
with multiple cloning sites for the insertion of structural gene
sequences there between. The plasmid further comprises the ColE1
and fl origins of replication and neomycin-resistance and
ampicillin-resistance genes.
[0288] 2. Intermediate plasmids
[0289] Plasmid pcDNA3.Galt
[0290] Plasmid pcDNA3.Galt (BresaGen Limited, South Australia,
Australia: FIG. 28) is plasmid pcDNA3 (Invitrogen) and comprises
the cDNA sequence encoding porcine gene
alpha-1,3-galactosyltransferase (Galt) operably under the control
of the CMV-IE promoter sequence such that it is capable of being
expressed therefrom. To produce plasmid pcDNA3.Galt, the porcine
gene alpha-1,3-galactosyltransferase cDNA was cloned as an EcoRI
fragment into the EcoRI cloning site of pcDNA3. The plasmid further
comprises the ColE1 and fl origins of replication and the neomycin
and ampicillin-resistance genes.
[0291] 3. Control Plasmids
[0292] Plasmid pCMV.Galt
[0293] Plasmid pCMV.Galt (FIG. 29) is capable of expressing the
Galt structural gene under the control of the CMV-IE promoter
sequence. To produce plasmid pCMV.Galt, the Galt sequence from
plasmid pcDNA3.Galt was excised as an EcoRI fragment and cloned in
the sense orientation into the EcoRI site of plasmid pCMV.cass
(FIG. 2). Plasmid pCMV.EGFP.Galt
[0294] Plasmid pCMV.EGFP.Galt (FIG. 30) is capable of expressing
the Galt structural gene as a Galt fusion polypeptide under the
control of the CMV-IE promoter sequence. To produce plasmid
pCMV.EGFP.Galt, the Galt sequence from pCMV.Galt (FIG. 29) was
excised as a BglII/BamHI fragment and cloned into the BamHI site of
pCMV.EGFP.
[0295] Plasmid pCMV.Galt.GFP
[0296] Plasmid pCMV.Galt.GFP (FIG. 31) was produced by cloning the
Galt cDNA as an EcORI fragment from pCDNA3 into EcoRI-digested
pCMV.EGFP in the sense orientation. This plasmid serves as both a
control and construct intermediate.
[0297] Plasmid pCMV.Gait.SV40L.0
[0298] The plasmid pCMV.Galt.SV40L.0 (FIG. 32) comprises a Galt
structural gene cloned downstream of the CMV promoter present in
.pCMV.SV40L.cass. To produce the plasmid the Galt cDNA fragment
from pCMV.Galt was cloned as a BglII/BamHI into BglII-digested
pCMV.SV40L.cass in the sense orientation.
[0299] Plasmid pCMV.O.SV40L.tlaG
[0300] The plasmid pCMV.O.SV40L.tlaG (FIG. 33) comprises a Galt
structural gene clones in an antisense orientation downstream of
the SV40L promoter present in pCMV.SV40L.cass. To produce this
plasmid the Galt cDNA fragment from pCMV.Galt was cloned as a
BglII/BamHI into BamHI-digested pCMV.SV40L.cass in the antisense
orientation.
[0301] Plasmid pCMV.O.SV40L.Galt
[0302] The plasmid pCMV.O.SV40L.Galt (FIG. 34) comprises a Galt
structural gene cloned downstream of the SV40L promoter present in
pCMV.SV40L.cass. To produce the plasmid the Galt cDNA fragment from
pCMV.Galt was cloned as a BglII/BamHI into BamHI-digested
pCMV.SV40L.cass in the sense orientation.
[0303] 4. Test Plasmids
[0304] Plasmid pCMV.Gattx2
[0305] Plasmid pCMV.Galtx2 (FIG. 35) comprises a direct repeat of a
Galt open reading frame under the control of the CMV-IE promoter
sequence. In eukaryotes cells at least, the open reading frame
located nearer the CMV-IE promoter is translatable. To produce
pCMV.Galtx2, the Galt structural gene from pCMV.Galt was excised as
a BglII/BamHI fragment and cloned in the sense orientation into the
BamHI cloning site of pCMV.Galt.
[0306] Plasmid pCMV.Galtx4
[0307] Plasmid pCMV.Galtx4 (FIG. 36) comprises a quadruple direct
repeat of a Galt open reading frame under the control of the CMV-IE
promoter sequence. In eukaryotes cells at least, the open reading
frame located nearer the CMV-IE promoter is translatable. To
produce pCMV.Galtx4, the Galtx2 sequence from pCMV.Galtx2 was
excised as a BglII/BamHI fragment and cloned in the sense
orientation into the BamHI cloning site of pCMV.Galtx2.
[0308] Plasmid pCMV.GaltSV40L.Galt
[0309] The plasmid pCMV.Galt.SV40L.Galt (FIG. 37) is designed to
express two sense transcripts of Galt, one driven by the CMV
promoter, the other by the SV40L promoter. To produce the plasmid a
Galt cDNA fragment from pCMV.Galt was cloned as a BglII/BamHI
fragment into BglII-digested pCMV.O.SV40.Galt in the sense
orientation.
[0310] Plasmid pCMV.Galt.SV40L.tlaG
[0311] The plasmid pCMV.GaltSV40.tlaG (FIG. 38) is designed to
express a sense transcript of Galt driven by the CMV promoter and
an antisense transcript driven by the SV40L promoter. To produce
the plasmid a Galt cDNA fragment from pCMV.Galt was cloned as a
BglII/BamHI fragment into BglII-digested pCMV.O.SV40.taIG in the
sense orientation.
[0312] Plasmid pCMV.Galt.GFP.daG
[0313] Plasmid pCMV.Gatt.GFP.ttaG (FIG. 39) comprise a Galt
palindrome, interrupted by the insertion of a GFP sequence between
each Galt structural gene in the inverted repeat. To produce this
plasmid the BglII/BamHI Galt cDNA fragment from pCMV.Galt was
cloned into the BamHI site of pCMV.Galt.GFP in the antisense
relative to the CMV promoter.
[0314] Plasmid pCMV.EGFP.GaItPFG
[0315] The plasmid pCMV.EGFP.Galt.PFG (FIG. 40) comprises a GFP
palindrome, interrupted by the insertion of a Galt sequence between
each GFP structural gene of the inverted repeat, expression of
which is driven by the CMV promoter. To produce this plasmid the
Galt sequences from pCMV.Galt were cloned as a BglII/BamHI fragment
into BamHI-digested pCMV.EGFP in the sense orientation to produce
the intermediate pCMV.EGFP.Galt (not shown): following this further
GFP sequences from pCR.Bgl-pCMV.EGFP.Galt in the antisense
orientation.
[0316] Plasmid pCMV.Galt.SV40LR
[0317] The plasmid pCMV.Galt.SV40LR (FIG. 41) is designed to
express GalT cDNA sequences cloned between the opposing CMV and
SV40L promoters in the expression cassette pCMV.SV40LR.cass. To
produce this plasmid Gall sequences from ID pCMV.Galt were cloned
as a BglII/BamHI fragment in BglII-digested pCMV.SV40LR.cass in the
sense orientation relative to the 35S promoter.
EXAMPLE 3
Genetic Constructs Comprising PVY Nia Sequences Operably Linked to
the 35S Promoter Sequence and/or the SCBV Promoter Sequence
[0318] 1: Binary Vector
[0319] Plasmid pART27
[0320] Plasmid pART27 is a binary vector, specifically designed to
be compatible with the pART7 expression cassette. It contains
bacterial origins of replication for both E. coli and Agrobacterium
tumefaciens, a spectinomycin resistance gene for bacterial
selection, left and right T-DNA borders for transfer of DNA from
Agrobacterium to plant cells and a kanamycin resistance cassette to
permit selection of transformed plant cells. The kanamycin
resistance cassette is located between the T-DNA borders, pART27
also contains a unique NotI restriction site which permits cloning
of constructs prepared in vectors such as pART7 to be cloned
between the T-DNA borders. Construction of pART27 is described in
Gleave, A P (1992).
[0321] When cloning NotI inserts into this vector, two insert
orientations can be obtained. In all the following examples the
same insert orientation, relative to the direction of the 35S
promoter in the described pART7 constructs was chosen; this was
done to minimise any experimental artefacts that may arise from
comparing different constructs with different insert
orientations.
[0322] 2. Commercial Plasmids
[0323] Plasmid pBC (KS-)
[0324] Plasmid pBC (KS-) is commercially available from Stratagene
and comprises the LacZ promoter sequence and lacZ-alpha
transcription terminator, with a multiple cloning site for the
insertion of structural gene sequences therein. The plasmid further
comprises the ColE1 and fl origins of replication and a
chloroamphenicol-resistance gene.
[0325] Plasmid pSP72
[0326] Plasmid pSP72 is commercially available from Promega and
contains a multiple cloning site for the insertion of structural
gene sequences therein. The plasmid further comprises the ColE1
origin of replication and an ampicillin-resistance gene.
[0327] 3. Expression Cassettes
[0328] Plasmid pART7
[0329] Plasmid pART7 is an expression cassette designed to drive
expression of sequences cloned behind the 35S promoter. It contains
a polylinker to assist cloning and a region of the octipine
synthase terminator. The 35S expression cassette is flanked by two
Not I restriction sites which permits cloning into binary
expression vectors, such as pART27 which contains a unique NotI
site. Its construction as described in Gleave, A P (1992), a map is
shown in FIG. 43.
[0330] Plasmid pART7.35S.SCBV.cass
[0331] Plasmid p35S.CMV.cass was designed to express two separate
gene sequences cloned into a single plasmid. To create this
plasmid, sequences corresponding to the nos terminator and the SCBV
promoter were amplified by PCR then cloned in the polylinker of
pART7 between the 35S promoter and OCS.
[0332] The resulting plasmid has the following arrangement of
elements:
[0333] 35S promoter-polylinker 1-NOS terminator-SCBV
promoter-polylinker 2-OCS terminator.
[0334] Expression of sequences cloned into polylinker 1 is
controlled by the 35S promoter, expression of sequences cloned into
polylinker 2 is controlled by the SCBV promoter.
[0335] The NOS terminator sequences were amplified from the plasmid
pAHC27 (Christensen and Quail, 1996) using the two
oligonucleotides;
TABLE-US-00002 NOS 5' (forward primer; SEQ ID ??)
5'-GGATTCCCGGGACGTCGCGAATTTCCCCCGATCGTTC-3'; and NOS 3' (reverse
primer, SEQ ID ??) 5'-CCATGGCCATATAGGCCCGATCTAGTAACATAG-3'
[0336] Nucleotide residues 1 to 17 for NOS 5' and 1 to 15 for NOS
3' represent additional nucleotides designed to assist in construct
preparation by adding additional restriction sites. For NOS 5'
these are BamHI, SmaI, AatII and the first 4 bases of an NruI site,
for NOS 3' these are NcoI and SfiI sites. The remaining sequences
for each oligonucleotide are homologous to the 5' and 3' ends
respectively of NOS sequences in pAHC 27.
[0337] The SCBV promoter sequences were amplified from the plasmid
pScBV-20 (Tzafir et al, 1998) using the two oligonucleotides:
TABLE-US-00003 SCBV 5': 5'-CCATGGCCTATATGGCCATTCCCCACATTCAAG-3';
and SCBV 3': 5'AACGTTAACTTCTACCCAGTTCCAGAG-3'
[0338] Nucleotide residues 1 to 17 of SCBV 5 encode NcoI and SfiI
restriction sites designed to assist in construct preparation, the
remaining sequences are homologous to upstream sequences of the
SCMV promoter region, Nucleotide residues 1 to 9 of SCBV 3' encode
Psp10461 and HpaI restriction sites designed to assist in construct
5 preparation, the remaining sequences are homologous to the
reverse and complement of sequences near the transcription
initiation site of SCBV.
[0339] Sequences amplified from pScBV-20 using PCR and cloned into
pCR2.1 (Invitrogen) to produce pCR.NOS and pCR.SCBV respectively.
Smal I/SfiI cut pCR.NOS and SfiI/HpaI cut pCR.SCBV were ligated
into Sma I cut pART7 and a plasmid with a suitable orientation was
chosen and designated pART7.35S.SCBV.cass, a map of this construct
is shown in FIG. 43.
[0340] 4. Intermediate Constructs
[0341] Plasmid pBC.PVY
[0342] A region of the PW genome was amplified by PCR using
reverse-transcribed RNA isolated from PVY-infected tobacco as a
template using standard protocols and cloned into a plasmid pGEM 3
(Stratagene), to create pGEM.PVY. A SalI/HindII fragment from
pGEM.PVY, corresponding to a SalI/HindIII fragment positions
1536-2270 of the PVY strain O sequence (Acc. No 012539, Genbank),
was then subcloned into the plasmid pBC (Stratagene Inc.) to create
pBC.PVY (FIG. 44).
[0343] Plasmid pSP72.PVY
[0344] Plasmid pSP72.PVY was prepared by inserting an EcoRI/SalI
fragment from pBC.PVY into EcoRI/SalI cut pSP72 (Promega). This
construct contains additional restriction sites flanking the PVY
insert which were used to assist subsequent manipulations. A map of
this construct is shown in FIG. 45.
[0345] Plasmid ClapBC.PVY
[0346] Plasmid Cla pBC.PVY was prepared by inserting a ClaI/SalI
fragment from pSP72.PVY into ClaI/Sal I cutpBC tagene). This
construct contains additional restriction sites flanking the PVY
insert which were used to assist subsequent manipulations. A map of
this construct is shown in FIG. 46.
[0347] Plasmid pBC.PVYx2
[0348] Plasmid pBC.PVYx2 contains two direct head-to-tail repeats
of the PVY sequences derived from pBC.PVY. The plasmid was
generated by cloning an AccI/ClaI PVY fragment from pSP72.PVY into
AccI cut pBC.PVY and is shown in FIG. 47.
[0349] Plasmid pSP72.PVYx2
[0350] Plasmid pSP72.PVYx2 contains two direct head-to-tail repeats
of the PVY sequences derived from pBC.PVY. The plasmid was
generated by cloning an AccI/ClaI PVY fragment from pBc.PVY into
AccI cut pSP72.PVY and is shown in FIG. 48.
[0351] Plasmid pBC.PVYx3
[0352] Plasmid pBC.PVYx3 contains three direct head-to-tail repeats
of the PVY sequences derived from pBC.PVY. The plasmid was prepared
by cloning an AccI/ClaI PVY fragment from pSP72.PVY into AccI cut
pBC.PVYx2 and is shown in FIG. 49.
[0353] Plasmid pBC.PVYx4
[0354] Plasmid pBC.PVYx4 contains four direct head-to-tail repeats
of the PVY sequences derived from pBC.PVY. The plasmid was prepared
by cloning the direct repeat of PVY sequences from pSP72.PVYx2 as
an AccI/ClaI fragment into AccI cut pBC.PVYx2 and is shown in FIG.
50.
[0355] Plasmid pBC.PVY.LNYV
[0356] All attempts to create direct palindromes of PVY sequences
failed, presumably such sequence arrangements are unstable in
commonly used E. coli cloning hosts. Interrupted palindromes
however proved stable.
[0357] To create interrupted palindromes of PVY sequences a
"stuffer" fragment of approximately 360 by was inserted into Cla
pBV.PVY downstream of the PVY sequences. The stuffer fragment was
made as follows:
[0358] A clone obtained initially from a cDNA library prepared from
lettuce necrotic yellows virus (LNYV) genomic RNA (Deitzgen el al,
1969), known to contain the 4b gene of the virus, was amplified by
PCR using the primers:
TABLE-US-00004 LNYV 1: 5'-ATGGGATCCGTTATGCCAAGAAGAAGGA-3'; and LNYV
2: 5'-TGTGGATCCCTAACGGACCCGATG-3'
[0359] The first 9 nucleotide of these primers encode a BamHI site,
the remaining nucleotides are homologous to sequences of the LNYV
4b gene.
[0360] Following amplification, the fragment was cloned into the
EcoRI site of pCR2.1 (Stratagene). This EcoRI fragment was cloned
into the EcoRI site of Cla pBC.PVY to create the intermediate
plasmid pBC.PVY.LNYV which is shown in FIG. 51.
[0361] Plasmid pBC.PVY.LNYV.PVY
[0362] The plasmid pBC.PVY.LNYVYVP contains an interrupted direct
repeat of PVY sequences. to create this plasmid a HpaI/Hincll
fragment from pSP72 was cloned into SmaI-digested pBC.PVY.LNYV and
a plasmid containing the sense orientation isolated, a map of this
construct is shown in FIG. 52.
[0363] Plasmid pBC.PVY.LNYV.YVP.sub..DELTA.
[0364] The plasmid pBV.PVY.LNYVYVP.sub..DELTA. contains a partial
interrupted palindrome of PVY sequences. One arm of the palindrome
contains all the PVY sequences from pBC.PVY, the other arm contains
part of the sequences from PVY, corresponding to sequences between
the EcoRV and HincII sites of pSP72.PVY. To create this plasmid an
EcoRV/HincII fragment from pSP72.PVY was cloned into SmaI-digested
pBC.PVY.LNYV and a plasmid containing the desired orientation
isolated, a map of this construct is shown in FIG. 51
[0365] Plasmid pBC.PVY.LNYV.YVP
[0366] The plasmid pBCPVY.LNYV.YVP contains an interrupted
palindrome of PVY sequences. To create this plasmid a HpaI/HincII
fragment from pSP72. was cloned into Sma-digested pBC.PVY.LNYV and
a plasmid containing the antisense orientation isolated, a map of
this construct is shown in FIG. 54.
[0367] 5. Control Plasmids
[0368] Plasmids pART7.PVY & pART7.PVY
[0369] Plasmid pART7.PVY (FIG. 55) was designed to express PVY
sequences driven by the 35S promoter. This plasmid serves as a
control construct in these experiments, against which all other
constructs was compared. To generate this plasmid a ClaI/AccI
fragment from ClapBC.PVY was cloned into ClaI-digested pART7 and a
plasmid with expected to express a sense PVY sequence with respect
to the PVY genome, was selected. Sequences consisting of the 35S
promoter, PVY sequences and the OCS terminator were excised as a
NotI fragment and cloned into Noti-digested pART27, a plasmid with
the desired insert orientation was selected and designated
pART27.
[0370] Plasmids pART7.35S.PVY.SCBV.O &
pART27.35S.PVY.SCBV.O
[0371] Plasmid pART7.35S.PVY.SCBV.0 (FIG. 56) was designed to act
as a control for co-expression of multiple constructs from a single
plasmid in transgenic plants. The 35S promoter was designed to
express PVY sense sequences, whilst the SCBV promoter was empty. To
generate this plasmid, the PVY fragment from Cla pBC.PVY was cloned
as a XhoI/EcoRI fragment into XhoI/EcoRI-digested
pART7.35S.SCBV.cass to create p35S.PVY.SCBV>O. Sequences
consisting of the 35S promoter driving sense PVY sequences and the
1405 terminator and the SCBV promoter and OCS terminator were
excised as a Noll fragment and cloned into pART27, a plasmid with
the desired insert orientation was isolated and designated
pART27.35S.PVY.SCBV.O.
[0372] Plasmids pART7.35S.O.SCBV.PVY 8 pART27.35S.O.SCBV.PVY
[0373] Plasmid pART27.35S.O.SCBV.PVY (FIG. 57) was designed to act
as an additional control for co-expression of multiple constructs
from a single plasmid in transgenic plants. No expressible
sequences were cloned behind the 35S promoter, whilst the SCBV
promoter drove expression of a PVY sense fragment. To generate this
plasmid, the PVY fragment from Cla pBC.PVY was cloned as a ClaI
fragment into ClaI-digested pART7.35S.SCBV.cass, a plasmid
containing PVY sequences in a sense orientation was isolated and
designated p35S.O.SCBV.PVY. Sequences, consisting of the 35S
promoter and NOS terminator, the SCBV promoter driving sense PVY
sequences and the OCS terminator were excised as a Noll fragment
and cloned into pART27, a plasmid with the desired insert
orientation was isolated and designated pART27.35S.O.SCBV.PVY.
[0374] Plasmids pART7.35S.O.SCBV.YVP & pART7.35S.O.SCBV.YVP
[0375] Plasmid pART7.35S.O.SCBV.YVP (FIG. 58) was designed to act
as an additional control for co-expression of multiple constructs
from a single plasmid in transgenic plants. No expressible
sequences were cloned behind the 35S promoter, whilst the SCBV
promoter drove expression of a PW antisense fragment. To generate
this plasmid, the PVY fragment from Cla pBC.PVY was cloned as a
ClaI fragment into ClaI-digested p35S.SCBV.cass, a plasmid
containing PCY sequences in an antisense orientation was isolated
and designated p35S,O.SCBV.YVP. Sequences, consisting of the 35S
promoter and NOS terminator, the SCBV promoter driving sense PVY
sequences and the OCS terminator were excised as a NotI fragment
and cloned into pART27, a plasmid with the desired insert
orientation was isolated and designated pART27.35S.O.SCBV.YVP.
[0376] 6. Test Plasmids
[0377] Plasmids pART7.PVYx2 & pART27.PVYx2
[0378] Plasmid pART7.PVYx2 (FIG. 59) was designed to express a
direct repeat of PVY sequences driven by the 35S promoter in
transgenic plants, To generate this plasmid, direct repeats from
pBC.PVYx2 were cloned as a XhoI/BamHI fragment into XhoI/BamHI cut
pART7. Sequences consisting of the 35 S promoter, direct repeats of
PVY and the OCS terminator were excised as a NotI fragment from
pART7.PVYx2 and cloned into NotI-digested pART27, a plasmid with
the desired insert orientation was selected and designated
pART27.PVYx2.
[0379] Plasmids pART7.PVYx3 & pART27.PVYx3
[0380] Plasmid pART7.PVYx3 (FIG. 60) was designed to express a
direct repeat of three PVY sequences driven by the 355 promoter in
transgenic plants. To generate this plasmid, direct repeats from
pBC.PVYx3 were cloned as a XhoI/BamHI fragment into XhoI/BamHI cut
pART7. Sequences consisting of the 35S promoter, direct repeats of.
PVY and OCS terminator were excised as a NotI fragment from
pART.PVYx3 and cloned into NotI-digested pART27, a plasmid with the
desired insert orientation was selected and designated
pART27.PVYx3.
[0381] Plasmids pART7.PVYx4 & pART27.PVYx4
[0382] Plasmid pART7.PVYx4 (FIG. 61) was designed to express a
direct repeat of four PVY sequences driven by the 35S promoter in
transgenic plants, To generate this plasmid, direct repeats from
pBC.PVYx4 were cloned as a XhoI/BamHI fragment into xhoI/BamHI cut
pART7. Sequences consisting of the 35S promoter, direct repeats of
PVY and the OCS terminator were excised as a NotI fragment from
pART7.PVYx3 and cloned into NotI-digested pART27, a plasmid with
the desired insert orientation was selected and designated
pART27.PVYx3.
[0383] Plasmids pART7.PVY.LNYV.PVY & pART27.PVY.LNYV.PVY
[0384] Plasmid pART7.PVY.LNYV.PVY (FIG. 62) was designed to express
the interrupted direct repeat of PVY sequences driven by the 35S
promoter in transgenic plants. This construct was prepared by
cloning the interrupted direct repeat of PVY from pBC.PVY.LNYV.PVY
as a XhoI/XbaI fragment into pART7 digested with XhoI/XbaI.
Sequences consisting of the 35S promoter, the interrupted direct
repeat of PVY sequences and the OCS terminator were excised from
pART7.PVY.LNYV.PVY as a NotI fragment and cloned into Notf-digested
pART27, a plasmid with the desired insert orientation was selected
and designated pART27.PVY.LNYV.PVY.
[0385] Plasmids pART7.PVY.LNYV.YVP.sub..DELTA. &
pART27.PVY.LNYV.YVP.sub..DELTA.
[0386] Plasmid pART7.PVY.LNYV.YVP.sub..DELTA. (FIG. 63) was
designed to express the partial interrupted palindrome of PVY
sequences driven by the 35S promoter in transgenic plants. This
construct was prepared by cloning the partial interrupted
palindrome of PVY sequences from pBC.PVY.LNYV.YVP.sub..DELTA. as a
XhoI/XbaI fragment into pART7 digested with XhoI/XbaI. Sequences
consisting of the 355 promoter, the partial interrupted palindrome
of PVY sequences and the OCS terminator were excised from
pART7.PVY.LNYV.YVP.sub..DELTA. as a NotI fragment and cloned into
NotI-digested pART27, a plasmid with the desired insert orientation
was selected and designated pART27.PVY.LNYV.YVP.
[0387] Plasmids pART7.PVY.LNYV.YVP & pART27.PVY.LNYV.YVP
[0388] Plasmid pART7.PVY.LNYV.YVP (FIG. 64) was designed to express
the interrupted palindrome of PVY sequences driven by the 35S
promoter in transgenic plants. This construct was prepared by
cloning the interrupted palindrome of PVY sequences from
pBC.PVY.LNYV.YVP.sub..DELTA. as a XhoI/XbaI fragment into pART7
digested with XhoI/XbaI. Sequences consisting of the 35S promoter,
the interrupted palindrome of PVY sequences and the OCS terminator
were excised from pART7.PVY.LNYV.YVP as a NotI fragment and cloned
into pART27, a plasmid with the desired insert orientation was
selected and designated pART27.PVY.LNYV.YVP.
[0389] Plasmids pART7.35S.PVY.SCBV.YVP &
pART27.35S.PVY.SCBV.YVP
[0390] Plasmid pART7.35S.PVY.SCBV.YVP (FIG. 65) was designed to
co-express sense and antisense constructs in transgenic plants. To
generate this plasmid the PVY fragment from Cla pBC.PVY was cloned
as a XhoIlEcoRI fragment into xhol/EcoRI-digested
p35S.SCBV.O.SCBV.YVP. Sequences, consisting of the 35S promoter
driving sense PVY sequences and the NOS terminator and the SCBV
promoter driving antisense PVY and the OCS terminator were excised
as a Noll fragment and cloned into pART27, a plasmid with the
desired insert orientation was isolated and designated
pART27.35S.PVY.SCBV.YVP.
[0391] Plasmids pART7.35S.PVYx3.SCBV.YVPx3 &
pART27.35S.PVYx3.SCBV.YVPx3
[0392] Plasmid pART7.35S.PVYx3.SCBV.YVPx3 (FIG. 66) was designed to
co-express sense and antisense repeats of PVY in transgenic plants.
to generate this plasmid, the intermediate pAR17.35S.O.SCBV.YVPx3
was constructed by cloning the triple direct PVY repeat from
ClapBC.PVYx3 as a ClaI/AccI fragment into Cla-digested
p35S.SCBV.cass and isolating a plasmid with an antisense
orientation. for p35S.PVYx3.SCBV.YVPx3 the triple direct PVY repeat
from Cla pBC.PVYx3 was cloned as a KpnI/SmaI fragment into
KpnI/SmaI-digested p35S.O.SCBV.YVPx3 to create
p35S.PVYx3.SCBV.YVPx3. Sequences including both promoters,
terminators and direct PVY repeats were isolated as a NotI fragment
and cloned into pART27. A plasmid with an appropriate orientation
was chosen and designated pART27.35S.PVYx3.SCBV.
[0393] Plasmids pART7.PVYx3.LNYV.YVPx3 &
pART27.PVYx3.LNYV.YVPx3
[0394] Plasmid pART7.PVYx3.LNYV.YVPx3 (FIG. 67) was designed to
express triple repeats of PW sequences as an interrupted
palindrome. To generate this plasmid an intermediate,
pART7x3.PVY.LNYV.YV was constructed by cloning a PVY.LNYV.YVP
fragment from pBC.PVY.LNYV.YVP as an AccI/ClaI fragment into the
plasmid pART7.PVYx2. pAR17.35S.PVYx3.LNYV.YVPx3, was made by
cloning an additional PVY direct repeat from pBC.PVYx2 as an
AccI/ClaI fragment into ClaI digested pART7x3.PVY.LNYV.YVP.
Sequences from pART7.35S.PVYx3.LNYV.YVPx3, including the 35S
promoter, all PVY sequences and the OCS terminator were excised as
a NotI fragment and cloned into NotI-digested pART27, a plasmid
with an appropriate orientation was chosen and designated
pART27.35S.PVYx3.LNYV.
[0395] Plasmids pART7.PVY multi & pART27.PVY Multi
[0396] Plasmid pART7.35SPVY multi (FIG. 68) was designed to express
higher order direct repeats of regions of PVY sequences in
transgenic plants. Higher order direct repeats of a 72 by of the
PVY Nia region from PVY were prepared by annealing two partially
complementary oligonucleotides as follows:
TABLE-US-00005 PVYt
5'-TAATGAGGATGATGTCCCTACCTTTAATTGGCAGAAATTTCTGTGGA
AAGACAGGGAAATCTTTCGGCATTT-3'; and PVY2:
5'-TTCTGCCAATTAAAGGTAGGGACATCATCCTCATTAAAATGCCGAAA
GATTTCCCTGTCTTTCCACAGAAAT-3'
[0397] The oligonudeotides were phosphorylated with T4
polynudeotide kinase, heated and cooled slowly to permit
self-annealing, ligated with T4 DNA ligase, end-filled with Klenow
polymerase and cloned into pCR2.1 (Invitrogen). Plasmids containing
multiple repeats were isolated and sequences were cloned as EcoRI
fragments in a sense orientation into EcoRI-digested pART7, to
create the intermediate pART7.PVY multi. to create pART27.PVY
multi, the 35S promoter, PVY sequences and the OCS terminator were
excised as a NotI fragment and cloned into NotI-digested pART27. A
plasmid with an appropriate insert orientation was isolated and
designated pART27.PVY multi.
Example 6
Inactivation of Virus Gene Expression in Mammals
[0398] Viral immune lines are created by expressing viral sequences
in stably transformed cell lines.
[0399] In particular, lytic viruses are used for this approach
since cell lysis provides very simple screens and also offer the
ability to directly select for potentially rare transformation
events which might create viral immunity. Sub-genomic fragments
derived from a simple single stranded RNA virus (Bovine
enterovirus--BEV) or a complex double stranded DNA virus, Herpes
Simplex Virus I (HSV I) are cloned into a suitable vector and
expressed in transformed cells. Mammalian cell lines are
transformed with genetic constructs designed to express viral
sequences driven by the strong cytomegalovirus (CMV-IE) promoter.
Sequences utilised include specific viral replicase genes. Random
"shotgun" libraries comprising representative viral gene sequences,
may also be used and the introduced dispersed nucleic acid
molecule, to target the expression of virus sequences.
[0400] Exemplary genetic constructs for use in this procedure,
comprising nucleotide sequences derived from the BEV RNA-dependent
RNA polymerase gene, are presented herein.
[0401] For viral polymerase constructs, large numbers
(approximately 100) of transformed cell lines are generated and
infected with the respective virus. For cells transformed with
shotgun libraries very large numbers (hundreds) of transformed
lines are generated and screened in bulk for viral immunity.
Following virus challenge, resistant cell lines are selected and
analysed further to determine the sequences conferring immunity
thereon.
[0402] Resistant cell lines are supportive of the ability of the
introduced nucleotide sequences to inactivate viral gene expression
in a mammalian system.
[0403] Additionally, resistant lines obtained from such experiments
are used to more precisely define molecular and biochemical
characteristics of the modulation which is observed.
EXAMPLE 8
Induction of Virus Resistance in Transgenic Plants
[0404] Agrobacterium tumefaciens, strain LBA4404, was transformed
independently with the constructs pART27.PVY, pART27.PVYx2,
pART27.PVYx3, pART27.PVYx4, pART27.PVY.LNYV.PVY,
pART27.PVY.LNYV.YVP.sub..DELTA., pART27.PVY.LNYV.YVP,
pART27.35S.PVY.SCBV.O, pART27.35S.O.SCBV.PVY,
pART27.35S.O.SCBV.YVP, pART27.35S.PVY.SCBV.YVP,
pART27.35S.PVYx3.SCBV.YPVx3, pART27.PVYx3.LNYV.YVPx3 and
pART27.PVYx10, using tri-parental matings. DNA mini-preps from
these strains were prepared and examined by restriction with NotI
to ensure they contained the appropriate binary vectors.
[0405] Nicotiana tabaccum (cultivar W38) were transformed with
these Agrobacterium strains using standard procedures. Putative
transformed shoots were excised and rooted on media containing
kanamycin. Under these conditions we have consistently observed
that only transgenic shoots will root on kanamycin plates. Rooted
shoots were transferred to soil and allowed to establish. After two
to three weeks, vigorous plants with at least three sets of leaves
were chosen and infected with PVY.
[0406] Viral inoculum was prepared from W38 tobacco previously
infected with the virus, approximately 2 g of leaf material,
showing obvious viral symptoms were ground with carbarundum in 10
ml of 100 mM Na phosphate buffer (pH 7.5). the inoculum was diluted
to 200 ml with additional Na phosphate buffer. Two leaves from each
transgenic plant were sprinkled with carbarundum, then 0.4 ml of
inoculum was applied to each leaf and leaves rubbed fairly
vigorously with fingers. Using this procedure 100% of
non-transgenic control plants were infected with PVY.
[0407] To assay for viral resistance and immunity transgenic plants
are monitored for symptom development. The PVY strain (PVY-D, an
Australian PVY isolate) gives obvious symptoms on W38 tobacco, a
vein clearing symptom is readily observed on the two leaves above
the inoculated leaves, subsequent leaves show uniform chlorotic
lesions. Symptom development was monitored over a six week
period.
[0408] Transgenic lines were described as resistant if they showed
reduced viral symptoms, which manifests as a reduction in the leaf
are showing chlorotic lesions. Resistance ranges from very strong
resistance where only a few viral lesions are observed on a plant
to weak resistance which manifects as reduced symptoms on leaves
that develop late in plant growth.
[0409] Transgenic plants which showed absolutely no evidence of
viral symptoms were classified as immune. To ensure these plants
were immune they were re-inoculated with virus, most plants
remained immune, the few that showed symptoms were re-classified as
resistant.
[0410] For plant lines generated Southern blots are performed,
resistance in subsequent generations is monitored to determine that
resistance/immunity is transmissable. Additionally, the breadth of
viral resistance is monitored by challenging lines with other PVY
strains, to determine whether host range susceptibility is
modified.
[0411] Results from these experiments are described in Table 2 .
These data indicate that constructs comprising tandem repeats of
target gene sequence, either in the configuration of palindromes,
interrupted palindromes as direct repeat sequences, are capable of
conferring viral resistance and/or immunity in transgenic
plants.
[0412] Accordingly, such inverted and/or direct repeat sequences
modulate expression of the virus target gene in the transgenic
plant.
[0413] Constructs combining the use of direct and inverted repeat
sequences, namely pART27.35S.PVYx3.SCBV.YVPx3 and
pART27.PVYx3.LNW.YVPx3, are also useful in modulating gene
expression.
EXAMPLE 9
Inactivation of Galt in Animal Cells
[0414] To assay for Galt inactivation, porcine PK2 cells were
transformed with the relevant constructs. PK2 cells constitutively
express Galt enzyme, the activity of which results in the addition
of a variety of .alpha.-1,3-galactosyl groups to a range of
proteins expressed on the cell surface of these cells. Cells were
transformed using lipofectin and stably transformed lines were
selected using genetecin.
[0415] As an initial assay cell lines were probed for the presence
of the Galt-encoded epitope, i.e. .alpha.-1,3-galactosyl moieties
decorating cell surface proteins, using the lectin IB4. IB4 binding
was assayed either in situ or by FACS sorting.
[0416] For in situ binding, cells were fixed to solid supports with
cold methanol for 5 mins, cells were rinsed in PBS (phosphate
buffered saline) and non-specific IB4 binding was blocked with 1%
BSA in PBS for 10 mins. Fixed cells were probed using 20 ug/ml
IB4-biotin (Sigma) in 1% BSA, PBS for 30 mins at room temperature,
cells were washed in PBS then probed with a 1:200 dilution of
ExtrAvidin-FITC (Sigma) in PBS for 30 mins followed by further
rinses in PBS. Cells were then examined using fluorescence
microscopy, under these conditions the outer surface of PK2 control
cells uniformly stained green.
[0417] For FACS analysis, cells were suspended after treatment with
trypsin, washed in HBSS/Hepes (Hank's buffered saline solution with
20 mM Hepes, pH7.4) and probed with 10 ug/ml IB4-biotin (Sigma) in
HBSS/Hepes for 45 mins at 4.degree. C. Cells were washed in
HBSS/Hepes, probed with a 1:200 dilution of ExtrAvidin-FITC (Sigma)
in HBSS/Hepes for 45 mins at 4.degree. C. at and rinsed in cold
HBSS/Hepes prior to FACS soiling.
[0418] Using this approach transformed cell lines are assayed for
Galt inactivation and quantitative assessment of construct
effectiveness is determined. Moreover cell lines showing Galt
inactivation are isolated and subject to further molecular analyses
to determine the mechanism of gene inactivation.
TABLE-US-00006 No. OF PERCENTAGE OF PLANTS SHOWING PLANTS SPECIFIED
PHENOTYPE PLASMID CONSTRUCT TESTED SUSCEPTIBLE IMMUNE RESISTANT
pART27.PVY 19 16 1 2 pART27.PVYx2 13 5 4 4 pART27.PVYx3 21 2 5 14
pART27.PVYx4 21 5 7 9 pART27.35S.PVY.SCBC.0 25 8 0 17
pART27.35S.O.SCBV.PVY 22 8 0 14 pART27.35S.O.SCBV.YVP 18 14 0 4
pART27.35S.PVY.SCBV.YVP 17 3 8 6 pART27.PVY.LNYV.PVY 26 18 2 6
pART27.PVY.LNYV.YVP 20 6 10 4 pART27.PVY.LNYV.YVP.DELTA. 18 7 11
0
REFERENCES
[0419] 1. An et al. (1985) EMBO J 4:277-284. [0420] 2. Armstrong,
et al. Plant Cell Reports 9 335-339, 1990. [0421] 3. Ausubel, F. M.
et al. (1987) In: Current Protocols in Molecular Biology, Wiley
Interscience (ISBN 047140338). [0422] 4. Chalfie,M. el al (1994)
Science 263: 802-805. [0423] 5. Christensen, A. H. and Quail, P. H.
(1996) Transgenic Research 5: 213-218. [0424] 6. Christou, P., et
al. Plant Physiol 87: 671-674, 1988. [0425] 7. Cormack, B. et al
(1996) Gene 173: 33-38. [0426] 8. Crossway et al., Mol. Gen. Genet.
202:179-185, 1986. [0427] 9. Dorer, D. R., and Henikoff, S. (1994)
Cell 7: 993-1002. [0428] 10. Fromm et al. Proc. Natl. Acad. Sci.
(USA) 82:5824-5828, 1985. [0429] 11. Gleave, A. P. (1992) Plant
Molecular Biology 20:1203-1207. [0430] 12. Hanahan, D. (1983) J.
Mol. Biol. 166: 557-560. [0431] 13. Herrera-Estella et al., Nature
303: 209-213, 1983a. [0432] 14. Herrera-Estella et al., EMBO J. 2:
987-995, 1983b. [0433] 15. Herrera-Estella of al. In: Plant Genetic
Engineering, Cambridge University Press, N.Y., pp 63-93, 1985.
[0434] 16. Inouye, S. and Tsuji, F. I. (1994) FEBS Letts. 341:
277-280. [0435] 17. Jackson, I. J. (1995) Ann. Rev. Genet. 28:
189-217. [0436] 18. Krens, F.A., of al., Nature 296: 72-74, 1982.
[0437] 19. Kwon, B. S. et al. (1988) Biochem. Biophys. Res. Comm.
153:1301-1309. [0438] 20. Pal-Bhadra, M. et al. (1997) Cell 90:
479-490. [0439] 21. Paszkowski et al., EMBO J. 3:2717-2722, 1984
[0440] 22. Prasher, D. C. et al. (1992) Gene 111: 229-233. [0441]
23. Sanford, J. C., et al., Particulate Science and Technology 5:
27-37, 1987.
Sequence CWU 1
1
16126DNAArtificial SequencePrimer Bgl-GFP for Green Fluorescent
Protein in jellyfish. 1agatctgtaa acggccacaa gttcag
26226DNAArtificial SequencePrimer GFP-Bam for Green Fluorescent
Protein in jellyfish. 2ggatccttgt acagctcgtc catgcc
26374DNAArtificial SequencePrimer SV40-1 for SV40 late promoter.
3gtcgacaata aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgatttt
60tgcaaaagcc tagg 74431DNAArtificial SequencePrimer SV40-2 for SV40
late promoter. 4gtcgacgttt agagcagaag taacacttcc g
31538DNAArtificial SequencePrimer BEV-1 for the BEV RNA-dependant
RNA polymerase from virus. 5cggcagatct aacaatggca ggacaaatcg
agtacatc 38631DNAArtificial SequencePrimer BEV-2 for the BEV
RNA-dependant RNA polymerase from virus. 6cccgggatcc tcgaaagaat
cgtaccactt c 31729DNAArtificial SequencePrimer BEV-3 for the BEV
RNA-dependant RNA polymerase from virus. 7gggcggatcc ttagaaagaa
tcgtaccac 29828DNAArtificial SequencePrimer BEV-4 for the BEV
RNA-dependant RNA polymerase from virus. 8cggcagatct ggacaaatcg
agtacatc 28937DNAArtificial SequencePrimer NOS 5' for the NOS
terminator sequence from agrobacterium. 9ggattcccgg gacgtcgcga
atttcccccg atcgttc 371033DNAArtificial SequencePrimer NOS 3' for
the NOS terminator sequence from agrobacterium. 10ccatggccat
ataggcccga tctagtaaca tag 331133DNAArtificial SequencePrimer SCBV
5' for the SCBV promoter sequence from virus. 11ccatggccta
tatggccatt ccccacattc aag 331227DNAArtificial SequencePrimer SCBV
3' for the SCBV promoter sequence from virus. 12aacgttaact
tctacccagt tccagag 271328DNAArtificial SequencePrimer LNYV 1 for
the LNYV 4 KB gene from virus. 13atgggatccg ttatgccaag aagaagga
281424DNAArtificial SequencePrimer LNYV 2 for the LNYV 4 KB gene
from virus. 14tgtggatccc taacggaccc gatg 241572DNAArtificial
SequencePrimer PVY1 for the PVY Nia region from virus. 15taatgaggat
gatgtcccta cctttaattg gcagaaattt ctgtggaaag acagggaaat 60ctttcggcat
tt 721672DNAArtificial SequencePrimer PVY2 for the PVY Nia region
from virus. 16ttctgccaat taaaggtagg gacatcatcc tcattaaaat
gccgaaagat ttccctgtct 60ttccacagaa at 72
* * * * *