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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/11—Antisense
  • C12N2310/111—Antisense spanning the whole gene, or a large part of it

Definitions

• the present invention provides methods for introducing variegation into plants and animals by manipulation of genetic material.
• Variegation encompasses variation in appearance or colour of an organism as well as variation in biochemical properties of an organism.
• Variegation is due to several causes, namely: a. Segregation of mutant and normal chloroplasts (in plants) b. Nuclear chimaeras c. Transposon instability causing somatic mutations d. Virus infections e. Position-effect variegation (PEV)
• Pattern genes cause sectoring, spotting and other variable patterns but do not normally produce typical variegation.
• PEV is genetically stable.
• PEV is the mosaic expression of a gene near the breakpoint of a chromosomal rearrangement. It has been described in several species of Drosophila, in a plant and in a fungus. This suggests that the phenomenon is probably ubiquitous. The molecular basis of PEV has been unknown since the discovery of the phenomenon by H. J. Muller in 1930.
• PEV and some other forms of variegation, arise from either anti-sense transcription or promoter occlusion (transcription readthrough), the former operating when a breakpoint is on the 3' side of affected genes and the latter when a breakpoint is on the 5' side of affected genes.
• promoter occlusion transcription readthrough
• Anti-sense RNA has been widely used previously to modify gene functioning (Izant and Weintraub 1985 Science 221, 345-352; Green et al 1986 Annual Review of Biochemistry 55, 569-597). However, it has not previously been used in the context of producing variegation.
• Anti-sense transcription may be used to produce phenotypic variegation by producing, using genetic engineering, genotypes in which both sense mRNA and anti-sense RNA are produced for the gene of interest.
• the anti-sense RNA may be for all or part of the gene. Because the anti-sense RNA transcript has a complementary base sequence to the sense mRNA, they will hybridize, thus inactivatirrg much or all of the sense mRNA.
• Variegation in phenotype arises when anti-sense/sense transcription is at a threshold where all sense mRNA is hybridized and inactivated in some cells, while in others sufficient sense mRNA escapes hybridization to lead to functional product. Differences among cells will arise due to slight variation in the ratio of anti-sense to sense transcription and from the random nature of the hybridization reaction.
• the phenotypic expression of different genotypes depends on the amount of wild type mRNA that remains unhybridized. Consequently, it depends on the ratio of sense to anti-sense transcription.
• Promoter occlusion is well known as a means for depressing gene function in prokaryotes (Adhya and Gottesman 1982 Cell 29, 939-944). However, it has not previously been applied to the deliberate production of variegated phenotypes.
• Promoter occlusion occurs when a gene without a transcription termination signal lies upstream of another gene and in the same transcriptional orientation. Transcription from the upstream promoter may interfere with normal transcription of the downstream gene by producing a non-functional read through transcript. A variegated phenotype may result when the downstream gene produces occasional normal transcripts such that by chance some cells have no transcript (and have mutant phenotypes) while other cells have functional transcripts (and have wild-type phenotypes).
• the present invention provides methods for the genetic engineering of stable, defined variegation in any species of living organism.
• the invention provides a genetically engineered organism in which the phenotypic expression of at least one gene is variegated, characterized by at least one functional gene and at least one inserted DNA sequence transcribable in the organism and capable of interfering with the expression of said gene or genes such that said gene or genes is(are) intermittently expressed.
• the inserted DNA sequence codes for mRNA complimentary to at least a part of mRNA transcribed from the functional gene and that it includes a promoter such as the S35 promoter of the cauliflower mosaic virus.
• Also within the scope of the invention is a process for genetically engineering variegation in an organism characterized in that at least one DNA sequence which is transcribable in said organism and which is capable of interfering with expression of at least one functional gene is inserted into said organism or into a gamete therefor, such that said gene is intermittently expressed in said organism.
• a vector system comprising a replicon suitable for insertion into chromosomal DNA of the species of interest, a gene cloning site and a DNA sequence capable of interfering with the expression of at least one gene inserted into said gene cloning site.
• a preferred organism according to the invention contains at least one gene associated with a promoter either 3' to the gene such that the promoter is oriented to promote transcription of the anti-sense copy of the gene to generate message capable of hybridizing to mRNA transcribed from the sense copy of the gene or 5' to the gene such that the promoter lies upstream of any promoter sequence associated with the gene and can cause transcription readthrough to generate an elongated non-functional mRNA resulting in variegated expression of the gene in the organism.
• the inserted DNA sequence may be inserted into an intron of the functional gene.
• an organism is defined as multi cell body or population of single cell bodies that share the same genotype.
• a number of genes it should be possible to cause a number of genes to variegate simultaneously by placing more than one gene in the construct, by transforming with a number of constructs containing different genes, or by making a construct such that genes in one orientation are subject to sense and anti-sense transcription and another in the opposite orientation is subject to promoter occlusion (transcription readthrough).
• Figure 1 illustrates sense/anti-sense constructs that can be used to produce variegation when inserted using a transformation vector into a variety lacking a functional form of the gene of interest.
• Figure 2 illustrates a sense/anti-sense construct for a portion of its length that can be used to produce variegation when inserted, using a transformation vector, into a variety lacking a functional form of the gene of interest.
• Figure 3 illustrates an anti-sense construct that can be used to produce variegation when inserted, using a transformation vector, into a variety containing one or more functional copies of the gene of interest.
• Figure 4 illustrates an anti-sense construct that can be used to produce variegation when inserted, using a transformation vector, into a variety containing one or more functional copies of the gene of interest.
• Figure 5 illustrates a promoter occlusion construct that can be used to produce variegation when inserted, using a transformation vector, into a variety lacking a functional allele of the gene of interest.
• Figure 6 illustrates the pW6 hsp70-w P element transformation vector plasmid.
• Figures 1 to 4 are illustrative of constructs of the anti-sense RNA/sense mRNA hybridization type.
• Figure 5 is illustrative of a construct of the promoter occlusion type.
• Constructs of the types illustrated in Figures 1, 2 and 5 can be inserted into a plant, microbe or animal species (as appropriate) lacking a normal functional form of the gene of interest.
• Constructs of the types illustrated in Figures 3 and 4 can be inserted into a plant, microbe or animal species (as appropriate) containing a functional form of the gene of interest. These insertions are carried out via standard methods such as Ti plasmid mediated transformation in dicotyledon plants (Horsch et al. 1985 Science 227, 1229-1231), microinjection of DNA into the male pronucleus in mammalian embryos, (Palmiter et al. 1982, Nature, 300, 611-615) P element mediated transformation in Drosophila (Spradling 1986 in "Drosophi la, A Practical Approach, ed by D.B. Roberts, IRL Press Oxford pp 175-199; Karess R.E. 1985 DNA Vol. II, Glover D.A. ed, IRL Press Oxford pp 121-141).
• Figures 1 and 2 illustrate genes with a promoter ( ⁇ ), and transcription termination signal ( ⁇ ).
• Figures 2 and 4 illustrate genes with introns and exons.
• the genes in Figures 1, 3 and 5 may or may not have introns.
• Figure 1 illustrates sense/anti-sense constructs that can be used to produce variegation when inserted using a transformation vector into a variety lacking a functional form of the gene of interest.
• the gene construct must lead to both sense mRNA and anti-sense transcription.
• the construct is made by taking the gene of interest with a 5' promoter and inserting an additional promoter beyond the 3' end of the gene in an orientation such that it will cause anti-sense transcription. This transcription is in addition to the mRNA transcribed from the sense strand. A transcription termination signal is shown inserted beyond the 5' promoter of the gene to terminate anti-sense transcription. The use of this is optional.
• An alternative construct in (b) has the gene of interest present in both sense and anti-sense orientations. This construct also leads to both sense mRNA and anti-sense RNA for the gene of interest.
• Figure 2 illustrates a sense/anti-sense construct for a portion of its length that can be used to produce variegation when inserted, using a transformation vector, into a variety lacking a functional form of the gene of interest.
• the gene construct must lead to the production of RNA molecules that are complementary for part of their lengths. A gene with introns and exons is shown above.
• the insert consists of a promoter and a transcription termination signal with transcribable DNA between.
• the insert is placed into an intron of the gene in the opposite transcriptional orientation to the gene itself, so that the gene promoter directs transcription of the anti-sense strand of the insert, while the inserted promoter directs transcription of the sense strand of the insert, leading to two RNA molecules that are partially complementary and capable of hybridising (as shown).
• Figure 3 illustrates an anti-sense construct that can be used to produce variegation when inserted, using a transformation vector, into a variety containing one or more functional copies of the gene of interest.
• the gene construct must produce anti-sense RNA capable of hybridising to the sense mRNA from the gene of interest.
• the construct contains an inversion of the transcribed region between the promoter and the transcription termination signal, such that anti-sense RNA is produced.
• a construct with a large inversion extending into or beyond the transcription termination signal is also suitable.
• a construct with an inversion within the gene that results upon transcription in an RNA that is anti-sense for a portion of the gene is also suitable. Several copies of such inserts may need to be introduced to produce variegation.
• Figure 4 illustrates an anti-sense construct that can be used to produce variegation when inserted, using a transformation vector, into a variety containing one or more functional copies of the gene of interest.
• the gene construct must produce anti-sense RNA for a portion of the gene, capable of hybridising to the sense mRNA from the gene of interest.
• the gene above is shown with introns and exons.
• the construct consists of a promoter and transcription termination signal with a segment of the intron inserted between them in the anti-sense orientation.
• the transcripts from the gene and the construct are shown as hybridising as they have regions with complementary base sequences. The same end may be achieved if the construct contains any portion of the transcribed portion of the gene (part or all of an exon or portions of both exon and intron) between the promoter and the transcription termination signal.
• Figure 5 illustrates a promoter occlusion construct that can be used to produce variegation when inserted, using a transformation vector, into a variety lacking a functional allele of the gene of interest.
• the gene construct must produce a non-functional readthrough transcript capable of inhibiting most normal transcription from the gene of interest.
• the gene of interest is shown above, a promoter plus transcribable DNA, without a transcription termination signal in the centre and the construct, made by joining these two DNA fragments at the bottom.
• the readthrough transcript is shown for the construct.
• an upstream promoter using a different RNA polymerase can be used to overcome this (e.g. rRNA gene).
• FIG 6 is a diagrammatic representation of the salient features of the pW6 hsp70-w P element transformation vector plasmid (not to scale).
• the hsp70 promoter (P) and portion of the leader sequence are fused to a truncated white gene (without its own promoter) and located within a P element transformation vector plasmid.
• the truncated first intron of the white gene is indicated, as is the polylinker (PL) at the 3' end of the white gene.
• Ndel, Nhel, SphI, AccI, Hindlll, Clal, PstI, StuI and BamHI are restriction sites referred to in Examples 1-3.
• P element sequences (not shown) are 5' to the hsp70 promoter and 3' to the polylinker (PL).
• Variegation of eye colour in Drosophila melanogaster by variegation of white gene expression using an anti-sense/sense construct inserted into a white mutant stock.
• the first step is to remove the hsp70 promoter from pW6 [an hsp70-white gene P element vector plasmid (Klemenz, Weber and Gehring 1987 NAR 15, 3947-3959) see Fig. 6]. This is achieved by digesting pW6 DNA with the restriction enzyme Nhel, allowing the cut plasmid to religate, and using it to transform Escherichia coli. The resulting plasmid is pW6hP-. Plasmid DNA from transformants is subjected to gel electrophoresi s to check that it is reduced in size from 8.3 kb to 7.5 kb. Plasmid DNA from a suitable transformant is then purified by CsCl density gradient centrifugation.
• the second step is to isolate the hsp70 promoter fragment.
• a double restriction enzyme digest of pW6 DNA with Nhel and Accl is performed followed by gel electrophoresis and extraction of the 231 bp fragment (containing the hsp70 promoter and' a portion of the leader sequence).
• the extracted fragment is purified using Gene clean (Trade Mark) and the ends filled in using standard methods (Maniatis et al. Cold Spring Harbor 1982).
• the third step is to insert the hsp70 promoter fragment into the pW6hP- plasmid in the anti-sense orientation.
• pW6hP- DNA is cut in the 3' polylinker with Stul, treated with alkaline phosphatase and ligated to the hsp70 promoter fragment isolated in step two above. This DNA is used to transform E. coli and DNA isolated from several transformants. Restriction mapping of these transformants is used to identify plasmids (pW6A) with the hsp70 promoter fragment in the required anti-sense orientation to the white gene fragment. Double digestion with Sphl and PstI yields an approximately 250 bp fragment from the desired plasmids.
• the fourth step is to isolate the white gene promoter along with a portion of the gene running into the first intron.
• C3W9 (a P element vector containing the complete white locus) is double digested with Hindi and Hindlll restriction enzymes followed by gel electrophoresis and extraction of the 1.6 kb white promoter fragment. The extracted fragment is purified using Gene clean (Trade Mark) and the ends filled in as above.
• the fifth step is to insert the whi te gene promoter fragment into the front of the white gene fragment in the P element transformation vector.
• the pW6A plasmid DNA from step three is cut with Nhel, the ends filled in as above, treated with alkaline phosphatase and ligated with the white gene promoter fragment from step four.
• the resulting plasmids are used to transform E. coli and DNA isolated from several transformants. Restriction mapping using double digests with Ndel and Nhel yields a 2.25 kb fragment from plasmids with the white gene promoter in the correct sense orientation.
• the resulting P element transformation vector plasmid (pW6AS) has the normal white gene promoter in a position to drive sense transcription of the truncated white gene and the hsp70 promoter i n a pos i tion to produce anti-sense transcription of it.
• the sense/anti-sense white construct within pW6AS is inserted into Drosophila melanogaster stock w 1118 using P element mediated transformation (Spradling 1986 in "Drosophila a practical approach", ed. D.
• EXAMPLE 2 Variegation of eye colour in Drosophila melanogaster by variegation of white gene expression using an anti-sense construct inserted into a white + stock.
• the first step is to remove the hsp70 promoter from pW6 [an hsp70-white gene P element vector plasmid (Klemenz, Weber and Gehring 1987 NAR 15, 3947-3959) see Fig. 63. This is achieved by digesting pW6 DNA with the restriction enzyme Nhel, allowing the cut plasmid to religate, and using it to transform Escherichia coli. The resulting plasmid is pW6hP-. Plasmid DNA from transformants is subjected to gel electrophoresis to check that it is reduced in size from 8.3 kb to 7.5 kb.
• the second step is to isolate the hsp70 promoter fragment.
• a double restriction enzyme digest of pW6 DNA with Nhel and Accl is performed followed by gel electrophoresis and extraction of the 231 bp fragment (containing the hsp70 promoter and a portion of the leader sequence).
• the extracted fragment is purified using Gene clean (Trade Mark) and the ends filled in using standard methods (Maniatis et al. Cold Spring Harbor 1982).
• the third step is to insert the hsp70 promoter fragment into the pW6hP- plasmid in the anti-sense orientation.
• pW6hP- DNA is cut in the 3' polylinker with Stul, treated with alkaline phosphatase and ligated to the hsp70 promoter fragment isolated in step two above. This DNA is used to transform E. coli and DNA isolated from several transformants. Restriction mapping of these transformants is used to identify plasmids (pW6A) with the hsp70 promoter fragment in the correct anti-sense orientation to the white fragment. Double digestion with Sphl and PstI yields an approximately 250 bp fragment from the desired plasmids.
• the fourth step is to remove the segment between the hsp70 promoter and the 3' end of the white gene fragment.
• DNA from pW6A is double digested with PstI and Clal, the restricted DNA treated with SI nuclease to remove overhangs, and religated to yield pW6A2.
• the fifth step is to isolate the rosy gene.
• DNA from the plasmid pryl (Rubin and Spradling 1982 Science 218, 348-353) is digested with Hindlll, the restricted DNA subjected to gel electrophoresis and the 7.2 kb fragment containing the rosy locus isolated, purified using Gene clean (Trade Mark) and the ends filled in as described above.
• the sixth step is to insert the rosy gene into the construct in the 3' polylinker.
• DNA from pW6A2 is digested with BamHll , the ends of the restricted DNA filled in as described above and ligated to the rosy fragment.
• the resulting P element transformation vector (p[W6A2, ry]) contains an hsp70 promoter attached in the anti-sense orientation to the 3' end of a fragment the white locus (consisting of most of the coding sequence but lacking the first exon, most- of the first intron and. a portion of the last exon) plus the fully functional rosy gene.
• the anti-sense white construct and the rosy gene within p[W6A2,ry] is inserted into Drosophila melanogaster stock ry 506 using P element mediated transformation (Spradling 1986 in “Drosophi la a practical approach", ed. D. B. Roberts, IRL press Oxford pp. 175-199; Karess, R. E. 1985 in "DNA Cloning Vol. II” ed. D. A. Glover, IRL press Oxford pp. 121-141).
• Purified p[W6A2, ry] DNA is coinjected with the wings-clipped P element helper (pII25.7wc) DNA into dechorionated embryos.
• progeny are mated to ry 506 flies of the opposite sex and their progeny raised under normal conditions.
• Successful transformants would be selected as those progeny with normal wild-type red eye colouration (i.e. ry + ).
• Successful transformants are used to found homozygous or balanced stocks using standard methods as described by Spradling and by Karess (see above for citations). Some stocks will not yield homozygous viable transformants and will have to be maintained as balanced stocks.
• Temperature shock (37°C for 1 hour) or temperature shift (grown at 18°C for the larval period and shifted to 29°C for the pupal period) treatments will normally be required for these stocks to produce variegated eyes, the details depending on the chromosomal site of insertion of the white anti-sense P element construct. In many cases increased copy numbers of the anti-sense white construct will be required to produce variegated white expression. Increased copy numbers of the construct are produced by re-mobilizing the P element anti-sense white gene construct by microinjecting embryos of transformed stocks with the helper P element (pII25.7wc), raising progeny, mating them to ry 506 and producing stocks. These stocks are tested for variegated eyes using heat shocks or shifts to higher temperatures during the pupal period as described above.
• Variegation of eye colour in Drosophila melanogaster by variegation of white gene expression using a promoter occlusion construct inserted into a white mutant stock.
• the first step is to isolate the rRNA gene spacer that contains its promoter.
• pDm238 a plasmid containing a full rRNA gene
• Hindlll a plasmid containing a full rRNA gene
• the extracted fragment is purified using Gene clean and the ends filled in as above.
• the second step is to insert this rRNA spacer fragment into pW8 [an hsp70 White gene P element vector plasmid differing from pW6 in having the polylinked at the 5' end) Klemenz et al, 1987, NAR, 15, 3947-3959)3.
• DNA from pW8 is cut with Stul, treated with alkaline phosphatase and ligated with the rRNA spacer fragment.
• the resulting plasmids are used to transform E. coli and DNA isolated from several transformants.
• the white gene promoter occlusion construct within pW8rPO is inserted into Drosophi la melanogaster stock w 1118 using P element mediated transformation (Spradling 1986 in "Drosophi la a practical approach” ed. D.
• Successful transformants are selected as those progeny with some eye colouration (i.e. not white). Successful transformants are used to found homozygous or balanced stocks using standard methods as described by Spradling and by
• Temperature treatment required for these stocks to produce variegated eyes will depend on the chromosomal site of insertion of the white gene promoter occlusion P element construct. Variegation is more likely to be found when the stocks are raised at 18°C uncrowded, ideal conditions. Lower proportions of mutant (white) cells will be found when the hsp70 promoter is induced with heat shock or a shift to a higher temperature is imposed during the pupal period. Stocks are tested with w 1118 on the X chromosomes.
• the present invention may be used to provide stable defined phenotypic variegation in plant and animal species.
• the most important applications of these methods are likely to be to produce variegated leaves and flowers in plants and to produce variegated coats in cats, dogs and other mammals.
• the invention can also be applied to other phyla of the animal kingdom, especially to birds and insects.

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• 1987
  • 1987-08-26 WO PCT/AU1987/000291 patent/WO1988001645A1/en not_active Application Discontinuation
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