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At least 109 records · Page 6Linked to original sources
PubMed2004

Cdx1 induced intestinal metaplasia in the transgenic mouse stomach: comparative study with Cdx2 transgenic mice.

BACKGROUND AND AIMS: Gastric intestinal metaplasia, which is mainly induced by Helicobacter pylori infection, is thought to be a precancerous lesion of gastric adenocarcinoma. Intestinal metaplastic mucosa expresses intestine specific homeobox genes, Cdx1 and Cdx2, in the human gastric mucosa. We and others have reported that ectopic expression of Cdx2 in the gastric epithelium generates intestinal metaplasia in the transgenic mouse model. METHODS: To clarify the differences in the roles of Cdx1 and Cdx2 in intestinal metaplasia, we generated transgenic mice expressing Cdx1 in the gastric mucosa and compared Cdx1 induced gastric mucosal morphological changes with Cdx2 induced intestinal metaplasia. RESULTS: The gastric mucosa in Cdx1 transgenic mice was completely replaced by intestinal metaplastic mucosa, consisting of all four intestinal epithelial cell types: absorptive enterocytes, goblet, enteroendocrine, and Paneth cells. Paneth cells, which were not recognised in Cdx2 transgenic mice, were in the upper portion of the intestinal metaplastic mucosa. Pseudopyloric gland metaplasia, which was induced in Cdx2 transgenic mice, was not recognised in Cdx1 transgenic mice. Proliferating cell nuclear antigen (PCNA) positive cells were diffusely scattered in Cdx1 induced intestinal metaplastic mucosa while PCNA positive cells in Cdx2 induced intestinal metaplastic mucosa were in the base of the metaplastic mucosa. Intestinal metaplastic mucosa of Cdx1 transgenic mouse stomach was significantly thicker than that of wild-type or Cdx2 transgenic mouse stomach. CONCLUSIONS: We have confirmed that Cdx1 induced gastric intestinal metaplasia but that it differed from Cdx2 induced intestinal metaplasia in differentiation, structure, and proliferation.

Animals↗
PubMed1994

Variation in expression of a bovine alpha-lactalbumin transgene in milk of transgenic mice.

Transgenic mice were produced to study the production of bovine alpha-LA in their milk. A 7.6-kb fragment containing a bovine alpha-LA gene was purified and microinjected into pronuclear stage mouse embryos. This fragment contained 2.0 kb of 5' flanking region, the 1.7-kb coding region, and 2.7 kb of 3' flanking region. Out of 121 potential transgenic founder mice, 3 were identified as being transgenic by the polymerase chain reaction. Multiple mice from the second, third, and fourth generation from each line were milked, and the milk was analyzed using an ELISA assay and Western blots to determine the presence of bovine alpha-LA. Bovine alpha-LA was present at concentrations up to 1.5 mg of protein/ml of mouse milk. The high degree of expression variation between mice within each of the transgenic lines was a characteristic that has not been reported in other studies of transgene expression in milk. Production of bovine alpha-LA in the milk of these transgenic mice showed a high degree of variation both within a lactation and between mice within a line. The bovine alpha-LA concentration in a single line of transgenic mice exhibited as much as a 10-fold variation between mice. Variations as high as 3-fold were detected within a single lactation in the same mouse. These differences in expression appeared to be correlated with mouse milk production; bovine alpha-LA was higher on d 10 and 15 of lactation than on d 5. Transgenic mice that show variation in expression of a bovine gene might offer a unique system for studying quantitative traits in a laboratory model.

Animals↗
PubMed2001

Genomic interspersions determine the size and complexity of transgene loci in transgenic plants produced by microprojectile bombardment.

The structure of transgene loci in six transgenic allohexaploid oat (Avena sativa L.) lines produced using microprojectile bombardment was characterized using fluorescence in situ hybridization (FISH) on extended DNA fibers (fiber-FISH). The transgene loci in five lines were composed of multiple copies of delivered DNA interspersed with genomic DNA fragments ranging in size from ca. 3 kb to at least several hundred kilobases, and in greater numbers than detected using Southern blot analysis. Although Southern analysis predicted that the transgene locus in one line consisted of long tandem repeats of the delivered DNA, fiber-FISH revealed that the locus actually contained multiple genomic interspersions. These observations indicated that transgene locus size and structure were determined by the number of transgene copies and, possibly to a greater extent, the number and the length of interspersing genomic DNA sequences within the locus. Large genomic interspersions detected in several lines were most likely the products of chromosomal breakage induced either by tissue culture conditions or, more likely, by DNA delivery into the nucleus using microprojectile bombardment. We propose that copies of transgene along with other extrachromosomal DNA fragments are used as patches to repair double-strand breaks (DSBs) in the plant genome resulting in the formation of transgene loci.

Avena↗
PubMed1990

Most tumors in transgenic mice with human c-Ha-ras gene contained somatically activated transgenes.

Two independent transgenic mouse lines carrying human hybrid c-Ha-ras genes with their own promoter region encoding prototype products, were established. In these lines, about 50% of transgenic offspring had tumors within 18 months. The tumors developed in restricted tissues and about 60% of affected mice had angiosarcomas. The transgenes were expressed both in the tumors and in all normal tissues. However, somatic mutational activation was detected only in the transgenes of the tumors. The point mutation at the 61st codon, from CAG(Gln) to CTG(Leu), was detected in all angiosarcomas (22/22), some lung adenocarcinomas (3/11) and Harderian gland adenocarcinomas (4/7) in both lines. The other point mutation at the 12th codon from GGC(Gly) to GTC(Val) was detected in two of the four skin papillomas. No mutations on these codons were detected in normal tissues of transgenic mice. Nontransgenic littermates had no tumors at all. From these results, it was strongly suggested that the mouse tumors do not develop only by the expression of the transgenes, and that definite somatic point mutation of the human c-Ha-ras transgenes in certain cell types may be a causative event in tumorigenesis in these transgenic mice.

Animals↗
PubMed1993

Transgenic tumor necrosis factor (TNF)-alpha production in pancreatic islets leads to insulitis, not diabetes. Distinct patterns of inflammation in TNF-alpha and TNF-beta transgenic mice.

To understand the role of TNF in the regulation of inflammation and the development of autoimmune diseases such as insulin-dependent diabetes mellitus, we produced transgenic mice in which the synthesis of murine TNF-alpha was directed by the rat insulin II promoter. The expression of the TNF-alpha transgene was restricted to the pancreas, in contrast to TNF-beta expression from the same promoter, in which the transgene was expressed in the pancreas, kidney, and skin. The expression of TNF-alpha in the pancreas of transgenic mice resulted in an overwhelming insulitis, composed of CD4+ and CD8+ T cells and B220+ B cells, considerably greater than that of TNF-beta transgenics. Moreover, in contrast to the predominant peri-insulitis observed in TNF-beta transgenic mice, the majority of the infiltrate in the TNF-alpha transgenic mice was within the islet itself. These unique patterns of infiltration were observed in the F1 progeny of crosses with C57BL/6 as well as NOD. Both TNF-alpha and TNF-beta transgenic mice show elevated expression of leukocyte adhesion molecules VCAM-1 and ICAM-1 in islet endothelia and increased expression of MHC class I on islet cells. This inflammation did not result in reduced insulin content of the islets, nor did it lead to diabetes. These data suggest that additional stimuli are necessary to initiate the process of islet destruction.

Animals↗
PubMed2001

Reinitiated expression of EJras transgene in targeted epidermal cells of transgenic rabbits by cottontail rabbit papillomavirus infection.

Transgenic rabbits carrying the EJras oncogene have been established in our laboratory (Am. J. Pathol. 155 (1999) 315). The expression of the ras gene is targeted to the epidermal keratinocytes using the upstream regulatory region (URR) of the cottontail rabbit papillomavirus (CRPV). All of the transgenic rabbits develop keratoacanthomas at multiple sites in the skin at 2-3 days after birth, and the tumors spontaneously regress in 1.5-2 months. With regression of the keratoacanthomas, the rabbits appear normal and EJras expression is undetectable in their skin. To determine if CRPV infection would reinitiate the expression of the EJras transgene and make the rabbits more sensitive to tumorigenesis, the rabbits were infected with CRPV at 2 months of age when the keratoacanthomas had regressed. This study shows that CRPV infection of the transgenic rabbit skin could shorten the latency required for CRPV papilloma initiation, and significantly increase the tumor growth and persistence rate compared with non-transgenic rabbits. Furthermore, EJras expression became detectable in the CRPV induced papillomas in transgenic rabbits, but not in the papillomas of non-transgenic rabbits. These results indicate that CRPV infection is able to reinitiate the expression of the CRPV URR controlled EJras oncogene carried by the transgenic rabbits and that the expression of EJras can enhance the tumorigenesis of CRPV infection.

Animals↗
PubMed2002

Transgenic 14-3-3 isoforms in plants: the metabolite profiling of repressed 14-3-3 protein synthesis in transgenic potato plants.

14-3-3 proteins are abundant eukaryotic proteins that interact with many other proteins, thereby modulating their function and thus cell metabolism. The data from mRNA analysis confirm the developmental regulation of 14-3-3 isoform expression in potato plants. In order to test whether or not 14-3-3 protein expression affects plant phenotype and metabolism, transgenic potato plants either overexpressing Cucurbita pepo 14-3-3 or underexpressing endogenous 14-3-3 isoforms were analysed. An increase in tuber number and a decrease in tuber size in the overexpressed transformant was observed; the transgenic plants contain more chlorophyll than the control and they lose it more slowly than the control when transferred to the dark. The 14-3-3-repressed transgenic plants showed a decrease in tuber number and an increase in tuber size; an increase in the fresh weight of the transgenic tubers was also detected. The increased catecholamine level was accompanied by an increased ratio of soluble sugars to starch in overexpressed transformant. The opposite effect was detected in 14-3-3-repressed transgenic plants. All the repressed plants showed significant increases in nitrate reductase (NR) activity, suggesting that the regulation of NR occurs in vivo, and is not isoform-dependent. The increase in NR activity resulted in a significant decrease in nitrate level. The level of sucrose phosphate synthase activity was also significantly increased in all 14-3-3-underexpressed transgenes, and remarkably the increase in enzyme activity was accompanied by respective changes in sucrose levels in the tubers. The most intriguing finding was the significant (2-3-fold) increase in ethylene content in all the 14-3-3-repressed transgenic lines, which probably resulted from a methionine level increase. The substantial increase of ethylene level in the repressed forms might explain the significant shortening of the vegetation period of the analysed transgenic plants.

14-3-3 Proteins↗
PubMed1999

Silencing of an aleurone-specific gene in transgenic rice is caused by a rearranged transgene.

In rice, silencing of the aleurone-specific Ltp2-gus transgene, causing easily detectable staining patterns on the grain surface, offers a convenient tool to study quantitative aspects of gene silencing in monocots. In this paper we analyzed phenotypes, occurrence, inheritance and environmental effects on the silencing. We also report on the cloning of transgenes, determination of their structure and analysis of transcripts from the transgene loci. The results show that various patterns of silencing appeared in the R2 generation at which most of the transgenes became homozygous and that they were inherited for five generations. In addition, silencing independently occurred in three generations and reversion to full expression was also found. Cloning of transgenes from a silenced L3.3 line demonstrated that this line carried two transgene loci: one carried an intact Ltp2-gus gene and the other carried a rearranged transgene in which part of the gus gene was in the antisense orientation. Analysis of gus transcripts indicated that partial antisense RNA derived from the rearranged transgene was present in silenced lines and was polyadenylated but that it was absent in non-silenced lines. RNA analyses suggested that the Ltp2-gus silencing in the aleurone layer was post-transcriptional and that it may be caused by interaction of partial antisense gus transcripts with normal sense transcripts. Possible involvement of antisense transcripts in post-transcriptional silencing is discussed.

Base Sequence↗
PubMed1994

Transgenic cattle resulting from biopsied embryos: expression of c-ski in a transgenic calf.

Producing transgenic cattle by microinjection of DNA into pronuclei has been inefficient and costly, in large part because of the cost of maintaining numerous nontransgenic pregnancies to term. We designed a system for early identification of transgenic embryos in which biopsies of embryos were assayed by polymerase chain reaction for presence of the transgene before embryo transfer. A total of 2555 embryos were microinjected with one of two DNA constructs. Of the 533 embryos biopsied, 112 were judged to be potentially transgenic and were transferred nonsurgically to recipients, resulting in production of 29 putative transgenic fetuses. One fetus and one calf (7% of offspring) were subsequently shown to be definitively transgenic. The calf was transgenic for a chicken c-ski cDNA, and several months after birth developed dramatic muscular hypertrophy followed by muscle degeneration. This phenotype was associated with expression of high levels of mRNA from the transgene.

Animals↗
PubMed1995

Promoter demethylation in MMTV/N-rasN transgenic mice required for transgene expression and tumorigenesis.

We studied demethylation within the transgene promoter in transgenic mice carrying the N-ras proto-oncogene driven by the mouse mammary tumor long terminal repeat (MMTV/N-rasN) and the relationship of demethylation to transgene overexpression and tumorigenesis. Demethylation at Fspl or Clal sites correlated with age of the animal and transgene expression in nontumorous mammary gland. Demethylation preceded expression in this tissue. In lymphomas and mammary tumors, the promoter Fspl and Clal sites were significantly more demethylated than in nontumorous control tissues. The Aval, Cfol, and Hpall sites were also found to be undermethylated in older animals and showed differences between tumor and control tissues. Two additional sites (Eagl and Narl) remained fully methylated in all tissues. In contrast with normal tissue, demethylation at the Fspl and Clal sites and expression were not correlated in tumor tissue. An increase in expression in normal tissue initially occurred and was correlated with the level of promoter demethylation; this increase was followed by a further increment in transgene expression when tumors developed. Thus, promoter demethylation leading to transgene overexpression was associated with long-latency tumorigenesis in MMTV/N-rasN transgenic mice. Demethylation of proto-oncogene promoters may therefore be a mechanism of carcinogenesis that requires further investigation in human tumors.

5-Methylcytosine↗
PubMed2001

Use of the tetracycline-controlled transcriptional silencer (tTS) to eliminate transgene leak in inducible overexpression transgenic mice.

The doxycycline-inducible reverse tetracycline transactivator (rtTA) is frequently used to overexpress transgenes in a temporally regulated fashion in vivo. These systems are, however, often limited by the levels of transgene expression in the absence of dox administration. The tetracycline-controlled transcriptional silencer (tTS), a fusion protein containing the tet repressor and the KRAB-AB domain of the kid-1 transcriptional repressor, is inhibited by doxycycline. We hypothesized that tTS would tighten control of transgene expression in rtTA-based systems. To test this hypothesis we generated mice in which the CC10 promoter targeted tTS to the lung, bred these mice with CC10-rtTA-interleukin 13 (IL-13) mice in which IL-13 was overexpressed in an inducible lung-specific fashion, and compared the IL-13 production and phenotypes of parental mice and the triple transgenic CC10-rtTA/tTS-IL-13 progeny of these crosses. In the CC10-rtTA-IL-13 mice, IL-13, mucus metaplasia, inflammation, alveolar enlargement, and enhanced lung volumes were noted at base line and increased greatly after doxycycline administration. In the triple transgenic tTS animals, IL-13 and the IL-13-induced phenotype could not be appreciated without doxycycline. In contrast, tTS did not alter the induction of IL-13 or the generation of the IL-13 phenotype by doxycycline. Thus, tTS effectively eliminated the baseline leak without altering the inducibility of rtTA-regulated transgenes in vivo. Optimal "off/on" regulation of transgene expression can be accomplished with the combined use of tTS and rtTA.

Animals↗
PubMed1996

Beta-MHC and SMLC1 transgene induction in overloaded skeletal muscle of transgenic mice.

The hypertrophic responses of white fast-twitch muscle to mechanical overload has been investigated using transgenic mice. After 7 wk of overload, endogenous beta-myosin heavy chain (MHC) and slow myosin light chain 1 and 2 (SMLC1, SMLC2) protein were increased in the overloaded plantaris (OP) muscle compared with sham-operated control plantaris (CP)muscle. Concurrently, the levels of endogenous beta-MHC, SMLC1, SMLC2, and cardiac/slow troponin C (CTnC) mRNA transcripts were significantly increased in OP muscles, whereas skeletal troponin C (sTnC) mRNA transcript levels decreased. As an initial attempt to locate DNA sequence(s) that governs beta-MHC induction in response to mechanical overload, multiple independent transgenic lines harboring four different human beta-MHC transgenes (beta 1286, beta 988, beta 450, beta 141) were generated. Except for transgene beta 141, muscle-specific expression and induction (3- to 22-fold) in OP muscles were observed by measuring chloramphenicol acetyltransferase activity (CAT assay). Induction of a SMLC1 transgene (3920SMLC1) in OP muscles was also observed. Collectively, these in vivo data provide evidence that 1) a mechanical overload inducible element(s) is located between nucleotides -450 and +120 of the human beta-MHC transgene, 2) 3,900 bp of 5' sequence is sufficient to confer mechanical overload induction of a SMLC1 transgene, and 3) the increased expression of slow/type I isomyosin (beta-MHC, SMLC1, SMLC2) in response to mechanical overload is regulated, in part, transcriptionally.

Animals↗
PubMed2004

Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals.

In this paper, we describe a rapid and accurate real-time quantitative PCR-based system to determine transgene copy number in transgenic animals. We used the 2(-deltadeltaCt) method to analyze different transgenic lines without the requirement of a control sample previously determined by Southern blot analysis. To determine the transgene copy number in several mouse lines carrying a goat beta-Lactoglobulin transgene, we developed a TaqMan assay in which a goat genomic DNA sample was used as a calibrator. Moreover, we used the glucagon gene as a reference control because this gene is highly conserved between species and amplifies with the same efficiency and sensitivity in goat as in mouse. With this assay, we provide an alternative simple method to determine the transgene copy number, avoiding the traditional and tedious blotting techniques. The assay's discrimination ability from our results is of at least six copies and, similar to the limitations of the blotting techniques, the accuracy of the quantification diminishes when the transgene copy number is high.

Animals↗
PubMed1999

[Generation and analyses of transgenic mice containing mutant p53 transgene].

p53 is known to be a suppressor oncogene product regulating the cell cycle as a check point protein. Loss of function mutations were frequently observed in various human cancers. To investigate the role of mutant form of p53 in the presence of wild-type p53 in vivo, we generated two types of transgenic mice carrying p53 transgenes with mutation at the codon 248 from Arg to Trp or at the codon 249 from Arg to Ser. We obtained seven transgenic mouse lines with Trp-mutant type and eight lines from Ser-mutant type which express mutant p53 transgenes. These transgenic mice were observed for tumor formation over a period of one year. However, no tumors were developed within the monitoring period. To test whether the mutant p53 has a dominant negative effect on the wild type p53 or not, transgenic mice were crossed with p53 knockout mice which were heterozygous for the endogenous wild type p53 gene. Lymphocytes of these mice were cultured after gamma irradiation to induce apoptosis. Viability of lymphocytes as well as fragmentation of DNAs were measured. Apoptosis of lymphocytes from p53 +/- mutant transgenic mice was not suppressed compared with lymphocytes from p53 +/- mice. From these results, the mutant p53 with point mutation at the codon 248 from Arg to Trp or at the codon 249 from Arg to Ser, has a loss of function mutation, but not a dominant negative mutation over the wild type p53.

Animals↗
PubMed1997

Large fragment of the probasin promoter targets high levels of transgene expression to the prostate of transgenic mice.

BACKGROUND: Androgen regulation and prostate-specific expression of targeted genes in transgenic mice can be controlled by a small DNA fragment of the probasin (PB) promoter (-426 to +28 base pairs, bp). Although the small PB fragment was sufficient to direct prostate-specific expression, the low levels of transgene expression suggested that important upstream regulatory sequences were missing. METHODS: To enhance transgene expression, a large fragment of the PB promoter (LPB, -11,500 to +28 bp) was isolated, linked to the bacterial chloramphenicol acetyl transferase (CAT) gene, and microinjected into CD1 mouse oocytes to generate transgenic mouse lines. RESULTS: As shown by the immunohistochemical studies, CAT gene expression was restricted to the prostatic epithelial cells in a tissue-specific manner. High levels of CAT gene expression were observed in two of the six LPB-CAT transgenic lines. In Line 1, developmental regulation of LPB-CAT was detected early, from 1 to 4 weeks of age, with the activity of CAT increasing from 3 to 40,936 dpm/min/mg protein. Upon sexual maturation and elevated serum androgen levels (7 weeks of age), a further 18-fold rise in CAT activity occurred. Hormone ablation by castration in mature mice dramatically reduced transgene expression, whereas treatment with androgens returned LPB-CAT expression to precastration levels. In contrast, treatment with glucocorticoids had no significant effect on CAT gene expression. Zinc treatment of the castrated animals also increased LPB-CAT expression three- to four-fold in two prostatic lobes. CONCLUSIONS: This study demonstrates that important regulatory DNA sequences located in the LPB fragment contribute to tissue-specific expression and greatly increase levels of transgene expression induced by androgens and zinc.

Aging↗
PubMed1995

Developmental regulation of the ovine beta-lactoglobulin/human serum albumin transgene is distinct from that of the beta-lactoglobulin and the endogenous beta-casein genes in the mammary gland of transgenic mice.

We compared the developmental pattern of expression of the sheep beta-lactoglobulin (BLG), the chimeric BLG/human serum albumin (HSA), and the endogenous murine beta-casein genes in the mammary gland of virgin, pregnant and lactating transgenic mice, both at the RNA (expression) and protein (synthesis and secretion) levels. The BLG and casein genes were expressed at very low levels in virgin animals and during early stages of pregnancy. The increase in the expression of these genes started at the second half of pregnancy and reached a peak between the end of pregnancy and day 10 of lactation. The accumulation of their RNA coincided with that of the corresponding proteins, indicating a transcriptional control of expression of these genes. The expression and secretion patterns of the endogenous casein gene in transgenic and nontransgenic mice were indistinguishable. The hybrid BLG/HSA gene constructs displayed distinct patterns of expression in virgin animals and at early stage of pregnancy, from that of the BLG transgene or the endogenous mouse milk protein gene. High levels of expression (17-60% of that on day 18 of pregnancy) were detected in the mammary gland of virgin animals. At day 5 of pregnancy there was a dramatic decrease in HSA synthesis and secretion in all transgenic strains tested. The down-regulation, revealed by immunoprecipitation and immunohistochemical studies, demonstrated that at that stage of pregnancy only 10-18% of ductal structures contained HSA expressing cells in contrast to the majority of ducts expressing HSA in virgin animals. These morphological studies also demonstrated that the down-regulation in HSA synthesis and secretion was correlated with the transition from ducts comprised of a single layer of epithelial cells (characteristic of the virgin state) to ducts composed of multilayers of such cells. In two of the three transgenic strains tested, the down-regulation at the protein level was associated with a similar decrease in HSA transcripts. In the exceptional strain no. 23, HSA transcripts continued accumulating even at this stage. The differences in the control of expression at the RNA level between these transgenic strains were also confirmed by in situ hybridization. Our results suggest the involvement of at least two regulatory mechanisms effective at early stages of gestation in the control of expression/secretion of the HSA transgene targeted for expression in the mammary gland by the BLG milk protein promoter. These putative mechanisms may play key roles in the interplay between normal mammogenesis and lactogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2002

Expression of the gene of interest fused to the EGFP-expressing gene in transgenic mice derived from selected transgenic embryos.

The present paper describes the expression of a target fusion gene, WAP/hGH fused to the EGFP-expressing gene in transgenic mice derived from the transfer of transgenic embryos selected because of their expression of enhanced green fluorescent protein (EGFP). The 6.7-kb fusion gene was microinjected as a single cassette gene construct into the pronuclei of mouse zygotes. The surviving embryos were cultured and were classified according to the EGFP expression patterns at the morula or blastocyst stage. After the transfer of embryos with uniform-expression or mosaic-expression of EGFP, transgenesis occurred in 85.7% to 86% or 44.1% to 44% of the pups, respectively. No transgenic pups were derived from EGFP negative embryos. In the transgenic females, EGFP was ubiquitously expressed under the control of the CAG promoter, and hGH was expressed under the control of the WAP promoter in an appropriate fashion: hGH was secreted into the milk of lactating transgenic females. The presence or absence of the expression of EGFP coincided with that of the hGH gene in the transgenic mice. The present cassette gene construct is a useful example for circumventing the routine analyses of DNA and RNA required for the generation and maintenance of transgenic lines.

Animals↗
PubMed1996

Deletions in the 3' untranslated region of cowpea chlorotic mottle virus transgene reduce recovery of recombinant viruses in transgenic plants.

The 3' untranslated region (UTR) adjacent to the capsid gene is frequently included with the transgene in the construction of capsid protein mediated virus resistant transgenic plants. Since ribonucleotide sequences within the 3' UTR are involved in the initiation of viral replication, the presence of this sequence may encourage the participation of the transgene in RNA recombination. Experiments were designed to explore the involvement of the 3' UTR of cowpea chlorotic mottle virus (CCMV) in RNA recombination events between transgenes and challenging viruses. Nicotiana benthamiana was transformed with CCMV transgenes consisting of the 3' two-thirds of the capsid gene and fragments of the associated 3' UTR lacking the terminal 69, 83, or 214 nucleotides. Plants were inoculated with wild-type CCMV transcripts for RNAs 1 and 2 and a movement defective RNA 3 transcript lacking the 3' third of the capsid gene. While no recombinant virus was detected in plants expressing 3' deletion constructs, 3% of control transformants containing an identical segment of the capsid gene with the complete 3' UTR yielded viable recombinant virus. These results suggest that RNA recombination between transgenic RNA and viral RNA can be reduced significantly by omitting or disrupting the 3' UTR in the transgene.

Bromovirus↗