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At least 307 records · Page 17Linked to original sources
PubMed1991

Cell-specific, developmentally and hormonally regulated expression of the rabbit uteroglobin transgene and the endogenous mouse uteroglobin gene in transgenic mice.

We have generated a transgenic mouse line by introducing the rabbit uteroglobin gene with 4 kb of 5'-flanking DNA and 1 kb of 3'-flanking DNA into the mouse germ line via microinjection into fertilized oocytes. Expression of the rabbit uteroglobin transgene was examined and compared with the endogenous mouse uteroglobin gene. Both genes are expressed in the lung, male genital tract and uterus. In the lung, mRNA expression is enhanced by glucocorticoids and restricted to the Clara cells that line terminal and respiratory bronchioli. During embryonic lung development, transcripts are first detected at day 17. Expression in the uterus is restricted to the glandular epithelium and can be induced by sequential treatment with estrogens and progesterone. In the uterus of these pseudopregnant mice the level of rabbit uteroglobin transcripts is higher than that of the mouse endogenous uteroglobin transcripts. In the male genital tract, expression of both genes is restricted to the epithelial layers of the vesicular gland, vas deferens and epididymis. Our results indicate that the rabbit uteroglobin gene together with 4 kb of 5'-flanking DNA and 1 kb of 3'-flanking DNA contains the information required for cell type-specific, developmentally, and hormonally regulated expression.

Animals↗
PubMed2001

Intravitreal adenoviral gene transfer evokes an immune response in the retina that is directed against the heterologous lacZ transgene product but does not limit transgene expression.

Recombinant E1-deleted adenoviral vectors (DeltaE1-Ad) are promising tools for in vivo gene transfer into the mammalian CNS including the retina. However, the duration of transgene expression is limited, and this limitation has partly been attributed to an immune response directed against vector-derived proteins. Here, we employed immunocytochemistry to assess the immune response to intravitreously injected DeltaE1-Ad encoding the lacZ gene or various neurotrophins (NTs). beta-Galactosidase was expressed by retinal cells for up to 4 weeks. Following intravitreal inoculation of AdCMV-lacZ, microglial and T cells were detected with a panel of antibodies in the retinal cell layers after 2 days (D2). The inflammatory response reached a maximum between D7 and D14. In contrast, no immune response was seen following injection of Ad encoding NTs. Yet, like with Ad-CMV-lacZ, their expression was also limited to approximately 4 weeks. Thus, beta-galactosidase seems to trigger a host immune response following intravitreal adenoviral lacZ gene transfer, but immune responses are not the cause of limited NT transgene expression from the CMV promoter in the inner retina.

Adenoviridae↗
PubMed1999

Tissue folate binding protein levels in transgenic mice with tumors and in non-transgenic controls.

Localized folate deficiency may be a risk factor for cancer. Since, folate binding proteins (FBP) and reduced folate carrier proteins (RFC) mediate cellular transport of folate, we compared FBP concentrations in several organs from tumor-bearing transgenic (TBT) mice and tumor-free non-transgenic controls (NTC) of the same strain, age, and fed identical diets. Liver, spleen, brain, small intestine and kidney were individually homogenized in phosphate-buffered saline (PBS) and separated into membrane, cytoplasmic, mitochondrial/lysomal and nuclear fractions (confirmed with marker enzymes). Homogenates and fractions was analyzed for total protein, and FBP. We used rabbit anti-bovine milk antibody and ELISA to measure FBP. FBP concentrations in kidney, small intestine, and spleen of TBT mice were higher than those of NTC mice; the opposite was true in liver and lung. FBP seemed to be upregulated in kidneys (all fractions), small intestine (all fractions), and spleen (cytoplasmic and nuclear fractions only) of TBT mice compared to NTC mice; the opposite appeared true in liver (all fractions) and lung (all fractions). FBP concentrations in brain, heart, and muscle of TBT mice were not different from those in brain, heart and muscle of NTC mice. A longitudinal study will determine if these changes in FBP concentrations precede tumor onset.

Animals↗
PubMed1997

RT-PCR method specific for the detection of transgenic CFTR mRNA in the presence of transgene plasmid DNA and endogenous CFTR mRNA.

A major problem with experiments involving the correction of cystic fibrosis by gene transfer has been reliably detecting the transgenic message and distinguishing this from an endogenous message and from vector DNA. We have exploited the SV40 small T antigen intron present in an expression vector containing the CFTR cDNA (pCFAS) using a primer directed to the spliced sequence to allow specific and precise detection of the transgeneic message.

Antigens, Polyomavirus Transforming↗
PubMed2001

The left ventricular stress-velocity relation in transgenic mice expressing a dominant negative CREB transgene in the heart.

OBJECTIVE: CREB(A133) transgenic mice that express a dominant negative CREB transcription factor in cardiomyocytes develop a dilated cardiomyopathy that is anatomically, physiologically, and clinically similar to human idiopathic dilated cardiomyopathy. The goals of this study were to quantitate left ventricular (LV) contractility and measure cardiac reserve in CREB(A133) mice by using the relation of end-systolic wall stress to the velocity of fiber shortening. METHODS: A total of 37 adult CD-1 mice (including both nontransgenic and CREB(A133) transgenic mice) were studied with simultaneously acquired high-fidelity instantaneous aortic pressures and 2-dimensionally targeted M-mode echocardiograms. RESULTS: CREB(A133) mice displayed significantly lower values of LV fiber shortening velocities over a wide range of afterloads, and they displayed smaller dobutamine-induced shifts from baseline contractility relations. Counterbalancing effects of differences in LV geometry and aortic pressures resulted in comparable levels of LV wall stress during ejection in both groups. CONCLUSION: These results demonstrate directly that CREB(A133) mice display reduced LV contractility at baseline and decreased cardiac reserve.

Animals↗
PubMed1996

In the absence of endogenous mouse apolipoprotein E, apolipoprotein E*2(Arg-158 --> Cys) transgenic mice develop more severe hyperlipoproteinemia than apolipoprotein E*3-Leiden transgenic mice.

Apolipoprotein E*2(Arg-158 --> Cys) (APOE*2) transgenic mice were generated and compared to the previously generated apolipoprotein E*3-Leiden (APOE*3-Leiden) transgenic mice to study the variable expression of hyperlipoproteinemia associated with these two APOE variants. In the presence of the endogenous mouse Apoe gene, the expression of the APOE*3-Leiden gene resulted in slightly elevated levels of serum cholesterol as compared with control mice (2.7 +/- 0. 5 versus 2.1 +/- 0.2 mmol/liter, respectively), whereas the expression of the APOE*2(Arg-158 --> Cys) gene did not affect serum cholesterol levels, even after high/fat cholesterol feeding. The extreme cholesterol level usually found in apoE-deficient mice (Apoe-/- mice; 23.6 +/- 5.0 mmol/liter) could be rescued by introducing the APOE*3-Leiden gene (APOE*3-Leiden.Apoe-/-; 3.6 +/- 1. 5 mmol/liter), whereas the expression of the APOE*2(Arg-158 --> Cys) gene in Apoe-/- mice minimally reduced serum cholesterol levels (APOE*2.Apoe-/-; 16.6 +/- 2.9 mmol/liter). In vivo very low density lipoprotein (VLDL) turnover studies revealed that APOE*2.Apoe-/- VLDL and APOE*3-Leiden.Apoe-/- VLDL display strongly reduced fractional catabolic rates as compared with control mouse VLDL (4.0 and 6.1 versus 22.1 pools/h). In vitro low density lipoprotein (LDL) receptor binding studies using HepG2 and J774 cells showed that APOE*2. Apoe-/- VLDL is completely defective in binding to the LDL receptor, whereas APOE*3-Leiden.Apoe-/- VLDL still displayed a considerable binding activity to the LDL receptor. After transfection of APOE*2.Apoe-/- and APOE*3-Leiden.Apoe-/- mice with adenovirus carrying the gene for the receptor-associated protein (AdCMV-RAP), serum lipid levels strongly increased (15.3 to 42.8 and 1.4 to 15.3 mmol/liter for cholesterol and 5.0 to 35.7 and 0.3 to 20. 7 mmol/liter for triglycerides, respectively). This indicates that RAP-sensitive receptors, possibly the LDL receptor-related protein (LRP), mediate the plasma clearance of both APOE*2.Apoe-/- and APOE*3-Leiden. Apoe-/- VLDL. We conclude that in vivo the APOE*2 variant is completely defective in LDL receptor binding but not in binding to LRP, whereas for the APOE*3-Leiden mutant both LRP and LDL receptor binding activity are only mildly affected. As a consequence of this difference, APOE*2.Apoe-/- develop more severe hypercholesterolemia than APOE*3-Leiden.Apoe-/- mice.

Animals↗
PubMed1996

Rapid induction of more malignant tumors by various genotoxic carcinogens in transgenic mice harboring a human prototype c-Ha-ras gene than in control non-transgenic mice.

In this study, we investigated the carcinogenic response of transgenic mice carrying the human prototype c-Ha-ras gene, namely Tg rasH2/CB6F1 mice, to various genotoxic carcinogens and compared it with that of control non-transgenic CB6F1 mice (non-Tg mice). The present studies were conducted as the first step in the evaluation of the Tg rasH2/CB6F1 mouse as a model for the rapid carcinogenicity testing system. Short-term (< or = 6 months) rapid carcinogenicity tests of various genotoxic carcinogens, 4-nitroquinoline-1-oxide, cyclophosphamide, N,N-diethylnitrosamine, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and methylazoxymethanol, revealed that Tg rasH2/CB6F1 mice are more susceptible to these genotoxic carcinogens than control non-Tg mice. Tg rasH2/CB6F1 mice developed tumors more rapidly compared with non-Tg mice. Malignant tumors were observed only in the carcinogen-treated Tg rasH2/CB6F1 mice, but not in non-Tg mice treated with the same carcinogens. Each carcinogen induced tumors in corresponding target tissues of the Tg rasH2/CB6F1 mice. Only a very few lung adenomas but no other tumors were seen as spontaneous tumors during the 6 months of carcinogenicity tests. These results demonstrate that more rapid onset and higher incidence of more malignant tumors can be expected with high probability after treatment with various genotoxic carcinogens in the Tg rasH2/CB6F1 mice than in control non-Tg mice. The Tg rasH2/CB6F1 mouse seems to be a promising candidate as an animal model for the development of a rapid carcinogenicity testing system.

4-Nitroquinoline-1-oxide↗
PubMed2000

CD46 transgene expression in pig peripheral blood mononuclear cells does not alter their susceptibility to measles virus or their capacity to downregulate endogenous and transgenic CD46.

CD46 (or membrane cofactor protein) protects autologous cells from complement-mediated lysis and has been expressed as a transgene in pigs to overcome complement-mediated hyperacute rejection of porcine organs upon transplantation into primates. Since CD46 has been identified as a receptor for measles virus (MV), the susceptibility of CD46-transgenic (tg) pig peripheral blood mononuclear cells (PBMC) to infection with MV strains which do and do not use CD46 as receptor was investigated. Surprisingly, it was found that MV vaccine strains (e.g. Edmonston) bound to tg as well as non-tg pig PBMC. Phytohaemagglutinin-stimulated CD46-tg and non-tg pig PBMC were equally well infected with MV vaccine strains irrespective of CD46 expression. Upon infection, tg CD46 was downregulated from the cell surface. In contrast, the binding capacity for MV wild-type strains to pig and human PBMC was low, irrespective of CD46 expression. These MV strains did not infect tg or non-tg pig cells. Expression of endogenous pig CD46 was detected with polyclonal sera against human CD46. After infection of pig PBMC with MV strain Edmonston, endogenous pig CD46 was also downregulated. This suggests an interaction between MV Edmonston and pig CD46. However, polyclonal CD46 sera did not inhibit infection with MV Edmonston indicating that CD46 may not exclusively act as a receptor for MV on these cells. Interestingly, similar results were observed using human PBMC. Data suggest that CD46 downregulation after interaction with MV may also occur in porcine organs which express endogenous and/or human CD46 as a means of protection against complement-mediated damage.

Animals↗
PubMed2000

A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing.

We showed previously that 218 and 110 bp N gene segments of tomato spotted wilt virus (TSWV) that were fused to the non-target green fluorescent protein (GFP) gene were able to confer resistance to TSWV via post-transcriptional gene silencing (PTGS). N gene segments expressed alone did not confer resistance. Apparently, the GFP DNA induced PTGS that targetted N gene segments and the incoming homologous TSWV for degradation, resulting in a resistant phenotype. These observations suggested that multiple resistance could be obtained by replacing the GFP DNA with a viral DNA that induces PTGS. The full-length coat protein (CP) gene of turnip mosaic virus (TuMV) was linked to 218 or 110 bp N gene segments and transformed into Nicotiana benthamiana. A high proportion (4 of 18) of transgenic lines with the 218 bp N gene segment linked to the TuMV CP gene were resistant to both viruses, and resistance was transferred to R(2) plants. Nuclear run-on and Northern experiments confirmed that resistance was via PTGS. In contrast, only one of 14 transgenic lines with the TuMV CP linked to a 110 bp N gene segment yielded progeny with multiple resistance. Only a few R(1) plants were resistant and resistance was not observed in R(2) plants. These results clearly show the applicability of multiple virus resistance through the fusion of viral segments to DNAs that induce PTGS.

Artificial Gene Fusion↗
PubMed2001

HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal.

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region of the silenced gene and with accumulation of small RNAs (21 to 25 nucleotides) homologous to the silenced gene. In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction. Previously, we showed that the helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS. Here, we report that plants in which PTGS has been suppressed by HC-Pro fail to accumulate the small RNAs associated with silencing. However, the transgene locus of these plants remains methylated. Grafting experiments indicate that HC-Pro prevents the plant from responding to the mobile silencing signal but does not eliminate its ability to produce or send the signal. These results demonstrate that HC-Pro functions downstream of transgene methylation and the mobile signal at a step preceding accumulation of the small RNAs.

Algorithms↗
PubMed1995

Expression of osteopontin in a macrophage cell line and in transgenic mice with pulmonary fibrosis resulting from the lung expression of a tumor necrosis factor-alpha transgene.

The expression level of osteopontin (OPN) mRNA was found to be increased in a macrophage cell line in the presence of recombinant tumor necrosis factor-alpha (TNF-alpha). OPN mRNA level was also explored in the lungs of transgenic mice which were expressing TNF-alpha in type II pneumocytes, a condition leading to pulmonary alveolitis and progressive fibrosis. OPN mRNA was significantly increased in the lungs of these transgenic mice. In situ hybridization showed that it was localized mostly in alveolar macrophages. Since OPN can be induced in macrophages by TNF-alpha stimulation and since on the other hand osteopontin appears to decrease the level of nitric oxide synthase, and thus the production of nitric oxide, osteopontin might also indirectly have an antifibrotic effect. The role played by osteopontin in fibrotic lesions resulting from the release of TNF-alpha deserves further study, since it may be involved in the balance of opposite effects eventually leading to local tissue damage ending in fibrosis.

Animals↗
PubMed1998

Expression of an AQP2 Cre recombinase transgene in kidney and male reproductive system of transgenic mice.

A transgenic mouse approach was used to examine the mechanism of principal cell-specific expression of aquaporin-2 (AQP2) within the renal collecting duct. RT-PCR and immunocytochemistry revealed that murine AQP2 was expressed in principal cells in the renal collecting duct, epithelial cells of the vas deferens, and seminiferous tubules within testis. The vas deferens expression was confirmed in rats. RT-PCR and immunocytochemistry showed that 14 kb of the human 5'-flanking region confers specific expression of a nucleus-targeted and epitope-tagged Cre recombinase in the principal cells within the renal collecting duct, in the epithelial cells of the vas deferens, and within the testis of transgenic mice. These results suggest that cell-specific expression of AQP2 is mediated at the transcriptional level and that 14 kb of the human AQP2 5'-flanking region contain cis elements that are sufficient for cell-specific expression of AQP2. Finally, renal principal cell expression of Cre recombinase is the first step in achieving cell-specific gene knockouts, thereby allowing focused examination of gene function in this cell type.

Animals↗
PubMed2004

Adrenocortical tumorigenesis in transgenic mice expressing the inhibin alpha-subunit promoter/simian virus 40 T-antigen transgene: relationship between ectopic expression of luteinizing hormone receptor and transcription factor GATA-4.

We have analyzed the ontogeny and putative mechanisms of transregulation of LH receptor (LHR) and transcription factor GATA-4, coexpressed during the adrenocortical tumorigenesis of prepubertally gonadectomized transgenic (TG) mice expressing the inhibin alpha-subunit promoter/simian virus 40 T-antigen (inhalpha/Tag) transgene. The onset of adrenal LHR mRNA and protein expression coincided with that of GATA-4 at the age of 4 months and preceded the appearance of discernible adrenal tumors at about 6 months. In situ hybridization and double-immunohistochemistry demonstrated colocalization of the LHR and GATA-4 messages and proteins in the adrenal cortex. A GATA-4 expression plasmid cotransfected with a murine LHR promoter-driven luciferase reporter plasmid, containing a consensus GATA-binding site, induced a dose-dependent significant transactivation of the LHR promoter in nonsteroidogenic human embryonic kidney 293, steroidogenic murine mLTC-1 Leydig cells and in murine adrenal Y-1 cells. The Calpha1 cells derived from an Inhalpha/Tag adrenal tumor did not show this response, apparently due to their high endogenous GATA-4 expression. However, an additional link between GATA-4 and LHR in Calpha1 cells was provided upon the LH/human chorionic gonadotropin stimulation of LHR promoter activity; mutations or deletion of the consensus GATA-4 binding site of the LHR promoter abolished this transactivation. EMSAs further proved GATA-4 binding to the putative consensus GATA recognition site. Our results demonstrate direct interrelationship between LHR and GATA-4 expression during adrenocortical tumorigenesis of the inhalpha/Tag mice. There is apparently a positive and reciprocal feed-forward amplification link between LHR and GATA-4 expression. This mechanism gradually and in synergy with Tag expression leads to formation of the LH-dependent adrenocortical tumors.

Adrenal Cortex Neoplasms↗
PubMed2004

Myosin 3A transgene expression produces abnormal actin filament bundles in transgenic Xenopus laevis rod photoreceptors.

Myo3A, a class III myosin, localizes to the distal (plus) ends of inner segment actin filament bundles that form the core of microvillus-like calycal processes encircling the base of the photoreceptor outer segment. To investigate Myo3A localization and function, we expressed green fluorescent protein-tagged bass Myo3A and related constructs in transgenic Xenopus rods using a modified opsin promoter. Tagged intact Myo3A localized to rod calycal processes, as previously reported for native bass Myo3A. Transgenic rods developed abnormally large calycal processes and subsequently degenerated. Modified Myo3A expression constructs demonstrated that calycal process localization required an active motor domain and the tail domain. Expressed tail domain alone localized to actin bundles along the entire inner segment length, rather than to the distal end. This tail domain localization required the conserved C-terminal domain (3THDII) previously shown to possess an actin-binding motif. Our findings suggest that Myo3A plays a role in the morphogenesis and maintenance of calycal processes of vertebrate photoreceptors.

Actin Cytoskeleton↗
PubMed1999

Reorganization of cholinergic terminals in the cerebral cortex and hippocampus in transgenic mice carrying mutated presenilin-1 and amyloid precursor protein transgenes.

Cholinergic deficits are one of the most consistent neuropathological landmarks in Alzheimer's disease (AD). We have examined transgenic mouse models (PS1M146L, APPK670N,M671L) and a doubly transgenic line (APPK670N,M671L + PS1M146L) that overexpress mutated AD-related genes [presenilin-1 (PS1) and the amyloid precursor protein (APP)] to investigate the effect of AD-related gene overexpression and/or amyloidosis on cholinergic parameters. The size of the basal forebrain cholinergic neurons and the pattern of cholinergic synapses in the hippocampus and cerebral cortex were revealed by immunohistochemical staining for choline acetyltransferase and the vesicular acetylcholine transporter, respectively. At the time point studied (8 months), no apparent changes in either the size or density of cholinergic synapses were found in the PS1M146L mutant relative to the nontransgenic controls. However, the APPK670N,M671L mutant showed a significant elevation in the density of cholinergic synapses in the frontal and parietal cortices. Most importantly, the double mutant (APPK670N,M671L + PS1M146L), which had extensive amyloidosis, demonstrated a prominent diminution in the density of cholinergic synapses in the frontal cortex and a reduction in the size of these synapses in the frontal cortex and hippocampus. Nonetheless, no significant changes in the size of basal forebrain cholinergic neurons were observed in these three mutants. This study shows a novel role of APP and a synergistic effect of APP and PS1 that correlates with amyloid load on the reorganization of the cholinergic network in the cerebral cortex and hippocampus at the time point studied.

Acetylcholine↗
PubMed1996

Ploidy and karyotypic alterations associated with early events in the development of hepatocarcinogenesis in transgenic mice harboring c-myc and transforming growth factor alpha transgenes.

The cooperation of the c-myc oncogene with the growth factor transforming growth factor (TGF)-alpha in development of liver tumors in transgenic mice has been demonstrated previously. In this study, we analyzed the ploidy and karyotype of c-myc, TGF-alpha, parental control, and the double transgenic c-myc/TGF-alpha hepatocytes at 3 weeks of age when the liver is histologically normal and at 10 weeks when the c-myc/TGF-alpha liver is dysplastic and contains basophilic foci. Eighty % of the 10-week hepatocytes were aneuploid, and 32% had chromosomal breakage. Statistically significant breakage was observed in six different chromosomes. Breakage at band A5 and at the border of bands C4/5 of chromosome 1 was observed. Fragile sets on chromosome 4 were most frequent in the middle of the chromosome at bands C2 and C6. Chromosome 6 was fragile at band F2. The region of chromosome 7 at bands B5 and D3 was frequently broken and involved in translocations. Chromosome 12 was broken at bands D1 and D3. The breakage sites on chromosomes 1, 4, 7, and 12 correspond to sites of tumor susceptibility genes in the mouse. Although there was no consistent change in copy number, recurrent translocations between chromosomes 1, 4, 7, 12 and 19 were also observed. These studies demonstrate that the development of dysplasia and basophilic foci in the liver is correlated with aneuploidy and chromosome breakage. The specific fragile sites indicate genetic regions that are altered during early stages of hepatocarcinogenesis. Due to the conservation of genetic linkage groups between mice and humans, the identification of genetic alterations in the mouse during hepatocarcinogenesis may provide critical information about tumor susceptibility genes that are important in the early development of human hepatocellular carcinoma.

Animals↗
PubMed1993

Induction of a tomato anionic peroxidase gene (tap1) by wounding in transgenic tobacco and activation of tap1/GUS and tap2/GUS chimeric gene fusions in transgenic tobacco by wounding and pathogen attack.

The anionic peroxidase genes of tomato, tap1 and tap2, are induced by wounding in tomato fruits and by elicitor treatment in cell suspension cultures. These homologous genes code for anionic peroxidases that are postulated to cause polymerization of the phenolic residues into wall polymers in wound-healing and pathogen-infected tissues. An expression construct containing the entire TAP1 gene with its 5' and 3' flanking sequences was introduced into tobacco by Agrobacterium tumefaciens-mediated gene transfer. Also, constructs containing the 5' upstream regions of tap1 and tap2 including sequences coding for their respective putative leader peptides fused translationally to the beta-glucuronidase (GUS) reporter gene were made and introduced into tobacco. Northern blot analysis of transcripts from wound-healing leaf tissues of transformants containing tap1 showed that the introduced gene was being transcribed in the heterologous host. The induction of tap1 transcripts in the wound-healing transgenic tobacco tissues was observed by 48 h and increased over time period of 84 h. Wounding also led to expression of GUS in tap1/GUS and tap2/GUS transformants and GUS activity was localized to the wound site. Activation of the tap1 and tap2 promoters in wound-healing transgenic tobacco tissues showed a GUS expression profile that correlated with the postulated role for anionic peroxidases in phenolic polymerization in suberizing tissues. Inoculation of tap1/GUS and tap2/GUS transformant leaves with fungal conidia from Fusarium solani f. sp. pisi caused expression of GUS in locally inoculated regions, and GUS expression increased over a period of four days.

Blotting, Northern↗
PubMed2000

The promoter of the Vicia faba L. gene VfEnod12 encoding an early nodulin is active in cortical cells and nodule primordia of transgenic hairy roots of Vicia hirsuta as well as in the prefixing zone II of mature transgenic V. hirsuta root nodules.

A full-length cDNA encoding the Vicia faba L. early nodulin VfEnod12 was isolated. The deduced protein sequence specified a 90 amino acid protein with a MW of 10206 and contained a putative signal peptide sequence followed by PPX(3) repeats characteristic of Enod12 proteins. The VfEnod12 gene was found to be expressed specifically in root nodules as early as 3 days post inoculation with Rhizobium leguminosarum bv. viciae. In mature nodules, VfEnod12 transcripts were confined to the prefixing zone II. A 3.3 kb genomic fragment carrying the complete VfEnod12 coding region was isolated. No intervening sequences were identified in the coding region. A promoter fragment carrying the -692/-41 region mediated reporter gene expression in root cortical cells, nodule primordia and the prefixing zone II of transgenic Vicia hirsuta root nodules. This fragment contained a putative binding site for the transcription factor ENBP1. In contrast to the highly conserved terminal AATAA motif of the ENBP1 binding site of known Enod12 promoters, the VfEnod12 promoter was characterized by an altered terminal AATAT sequence. This alteration did not interfere with VfEnod12 promoter activity in transgenic roots and nodules of V. hirsuta.

Journal Article↗