MULTIPLE TRANS-ACTING FACTORS MEDIATE THE INDUCTION OF C-FOS IN VASCULAR SMO

Previous work from this laboratory has demonstrated that the induction of c-fos by angiotensin (Ang) Ⅱ in vascular smooth muscle cells (VSMC) is mediated by at least two enhancer elements, the serum response element (SRE) and the cyclic AMP response element (CRE). These two elements appear to act independently and equally. The current study is aimed at identifying the trans-acting proteins that interact with these enhancer ele-.ments to mediate this response. Rat aortic smooth muscle (RASM) cells were grown in culture and made quiescent by placing them in a defined serum-free media for 48 hours.Aug Ⅱor vehicle was then added, the cells harvested 30 minutes later and nuclear extracts isolated. Gel mobility shift assay were then performed.gel shifts done with wild type SRE oligonucleotide demonstrated that two specific proteins bound to this element.Mutations in central CArG box of the SRE inhibited binding but mutations in the ets binding site in the SRE had no effect. Extracts from Aug Ⅱor vehicle treated cells demonstrated an identical pattern of binding. Similar experiments performed with CRE noligonucleotide identified three proteins which bound to CRE. Again,no differences were observed between vehicle and AugⅡtreated extracts. An antibody to serum response factor(SRF),which binds to the SRE, supershifted the proteins that bound to the SRE. When gel shifts with RASM cells extracts were compared to gel shifts from either NIH 3T3 fibroblast cell extraes of HeLa cell extracts,a different pattern of protein binding was observed using the SRE oligonucleotide.In contrast to the studies with the SRF antibody,an antibody to CRE binding protein (CREB)-43, the most common CREB protein, failed to recognize protein from RASM extract in a supershift assay. When the gel shift pattern with the CRE oligonucleotide between RASM, 3T3 and HeLa cell extracts was compared,no similarity was seen. Taken together,'these data suggest that stimulation of c-for in VSMC's occurs via activation of a unique set of proteins. Activation of these proteins is not due to new protein synthesis. Further work will be aimed at identifying these proteins and studying their mechanism of activation in VSMC's.

Anticancer Drug Resistance of HeLa Cells Transfected With Rat Glutathione S-transferase pi Gene

To establish a cytologic expressing system of rat glutathione S-transferase pi (GST-pi) cDNA for detecting the resistance of HeLa cells to anticancer drugs. Methods The assessment was made with various anticancer drugs (adriamycin, mitomycin, cisplatinum and vincristine) that showed different cytotoxicities in transfectant HeLa cells with pSV-GT containing rat GST-pi cDNA (HeLa/pSV-GT) or control pSV-neo (HeLa/pSV-neo). Expression levels of GST-pi mRNA in HeLa/pSV-GT and HeLa/pSV-neo were measured by in situ hybridization using Digoxin-labelled cDNA probe. Results HeLa/pSV-GT expressed significantly high degree of GST-pi mRNA, whereas both HeLa/pSV-neo and HeLa cells had very low expression. Cytotoxicities of HeLa/pSV-GT and HeLa/pSV-neo with 4 anticancer drugs were measured by MTT assay. Drug concentrations for yielding 50% inhibition (IC50) in HeLa/pSV-GT by adriamycin, mitomycin and cisplatinum were 70.13 靏/mL, 10.95 靏/mL and 16.52 靏/mL, respectively. In contrast, IC50 in HeLa/pSV-neo was 10.34 靏/mL, 7.48 靏/mL and 13.70 靏/mL, respectively. The cytotoxicities of vincristine on both HeLa/pSV-GT and HeLa/pSV-neo were not significantly different. Conclusions Our findings suggest that HeLa/pSV-GT containing rat GST-pi cDNA is resistant to some anticancer drugs due to overexpression of GST-pi. Also, HeLa/pSV-GT cell line could serve as a useful cytogenetic model for further research.

The enhancement effect of pp38 gene product on the activity of its upstream bi-directional promoter in Marek's disease virus

There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene and 1.8-kb mRNA transcript gene family in the genome of Marek’s disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) reporter plamids, pP(pp38)-EGFP and pP(1.8- kb)-EGFP, were constructed under this bi-directional promoter in two directions. The two plasmids were transfected into uninfected chicken embryo fibroblast (CEF), MDV clone rMd5 infected CEF (rMd5-CEF) and pp38-deleted derivative rMd5Δpp38 infected CEF (rMd5Δpp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF, and no EGFP could be detected in uninfected CEF or rMd5Δpp38-CEF, implying that pp38 was a factor influencing the activ-ity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to co- transfect CEF or rMd5Δpp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5Δpp38-CEF but still not in uninfected CEF, implying that pp38 needs other protein(s) to work together for the complete trans-acting activity. Another MDV gene, 24 kd phosphorylated protein pp24 gene was cloned into pcDNA3.1 as a pp24-expressing recombinant plasmid pcDNA-pp24. When uninfected CEF was co-transfected with pcDNA-pp38, pcDNA-pp24 and EGFP expressing plasmids pP(pp38)-EGFP or pP(1.8-kb)-EGFP, the EGFP could be detected. These results indicated that pp38 and pp24 could enhance the activity of the promoter when they worked together. DNA mobility shift assay showed that pp38 would bind to the bi-directional promoter with the co-existing of pp24, although neither of them alone influenced mobility of the promoter DNA. All the above suggested that MDV pp38 could transactivate the bi-directional promoter when combined with pp24. The results also indicated that the activity of the promoter in the direction of 1.8-kb mRNA was significantly stronger than that of pp38 direction.

A LIM Domain Protein from Tobacco Involved in Actin-Bundling and Histone Gene Transcription

The two LIM domain-containing proteins from plants （LIMs） typically exhibit a dual cytoplasmic-nuclear dis-tribution, suggesting that, in addition to their previously described roles in actin cytoskeleton organization, they partici-pate in nuclear processes. Using a south-western blot-based screen aimed at identifying factors that bind to plant histone gene promoters, we isolated a positive clone containing the tobacco LIM protein WLIM2 （NtWLIM2） cDNA. Using both green fluorescent protein （GFP） fusion-and immunology-based strategies, we provide clear evidence that NtWLIM2 local-izes to the actin cytoskeleton, the nucleus, and the nucleolus. Interestingly, the disruption of the actin cytoskeleton by latrunculin B significantly increases NtWLIM2 nuclear fraction, pinpointing a possible novel cytoskeletal-nuclear crosstalk. Biochemical and electron microscopy experiments reveal the ability of NtWLIM2 to directly bind to actin filaments and to crosslink the latter into thick actin bundles. Electrophoretic mobility shift assays show that NtWLIM2 specifically binds to the conserved octameric cis-elements （Oct） of the Arabidopsis histone H4A748 gene promoter and that this binding largely relies on both LIM domains. Importantly, reporter-based experiments conducted in Arabidopsis and tobacco proto-plasts confirm the ability of NtWLIM2 to bind to and activate the H4A748 gene promoter in live cells. Expression studies indicate the constitutive presence of NtWLIM2 mRNA and NtWLIM2 protein during tobacco BY-2 cell proliferation and cell cycle progression, suggesting a role of NtWLIM2 in the activation of basal histone gene expression. Interestingly, both live cell and in vitro data support NtWLIM2 di/oligomerization. We propose that NtWLIM2 functions as an actin-stabilizing protein, which, upon cytoskeleton remodeling, shuttles to the nucleus in order to modify gene expression.

Developmental stage-specific factors in the mouse haematopoietic tissues binding to the 5'-flanking as-acting elements of humanε-globin gene

The human ε-globin gene is expressed in a tissue-specific and developmental stage-specific manner. During the earliest stage of gestation, this gene is expressed in the yolksac, but is silenced completely at the 6th-8th weeks of gestation. Recently, several studieson the transgenic mice have shown that the 5′-flanking DNA sequences of human

Phasing analysis of the transcriptome and epigenome in a rice hybrid reveals the inheritance and difference in DNA methylation and allelic transcription regulation

Hybrids are always a focus of botanical research and have a high practical value in agricultural production.To better understand allele regulation and differences in DNA methylation in hybrids,we developed a phasing pipeline for hybrid rice based on two parental genomes(PP2PG),which is applicable for Iso-Seq,RNA-Seq,and Bisulfite sequencing(BS-Seq).Using PP2PG,we analyzed differences in gene transcription,alternative splicing,and DNA methylation in an allele-specific manner between parents and progeny or different progeny alleles.The phasing of Iso-Seq data provided a great advantage in separating the whole gene structure and producing a significantly higher separation ratio than RNA-Seq.The interaction of hybrid alleles was studied by constructing an allele co-expression network that revealed the dominant allele effect in the network.The expression variation between parents and the parental alleles in progeny showed tissue-or environment-specific patterns,which implied a preference for trans-acting regulation under different conditions.In addition,by comparing allele-specific DNA methylation,we found that CG methylation was more likely to be inherited than CHG and CHHmethylation,and its enrichment in genic regions was connected to gene structure.In addition to an effective phasing pipeline,we also identified differentiation in OsWAK38 gene structure that may have led to the expansion of allele functions in hybrids.In summary,we developed a phasing pipeline and provided valuable insights into alternative splicing,interaction networks,trans-acting regulation,and the inheritance of DNA methylation in hybrid rice.