The differentiation-dependent inhibition of SV40 early promoter/enhancer activity in 3T3-L1 cells is mediated through promoter and not enhancer sequences

Using a plasmid bearing chloramphenicol acetyltransferase (CAT) gene controlled by Simian virus 40 (SV40) early promoter/enhancer complex (pA0cat), we analyzed functional enhancer motifs in 3T3-L1 fibroblast and adipocyte cells. Deletion mutant series of pA0 at the enhancer complex showed that gene expression both in fibroblast and adipocyte cells was dependent on a similar set of enhancer motifs. When pA0 was introduced into 3T3-L1 fibroblasts and the cells were induced to differentiate into adipocytes, CAT activity expressed in fibroblasts was suppressed. Experiments with the deletion mutants at the enhancer complex showed that the suppression was not related to any enhancer motif, and CAT activity was observed with a plasmid having only the promoter sequence. When pA0cat was co-transfected with excess of promoter sequence, the suppression in adipocytes was counteracted. This suggested that negativetrans-acting factors of the promoter sequence were responsible for the suppression in adipocytes.Abbreviations CAT chloramphenicol acetyltransferase - CAT the gene encoding CAT - SV40 Simian virus 40 - Asc-P ascorbic acid phosphate     

Bioluminescence imaging of hepatitis B virus enhancer and promoter activities in mice

By bioluminescence imaging and hydrodynamic gene transfer technology, the activities of hepatitis B virus (HBV) promoters and the effects of HBV enhancers on these promoters in mice under true physiological conditions have been assessed. Our studies reveal that either of the two HBV enhancers can stimulate HBV major promoter activity in hepa 1-6 cells (in vitro) and in mouse liver (in vivo), and the enhancer effects on the three promoters (S1, S2 and X promoter) are markedly greater in vivo than in vitro. The two HBV enhancers have no cooperative action on HBV promoters in vitro or in vivo.     

肌肉增强子因子2对猪肌肉生长抑制素启动子活性的调节

肌肉生长抑制素(Myostatin,Mstn)是转化生长因子-β超家族的成员,在哺乳动物的骨骼肌生长和分化过程中起负调控作用,其转录调控受到多个基因的影响,其中肌肉增强子因子2(MEF2)是重要的调控因子之一。因此,对猪Mstn启动子上MEF2位点及其作用方式的探讨具有重要意义。首先,通过PCR方法扩增了猪Mstn基因上游1 969 kb的启动子序列,利用生物信息学方法分析该序列包含3个MEF2的结合位点;其次,采用逐步删除的方法获得5个长度不等的启动子,用荧光素酶报告系统评估了它们在小鼠成肌细胞C2C12中的活性。其次,转入含有MEF2结合位点的启动子片段和MEF2C表达载体,可以显著增强启动子活性2～6倍,高表达另一亚型MEF2A则启动子活性没有明显改变。最后,转入MEF2C表达载体,用实时定量PCR和Western blotting方法检测Mstn的转录和蛋白水平的变化,结果发现mRNA升高了2～4倍;在肌管细胞中,蛋白翻译水平也有显著升高。这些结果显示,MEF2C可以通过激活Mstn参与猪肌肉生长和发育阶段的调节。研究为Mstn基因的转录调控提供了有效的作用靶点和效应分子,为进一步探讨Mstn的功能调控提供了一种新的思路。     

Sulfoxide stimulation of chondrogenesis in limb mesenchyme is accompanied by an increase in type II collagen enhancer activity

We have utilized a modification of the limb bud mesenchyme micromass culture system to screen compounds that might stimulate chondrogenesis. Two compounds in the sulfoxide family (methylphenylsulfoxide and p-chlorophenyl methyl sulfoxide) were stimulatory at 10(-2) M and 10(-3) M, respectively; whereas other sulfoxides and organic solvents were not active at these concentrations. In addition, specific growth factors (basic FGF, IGF-I, IGF-II) were not chondroinductive at concentrations that are active in other cell systems. Both sulfoxide compounds stimulated cartilage nodule formation, [35S]sulfate incorporation, and activity of the regulatory sequences of the collagen II gene. In contrast, transforming growth factor beta-1 (10 ng/ml) stimulated sulfate incorporation but produced only a diffuse deposition of cartilage matrix and reduced the ability of the cells to utilize the regulatory sequences of the collagen II gene. The sulfoxides appear to promote the differentiation of limb bud cells to chondrocytes and thus exhibit chondroinductive activity.     

Mouse oocyte mitogenic activity is developmentally coordinated throughout folliculogenesis and meiotic maturation.

Oocytes secrete soluble factors that regulate the growth and differentiation of follicular cells, including maintenance of the distinctive cumulus cell phenotype. This study determines whether the mitogenic activity of oocytes is developmentally regulated and examines the responsiveness of follicular cells to oocytes at different stages of follicular development. Prepubertal SV129 mice of varying ages were primed with 5 IU equine chorionic gonadotropin (eCG) and oocytes/zygotes collected either 46 h post-eCG (immature oocytes), 12 h after administration of 5 IU human CG (hCG; ovulated ova), or 12 h post-hCG and mating (zygotes). Mural granulosa cells (MGC) from antral follicles and GC from preantral follicles were cultured +/- denuded oocytes (DO) for 18 h, followed by a 6-h pulse of [(3)H]thymidine as an indicator of cellular DNA synthesis. Coculturing MGC with meiotically maturing oocytes led to a dose-dependent increase in [(3)H]thymidine incorporation (20-fold above control levels at 0.5 DO/microl). However, [(3)H] counts remained unchanged from control levels when cultured with meiotically incompetent DO from 11- to 15-day-old mice (3% germinal vesicle breakdown; GVB), irrespective of dose of DO or developmental status of GC (MGC or preantral GC). In some treatments, spontaneous meiotic resumption of competent oocytes was prevented by culturing with 5 microM milrinone, a selective inhibitor of oocyte-specific cyclic nucleotide phosphodiesterase. The mitogenic capacity of oocytes was found to decline during and after oocyte maturation. [(3)H]Thymidine incorporation in MGC was highest (11-fold above controls) when cultured with meiotically inhibited (milrinone-treated) GV DO, stimulated 5.5-fold by culture with maturing oocytes, 3-fold with ovulated ova, and unstimulated by zygotes. [(3)H]Thymidine incorporation in MGC was not altered by the dose of milrinone, either in the presence or absence of DO. Metaphase I marked the beginning of the decline in the capacity of oocytes to promote MGC DNA synthesis. These results demonstrate that the capacity of oocytes to promote proliferation of granulosa cells follows a developmental program, closely linked to oocyte meiotic status, increasing with the acquisition of meiotic competence and declining during and after oocyte maturation.     

The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.