GENES ANCESTRAL TO THE THYROID/STEROID RECEPTOR FAMILY

甲状腺/类固醇受体家族的祖先基因

• 批准号: 6432139
• 负责人: JOSEPH EDWARD RALL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432139
• 关键词: Caenorhabditis elegans; Cnidaria; biochemical evolution; complementary DNA; gene mutation; genetic promoter element; genetically modified animals; genetics; hormone receptor; introns; invertebrate embryology; invertebrate hormone; molecular cloning; nucleic acid sequence; protein isoforms; protein structure function; reporter genes; skin; steroid hormone receptor; thyroid hormones

In continuation of work on the nuclear hormone receptor (NCR) CHR3, we were able to show that a new technique in which RANI is generated in the bacteria which the worms ingest is more reliable than soaking worms in RANI. The gene of interest is introduced into the bacteria with two T7 RNA polymerase promoters . IPTG will induce the gene of interest and the worms are fed this construct. We how have tens of thousands of worms which are synchronized so we can estimate the activity of genes which might be induced or repressed by CHR3 at two hourly intervals between L1 and L2. We have primers which we have shown to work for eight candidate genes so with RT PCR we should be able to answer these questions in a relatively few weeks. Additionally, we performed several molting screens and found a single mutation on chromosome 3 near the dpy-18 locus which reproduced the results of CHR3 inhibition by its RANI.. We are currently attempting to isolate this gene. (M Kostrouchova, M. Krause)Previous work on c intestinalis identified allowed us to identify and sequence a nCR gene analogous to the TR. It did not bind T3 and we have been trying to clone additional genes but have been hampered by a lack of good DNA and RNA from cina. Hence for the instant we have shifted to studying aldehyde dehydrogenase genes in the scallop since this protein makes up all of the scallop lens protein. The gene has been isolated and sequenced (Piatigorsky, Carosa) Now we have examined the activity of the promoter of this gene in mouse lens cells, L929 mouse fibroblasts, and Cos 7 monkey kidney cells. The +63/-2120 construct hooked up to the luciferase gene was active in both mouse cell lines but inactive in the Cos 7 cells. There is a CREB like site in the scallop ALDH promoter and each of five nucleotides in it were mutated. All except one of these single base pair mutations abolished activity. One mutation which actually created a sequence identical to the mouse alph a crystalline promoter reduced but did not abolish activity. Three mutations were introduced into the alpha a CRYBP 1 site found in mouse and scallop lens promoters. None of the mutations destroyed activity and one that recreated the mouse site, increased activity two fold. Mutations in an AP1 site did not affect promoter activity. Gel shift assays showed specific protein binding to the scallop CREB site sequence from nuclear extracts of the alpha TN4-1 mouse lens cells or the L929 mouse fibroblast cells. No binding was seen with COS 7 cell nuclear extracts. Some binding was seen with scallop stomach and gill cells--lens cells are present in too small a quantity for adequate nuclear extracts. There is also a putative PAX 6 sequence in the scallop promoter and double transfection experiments showed that with a PAX 6 vector, activity of the scallop promoter could be doubled. Analysis of the sequences of important regions in thirteen well studied promoters of different lens crystalline genes from eight different species failed to identify a common motif. Thus lens specificity might be a function of a specific combination of multiple common sequences or, more likely, greater message and protein stability in the lens. (Carosa and Piatigorsky)We have continued work on nhrs from trepedilia cystophora and have shown that transfection of the jRXR gene into lens cells enhanced the activity of the J1B promoter (of the lens crystalline in that species) attached to firefly lucifers as a reporter. Both 9-is and all-trans resinoid acid enhanced further the activity of jRXR. This is somewhat curious since we have previously shown that jRXR specifically binds with high affinity only the is isomer. There was no result if 3T3 cells, P19 embryonal carcinoma, or Cos-7 cells were used. We have continued work on growing cystophora so they may metamorphose. We then plan to study gene expression during development using the yeast two hybrid screen. (Kostrouch, Z.)

在继续对核激素受体 (NCR) CHR3 进行研究的过程中，我们能够证明，一种在蠕虫摄入的细菌中产生 RANI 的新技术比将蠕虫浸泡在 RANI 中更可靠。将感兴趣的基因引入带有两个 T7 RNA 聚合酶启动子的细菌中。 IPTG 将诱导感兴趣的基因，并将该构建体喂给线虫。 我们如何拥有数以万计的同步蠕虫，这样我们就可以在 L1 和 L2 之间每隔两个小时估计 CHR3 可能诱导或抑制的基因的活性。我们有引物，已证明对八个候选基因有效，因此通过 RT PCR，我们应该能够在相对几周内回答这些问题。此外，我们进行了几次蜕皮筛选，并在 dpy-18 位点附近的 3 号染色体上发现了一个单一突变，该突变再现了 CHR3 被 RANI 抑制的结果。我们目前正在尝试分离该基因。 （M Kostrouchova，M. Krause）先前对肠杆菌的研究使我们能够鉴定出与 TR 类似的 nCR 基因并对其进行测序。它不结合T3，我们一直在尝试克隆其他基因，但由于中国缺乏良好的DNA和RNA而受到阻碍。因此，我们目前已转向研究扇贝中的乙醛脱氢酶基因，因为该蛋白质构成了所有扇贝晶状体蛋白质。该基因已被分离并测序（Piatigorsky，Carosa）现在我们已经检查了该基因的启动子在小鼠晶状体细胞、L929小鼠成纤维细胞和Cos 7猴肾细胞中的活性。连接到荧光素酶基因的+63/-2120构建体在两种小鼠细胞系中都有活性，但在Cos 7细胞中没有活性。扇贝ALDH启动子中有一个CREB样位点，其中五个核苷酸均发生突变。除了其中一个单碱基对突变外，所有突变都消除了活性。一种突变实际上产生了与小鼠α晶状启动子相同的序列，但活性降低了，但并未消除。在小鼠和扇贝晶状体启动子中发现的 α a CRYBP 1 位点中引入了三个突变。没有一种突变破坏了活性，而一种重建了小鼠位点的突变使活性增加了一倍。 AP1 位点的突变不影响启动子活性。凝胶位移测定显示，特定蛋白质与来自 α TN4-1 小鼠晶状体细胞或 L929 小鼠成纤维细胞核提取物的扇贝 CREB ​​位点序列结合。未观察到与 COS 7 细胞核提取物的结合。在扇贝胃和鳃细胞中发现了一些结合——晶状体细胞的数量太少，无法获得足够的核提取物。扇贝启动子中还存在推定的PAX 6序列，双转染实验表明，使用PAX 6载体，扇贝启动子的活性可以加倍。对来自八个不同物种的不同晶状体晶体基因的十三个经过充分研究的启动子的重要区域的序列进行分析，未能识别出共同的基序。因此，晶状体特异性可能是多个常见序列的特定组合的函数，或更可能是晶状体中更大的信息和蛋白质稳定性的函数。 （Carosa 和 Piatigorsky）我们继续对来自 Trepedilia Cytophora 的 nhrs 进行研究，并表明将 jRXR 基因转染到晶状体细胞中增强了作为报告基因附着在萤火虫荧光素上的 J1B 启动子（该物种的晶状体晶体）的活性。 9-is 和全反式树脂酸均进一步增强了 jRXR 的活性。这有点奇怪，因为我们之前已经证明 jRXR 仅以高亲和力特异性结合异构体。如果使用3T3细胞、P19胚胎癌或Cos-7细胞则没有结果。我们继续致力于培养囊藻，以便它们能够变形。然后，我们计划使用酵母两种杂交筛选来研究发育过程中的基因表达。 （科斯特鲁奇，Z.）