GENES ANCESTRAL TO THE THYROID/STEROID RECEPTOR FAMILY

在对核激素受体(NCR)CHR3的继续工作中，我们能够证明一种新的技术，在这种技术中，在蠕虫摄入的细菌中产生Rani比在Rani中浸泡蠕虫更可靠。通过两个T7 RNA聚合酶启动子将感兴趣的基因导入细菌。IPTG将诱导感兴趣的基因，蠕虫被喂养这个结构。我们有数以万计的蠕虫是同步的，所以我们可以估计在L1和L2之间每小时两次可能被CHR3诱导或抑制的基因的活动。我们有对8个候选基因起作用的引物，所以通过RT-PCR，我们应该能够在相对较短的几周内回答这些问题。此外，我们还进行了几次换羽筛选，在dpy-18位点附近的3号染色体上发现了一个单一突变，该突变重现了Rani抑制ChR3的结果。我们目前正在尝试分离这种基因。(M Kostrouchova，M.Krause)之前在肠道线虫上所做的工作使我们能够鉴定和测序一个与tr类似的NCR基因。它没有结合T3，我们一直在试图克隆更多的基因，但由于缺乏来自中国的良好DNA和RNA而受阻。因此，我们暂时转向研究扇贝中的乙醛脱氢酶基因，因为这种蛋白构成了扇贝晶状体蛋白的全部。该基因已被分离和测序(Piatigorsky，Carosa)，现在我们已经在小鼠晶状体细胞、L929小鼠成纤维细胞和Cos-7猴肾细胞中检测了该基因启动子的活性。连接到荧光素酶基因的+63/-2120结构在两个小鼠细胞系中都是活跃的，但在Cos 7细胞中不活跃。在扇贝ALDH启动子中有一个CREB样位点，其5个核苷酸均发生突变。除了这些单碱基对突变中的一个之外，所有的突变都取消了活性。一种突变实际上产生了与小鼠α-晶体启动子相同的序列，这种突变减少了活性，但没有消除活性。三个突变被引入到在小鼠和扇贝晶状体启动子中发现的CRYBP 1位点的α。这些突变都没有破坏活性，其中一个突变重建了老鼠的部位，使活性增加了两倍。AP1位点的突变不影响启动子的活性。凝胶位移分析显示，αTn4-1小鼠晶状体细胞或L929小鼠成纤维细胞的核提取液中的扇贝CREB位点序列可与其特异结合。COS-7细胞核提取液未见结合。扇贝的胃和鳃细胞有一些结合--晶状体细胞的数量太少，不足以提取足够的核。在扇贝启动子中还含有一个可能的PAX-6序列，双转染实验表明，利用PAX-6载体，扇贝启动子的活性可以加倍。对来自8个不同物种的不同晶体基因的13个经过充分研究的启动子的重要区域序列的分析未能确定一个共同的基序。因此，晶状体特异性可能是多个共同序列的特定组合的函数，或者更有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)我们继续研究海燕的nhRs，并表明将jRXR基因导入晶状体细胞可以增强附着在萤火虫上的J1B启动子(该物种的晶状体)作为报告基因的活性。9-IS和全反式树脂酸均能进一步增强jRXR的活性。这有点奇怪，因为我们之前已经证明，jRXR只与IS异构体高亲和力特异结合。如果使用3T3细胞、P19胚胎癌细胞或Cos-7细胞，则没有结果。我们还在继续种植天花粉，这样它们就可以变质了。然后，我们计划使用酵母双杂交筛选来研究发育过程中的基因表达。(科斯特鲁奇，Z.)

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.)