Partial structure of the gene for chicken cartilage proteoglycan core protein

A genomic DNA fragment (gCORE-1), encoding a portion of the cartilage proteoglycan core protein, has been isolated from a phage library using cDNA as a probe. The genomic insert is about 17 kilobase pairs; two BamHI fragments of the insert (1.3 and 4.8 kilobase pairs) contain most of the hybridizable sequences found in the cDNA. Sequence analysis of these fragments shows that they contain a total of five exons that encompass 216 amino acid residues, all of which are identical to those of the corresponding cDNA sequence. Three of the exons, which are adjacent to one another, are very similar to the corresponding exons in the gene of a rat hepatic lectin as well as to an exon in the gene of human pulmonary surfactant-associated protein. There is a strong degree of conservation of amino acid sequences encoded in the three genes, although there is no similarity between their introns. The sizes of the five exons in gCORE-1, except for one (which is indeterminate because only a partial cDNA sequence is available), are less than 184 base pairs, whereas the sizes of the introns range from 218 to greater than 2629 base pairs. Four of the introns interrupt an exon codon at either their donor or acceptor sites, between the first and second nucleotides. Only one intron does not split a codon. Intron and exon boundary sites are in agreement with known consensus sequences for introns. The dispersed distribution and relatively small size of the exons, if representative of the entire gene, suggest that the complete gene which codes for the core protein may be quite sizable.     

Sequence of pig 17beta-hydroxysteroid dehydrogenase Type3 cDNA and its expression in mammalian cells

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) Type3 is an NADPH-dependent membrane-bound enzyme that is specifically expressed in testis and catalyzes the conversion of androstenedione to testosterone. To date, the sequence of Type3 enzymes has been clarified in humans, mice and rats; however, the sequence of the pig enzyme remains unknown. In this study, we determined the cDNA sequence of pig testicular 17beta-HSD Type3. PCR primers for partial pig testicular 17beta-HSD Type3 were designed from rat and human enzyme consensus sequences. Full-length cDNA was obtained by 3'- and 5'-RACE based on partial PCR products. The cDNA coding region was 933 bp in length, which is the same as the human enzyme, and shared 84.7% sequence identity with the human cDNA coding region. The monomer was estimated to have a molecular weight of 34,855 and to contain 310 amino acid residues. The predicted pig amino acid sequence showed 81.9, 75.5 and 72.9% sequence identity with the human, rat and mouse sequences, respectively. To elucidate 17beta-HSD Type3 activity, the expression vector pCMV/pig17beta-HSD3 was established and transfected into human embryo kidney 293 cells. Subsequently, 17beta-HSD activity (androstenedione conversion to testosterone) was strongly detected in cell lysates.     

Characterization of type 12 17beta-hydroxysteroid dehydrogenase, an isoform of type 3 17beta-hydroxysteroid dehydrogenase responsible for estradiol formation in women

A novel 17beta-hydroxysteroid dehydrogenase (17beta-HSD) chronologically named type 12 17beta-HSD (17beta-HSD12), that transforms estrone (E1) into estradiol (E2) was identified by sequence similarity with type 3 17beta-HSD (17beta-HSD3) that catalyzes the formation of testosterone from androstenedione in the testis. Both are encoded by large genes spanning 11 exons, most of them showing identical size. Using human embryonic kidney-293 cells stably expressing 17beta-HSD12, we have found that the enzyme catalyzes selectively and efficiently the transformation of E1 into E2, thus identifying its role in estrogen formation, in contrast with 17beta-HSD3, the enzyme involved in the biosynthesis of the androgen testosterone in the testis. Using real-time PCR to quantify mRNA in a series of human tissues, the expression levels of 17beta-HSD12 as well as two other enzymes that perform the same transformation of E1 into E2, namely type 1 17beta-HSD and type 7 17beta-HSD, it was found that 17beta-HSD12 mRNA is the most highly expressed in the ovary and mammary gland. To obtain a better understanding of the structural basis of the difference in substrate specificity between 17beta-HSD3 and 17beta-HSD12, we have performed tridimensional structure modelization using the coordinates of type 1 17beta-HSD and site-directed mutagenesis. The results show the potential role of bulky amino acid F234 in 17beta-HSD12 that blocks the entrance of androstenedione. Overall, our results strongly suggest that 17beta-HSD12 is the major estrogenic 17beta-HSD responsible for the conversion of E1 to E2 in women, especially in the ovary, the predominant source of estrogens before menopause.     

Frequent gene conversion between human red and green opsin genes

To study the evolution of human X-linked red and green opsin genes, genomic sequences in large regions of the two genes were compared. The divergences in introns 3, 4, and 5 and the 3′ flanking sequence of the two genes are significantly lower than those in exons 4 and 5. The homogenization mechanism of introns and the 3′ flanking sequence of human red and green opsin genes is probably gene conversion, which also occurred in exons 1 and 6. At least one gene conversion event occurred in each of three regions (1, 3, and 5) in the sequences compared. In conclusion, gene conversion has occurred frequently between human red and green opsin genes, but exons 2, 3, 4, and 5 have been maintained distinct between the two genes by natural selection. Received: 29 September 1997 / Accepted: 29 September 1997     

The compositional properties of human genes

Summary The present work represents the first attempt to study in greater detail previously proposed compositional correlations in genomes, based on a body of additional data relating to gene localizations as well as to extended flanking sequences extracted from gene banks. We have investigated the correlations that exist between (1) the GC levels of exons of human genes, and (2) the GC levels of either intergenic sequences or introns associated with the genes under consideration. In both cases, linear relationships with slopes close to unity were found. The similarity of the linear relationships indicates similar GC levels in intergenic sequences and introns located in the same isochores. Moreover, both intergenic sequences and introns showed GC levels 5–10% lower than the corresponding exons. The above findings considerably strengthen the previously drawn conclusion that coding and noncoding sequences (both inter- and intragenic) from the same isochores of the human genome are compositionally correlated. In addition, we find linear correlations between the GC levels of codon positions and of the intergenic sequences or introns associated with the corresponding genes, as well as among the GC levels of codon positions of genes.     

鳜鱼胰蛋白酶和淀粉酶与胃蛋白酶原基因的克隆与序列分析

采用RT-PCR及RACE法,克隆得到鳜鱼(Siniperca chuatsi)肝胰脏胰蛋白酶(trypsin, Try)、淀粉酶(amylase, Amy)基因 cDNA全序列.结果表明,鳜鱼Try基因cDNA全长为896 bp,其中开放阅读框 (open reading frame,ORF)为744 bp,编码247个氨基酸. 序列同源性分析发现,鳜鱼Try与 斑马鱼(Danio rerio)、非洲爪蟾(Xenopus laevis)、 小鼠Try和人TRY氨基酸序列同源性分别为81.4%、75.3%、74.5%和71.4%.鳜鱼Amy 基因cDNA全长为1 647 bp,其中ORF为1 539 bp,编码512个氨基酸.鳜鱼Amy与斑马鱼 、非洲爪蟾、小鼠Amy和人AMY氨基酸序列同源性分别为79.7%、75.4%、71.9%和70.9%. 同时对鳜鱼基因组进行PCR,获得鳜鱼Try、Amy与胃蛋白酶原(pepsinogen, Pep)全基因组DNA序列.序列分析表明,鳜鱼Try基因由4个内含子和5个外显子组成,全长1 362 bp;鳜鱼Amy基因由8个内含子和9个外显子组成,全长4 267 bp;鳜鱼Pep基因由8个内含子和9个外显子组成,全长 4 032 bp,与其它脊椎动物基因结构相似.应用Genome walker方法在鳜鱼克隆得到长度分别为1 189 bp、413 bp和527 bp的Try、Amy和Pep基因的5′侧翼区序列以及1段长为704 bp的Pep 基因3′侧翼区序列,并利用相关软件预测其中具有多个可调节其表达的调控元件.鳜鱼Try、Am y和Pep基因组全序列的克隆及其序列、结构分析和分子系统进化等的研究,为鱼类消化代谢相关基因的生理功能及表达调控机理进一步研究提供依据.     

Characterization of two members of the maize gene family, Incw3 and Incw4, encoding cell-wall invertases

Two maize putative cell-wall invertase genes (Incw3 and Incw4) have been isolated by screening a genomic DNA library (Zea mays L. W22) using the cDNA probes encoding the two maize cell-wall invertases Incw1 and Incw2. The Incw3 and Incw4 genes contain six exons/five introns and five exons/four introns, respectively. The protein sequences deduced from both genes revealed a beta-fructosidase motif and a cysteine catalytic site known to be conserved in invertase genes. A detailed analysis of the protein and nucleotide sequences provides evidence that the Incw3 and the Incw4 genes encode putative cell-wall invertases. Furthermore, the isoelectric point deduced from the INCW4 protein sequence suggested that the Incw4 gene may encode a unique type of cell-wall invertase unbound in the apoplast. Gene expression studies using RT-PCR and in-situ RT-PCR hybridization showed that the Incw3 expression is organ/tissue-specific and developmentally regulated. In contrast, the Incw4 gene is constitutively expressed in all vegetative and reproductive tissues tested.     

绵羊钙调蛋白调节基因Calponin h1 的分子克隆

在一项研究中我们发现雌激素体在胚胎发育后期对绵羊子宫平滑肌Calponin (CaP) 基因的活动有明显上调作用,而CaP一直被作为观察其他基因表达水平变化的基准参照基因（Reference Gene）。迄今为止, 绵羊CaP尚未完整克隆,为进一步了解其结构和功能,根据人、小鼠和家猪的同源保守区序列设计锚定寡核苷酸引物,通过5′-RACE及3′-RACE方法克隆了绵羊子宫平滑肌组织全长CaP h1 cDNA (GenBank登录号： AY327118), 在cDNA序列的基础上, 又通过PCR-SSP方法获得了CaP h1基因除内含子1、2之外的其余4个内含子全部序列 （GenBank登录号分别为：AY771807,AY771808, AY771809, AY771810） 。DNA序列测定和分析表明,绵羊子宫平滑肌CaP h1 cDNA全长1499bp, 编码297个氨基酸,5′-UTR及3′-UTR分别为79bp和529bp。CaP h1基因组DNA的克隆和序列分析表明,绵羊CaP全长约8kb,由 7个外显子和6个内含子组成。 同源序列比较发现,该基因外显子在不同物种间相对保守;与人类、野猪、小鼠、大鼠和鸡Calponin mRNA同源性分别为88％、92％、81％、79％和81％,但不同物种间内含子存在较大差异(>50%)。本研究填补了绵羊CaP基因分子克隆的空白,为进一步研究该基因的功能及子宫平滑肌收缩的调节机理奠定了基础。