应用RACE法克隆鸽恒定链基因的研究

为比较禽类恒定链的结构和功能, 应用RACE (Rapid Amplification of cDNA Ends) 技术首次克隆并鉴定了鸽恒定链基因。首先用一对含高度保守的DNA片段的简并引物, 从鸽脾细胞RNA扩增部分恒定链片段, 接着测序并设计新引物分别从5′和3′RACE扩增延长该片段。最后根据全基因的序列设计上、下游引物,获得大小为1 050 bp的全长cDNA。比较核苷酸序列, 鸽与鸡的Ii链同源性达到82.8％, 而与人等其它动物的同源性则在52.0％以上; 其中633 bp的开放阅读框编码211个氨基酸残基的前体蛋白。推导和分析氨基酸序列表明, 分子结构与鸡恒定链相似, 其中有些氨基酸残基表现出较高的保守性。     

Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4.

Screening of a genomic DNA library with a portion of the cDNA encoding the gamma-aminobutyric acid (GABA) receptor subunit rho1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to alpha, beta, gamma, and delta GABA receptor subunits and 74% similarity to the GABA rho1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho2. Polymerase chain reaction (PCR) amplification of rho1 and rho2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12-q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.     

Alternative transcriptional initiation and splicing of mouse Lamc2 message.

To investigate the mechanism of two forms of messages (5.1 and 2.4 kb) in mouse Lamc2, a gene encoding for the gamma2 chain of epithelial cell-specific laminin 5, we analyzed approximately 40 kb of genomic DNA containing the sequences from the first intron to the 23rd exon to the 3' untranscribed region. With the 5' rapid amplification of cDNA end (RACE) and primer extension technique using RNA from mouse kidney and thymic epithelial cells, we found that the smaller message starts at the 2895th base of the cDNA within the 19th exon. Upstream sequences of this alternative start site showed the promoter activity in the reporter assay with the secreted form of alkaline phosphatase (SEAP). We also identified a novel alternatively spliced exon (exon 19B) that includes two stop codons. These results revealed two possible additional open reading frame that are different from the previously described alternative human gamma2 peptide. Therefore, the mechanism for generating smaller message and the event of alternative splicing are quite distinct between mouse and human Lamc2, although genomic organization is highly conserved. The significance of the presence of alternative message is discussed.     

小地老虎UV视蛋白基因的克隆及序列分析

视蛋白是感光物质的重要组成成分,是动物感知周围光环境的重要途径之一。本文以小地老虎(Agrotis ypsilon)3日龄成虫为材料,利用RT-PCR和RACE末端扩增技术克隆得到小地老虎UV视蛋白基因的cDNA序列。序列分析表明,小地老虎视蛋白基因的cDNA序列1 632 bp,包括一个1 140 bp的完整开放阅读框架,编码379个氨基酸,理论蛋白分子量(Mw)41.50 ku,等电点(pI)7.56。GenBank登录号为JN185654。UV视蛋白包括7个跨膜拓扑结构和一个G蛋白偶联受体家族,第107位赖氨酸与UV视蛋白的紫外敏感性有重要关系。同源性分析显示,小地老虎UV视蛋白基因与其他昆虫的UV视蛋白基因具有较高同源性。本研究对深入探究UV视蛋白在动物夜行生活中的作用具有重要意义。     

Isolation and characterization of serine protease gene(s) from Perkinsus marinus

This study reports the first serine protease gene(s) isolated from Perkinsus marinus. Using universal primers, a 518 bp subtilisin-like serine protease gene fragment was amplified from P. marinus genomic DNA and used as a probe to screen a lambda-phage P. marinus genomic library; 2 different lambda-phage clones hybridized to the digoxigenin(DIG)-labeled subtilisin-like gene fragment. Following subcloning and sequencing of the larger DNA fragment, a 1254 bp open reading frame was identified and later confirmed, by 5' and 3' random amplification of cDNA ends (RACE) and northern blot analysis, to contain the entire coding-region sequence. Sequence analysis of the 3' RACE results from 2 isolate cultures, VA-2 (P-1) and LA 10-1, revealed multiple polymorphic sites within and among isolates. We identified 2 different types of cDNA clones with 95.53% nucleotide sequence similarity, suggesting the possibility of 2 closely related genes within the P. marinus genome. Southern blot analysis of genomic DNA from 12 genetically distinct P. marinus isolate cultures revealed 2 different banding patterns among isolates.     

Identification of two processed pseudogenes of the human Tom20 gene

The open reading frame (ORF) of the human Tom20 gene (hTom20) was amplified by PCR from a HeLa cDNA library using primers based on the sequence of HUMRSC145 and cloned into a pET15b vector. Amplification of human genomic DNA using these primers yielded a DNA fragment of the same size as that of the ORF of hTom20 cDNA. Sequencing of this fragment revealed that: (1) it has the same number of base pairs as the ORF of hTom20 cDNA (438 bp); and (2) the two sequences differ by 14 single base pair substitutions (97% similarity) causing eight changes in the amino acid sequence and two premature stop codons. Further amplification of human genomic DNA adaptor-ligated libraries using primers based on HUMRSC145 revealed three different sequence-related genomic regions; one corresponding to the fragment referred above, another corresponding to the hTom20 gene, and a third fragment of which the sequence differs from the ORF of hTom20 cDNA by only 22 base pair substitutions and a deletion of 4 bp. We conclude that, in addition to the hTom20 gene, there are two genomic DNA sequences (Ψ1Tom20 and Ψ2Tom20) that are processed pseudogenes of hTom20. Aspects concerning their evolutionary origin are discussed. Received: 12 September 1997 / Accepted: 29 November 1997     

Cloning and sequence analysis of cDNA encoding thiamin-binding proteins from sesame seeds

The amino acid sequences of the large polypeptides of thiamin-binding proteins (TBPs) from sesame ( Sesamum indicum L.) seeds (STBP-I, -II and -III) were analyzed. The large polypeptides of STBP-I, -II and -III had the same amino acid sequences as did their small polypeptides. The peptide sequence information obtained from STBPs was used to synthesize DNA primers for amplification of the gene(s) encoding STBPs. A 200-bp fragment was amplified from cDNA synthesized from RNA from sesame seeds 4 weeks after flowering. The 200-bp fragment was used to clone full-length cDNA(s) encoding STBP(s) with RACE techniques. A 644-bp fragment was amplified, cloned and sequenced. The cDNA was a full-length clone encoding STBP(s). It contained an open reading frame, which defined a 143-residue polypeptide. The identified small and large polypeptide sequences of STBPs exactly matched the sequence encoded within the cDNA clone. These results indicated that the small and large polypeptides of STBPs were encoded on the mRNA as a single large proprotein precursor and that the final mature forms were generated by post-translational processing in the same manner as the other 2S albumins of plant seeds.     

棉铃虫β-微管蛋白基因cDNA序列的克隆与序列分析

β-微管蛋白是构成细胞骨架的重要组成性蛋白,对昆虫的蜕皮、器官形成等生长发育阶段均能产生重要影响。本文以棉铃虫Helicoverpa armigera(Hübner)3日龄成虫为材料,利用RACE末端扩增技术克隆得到棉铃虫的β-微管蛋白基因的cDNA序列。序列分析表明:棉铃虫β-微管蛋白基因的cDNA序列包含1775个碱基,包括一个1347个碱基的开放阅读框,编码448个氨基酸组成的多肽。GenBank登录号:JF767013。同源性分析表明,棉铃虫的微管蛋白基因与本研究所比对其它昆虫的β-微管蛋白基因具有高度的同源性,达到90%左右。本研究克隆得到棉铃虫的β-微管蛋白基因的cDNA序列,对进一步深入研究该基因功能有重要意义。     

Isolation and characterization of a human p53 cDNA clone: expression of the human p53 gene.

A cDNA clone for human p53 cellular tumor antigen has been isolated and characterized. This clone contains the complete 3'-untranslated region and most of the open reading frame for the protein. Nucleotide sequence analysis revealed that p53 mRNA contains an Alu repeat in the 3'-untranslated region. Hybridization selection experiments showed this clone was capable of selectively binding p53 mRNA. In vitro translation of SV80 mRNA resulted in the synthesis of two immunoreactive p53 polypeptide species. Northern blot analysis showed that human p53 mRNA was 2.8 kb in length and was present in cell lines containing high and low levels of p53 protein. There appears to be only a single p53 gene in human cells and Southern blot analysis demonstrated no major genomic rearrangements or amplification of the p53 gene in the transformed cell lines examined.     

Expression of the split gene cob in yeast: Evidence for a precursor of a “maturase” protein translated from intron 4 and preceding exons

Intron 4 (14) of the split gene cob in mitochondrial DNA contains a long open reading frame in phase with the preceding exon. Mutations in this intron block the excision of the 14 sequence from the cob precursor RNA and, at the same time, generate a series of new polypeptides, parts of which apparently result from translation of 14 sequences. We sequenced six mutations clustered in the upstream part of the open reading frame, about 340 bp from the exon-intron boundary (box9 cluster). Four are base pair exchanges in the same triplet of this region; these form the polypeptides typical for 14 plus a trans-acting product encoded by 14, as shown by complementation studies. The other two mutations—a -2 bp deletion at the same site, causing frameshift with a chain-terminating codon within a few triplets, and a base pair exchange at a nearby site-affect both the formation of 14 typical translation products and the trans-acting function. These results on box9 mutants combined with results on box7 mutants suggest that an 14-encoded “maturase” protein (apparent molecular weight, 27,000) is cleaved off a precursor protein (apparent molecular weight, 55,000) encoded by exon sequences B1 to B4 and the intron open reading frame. We further discuss the role of the box9 nucleotide sequence in the maturation of cob-specific RNA.     

烟草黄酮醇合成酶基因的克隆及其序列分析

根据已知的黄酮醇合成酶cDNA保守序列设计引物,用RT-PCR技术从烟草叶片中扩增获得黄酮醇合成酶cDNA片段,再用RACE方法得到其两端序列。根据获得的序列,设计引物分离得到完整的1188bp的黄酮醇合成酶基因,其开放阅读框编码346个氨基酸。序列分析显示,烟草黄酮醇合成酶与高杯花、矮牵牛和马铃薯的同源性分别为87％、86％和84％,与其它物种中的同源性也在80％左右,表明不同物种中黄酮醇合成酶基因具有高度同源性。此外,在氨基酸水平上,该酶与其它依赖于2-酮戊二酸的双加氧酶及其相关酶也具有同源性。     

玉米大斑病菌小柱孢酮脱水酶基因(scd)的克隆与功能分析

根据已知植物病原真菌黑色素生物合成相关基因scd(scytalone dehydratase)氨基酸序列保守区域设计简并引物,分别以玉米大斑病菌基因组DNA和cDNA为模板,通过PCR技术获得scd基因的同源片段,利用SMART-RACE技术和3′RACE技术获得了scd的cDNA全长序列。并根据scd基因cDNA全长序列设计基因特异性引物扩增玉米大斑病菌基因组DNA获得了该基因DNA全长。通过DNA序列和cDNA序列对比分析发现scd基因编码一个180个氨基酸的开放阅读框架,DNA序列含有两个分别为50bp和78bp的内含子。生物信息学分析表明其氨基酸序列与水稻胡麻叶斑病菌的scd基因的相似性很高。DHN黑色素生物合成途径特异性抑制剂—Carpropamid处理玉米大斑病菌,在12~24h之内可以抑制病菌分生孢子的萌发和附着胞的产生,但随着处理时间的延长抑制剂的抑制作用变弱,并且经过抑制剂处理的病菌不能侵入寄主组织或不能在寄主组织内扩展。初步明确了scd与玉米大斑病菌黑色合成途径及致病性的关系。