Molecular evolution of the carbonic anhydrase genes: Calculation of divergence time for mouse carbonic anhydrase I and II

Summary A cDNA clone in pBR322 that cross-hybridizes with a mouse carbonic anhydrase form II (CAII) probe has been sequenced and identified as mouse carbonic anhydrase form I (CAI). The 1224-base-pair clone encodes the entire 260-amino-acid protein and appears to contain an Alu-like element in the 3 untranslated region. The deduced amino acid sequence exhibits 77% homology to human CAI and contains 17 of the 20 residues that are considered unique to and invariant for all mammalian CAI isozymes. The results of a detailed comparison of the nucleic acid sequences spanning the coding regions of mouse CAI and rabbit CAI have been used to calibrate an evolutionary clock for the carbonic anhydrases (CAs). These data have been applied to a comparison of the mouse CAI and CAII nucleic acid sequences to calculate the divergence time between the two genes. The divergence-time calculation provides the first estimation of the evolutionary relationship between CAs based entirely on nucleotide sequence comparison.     

Mouse retinol binding protein gene: Cloning,expression and regulation by retinoic acid

A full-length cDNA clone encoding the retinol binding protein (RBP) was isolated from a mouse liver cDNA library by hybridization screening. The nucleotide sequence of murine RBP is 85 and 95% homologous to that of human and rat RBP, respectively, with a deduced amino acid sequence 83% homologous to both species. Analysis of the tissue expression pattern of RBP mRNA in the female mouse indicated relatively abundant expression in the liver, with lesser amounts in extrahepatic tissues including adipose, kidney, spleen and uterus, suggesting that these tissues may have a significant role in retinol homeostasis. Mouse liver cell RBP regulation by retinoids was also investigated. Both all-trans retinoic acid (AT-RA) and 9-cis retinoic acid (9c-RA) induced RBP mRNA expression in a dose- and time-dependent manner. Maximal levels (up to 4-fold above controls) were observed at 48h following treatment of both mouse hepatoma cells in vitro and in vivo in mice receiving a single, oral dose of either retinoid. Interestingly, 9c-RA was more potent at RBP induction in both in vivo and in vitro systems. Given the extent and temporal pattern of RBP induction, we suggest that the RA-mediated increase in liver RBP is part of a cellular protection mechanism. Increased levels of RBP would facilitate sequestration and possibly cellular export of RA in cells receiving prolonged exposure to high levels of RA, thus minimizing toxicity.     

Cloning and Sequencing of the Gene Encoding 10 kD Zein of Maize

The results of cloning and sequencing of the gene encoding 10 kD zein of maize (Zea mays L. ) with polymerase chain reaction (PCR) technique are here with presented. The genomic DNA template was extracted from sterilized seedlings of maize. Primered with a pair of synthetic 5′and 3′ PCR primers, a 0.57 kb DNA fragment was obtained after 30 PCR amplification cycles. The restriction map of the DNA fragment has been determined. The result indicated that the entire coding sequence of 10 kD zein gene has been cloned. The homologies of the DNA sequence and deduced amino acid sequences between our result and those published abroad are 96% and 90% respectively. As l0 kD zein is rich in sulphur, this gene might be used to improve the quality of crops, especially, the forage legumes by genetic engineering.     

HCV基因组NS1区的分子克隆及序列测定

对广东省一名慢性丙型肝炎病人血清中的ＨＣＶ基因组ＮＳ１区进行分子克隆及序列测定。采用微粒吸附法提取ＨＣＶ ＲＮＡ,随机引物逆转录后进行聚合酶链反应。所用引物位于ＮＳ１区,扩增产物７８０ｂｐ在低熔点琼脂糖中电泳,加嘏相应条带处凝胶,与ｐＵＣ１８的连接批应直接在低熔点琼脂糖中完成。重组体转化ＪＭ１０９,挑取菌落增殖后提取的质粒采用ＰＣＲ和酶切法鉴定阳性克隆。将其中３２０ｂｐ的片段亚克隆到ｐＵＣ１８和ｐ     

用PCR突变技术克隆艾滋病病毒蛋白酶基因

作者设计并合成了一对用于PCR技术的突变引物HIV-1 Pr1和HIV-1Pr2,分别在两引物中设计了两个突变点,使突变后基因含有EcoRI、HindⅢ和TAA序列,便于HIV-1 Pr基因的定向克隆和表达。用HIV-1 Pr1和HIV-1 Pr2作引物,采用PCR方法从HIV-1基因组DNA中扩增出了一个360bp长的DNA片段,用EcoRI和HindⅢ双酶切法将此片段定向克隆入pUC19质粒,将克隆基因插入M13mp18进行DNA序列分析。结果表明,该基因序列的读框完全正确,从而为HIV-1 Pr基因的表达及抑制剂的研究奠定了基础。     

The cDNA Cloning and Nucleotide Sequence of the Gene Encoding Nonstrucure Protein of Rice Dwarf Virus Genome Segment 10

The results of cloning and sequencing the gene encoding nonstructure protein of the rice dwarf virus (RDV) gtnome segment 10 with polymerase chain reaction(PCR) technique were reported. The amplified PGR product was cloned into Hind Ⅱ site of plasmid pGEM3zf(-) and analysed with restriction enzymes. The physical map of the cloned fragment has been constructed, the insert is 1150 bp in length with restriction enzyme sites of Sac Ⅰ, Hind Ⅲ, NdeⅠ, BamH Ⅰ, etc. Besides, two restriction enzyme sites Bgl Ⅱ and EcoR Ⅰ have been separetely added in the 5 and 3 end of the segment in order to be cloned into plant intermediate vector in a convenient way. The fragments cleaved by the above-mentioned restriction enzymes were subcloned and the DNA sequence of full length of segment 10 was determined. In comparison with the RDV epidemic in Japan, the nucleotide sequence and deduced amino acid sequence of cloned segment 10 are 96.03% and 97.17% in homology respectively.     

Cloning, expression and tissue distribution of the gene encoding rat fibroblast growth factor receptor subtype 4

The rat homologue of the gene encoding the fibroblast growth factor receptor subtype 4 (FGFR4) was cloned from rat lung mRNA, and the cDNA sequence was found to be 95% similar and 92% identical to the human homologue. Northern blot analysis of adult rat tissues demonstrated that a 3.1-kb mRNA encoding FGFR4 is detectable only in the lung and kidney. The receptor variant described here encodes two potential immunoglobulin-like domains, 21 hydrophobic amino acids encoding a potential transmembrane domain, and a split tyrosine kinase motif. However, the acidic box and hydrophobic signal peptide domains are not present in this cDNA isolate.     

Cloning and sequencing of the gene encoding the cell surface glycoprotein of Haloarcula japonica strain TR-1

The triangular disk-shaped halophilic archaeon Haloarcula japonica strain TR-1 has a glycoprotein on its cell surface. The complete gene encoding the cell surface glycoprotein (CSG) was cloned and sequenced. The gene has an open reading frame of 2586 bp, and a potential archaeal promoter sequence approximately 150 bp upstream of the ATG initiation codon. The mature CSG is composed of 828 amino acids and is preceded by a signal sequence of 34 amino acid residues. A hydropathy analysis showed a hydrophobic stretch at the C-terminus, that probably serves as a transmembrane domain. The amino acid sequence of the Ha. japonica CSG showed 52.1% and 43.2% identities to those from the Halobacterium halobium and Haloferax volcanii CSGs, respectively. Five potential N-glycosylation sites were found in the mature Ha. japonica CSG, sites that were distinctly different from those in Hb. halobium and Hf. volcanii. The Ha. japonica CSG gene was expressed in Escherichia coli. Received: 20 September 1996 / Accepted: 9 October 1996     

Detection of human papillomavirus types by the polymerase chain reaction and the differentiation between high-risk and low-risk cervical lesions

By means of a consensus polymerase chain reaction (PCR) method, the prevalence of HPV types was determined in cervical biopsies from 137 women referred to the gynecological outpatient clinic for colposcopy because of an abnormal cervical smear. The prevalence of HPV was 80.3%. There was a statistically highly significant rise in the prevalence of the oncogenic HPV types (16, 18, 31, 33) with increasing severity of cervical intraepithelial neoplasia (CIN I to III), indicating a role for these HPV types in the pathogenesis of cervical cancer. The prevalence of other HPV types decreased significantly with the severity of the lesion, suggesting that these HPV types play a less significant role in this process. These data indicate that HPV typing with PCR may be a valuable tool for distinguishing between highrisk and low-risk cervical lesions. Furthermore, our results suggest that the detection of HPV types by consensus PCR in the cervix of patients with an abnormal smear but without histologically detectable CIN is a useful tool for predicting which of these patiens will eventually develop CIN. Finally, a relatively low percentage (3%) of HPV double infections is reported in this study.     

天花粉蛋白基因的克隆及序列分析

本文应用ＤＮＡ多聚酶链式反应（ＰＣＲ）技术,从括楼基因组ＤＮＡ中扩增并克隆了天花粉蛋白（ＴＣＳ）基因。核酸序列分析结果表明,克隆片段包括ＴＣＳ的前原蛋白的编码序列和５＇一侧翼区段。其编码序列与已发表的不同来源的３种ＴＣＳ基因的核苷酸序列的同源性分别为９９．２０％,９８．７４％和９８．６４％。推导出的氨基酸序列与已发表的４种ＴＣＳ的氨基酸序列的同源性分别为９８．６２％、９８．６２％、９７．４１％和９     

HGV广东株和香港株NS5区部分基因的克隆及序列分析

采用ＨＧＶＮＳ５特异的２对引物,对两个香港株和一个广东株ＨＧＶＲＮＡ进行逆转录套式ＰＣＲ扩增,ＰＣＲ产物克隆入ｐＵＣ１９,重组质粒转化ＤＨ５α和ＪＭ１０９菌株。ＰＣＲ和酶切法鉴定阳性克隆,双脱氧链末端终止法测定核苷酸序列并进行同源性分析。结果发现核苷酸变异呈散在分布,三株间核苷酸和氨基酸序列同源性分别为９３．３％～９４％及９７％～９９．２％,与已报道的中国株（ＣＮ）相比,则同源性分别为９０％～９１．２％和９４％～９６．３％,与美国株（ＰＮＦ２１６１及Ｒ１０２９１）相比,为８７．１％～８９．５％和９５．２％～９７％,而与西非株（ＧＢＶＣ）相比,则达９１．４％～９３．８％和９７％～９７．９％。提示ＨＧＶＮＳ５区核苷酸和氨基酸序列相对保守,不同ＨＧＶ株存在一定的地区差异。     

Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5

The nucleotide sequence of the gene encoding the cellulose-binding protein A (CBPA) of Eubacterium cellulosolvens 5 was determined. The gene consists of an open reading frame of 3453 nucleotides and encodes a protein of 1151 amino acids with a molecular mass of 126408 Da. The deduced amino acid sequence of CBPA contained one domain highly similar to a catalytic domain of glycosyl hydrolases belonging to family 9, two linker-like domains and four domains of unknown function. Among the four domains of unknown function, the domains 1 and 2 region had significant homology in amino acid sequence with the cellulose-binding domains in the family 9 glycosyl hydrolases. The cloned gene was inserted into an expression vector, pBAD-TOPO, and expressed in Escherichia coli as a fused protein. The fused protein was detected by immunoblotting using antiserum against CBPA.