Molecular cloning and characterization of the DNA mismatch repair gene class 2 from the Trypanosoma cruzi

Genes with homology to the bacterial mutS gene, which encodes a protein involved in post-replication DNA mismatch repair, are known in several organisms. Using a degenerate PCR strategy, we cloned a Trypanosoma cruzi genomic DNA fragment homologous to the mutS gene class two (MSH2). This fragment was used as a probe to select the corresponding cDNAs from a T. cruzi cDNA library. The complete sequence of the gene (3304 bp), denominated TcMSH2, was obtained. The sequence contained an open reading frame of 2889 bp coding for a putative protein of 962 amino acids. Computational analyses of the amino acid sequence showed 36% identity with MSH2 proteins from other eukaryotes and revealed the presence of all functional domains of MutS proteins. Hybridization analyses indicated that the TcMSH2 gene is present as a single copy gene that is expressed in all forms of the T. cruzi life cycle. The role of the product of the TcMSH2 gene in mismatch repair was investigated by negative dominance phenotype analyses in Escherichia coli. When eukaryotic muts genes are expressed in a prokaryotic system, they increase the bacterial mutation rate. The same phenomenon was observed with the TcMSH2 cDNA, indicating that T. cruzi MSH2 interferes with the bacterial mismatch system. Phylogenetic analyses showed that the T. cruzi gene grouped with the MSH2 clade confirming the nature of the gene isolated in this work.     

Molecular cloning of an alpha-glucosidase-like gene from Penicillium minioluteum and structure prediction of its gene product

The dexC cDNA, which is expressed in dextran-containing medium by the filamentous fungus Penicillium minioluteum, was cloned and sequence characterized. The cDNA sequence comprises 1859 bp plus a poly (A) tail, coding for a predicted protein of 597 amino acids. The genomic counterpart was isolated by PCR, finding three introns in its sequence. The dexC gene was located by Southern blot in the same 9-kb fragment that the previously isolated dextranase-encoding gene (dexA). Sequence analysis revealed that the deduced DexC protein belongs to glycosyl hydrolase family 13, showing a high sequence identity (58%) with Aspergillus parasiticus alpha-1,6-glucosidase. In addition, the high sequence identity (51%) between DexC protein and oligo-1,6-glucosidase of Bacillus cereus, with three-dimensional (3D) structure determined, leads us to proposed a 3D model for the structural core of DexC protein.     

山蛭基因组DNA的提取及其PCR扩增产物的分析

利用PCR技术从海南山蛭体内分离山蛭素（抗凝血蛋白）基因,首先需获得不带有山蛭体表色素且完整的山蛭基因组DNA,本试验通过结合使用SDS-蛋白酶裂解法和CTAB法,有效的去除了山蛭基因组DNA提取过程中难以去除的色素,得到的基因组DNA保持完整,无降解,以之作为模板,进行PCR扩增,获得一个长度约为237bp的特异性片段,此片段与印度山蛭蛭素的cDNA大小几乎一致。初步表明,这个序列没有内含子,而仅是海南山蛭蛭素的编码序列。     

The single-copy gene encoding high-mobility-group protein HMG-I/Y from pea contains a single intron and is expressed in all organs

The coding and 3-downstream regions of the gene encoding the high mobility group protein HMG-I/Y from pea have been isolated, sequenced and characterised. A 795 bp pea genomic fragment containing the coding region of the pea HMG-I/Y gene with a single intron of 201 bp was isolated by PCR. The gene encodes a protein of 197 amino acid residues with four copies of the AT-hook DNA-binding motif encoded by exon 2. Southern blot analysis on genomic DNA revealed the presence of a single copy of the HMG-I/Y gene in the haploid genome. The pea HMG-I/Y gene is expressed in all organs of pea including roots, stems, leaves, flowers, tendrils and developing seeds, as determined by northern blot analysis.     

Characterization of the CYP4A11 gene,a second CYP4A gene in humans

Comparison between the cDNA sequence of CYP4A11 and that deduced from a published genomic clone suggested the presence of an additional CYP4A gene in humans, CYP4A22. PCR amplification of genomic DNA yielded overlapping clones covering 13kb of genomic DNA and extending from 1003bp upstream from CYP4A11 translation initiation to 135bp upstream of the mRNA polyadenylation signal. Sequence and Southern blot analysis showed the presence in humans of two highly homologous CYP4A genes, CYP4A11 and CYP4A22. These two genes share 96% sequence identity and have similar intron/exon sizes and distribution. Short nucleotide insertions (< or =10bp) in introns 1, 3, 9, and 11, and deletions (< or =18bp) in introns 4, 6, and 11 differentiate the two genes. RT-PCR amplification of human kidney RNA followed by restriction fragment analysis showed that CYP4A11 is the predominant isoform expressed in kidney.     

Chitin synthase homologs in three ectomycorrhizal truffles

Abstract Degenerate PCR primers were used to amplify a conserved gene portion coding chitin synthase from genomic DNA of six species of ectomycorrhizal truffles. DNA was extracted from both hypogeous fruitbodies and in vitro growing mycelium of Tuber borchii . A single fragment of about 600 bp was amplified for each species. The amplification products from Tuber magnatum, T. borchii and T. ferrugineum were cloned and sequenced, revealing a high degree of identity (91.5%) at the nucleotide level. On the basis of the deduced amino acid sequences these clones were assigned to class II chitin synthase. Southern blot experiments performed on genomic DNA showed that the amplification products derive from a single copy gene. Phylogenetic analysis of the nucleotide sequences of class II chitin synthase genes confirmed the current taxonomic position of the genus Tuber , and suggested a close relationship between T. magnatum and T. uncinatum .     

小麂Sry基因的克隆和测序

应用人的性别决定基因SRY(Sex-determining Region Y gene,SRY)中HMG框内的一对引物,对小麂细胞株的基因组DNA进行PCR扩增,得到雄性小麂细胞的220bp扩增产物,而在雌性小麂细胞中未发现扩增产物。将雄性小麂细胞的220bp扩增产物通过T-A互补法克隆到质粒pGEM-T 载体中,筛选阳性克隆进行DNA测序。测序结果表明小麂Sry基因保守序列与人的SRY基因保守区相同碱基的比值为152/184,达到82.6%。提示小麂Sry基因与人的SRY基因存在着较高的同源性,说明SRY基因在进化过程中高度保守。 Abstract:Using the primers from SRY gene——HMG Box for PCR amplification in genomic DNA of Muntiacus reevesi cell strains,a 220bp fragment was obtained in the male but not in the female.The 220bp fragment was cloned into the pGEM-T vector using T/A clone method.The identified positive clone was sequenced.The result shows that 82.6% nucleotides(152bp/184bp) are homologous between Muntiacus Sry and human SRY gene.It suggests that SRY is highly conserved during evolution.     

Characterization of theHelicoverpa assulta nucleopolyhedrovirus genome and sequence analysis of the polyhedrin gene region

A local strain ofHelicoverpa assulta nucleopolyhedrovirus (HasNPV) was isolated from infectedH. assulta larvae in Korea. Restriction endonuclease fragment analysis, using 4 restriction enzymes, estimated that the total genome size of HasNPV is about 138 kb. A degenerate polymerase chain reaction (PCR) primer set for the polyhedrin gene successfully amplified the partial polyhedrin gene of HasNPV. The sequencing results showed that the about 430 bp PCR product was a fragment of the corresponding polyhedrin gene. Using HasNPV partial predicted polyhedrin to probe the Southern blots, we identified the location of the polyhedrin gene within the 6 kbEcoRI, 15 kbNcoI, 20 kbXhoI, 17 kbBgl II and 3 kbClaI fragments, respectively. The 3 kbClaI fragment was cloned and the nucleotide sequences of the polyhedrin coding region and its flaking regions were determined. Nucleotide sequence analysis indicated the presence of an open reading frame of 735 nucleotides which could encode 245 amino acids with a predicted molecular mass of 29 kDa. The nucleotide sequences within the coding region of HasNPV polyhedrin shared 73.7&#x0025; identity with the polyhedrin gene fromAutographa californica NPV but were most closely related toHelicoverpa andHeliothis species NPVs with over 99% sequence identity.     

一步法扩增克隆IBDV上海超强毒VP2—4—3基因

分离、纯化了鸡传染性法氏囊病病毒超强毒 (vvIBDV)上海株SH95的病毒核酸dsRNA ,应用随机引物将RNA反转录成cDNA ,以此为模板一步扩增出A片段前体融合蛋白基因即VP2 4 3基因 ,将其克隆入 pGEM T载体 ,并进行序列分析 ,其与超强毒株HK4 6的核苷酸序列的同源性达 98% ,整个基因有 5个氨基酸差异 ,同源性达99.5 1% (10 0 7/ 10 12 )。     

Isolation and characterization of a DNA probe for Staphylococcus aureus subspecies aureus biovar

The gene encoding for polynucleotide phosphorylase (pnp) of a new biovar of Staphylococcus aureus subsp. aureus (NBSA) has been isolated from a genomic library of strain M280(0). The coding region consisted of a 1094-bp HindIII-HindIII DNA fragment encoding for a protein of 277 amino acids with a calculated molecular mass of 29.5 kDa. The nucleotide sequence of the structural gene, contained a continuous open reading frame of 836 bp, showed significant homology with the genes of bacterial polynucleotide phosphorylase from Bacillus subtilis (67.7% identity), from Haemophilus influenzae (62.4% identity), from Pseudomonas luminescens (61.6% identity), and from Escherichia coli (59.7% identity). DNA-DNA and DNA-colony slot-blot hybridizations demonstrated that the pnp gene, employed as a molecular probe, is specific for the identification of NBSA strains.     

胰蛋白酶抑制剂基因siRNA表达体系的构建

以大豆基因组DNA为模板,利用聚合酶链式反应(PCR)技术克隆了大豆胰蛋白酶抑制剂基因KSTI3的全长DNA片段,并将其构建到pMD18-T vector上。核苷酸序列测定结果表明:该基因片段全长654bp,与已发表的KSTI3基因序列同源性达99%。将反义 正义基因片段插入到pBI121 35S启动子下,构建重组质粒pBIKSTI3。通过冻融法将该重组质粒转入农杆菌EHA105中,获得了siRNA表达体系。利用农杆菌介导法将带有pBIKSTI3的菌株转化大豆,从2棵再生植株中得到2 100bp的特异性扩增条带,而未转化的植株中无该片段的产生。     

特异性检测胰腺组织表达Cre重组酶转基因小鼠的方法

利用组织特异性分子标志物启动子调控Cre重组酶,研制了6种在不同组织中特异性表达Cre重组酶的转基因小鼠.这些转基因小鼠的基因型鉴定均使用设计在Cre基因编码区的通用引物.为了特异性检测胰腺组织表达Cre重组酶的转基因小鼠,在大鼠胰岛素RIP启动子上和Cre基因上设计1对引物进行PCR扩增,并通过凝胶电泳进行分析.PCR结果显示,设计在Cre基因上的通用引物可以从6种不同组织特异性Cre重组酶转基因小鼠基因组DNA中扩增获得480 bp产物;利用本研究设计的特异性引物可以从胰腺组织表达Cre重组酶转基因小鼠基因组DNA中扩增200 bp的目的条带.这一结果表明,利用特异性引物进行PCR反应,可有效地将胰腺组织表达Cre重组酶转基因小鼠与其他多种组织的Cm重组酶转基因小鼠鉴别开来.     

Human gastric intrinsic factor: characterization of cDNA and genomic clones and localization to human chromosome 11

A human gastric intrinsic factor (IF) cDNA clone was isolated using a rat cDNA clone as a probe. Comparison of the predicted amino acid sequence revealed 80% identity of human IF with rat IF. These cDNA clones were used to isolate and map two overlapping clones encoding the human IF gene. The first exon of the cloned region (exon 2) contains 30 bp of the 5' untranslated region, the signal peptide, and the first 8 amino acids of the mature protein. Exons 3-10 encode the remainder of the coding and 3' noncoding regions. Southern analysis of genomic DNA indicated the presence of a single human IF gene and also revealed the presence of strong hybridizing sequences in genomic DNA from monkey, rat, mouse, cow, and human, suggesting that the IF gene is well conserved. The IF gene was localized to human chromosome 11 by concurrent cytogenetic and cDNA probe analysis of a panel of human X mouse somatic cell hybrids. Southern analysis of genomic DNA from patients with congenital pernicious anemia (lacking intrinsic factor) revealed normal restriction fragment patterns, suggesting that a sizable gene deletion was not responsible for the deficiency.     

Cloning and characterization of the O-methyltransferase I gene (dmtA) from Aspergillus parasiticus associated with the conversions of demethylsterigmatocystin to sterigmatocystin and dihydrodemethylsterigmatocystin to dihydrosterigmatocystin in aflatoxin biosynthesis.

O-Methyltransferase I catalyzes both the conversion of demethylsterigmatocystin to sterigmatocystin and the conversion of dihydrodemethylsterigmatocystin to dihydrosterigmatocystin during aflatoxin biosynthesis. In this study, both genomic cloning and cDNA cloning of the gene encoding O-methyltransferase I were accomplished by using PCR strategies, such as conventional PCR based on the N-terminal amino acid sequence of the purified enzyme, 5' and 3' rapid amplification of cDNA ends PCR, and thermal asymmetric interlaced PCR (TAIL-PCR), and genes were sequenced by using Aspergillus parasiticus NIAH-26. A comparison of the genomic sequences with the cDNA of the dmtA region revealed that the coding region is interrupted by three short introns. The cDNA of the dmtA gene is 1,373 bp long and encodes a 386-amino-acid protein with a deduced molecular weight of 43,023, which is consistent with the molecular weight of the protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The C-terminal half of the deduced protein exhibits 76.3% identity with the coding region of the Aspergillus nidulans StcP protein, whereas the N-terminal half of dmtA exhibits 73.0% identity with the 5' flanking region of the stcP gene, suggesting that translation of the stcP gene may start at a site upstream from methionine that is different from the site that has been suggested previously. Also, an examination of the 5' and 3' flanking regions of the dmtA gene in which TAIL-PCR was used demonstrated that the dmtA gene is located in the aflatoxin biosynthesis cluster between (and in the same orientation as) the omtA and ord-2 genes. Northern blotting revealed that expression of the dmtA gene is influenced by both medium composition and culture temperature and that the pattern correlates with the patterns observed for other genes in the aflatoxin gene cluster. Furthermore, Southern blotting and PCR analyses of the dmtA gene showed that a dmtA homolog is present in Aspergillus oryzae SYS-2.     

Analysis of a gene (vch) encoding hemolysin isolated and sequenced from Vibrio campbellii

This study describes the amplification, localization, and sequence analysis of a hemolysin gene from type strain V. campbellii NBRC 15631--the first report of a full-length hemolysin gene for the species. An amplicon ( approximately 600 bp) of polymerase chain reaction performed using V. campbellii DNA template and primers previously designed to target a fragment of V. harveyi hemolysin gene (vhh) was shotgun-cloned and sequenced, generating 576 bp nucleotide sequences of the V. campbellii hemolysin gene. PCR primers designed based on these initial sequences were used to amplify a 551-bp V. campbellii hemolysin gene fragment that was used as probe in Southern hybridization, which localized the complete hemolysin gene within a 3.5-kb HindIII restriction fragment of the V. campbellii genomic DNA. To obtain the remaining DNA sequences upstream and downstream of the 576-bp hemolysin gene sequences, inverse PCR was performed using a self-ligated (circularized) V. campbellii HindIII restriction fragment as the template and PCR primers designed to amplify flanking regions of the 576-bp gene fragment. Nucleotide sequences from the terminal regions of the 3.1-kb product of inverse PCR provided the flanking sequences, resulting in the complete sequence for the V. campbellii hemolysin gene. A VCH PCR primer set was designed to amplify a 1.3-kb region containing the entire hemolysin gene even from other V. campbellii strains, which was sequenced to confirm the V. campbellii hemolysin gene sequence. An open reading frame (ORF) of 1,254 bp (designated as vch) was identified, sharing 79% sequence identity with V. harveyi hemolysin gene vhh, representing 262 base substitutions between V. campbellii and V. harveyi. The deduced amino acid sequence of V. campbellii hemolysin (VCH) shows homologies to the V. harveyi hemolysin (VHH), thermolabile hemolysin of V. parahaemolyticus, proteins such as phospholipase of V. vulnificus and lecithinases of V. mimicus and V. cholerae. The VCH primer set did not produce any amplicon in PCR using V. harveyi DNA, and may therefore be used to distinguish environmental strains of V. campbellii from V. harveyi.     

嗜热菌Aquifex pyrophilus中mbh 2基因簇的鉴定和部分基因的克隆及测序

基于嗜热菌Aquifex aeolicus的mbhS2基因,设计合成特异性引物,以Aquifex pyrophilus 的染色体DNA为模板,应用PCR技术扩增出目的片段.序列测定结果显示,其推导出的氨基酸序列与A.aeolicus的相应序列具有85%的同源性.以此PCR片段为探针,从A.pyrophilus 的Nco I部分基因组文库(4～6kb)中筛选出含5kb大小插入片段的阳性克隆,进而对其进行了亚克隆及测序.结果表明,该插入子包含A.pyrophilus mbj2基因簇的小亚基基因mbhS2、orf1基因和部分orf2基因.所推导出的氨基酸序列与A.aeolicus的MbhS2和Orf963相比较的同源性分别为81%和60%.