Molecular cloning, gene expression, and identification of a splicing variant of the mouse parkin gene

We have isolated mouse cDNA clones that are homologous to human Parkin gene, which was recently found to be responsible for the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP). One of these cDNA clones had the 1,392-bp open reading frame encoding a protein of 464 amino acids with presumed molecular weight of 51,615. The amino acid sequence of mouse parkin protein exhibits 83.2% identity to human Parkin protein, including the ubiquitin-like domain at the N-terminus (identity = 89.5%) and the RING finger-like domain at the C-terminus (identity = 90.6%). Two other clones had the 783-bp open reading frame encoding a truncated protein of 261 amino acids without RING finger-like domain. It was proved to be a novel splicing variant by 3′-RACE method. Northern blot analysis revealed that mouse parkin gene is expressed in various tissues including brain, heart, liver, skeletal muscle, kidney, and testis. It is notable that mouse parkin gene expression appears evident in 15th day mouse embryo and increases toward the later stage of development. These mouse parkin cDNA clones will be useful for elucidating the essential physiological function of parkin protein in mammals. Received: 5 May 1999 / Accepted: 11 February 2000     

The nucleotide sequence of the human int-1 mammary oncogene; evolutionary conservation of coding and non-coding sequences.

The mouse mammary tumor virus can induce mammary tumors in mice by proviral activation of an evolutionarily conserved cellular oncogene called int-1. Here we present the nucleotide sequence of the human homologue of int-1, and compare it with the mouse gene. Like the mouse gene, the human homologue contains a reading frame of 370 amino acids, with only four substitutions. The amino acid changes are all in the hydrophobic leader domain of the int-1 encoded protein, and do not significantly alter its hydropathic index. The conservation between the mouse and the human int-1 genes is not restricted to exons; extensive parts of the introns are also homologous. Thus, int-1 ranks among the most conserved genes known, a property shared with other oncogenes.     

Cloning and DNA sequence of the gene coding for Clostridium thermocellum cellulase Ss (CelS), a major cellulosome component.

Clostridium thermocellum ATCC 27405 produces an extracellular cellulase system capable of hydrolyzing crystalline cellulose. The enzyme system involves a multicomponent protein aggregate (the cellulosome) with a total molecular weight in the millions, impeding mechanistic studies. However, two major components of the aggregate, SS (M(r) = 82,000) and SL (M(r) = 250,000), which act synergistically to hydrolyze crystalline cellulose, have been identified (J. H. D. Wu, W. H. Orme-Johnson, and A. L. Demain, Biochemistry 27:1703-1709, 1988). To further study this synergism, we cloned and sequenced the gene (celS) coding for the SS (CelS) protein by using a degenerate, inosine-containing oligonucleotide probe whose sequence was derived from the N-terminal amino acid sequence of the CelS protein. The open reading frame of celS consisted of 2,241 bp encoding 741 amino acid residues. It encoded the N-terminal amino acid sequence and two internal peptide sequences determined for the native CelS protein. A putative ribosome binding site was identified at the 5' end of the gene. A putative signal peptide of 27 amino acid residues was adjacent to the N terminus of the CelS protein. The predicted molecular weight of the secreted protein was 80,670. The celS gene contained a conserved reiterated sequence encoding 24 amino acid residues found in proteins encoded by many other clostridial cel or xyn genes. A palindromic structure was found downstream from the open reading frame. The celS gene is unique among the known cel genes of C. thermocellum. However, it is highly homologous to the partial open reading frame found in C. cellulolyticum and in Caldocellum saccharolyticum, indicating that these genes belong to a new family of cel genes.     

Cloning and characterization of a novel gene SRG-L expressed in late stages of mouse spermatogenic cells

Spermatogenesis is a continuum of spermatogoniarenewal and proliferation, meiosis, and spermiogenesisin mammalian. First the stem cells (primitive type A sper-matogonia) divide to preleptotene primary spermato-cytes, which develop to leptotene primary spermatocytes,zygotene primary spermatocytes, and pachytene anddiplotene primary spermatocytes in sequence. Then thediplotene primary spermatocytes go through two meioticdivisions, and produce round spermatids. Subsequently,round spermatids enter…     

柞蚕核型多角体病毒泛素类似基因的克隆与序列分析

从感病的柞蚕Antheraea pernyi蛹中分离纯化柞蚕核型多角体病毒 (ApNPV),提取基因组DNA,分别构建ApNPV DNA的HindⅢ和SalⅠ酶切片段文库。对基因文库中1个克隆进行序列分析,得到1个长度为321 bp的序列,其中包含一个编码76个氨基酸的开放阅读框,预测的分子量为8.46 kD,系泛素类似基因。在读码框的上游调控序列中,具有典型的晚期基因启动子序列ataag。氨基酸序列同源性分析结果表明,ApNPV与黄杉毒蛾Orgyia pseudotsugata核型多角体病毒 (OpNPV)的同源性最高 (96.1%),与苜蓿尺蠖Autographa californica核型多角体病毒 (AcNPV) 的同源性为86.8%,与棉褐带卷蛾Adoxophyes orana 颗粒体病毒 (AoGV) 的同源性最低（71.1%）,但与人类、线虫和酵母的泛素同源性分别为77.6%、76.3%和76.3%。一些氨基酸残基在真核生物中保守,在杆状病毒中不保守,个别氨基酸残基是杆状病毒所特有的,这些氨基酸序列的改变对杆状病毒泛素基因的作用有待进一步研究。     

Isolation and chromosomal localization of the human En-2 gene

By low stringency hybridization we have isolated from a human cosmid genomic library sequences homologous with a probe from the Drosophila engrailed gene. Partial nucleotide sequence analysis shows a consensus splice acceptor site followed by an open reading frame (ORF) that can encode 104 amino acids; the first 94 amino acids have 71% identity with the Drosophila engrailed protein. The shared region contains a homeo domain and is within the region of engrailed shared with the Drosophila invected gene and the mouse En-1 and En-2 genes. At the amino acid level, the human sequence is 85% identical with the mouse En-1 gene and 100% identical with the mouse En-2 gene. Hybridization against a panel of human-hamster somatic cell hybrids maps this human En-2 gene to chromosome 7, and regional mapping by in situ hybridization to human chromosomes localizes it to region 7q36 at the end of the long arm.     

OsRALyase1, a putative F-box protein identified in rice,Oryza sativa,with enzyme activity identical to that of wheat RALyase

A rice gene, OsRALyase1, encoding a product similar to wheat ribosomal RNA apurinic site specific lyase (RALyase), was isolated and expressed in vitro. An open reading frame of the gene predicted a protein of 476 amino acid residues with 75% identity to RALyase and contained an F-box-like motif in its amino terminal region. The rice gene product expressed in a wheat-germ protein expression system had the same characteristics as its wheat counterpart, cleaving a specific depurinated site of the 28S rRNA sarcin-ricin domain.     

Nucleotide sequence of the type C3 staphylococcal enterotoxin gene suggests that intergenic recombination causes antigenic variation.

The nucleotide sequence of the structural gene for staphylococcal enterotoxin type C3 (entC3) was determined. This gene contains 798-base-pair open reading frame that encodes a protein of 266 amino acid residues. Sequence analysis suggests that staphylococcal enterotoxin type C3 is synthesized in a precursor form that is processed to yield a mature extracellular form of 238 amino acid residues (molecular weight, 27,438). The entC3 gene is closely related to the gene for staphylococcal enterotoxin type C1, with 98% nucleotide sequence identity. Sequence comparisons between the entC3, entC1, and entB genes suggest that an ancestral entC1-like gene was formed by recombination between the entC3 and entB genes.     

The Brucella abortus cyclic beta-1,2-glucan virulence factor is substituted with O-ester-linked succinyl residues

Brucella periplasmic cyclic beta-1,2-glucan plays an important role during bacterium-host interaction. Nuclear magnetic resonance spectrometry analysis, thin-layer chromatography, and DEAE-Sephadex chromatography were used to characterize Brucella abortus cyclic glucan. In the present study, we report that a fraction of B. abortus cyclic beta-1,2-glucan is substituted with succinyl residues, which confer anionic character on the cyclic beta-1,2-glucan. The oligosaccharide backbone is substituted at C-6 positions with an average of two succinyl residues per glucan molecule. This O-ester-linked succinyl residue is the only substituent of Brucella cyclic glucan. A B. abortus open reading frame (BAB1_1718) homologous to Rhodobacter sphaeroides glucan succinyltransferase (OpgC) was identified as the gene encoding the enzyme responsible for cyclic glucan modification. This gene was named cgm for cyclic glucan modifier and is highly conserved in Brucella melitensis and Brucella suis. Nucleotide sequencing revealed that B. abortus cgm consists of a 1,182-bp open reading frame coding for a predicted membrane protein of 393 amino acid residues (42.7 kDa) 39% identical to Rhodobacter sphaeroides succinyltransferase. cgm null mutants in B. abortus strains 2308 and S19 produced neutral glucans without succinyl residues, confirming the identity of this protein as the cyclic-glucan succinyltransferase enzyme. In this study, we demonstrate that succinyl substituents of cyclic beta-1,2-glucan of B. abortus are necessary for hypo-osmotic adaptation. On the other hand, intracellular multiplication and mouse spleen colonization are not affected in cgm mutants, indicating that cyclic-beta-1,2-glucan succinylation is not required for virulence and suggesting that no low-osmotic stress conditions must be overcome during infection.     

Identification and characterization of an endolysin encoded by the<Emphasis Type="Italic">Streptomyces aureofaciens</Emphasis> phage μ1/6

An open reading frame homologous to the genes encoding several cell-wall hydrolyzing enzymes was identified on the genome of actinophage mu 1/6. This open reading frame encoding the putative endolysin was amplified by polymerase chain reaction and cloned into the expression vector pET-21a. This gene consisted of 1182 bp encoding a 393 amino acid polypeptide with a molar mass of 42.1 kDa. The gene product was overexpressed in Escherichia coli, and then the lytic enzyme was purified by a two-step chromatographic procedure. When applied exogenously, the endolysin of phage mu 1/6 was active against all tested Streptomyces strains but did not affect other bacteria. The amino acid sequence showed a high homology with a putative amidase of the Streptomyces phase phi C31. Downstream of the endolysin gene, an open reading frame encoding an 88 amino acid protein was identified. Structural analysis of its sequence revealed features characteristics for holin.     

Molecular cloning and characterization of a transferrin cDNA from the white-spotted flower chafer, Protaetia brevitarsis.

A full-length cDNA clone with high homology to insect transferrin genes was cloned by screening a Protaetia brevitarsis cDNA library. This gene (PbTf) had a total length of 2338 bp with an open reading frame (ORF) of 2163 bp, and encoded a predicted peptide of 721 amino acid residues. Like known cockroach, termite, and beetle transferrins, PbTf appears to have residues comprising iron-binding sites in both N- and C-terminal lobes. The deduced amino acid sequence of the PbTf cDNA was closest in structure to the beetle Apriona germari transferrin (68% protein sequence identity). Northern blot analysis revealed that PbTf exhibited fat body-specific expression and was upregulated by wounding, bacterial or fungal infection and iron overload, suggesting a functional role for PbTf in defense and stress responses.     

Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen

Abstract The P39 antigen is a specific, highly conserved, and immunogenic protein of Lyne disease spirochetes, Borrelia burgdorferi sensu lato. The nucleotide sequence of the gene encoding this protein was determined and found to be the first of two tandemly arranged open reading frames located on the spirochete's chromosome. These two open reading frames were designated bmpA for the gene encoding P39 and bmpB for the gene encoding the putative protein ORF2 encoded by the second open reading frame. The nucleic acid sequence identity for the two open reading frames was 62% while their deduced amino acid sequences were 52% identical. Comparison to sequence data bases demonstrated that the deduced amino acid sequences of both P39 and ORF2 were homologous to TmpC, a putative outer or cytoplasmic membrane lipoprotein of the syphilis spirochete, Treponema pallidum .     

Cloning and sequencing of an Escherichia coli gene, nlp, highly homologous to the ner genes of bacteriophages Mu and D108.

An nlp (Ner-like protein) gene was isolated from Escherichia coli. The nucleotide sequence of a 1,342-base-pair chromosomal DNA fragment containing the nlp gene was analyzed. It contained two open reading frames; one encoded 91 amino acid residues with an Mr of 10,361, and the other (ORFX) encoded 131 amino acid residues of the carboxyl-terminal region of a truncated polypeptide. The amino acid sequence deduced from the DNA sequence of nlp was highly homologous (62 to 63%) to the Ner proteins of bacteriophages Mu and D108. The amino-terminal region of Nlp deduced from the complete open reading frame contained a presumed DNA-binding region. The nlp gene was located at 69.3 min on the E. coli genetic map.     

Cloning,sequencing, and heterologous expression of a cellobiohydrolase cDNA from the basidiomycete Corticium rolfsii

From a Corticium rolfsii cDNA library, a clone homologous to other fungal cellobiohydrolase (CBH1) genes was isolated using the polymerase chain reaction. In the nucleotide sequence, one 1.6 kb long open reading frame coding for a polypeptide of 530 amino acid residues was detected which showed 64% identity with CBH1 of Phanerochaete chrysosporium. With expression of the 1.8 kb cDNA using the Aspergillus oryzae expression system, we detected microcrystalline cellulose (Avicel) hydrolyzing activity in the culture supernatant. The secreted protein, accompanied by the activity, was 89 kDa by SDS-polyacrylamide gel electrophoresis.     

芒果APl同源基因的克隆及其生物信息学分析

我们采用RT-PCR方法克隆了2个APl同源基因全长cDNA,分别命名为MAPl-1(GenBank accession No.FJ529206)和MAPl-2(GenBank accession No.FJ529207).MAPl-1编码247个氨基酸,开放阅读框长度为741 bp,蛋白质分子量为28.54kD,等电点为8.31;MAPl-2编码248个氨基酸,开放阅读框长度为744 bp,蛋白质分子量为28.78 kD,等电点为8.70.同源性分析表明,它们的核苷酸序列与其它木本植物APl同源基因的一致性为72%～81%.实验分析表明,MAPl-1和MAPl-2第1至第61个氨基酸含有一个MADS盒结构域,第88至第178个为K盒结构域;两个基因均定位于细胞核,且功能位点分布存在着不同,推测这两个基因在花器官发育过程中的功能存在差异.蛋白二级结构预测显示,MAPl-1蛋白有12个a-螺旋,4个β折叠区,14个β-转角;而MAPl-2蛋白有11个a-螺旋,5个β折叠区,15个β-转角:其大多数氨基酸具有亲水性.本研究有助于进一步了解芒果的开花分子机理及成花的生物学发育阶段.     

甘蓝型油菜赤霉素受体基因的克隆与表达

采用同源克隆技术从甘蓝型油菜中克隆到1个赤霉素受体基因,命名为BnGID1B(GenBank登录号为HQ589349).该基因含有1个内含子和2个外显子,其cDNA序列全长1 077 bp,编码358个氨基酸,推测蛋白质的相对分子量是40 203.4 Da,理论等电点为6.26.序列比对结果显示,BnGID1B基因与拟南芥GID1B基因核苷酸序列的相似性为86.3%,氨基酸序列的相似性为91.64%.实时荧光定量PCR分析表明,BnGID1B基因在苗期不同组织以及不同激素处理下的表达量有所不同,主要在根中表达,下胚轴中的表达量明显低于根,可见该基因具有一定程度的组织表达特异性.