Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme.

We isolated and sequenced a clone for Candida albicans enolase from a C. albicans cDNA library by using molecular genetic techniques. The 1.4-kbp cDNA encoded one long open reading frame of 440 amino acids which was 87 and 75% similar to predicted enolases of Saccharomyces cerevisiae and enolases from other organisms, respectively. The cDNA included the entire coding region and predicted a protein of molecular weight 47,178. The codon usage was highly biased and similar to that found for the highly expressed EF-1 alpha proteins of C. albicans. Northern (RNA) blot analysis showed that the enolase cDNA hybridized to an abundant C. albicans mRNA of 1.5 kb present in both yeast and hyphal growth forms. The polypeptide product of the cloned cDNA, which was purified as a recombinant protein fused to glutathione S-transferase, had enolase enzymatic activity and inhibited radioimmunoprecipitation of a single C. albicans protein of molecular weight 47,000. Analysis of the predicted C. albicans enolase showed strong conservation in regions of alpha helices, beta sheets, and beta turns, as determined by comparison with the crystal structure of apo-enolase A of S. cerevisiae. The lack of cysteine residues and a two-amino-acid insertion in the main domain differentiated C. albicans enolase from S. cerevisiae enolase. Immunofluorescence of whole C. albicans cells by using a mouse antiserum generated against the purified fusion protein showed that enolase is not located on the surface of C. albicans. Recombinant C. albicans enolase will be useful in understanding the pathogenesis and host immune response in disseminated candidiasis, since enolase is an immunodominant antigen which circulates during disseminated infections.     

球毛壳菌60S核糖体蛋白L10a基因克隆与特性分析

用粗糙脉孢菌（Neurospora crassa）XP_322380和赤霉菌（Gibberella zeag）PH-1（EAA76971）的60S核糖体蛋白L10a基因（60S ribosomal protein L10a,RPL10a）蛋白序列对球毛壳菌（Chaetomium globosum）ESTs序列数据库进行tBlastn检索,获得了球毛壳菌RPL10a cDNA序列。cDNA序列长765bp,开放阅读框654bp,编码217个氨基酸组成的多肽,蛋白分了量为23．9kD。BlastP分析表明该基因氨基酸序列与粗糙脉胞菌相似最高为89％;与玉蜀黍黑粉菌（Ustilago maydis）相似性最低为78％。cDNA序列及推测的氨基酸序列在GenBank登录（登录号分别为AY669070,AAT74578）。     

Isolation and characterization of the GFA1 gene encoding the glutamine:fructose-6-phosphate amidotransferase of Candida albicans.

Glutamine:fructose-6-phosphate amidotransferase (glucosamine-6-phosphate synthase) catalyzes the first step of the hexosamine pathway required for the biosynthesis of cell wall precursors. The Candida albicans GFA1 gene was cloned by complementing a gfa1 mutation of Saccharomyces cerevisiae (previously known as gcn1-1; W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). GFA1 encodes a predicted protein of 713 amino acids and is homologous to the corresponding gene from S. cerevisiae (72% identity at the nucleotide sequence level) as well as to the genes encoding glucosamine-6-phosphate synthases in bacteria and vertebrates. In cell extracts, the C. albicans enzyme was 4-fold more sensitive than the S. cerevisiae enzyme to UDP-N-acetylglucosamine (an inhibitor of the mammalian enzyme) and 2.5-fold more sensitive to N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (a glutamine analog and specific inhibitor of glucosamine-6-phosphate synthase). Cell extracts from the S. cerevisiae gfa1 strain transformed with the C. albicans GFA1 gene exhibited sensitivities to glucosamine-6-phosphate synthase inhibitors that were similar to those shown by the C. albicans enzyme. Southern hybridization indicated that a single GFA1 locus exists in the C. albicans genome. Quantitative Northern (RNA) analysis showed that the expression of GFA1 in C. albicans is regulated during growth: maximum mRNA levels were detected during early log phase. GFA1 mRNA levels increased following induction of the yeast-to-hyphal-form transition, but this was a response to fresh medium rather than to the morphological change.     

The oxidative stress-sensitive yap1 null strain of Saccharomyces cerevisiae becomes resistant due to increased carotenoid levels upon the introduction of the Chlamydomonas reinhardtii cDNA, coding for the 60S ribosomal protein L10a

The Saccharomyces cerevisiae yap1 null strain was transformed with a Chlamydomonas reinhardtii cDNA expression library. A 688-bp cDNA fragment, coding for the 60S ribosomal protein L10a (RPL10a), restored the capacity of the S. cerevisiae yap1 null strain to resist oxidative stress. The rpl10a gene is a single-copy gene in C. reinhardtii and encodes a constitutively produced 1.35-kb mRNA. The deduced 214-residue amino acid sequence was highly related with RPL10a proteins from eukarya (between 46.1 and 63.7% identity) and archaea (between 24.5 and 29.2% identity). Resistant transformants were pink, due to increased carotenoid levels, with the same chemical structure as torularhodin, the main carotenoid of the pink yeast Rhodotorula mucilaginosa. The pink transformants showed high resistance levels against H(2)O(2), paraquat, menadione, and UV light. Partial inhibition of the carotenoid synthesis by diphenylamine reduced the resistance levels, demonstrating the role of excess carotenoid synthesis in the resistance mechanism.     

The primary structure of rat ribosomal protein S28.

The amino acid sequence of the rat 40S ribosomal subunit protein S28 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein S28 has 69 amino acids and has a molecular weight of 7,836. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 8-10 copies of the S28 gene. The mRNA for S28 is about 450 nucleotides in length. Rat S28 is homologous to Saccharomyces cerevisiae S33.     

The C1orf9 gene encodes a putative transmembrane member of a novel protein family

Here we report the characterization of a human mRNA encoding a novel protein denoted C1orf9 (chromosome 1 open reading frame 9). The cDNA sequence, derived from a testis cDNA library, contains 5700 bp which encodes an open reading frame of 1254 amino acids. The deduced protein contains a putative N-terminal signal peptide and one putative transmembrane region, indicating membrane localization. No significant homology was found with known characterized proteins. However, a 150 amino acid region has significant homology to deduced protein sequences from other organisms, including Caenorhabditis elegans (43% identity), Saccharomyces cerevisiae (47% identity), Schizosaccharomyces pombe (48% identity), and two proteins from Arabidopsis thaliana (42% and 40% identity), suggesting a novel family of conserved domains. The C1orf9 gene was assigned to chromosome 1q24. The gene spans approximately 78.7 kb and is organized into at least 24 exons. Expression analysis revealed a single C1orf9 mRNA species of approximately 6.0 kb with a predominant expression in pancreas and testis, and only low levels of expression in other tissues examined.     

粘虫核糖体蛋白S7 cDNA的克隆和表达分析

运用RT-PCR和RACE技术克隆了粘虫Mythimna separata(Walker)核糖体蛋白S7基因(RPS7)的全长cDNA序列(GenBank登录号:JN582331),并对其进行生物信息学分析。结果表明,粘虫RPS7全长cDNA序列为762 bp,包括5'非编码区32 bp和3'非编码区67 bp。其开放阅读框(573 bp)编码190氨基酸肽链,具有核糖蛋白S7e蛋白家族典型特征。该肽链理论分子量为21.924 ku,等电点为9.82,富含4种类型的特定功能位点。该蛋白序列与其他动物RPS蛋白序列具有96.8%～98.2%高度同源性。应用荧光实时定量技术建立了粘虫脑部胚后发育RPS7表达模式。RPS7表达量随胚后发育脑部重建呈现出动态变化,这一结果显示RPS7是在转录水平上呈现发育性调节。     

The primary structure of rat ribosomal protein S13

The covalent structure of the rat 40S ribosomal subunit protein S13 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Rat S13 contains 150 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 17,080. Hybridization of a S13 cDNA to digests of nuclear DNA suggests that there are 8-10 copies of the gene for the protein. The mRNA for the protein is about 620 nucleotides in length. Rat S13 is related to Saccharomyces cerevisiae YS15 and to Halobacterium marismortui S11. The protein contains a possible internal duplication of 12 residues.     

The primary structure of rat ribosomal protein S10

The amino acid sequence of rat ribosomal protein S10 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein S10 contains 165 amino acids and has a molecular mass of 18917 Da. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 17-20 copies of the S10 gene. The mRNA for the protein is about 750 nucleotides in length. Ribosomal protein S10 has several possible internal duplications; one is a tandem repeat of ten residues that is basic and contains two or three prolines.     

The primary structure of rat ribosomal protein S19

The covalent structure of rat ribosomal protein S19 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein. Ribosomal protein S19 contains 144 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 15,944. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 15-18 copies of the S19 gene. The mRNA for the protein is about 640 nucleotides in length. Rat S19 is related to Saccharomyces cerevisiae S16A and to Halobacterium marismortui S12.     

An archaebacterial gene from Methanococcus vannielii encoding a protein homologous to the ribosomal protein L10 family

An open reading frame upstream of the Methanococcus vannielii L12 gene has been detected. The beginning of this open reading frame agrees with the N-terminal region of a protein (MvaL10) which has been isolated from the 50 S ribosomal subunit of M. vannielii and sequenced. The length of this gene is 1008 nucleotides, coding for 336 amino acids. Excellent sequence similarities were found to the L10-like ribosomal proteins from Halobacterium halobium and man. The N-terminal part of the MvaL10 protein shows significant sequence similarities to the E. coli L10 protein. MvaL10 is more than twice as long as E. coli L10 but is of length similar to those of the homologous halobacterial and human proteins. Interestingly, the C-terminal region of MvaL10 shows exceptionally high similarity to the C-terminal sequence of the MvaL12 protein. This is not the case for the E. coli proteins but was also observed for the human, Halobacterium and Sulfolobus proteins.     

Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid.

An IMP dehydrogenase gene was isolated from Candida albicans on a approximately 2.9-kb XbaI genomic DNA fragment. The putative Candida IMP dehydrogenase gene (IMH3) encodes a protein of 521 amino acids with extensive sequence similarity to the IMP dehydrogenases of Saccharomyces cerevisiae and various other organisms. Like the S. cerevisiae IMH3 sequence characterized in the genome sequencing project, the open reading frame of the C. albicans IMH3 gene is interrupted by a small intron (248 bp) with typical exon-intron boundaries and a consensus S. cerevisiae branchpoint sequence. IMP dehydrogenase mRNAs are detected in both the yeast and hyphal forms of C. albicans as judged by Northern hybridization. Growth of wild-type (sensitive) C. albicans cells is inhibited at 1 microg of mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenases, per ml, whereas transformants hosting a plasmid with the IMH3 gene are resistant to MPA levels of up to at least 40 microg/ml. The resistance of cells to MPA is gene dosage dependent and suggests that IMH3 can be used as a dominant selection marker in C. albicans.     

Identification of the gene encoding DNA topoisomerase I from Candida albicans

Abstract A gene encoding a type I topoisomerase (TOP1) was isolated from Candida albicans , sequenced, and expressed in Saccharomyces cerevisiae . The TOP1 gene was identified from a C. albicans genomic library by hybridization with the product of a polymerase chain reaction with degenerate primer sets encoding regions conserved in other TOP1 genes. A clone containing an open reading frame of 2463 bp and predicted to encode a protein of 778 amino acids with sequence similarity to eukaryotic type I topoisomerases was identified. The C. albicans TOP1 gene restored camptothecin sensitivity and increased the topoisomerase activity in S. cerevisiae , indicating that the DNA fragment encodes a functional C. albicans topoisomerase I.     

粘虫核糖体蛋白S7cDNA的克隆和表达分析

运用RT—PCR和RACE技术克隆了粘虫Mythimnaseparata（Walker）核糖体蛋白s7基因（RPS7）的全长eDNA序列（GenBank登录号：JN582331）,并对其进行生物信息学分析。结果表明,粘虫RPS7全长eDNA序列为762bp,包括5’非编码区32bp和3’非编码区67bp。其开放阅读框（573bp）编码190氨基酸肽链,具有核糖蛋白S7e蛋白家族典型特征。该肽链理论分子量为21．924ku,等电点为9．82,富含4种类型的特定功能位点。该蛋白序列与其他动物RPS蛋白序列具有96．8％-98．2％高度同源性。应用荧光实时定量技术建立了粘虫脑部胚后发育RPS7表达模式。RPS7表达量随胚后发育脑部重建呈现出动态变化,这一结果显示RPS7是在转录水平上呈现发育性调节。