The mold-specific MS8 gene is required for normal hypha formation in the dimorphic pathogenic fungus Histoplasma capsulatum

The dimorphic fungus Histoplasma capsulatum is the etiologic agent of one of the most common systemic mycoses of humans, histoplasmosis. In the environment, H. capsulatum grows in a differentiated mold form and shifts to an undifferentiated yeast form after mold fragments or spores are inhaled. This mold-to-yeast shift is required for disease. Little is known about the molecular biology of dimorphism in Histoplasma, and most studies have been directed toward yeast-specific genes. While it is important to examine the role of genes upregulated in the yeast morphotype, genes which are silenced in the yeast (i.e., mold-specific genes) may also play a critical role in dimorphism. To begin to examine this hypothesis, we report here the first misexpression and knockout analysis of a mold-specific gene in Histoplasma. The strongly expressed MS8 gene encodes a predicted 21-kDa protein extremely rich in glycine and glutamine. Forced expression of MS8 driven by the TEF1 promoter in yeast did not alter the yeast morphology at 37°C or mold formation at 25°C. Yeast expressing MS8 did exhibit clumping in liquid medium and formed “sticky” colonies on agar plates. Allelic replacement of MS8 was accomplished by a positive-negative selection procedure. ms8 knockout mutants formed apparently normal yeast at 37°C but gave rise to aberrant mycelia at 25°C. The mold colonies of the knockouts were less than half as large as normal, had a granular surface, produced a dark-red pigment, and formed short hyphae which were 40% wider with a distinctive twisted “zig-zag” shape.     

猪丙型肝炎病毒核心蛋白结合蛋白6同源基因的克隆化研究

利用不同种属动物之间重要基因序列高度同源的理论,应用分子生物学与生物信息学技术和方法。克隆猪丙型肝炎病毒(HCY)核心蛋白结合蛋白6(HCBP6)的同源基因。首先应用酵母双杂交技术,以表达HCV核心蛋白的表达栽体作为曾饵,对于百万级的肝细胞cDNA文库酵母进行配合、筛选,首先获得人HCBP6的全长鳊码基因。然后应用美国国立生物工程中心(NCBI)建立的核苷酸序列数据库(CenBank)的同源基因的检索,搜索与之同源的来源于猪的表达序列标签(EST)。然后根据基因同泺性的原则,确定猪HCBP6的同源基因。获得了与HCC核心蛋白结合蛋白的36个基因片段,其中之一命名为HCBP6。根据基因同源性搜索,获得了来源于猪的EST基因序列片段。最终确立了猪HCBP6的同源基因。利用不同物种之间基因同泺性的原理、NCBI数据库GenBank同源基因的搜索,获得了猪HCBP6同源基因。生物信息学技术在后基因组时代具有重要地位和作用。     

Complementary DNA sequence of rabbit CAP18--a unique lipopolysaccharide binding protein

CAP18 is a novel 18 kDa cationic protein [pI approximately 10] originally purified from rabbit granulocytes using as an assay the agglutination of lipopolysaccharide (LPS) coated erythrocytes. cDNA clones encoding CAP18 were isolated from a rabbit bone marrow cDNA library using a PCR generated oligonucleotide probe derived from the N-terminal amino acid sequence. The deduced amino acid sequence reveals a putative signal sequence of 29 amino acids and a mature protein of 142 amino acid residues. The predicted size of the encoded protein is 16.6 kDa with a pI of 10. There are no N-linked glycosylation sites. The CAP18 sequence bears no homology with other known LPS-binding proteins including human bacterial permeability increasing protein (BPI)(1) and rabbit LPS binding protein (LBP)(2).     

人参植物皂苷生物合成相关新基因的筛选与鉴定

人参植物根进行的特定发育过程在药用次生物———人参皂苷生物合成和累积中发挥重要作用。为从人参根中分离出人参皂苷生物合成相关基因 ,采用抑制差减杂交技术 ,构建四年和一年生人参根组织mRNA群体间正向差减cDNA文库。对从差减文库中筛选的 4 0个阳性cDNA克隆进行酶切、PCR与逆向Northern斑点杂交鉴定、DNA测序以及核苷酸序列同源性比较。结果表明 ,获得的 6个差减克隆在GenBank/DDBJ/BMBL无对应的同源基因 ,代表新基因序列。与此同时 ,使用Northern印迹杂交验证及半定量RT PCR进一步确认 ,6个转录本为根发育阶段差异性表达基因。因而提示 ,它们可能在人参皂苷生物合成中发挥了重要作用。此外 ,在人参茎、叶与种子中亦能检测到上述基因转录本的表达。目前 ,6个新基因已被命名 ,在GenBank注册并获登录号 ,为克隆上述新基因cDNA全长序列及深入鉴定其在人参皂苷生物合成中的功能提供了重要实验依据。     

Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues

Eleven cDNA clones encoding UDP-glucose pyrophosphorylase (UGPase) have been isolated from cDNA libraries prepared from seed embryo, seed endosperm and leaves of barley (Hordeum vulgare L.). The sequences were identical, with the exception of positioning of the poly(A) tail; at least five clones with different polyadenylation sites were found. For a putative full-length cDNA [1775 nucleotides (nt) plus polyadenylation tail], isolated from an embryo cDNA library, an open reading frame of 1419 nt encodes a protein of 473 amino acids (aa) of 51.6 kDa. An alignment of the derived aa sequence with other UGPases has revealed high identity to UGPases from eukaryotic tissues, but not from bacteria. Within the aa sequence, no homology was found to a UDP-glucose-binding motif that has been postulated for a family of glucosyl transferases. The derived aa sequence of UGPase contains three putative N-glycosylation sites and has a highly conserved positioning of five Lys residues, previously shown to be critical for catalysis and substrate binding of potato tuber UGPase. A possible role for N-glycosylation in the intracellular targeting of UGPase is discussed.     

Molecular cloning and characteristics of rpc19+ and rpc40+ Schizosaccharomyces pombe genes, coding for common subunits of nuclear RNA polymerase I and III

Full-length copies of cDNAs of the rpc19+ and rpc40+ genes encoding the common subunits of nuclear RNA polymerases I and III and the corresponding fragments of chromosomes were isolated from genomic and cDNA libraries of Schizosaccharomyces pombe and characterized. It was established that the cloned genes are located on chromosomes III and II of the fission yeast, respectively. The rpc40+ gene lacks introns, and the rpc19+ gene contains two intervening sequences. The comparison of subunits Rpc19 (125 aa; M 13 722 Da; pI 4.51) and Rpc40 (348 aa; M 39 141 Da; pI 5.40) of Sz. pombe, whose characteristics were deduced from the sequences of their cDNAs, with the orthologous components of other eukaryotes allowed the most conserved structure-functional domains of these proteins to be identified.     

假密环菌cDNA文库的构建及其阿拉伯糖苷酶基因的克隆(英文)

用SMART技术构建了载体为λTriplEx2的假密环菌的表达型cDNA文库。文库滴度为1．0×106pfu／ml,重组率约为98．3％,扩增后滴度为3．1×108pfu／ml,容量约为4．2×1010。从文库中随机挑选176个克隆进行5’端测序,得到147条表达序列标签(EST),并将测序结果提交到EMBL数据库。随机测序结果表明:插入片段长度均在200-800之间。测序结果经过生物信息学分析,发现有43条序列与已知序列有明显同源性,其中序列AJ620046与多形拟杆菌的阿拉伯糖苷酶序列有很高的序列一致性。用SMART-RACE技术成功获得了AJ620046的全长cDNA,克隆了AJ620046的开放阅读框AF,并成功构建了重组质粒pHIL-S1-AF,在毕赤酵母菌中进行了初步表达。     

Molecular cloning of mouse thioredoxin reductases

We screened clones for thioredoxin reductase genes with a degenerate PCR-based strategy and have isolated two novel cDNA clones from a mouse thymocyte cDNA library. These encode two distinct thioredoxin reductases (TrxR1 and TrxR2) with 499 and 527 amino acid (aa) residues and calculated molecular masses of 54.5 kDa and 56.8 kDa respectively. These proteins share 90% and 50% aa sequence identity with those of previously cloned human TrxR, containing the redox-active cysteines, FAD binding domain, and the selenocysteine (SeCys) insertion sequence, which is composed of a putative stem-loop sequence located in the 3'-untranslated region (UTR). TrxR2 showing less homology to human TrxR has a mitochondrial translocation signal and a mitochondrial prepeptide protease cleavage site in the N-terminal domain. Transient expression experiments of each gene as fusion proteins with Xpress-tagged protein in NIH 3T3 cells indicated that TrxR1 was localized in the nucleus and cytoplasm and TrxR2 in the mitochondria. Furthermore, we mapped the TrxR1 gene to chromosome 10 (placed 1.71 cR from D10Mit42, lod>3.0) and the TrxR2 gene to chromosome 16 (placed 22.56 cR from D16Mit34, lod>3.0). Thus, the mouse has at least two distinct nuclear genes for TrxR that have different translocation sites in the cell.     

Bovine corneal protein 54K (BCP54) is a homologue of the tumor-associated (class 3) rat aldehyde dehydrogenase (RATALD)

Amino acid (aa) sequence data from Staphylococcus areas V8 protease-digested bovine corneal 54-kDa protein (BCP54) fragments were utilized to derive mixed oligodeoxyribonucleotide (oligo) primers complementary to the reverse translation products of these sequences. These degenerate oligo primers were used to prime the amplification of BCP54 sequence from bovine corneal epithelial cell cDNA. The cDNA probe generated by this mixed oligo-primed amplification of cDNA was cloned and dideoxy-sequenced. A search of the GenBank database (version 63.0) revealed extensive sequence similarity to the cDNA encoding tumor-associated rat liver (class 3) aldehyde dehydrogenase (RATALD). Nucleotide (nt) and aa sequence alignment of the BCP54 translation product reveals it is 78% and 84% homologous with RATALD at the nt and aa levels, respectively. Conservation of aa sequence elements common to the aldehyde dehydrogenase family thought to be of structural/functional significance is further substantiated by this analysis. Included in the discussion is the likelihood that gene sharing (genes encoding metabolic enzymes and other stable proteins) may extend to the cornea.     

Identification and sequence of human PKY, a putative kinase with increased expression in multidrug-resistant cells, with homology to yeast protein kinase Yak1

We have previously shown that several protein kinases are present in higher activity levels in multidrug resistant cell lines, such as KB-V1. We have now isolated a gene that codes for a putative protein kinase, PKY, of over 130 kDa that is expressed at higher levels in multidrug-resistant cells. RNA from KB-V1 multidrug-resistant cells was reverse-transcribed and amplified by using primers derived from consensus regions of serine–threonine kinases and amplified fragments were used to recover overlapping clones from a KB-V1 cDNA library. An open reading frame of 3648 bp of DNA sequence predicting 1215 aa, has been identified. This cDNA hybridizes to a mRNA of about 7 kb which is expressed at high levels in human heart and muscle tissue and overexpressed in drug-resistant KB-V1 and HL60/ADR cells. Because its closest homolog is the yeast serine/threonine kinase, Yak1, we have called this gene PKY. PKY is also related to the protein kinase family that includes Cdks, Gsk-3, and MAPK proline-directed protein kinases. This protein represents the first of its type known in mammals and may be involved in growth control pathways similar to those described for Yak1, as well as possibly playing a role in multidrug resistance.     

利用锌指基序的保守性从人脑组织分离新的C_2H_2型锌指蛋白基因表达序列

本文利用锌指基序的保守性设计引物,在低严谨条件下扩增人基因组总DNA,获得8个长度呈梯度的PCR扩增产物电泳条带。取其中第2和第3电泳条带,回收DNA片段并将它们克隆,经测序和查新比较,共获得60个含有锌指蛋白基因基序的单一的序列,其中23个为新的锌指蛋白基因DNA片段。以它们为探针,杂交筛选人脑组织cDNA文库,得到初筛cD-NA克隆44个。对其中28个初筛cDNA克隆进行复筛之后,得到20个cDNA单克隆。对这些阳性克隆进行测序,读出18个含有锌指蛋白基因基序的序列,国际联网查新之后,证明其中16个是新的锌指蛋白基因片段。这些新的锌指蛋白基因片段为今后克隆有意义的全长锌指蛋白cDNA提供了重要的实验材料。     

Isolation of novel expression sequences of C2H2 type zinc finger protein gene from human brain tissue according to the conservation of zinc finger motif]

By low stringency PCR amplification of genomic DNA using the primers designed based on the conservation of zinc finger motif, we got 8 gradient eletrophoretic bands. After recovery of the second and third bands, the DNA fragments in them were cloned and sequenced. Compared to the GenBank database, among these 60 segments containing zinc finger motif, 23 segments were novel zinc finger genes' genomic segments. Then the human brain tissue cDNA library was screened, using these segments as probes, and 44 positive clones were obtained. Rescreening 28 of them, we got 20 rescreened clones. All of them were sequenced and sent to the GenBank DNA database for sequence analysis, the results showed that 16 were novel C2H2 type zinc finger protein cDNA segments. The cDNA segments encoding the novel C2H2 type zinc finger proteins provide the basic materials for cloning of full length cDNA of valuable novel zinc finger protein genes.     

应用抑制性消减杂交技术克隆大鼠肝再生过程中特异表达基因

应用抑制性消减杂交技术成功地构建了高消减效率的正向消减cDNA文库,从随机挑取的50个克隆中有31个均检出了60~400bp插入片段,对这些插入cDNA片段进行测序后经Genbank同源性检索,表明其中7个片段为未知新序列。大鼠肝切除后肝再生cDNA正向消减文库的建立为进一步大批量筛选、克隆肝再生特异性表达的未知新基因奠定了基础,初步筛选出的特异性表达的序列标记为进一步研究肝再生中基因的功能提供了依据。 Abstract:The cDNA from rat regenerating liver tissue was used as the tester and that from normal liver was used as the driver.A highly efficient subtractive cDNA library was constructed by suppression subtractive hybridization(SSH).After screening,31 clones from 50 clones which were derived from the cDNA library were inserted by 60~400bp cDNA fragments.24 cDNA fragments corresponded to known genes and 7 cDNA fragments were unknown sequences(GenBank accession number:BG447490~447496).