Mapping and sequence of the gene for the pseudorabies virus glycoprotein which accumulates in the medium of infected cells.

RNA from pseudorabies virus (PRV)-infected cells was translated in a reticulocyte lysate with and without the addition of dog pancreas microsomes. Upon addition of the microsomes to the translation reaction, an additional prominent protein product was observed that was not present when microsomes were omitted. The gene coding for this processed protein and its lower-molecular-weight precursor was mapped within the small unique region of the genome by hybridization of mRNA to cloned fragments of PRV DNA and translation of the selected mRNAs. A fragment of the coding region of this gene was inserted into an open reading frame cloning vector to express part of this gene as a hybrid protein in Escherichia coli. This hybrid protein was injected into mice to raise an antiserum which was found to precipitate the glycoprotein which accumulates in the medium of PRV-infected cells. This allows us to conclude that the gene for the "excreted" glycoprotein (gX) maps to the small unique region of the genome, and that the precursor of this glycoprotein is readily processed by dog pancreas microsomes. The region of the PRV genome which codes for this glycoprotein was sequenced and found to include an open reading frame coding for 498 amino acids, flanked by sequences which contain features common to eucaryotic promoters and polyadenylation signals. The predicted protein sequence includes a hydrophobic sequence at the N-terminus which could be a signal sequence, and a hydrophobic sequence followed by a hydrophilic sequence at the C-terminus.     

Construction of phoE-caa, a novel PCR- and immunologically detectable marker gene for Pseudomonas putida.

In this paper we describe the construction and use in Pseudomonas putida WCS358 of phoE-caa, a novel hybrid marker gene, which allows monitoring both at the protein level by immunological methods and at the DNA level by PCR. The marker is based on the Escherichia coli outer membrane protein gene phoE and 75 bp of E. coli caa, which encode a nonbacteriocinic fragment of colicin A. This fragment contains an epitope which is recognized by monoclonal antibody (MAb) 1C11. As the epitope is contained in one of the cell surface-exposed loops of PhoE, whole cells of bacteria expressing the protein can be detected by using the MAb. The marker gene contains only E. coli sequences not coding for toxins and therefore can be considered environmentally safe. The hybrid PhoE-ColA protein was expressed in E. coli under conditions of phosphate starvation, and single cells could be detected by immunofluorescence microscopy with MAb 1C11. Using a wide-host-range vector the phoE-caa gene was introduced into P. putida WCS358. The gene appeared to be expressed under phosphate limitation in this species, and the gene product was present in the membrane fraction and reacted with MAb 1C11. The hybrid PhoE-ColA protein could be detected on whole cells of WCS358 mutant strains lacking (part of) the O-antigen of the lipopolysaccharide but not on wild-type WCS358 cells, unless these cells had previously been washed with 10 mM EDTA. In addition to immunodetection, the phoE-caa marker gene could be specifically detected by PCR with one primer directed to a part of the phoE sequence and a second primer that annealed to the caa insert.     

Aspects of energy-yielding metabolism in the aphid,Schizaphis graminum,and its endosymbiont: Detection of gene fragments potentially coding for the ATP synthaseβ-subunit and glyceraldehyde-3-phosphate dehydrogenase

Specialized cells within the aphid,Schizaphis graminum, contain intracellular, vesicleenclosed eubacterial endosymbionts (Buchnera aphidicola). Using oligonucleotide probes derived from conserved sequences of the ATP synthase -subunit and glyceraldehyde-3-phosphate dehydrogenase, and the polymerase chain reaction (PCR), we have amplified, cloned, and sequenced three DNA fragments. Amino acid sequence similarity indicated that two of these fragments corresponded to endosymbiont and host genes potentially coding for the -subunit of ATP synthase. The host gene fragment contained two putative introns. The third DNA fragment corresponded to a portion of a gene coding for a glyceraldehyde-3-phosphate dehydrogenase that was highly related to one of the enzymes fromEscherichia coli (GapA). These results indicate thatB. aphidicola may have an ATP synthase and consequently could synthesize ATP from a proton motive force generated within the intracellular vesicles of host cells containing the endosymbionts. The detection of a gene fragment coding for a protein similar to glyceraldehyde-3-phosphate dehydrogenase suggests the presence of this glycolytic enzyme in the endosymbiont and its involvement in energy-yielding metabolism.     

A general method of polymerase-chain-reaction-enabled protein domain mutagenesis: construction of a human protein S-osteonectin gene.

Polymerase chain reaction (PCR) amplification was employed to construct a mosaic gene consisting of the propeptide region of protein S and the glutamic acid-rich domain of osteonectin. The strategy is straightforward, results in large amounts of material, and is universally applicable for the generation of protein domain chimeras. In some cases 10% dimethyl sulfoxide aided the amplification. Four base CCGC "clamp" sequences adjacent to BamHI restriction sites at the ends of the PCR products were used to enhance the ligation of products. A hybrid inverse complement oligonucleotide primer composed of sequences containing 20 nucleotides of protein S and 16 nucleotides of osteonectin was used in the first round of PCR. An additional osteonectin sequence was added to the initial amplified product by performing PCR using a second "boot-strap" primer containing 18 nucleotides of osteonectin. Primers used to amplify osteonectin encompassed the 146-aminoacid NH2-terminal half of osteonectin. The double-stranded first-round fragments of protein S-osteonectin and osteonectin were subsequently mixed together and one elongation cycle of PCR was performed. Annealing occurred as the result of the 34-base-pair overlap region composed of osteonectin sequence. Taq polymerase was used for elongation with subsequent recombinant DNA synthesis. After elongation, external primers were added to amplify the protein S-osteonectin gene construct. The protocol we have developed allows noncoding and coding segments of DNA to be linked, GC-rich areas of DNA to be amplified, hybridization temperatures to be increased, annealing times to be reduced, and PCR of products to be subcloned.     

Cloning and sequence analysis of the gene encoding 3-hexulose-6-phosphate synthase from the methylotrophic bacterium, Methylomonas aminofaciens 77a, and its expression in Escherichia coli

Abstract A DNA fragment of 550 bp was specifically amplified by PCR with primers based on the N-terminal sequence of the purified 3-hexulose-6-phosphate synthase from Methylomonas aminofaciens 77a and on that of a lysyl endopep(idase-derived peptide. Using this PCR product as a probe, a gene coding for 3-hexulose-6-phosphate synthase in M. aminofaciens 77a chromosomal DNA was cloned in Escherichia coli JM109. Sequencing analysis revealed that the gene encoding 3-hexulose-6-phosphate synthase contained a 624-bp open reading frame, encoding a protein composed of 208 amino acid residues with a calculated relative molecular mass of 21 224.     

Cloning of human heparan sulfate proteoglycan core protein, assignment of the gene (HSPG2) to 1p36.1----p35 and identification of a BamHI restriction fragment length polymorphism.

We have isolated a cDNA coding for the core protein of the large basement membrane heparan sulfate proteoglycan (HSPG) from a human fibrosarcoma cell (HT1080) library. The library was screened with a mouse cDNA probe and one clone obtained, with a 1.5-kb insert, was isolated and sequenced. The sequence contained an open reading frame coding for 507 amino acid residues with a 84% identity to the corresponding mouse sequence. This amino acid sequence contained several cysteine-rich internal repeats similar to those found in component chains of laminin. The HSPG cDNA clone was used to assign the gene (HSPG2) to the p36.1----p35 region of chromosome 1 using both somatic cell hybrid and in situ hybridization. In the study of the polymorphisms of the locus, a BamHI restriction fragment length polymorphism was identified in the gene. This polymorphism displayed bands of 23 and 12 kb with allele frequencies of 76 and 24%, respectively.     

伪狂犬病病毒宿主关闭蛋白的基因克隆及结构分析

为研究伪狂犬病病毒 (pseudorabiesvires ,PRV)UL4 1基因编码的病毒宿主关闭蛋白 (VHS)的结构与功能 ,通过PCR扩增得到含UL4 1基因完整编码区的 1174bp片段 ,将该片段克隆到表达载体pGEX KG中GST下游 ,在大肠杆菌BL2 1(DE3)中实现了GST VHS融合蛋白的高效表达 .序列分析发现VHS蛋白具有 4个保守区 ,并且第 3个保守区与核酸内切酶结构域FEN 1(1A76 )高度同源 .PROSPECT软件预测的伪狂犬病病毒VHS蛋白三维结构中含有 10个α螺旋和 2 2个β折叠 ,与单纯疱疹病毒Ⅰ型的VHS蛋白三维结构十分相似 .     

Sequence analysis of hexon gene from adenovirus KR95 inducing hydropericardium syndrome in chickens

The nucleotide sequence of a part of the HindIII-D fragment (3300 b.p.) of adenovirus KR95 DNA has been determined. Analysis of the nucleotide sequence disclosed a continuous ORF for hexon gene (2814 b.p.) coding the 937 residue protein, part of ORF for the C-terminal region of pVI polypeptide, including 114 residues and the beginning of ORF coding 25 N-terminal residues for viral endoproteinase. Comparison of predicted KR95 hexon sequence and 8 mammalian and avian adenovirus hexon sequences revealed the highest homology between KR95 strain and avian adenoviruses FAV10 and FAV1 (91.1 and 80.1%, respectively). The results were used for creating a test system on the basis of the polymerase chain reaction. The system was used in analysis of fowl samples obtained from 12 poultry farms in Russia. The sequences of hexon gene amplified fragments in the isolated strains and similar fragments of other mammalian and avian adenoviruses have been compared.     

Construction and expression in Escherichia coli of a gene of hybrid hemagglutinin H1-H3 of influenza virus]

The hybrid gene of influenza virus hemagglutinin (HA) of the H1-subtype, carrying the sequence coding for the fragment of H3-subtype antigenic site B, was constructed. The product of expression of this gene in E. coli was obtained as a fusion protein with beta-galactosidase. The chimeric protein was shown to retain the antigenic properties of HA of H1-subtype and to interact specifically with antibodies against the synthetic peptide corresponding to the B site fragment of HA of the H3-subtype.     

Expression, secretion and processing of hirudin in E. coli using the alkaline phosphatase signal sequence

A DNA fragment coding for the E. coli phoA signal peptide was synthesized and inserted into the expression vector pKK223-3. A single HindIII restriction site is located just at the end of the signal sequence. A gene coding for the proteinase inhibitor hirudin, which has previously been synthesized, was inserted into this HindIII site. The hybrid protein was expressed under control of the tac-promoter and secreted into the periplasm of E. coli. From the periplasmic fraction two processed proteins were isolated. One of these was identical with desulfatohirudin and also had similar biological properties.     

Isolation of human retinal genes: recoverin cDNA and gene.

A human retina cDNA library enriched for retina-specific clones was prepared by subtraction with a non-retina population of cDNA in combination with polymerase chain reaction (PCR) amplifications. A highly retina-specific cDNA clone (1190 bp) was obtained through this library encoding a 200 amino acid protein with three calcium binding sites and 87% homology to the bovine photoreceptor protein, recoverin, which has been shown to mediate the recovery of the dark current after photoactivation, and 58% homology to the calcium-binding chick cone protein, visinin. Analysis of the gene indicated a 9-10 kb single-copy gene with at least three exons and two introns. The three exons contained the entire coding sequence, and all of the calcium-binding EF-hand regions were in putative exon 1. The recoverin gene was mapped to human chromosome 17 by hybridization to a panel of human-rodent hybrid DNAs.     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

Cloning and expression of a yeast protein tyrosine phosphatase.

To study the regulation of tyrosine phosphorylation/dephosphorylation in Saccharomyces cerevisiae, a protein tyrosine phosphatase (PTPase) was cloned by the polymerase chain reaction (PCR). Conserved amino acid sequences within the mammalian PTPases were used to design primers which generated a yeast PCR fragment. The sequence of the PCR fragment encoded a protein with homology to the mammalian PTPases. The PCR fragment was used to identify the yeast PTP1 gene which has an open reading frame encoding a 335-amino acid residue protein. This yeast PTPase shows 26% sequence identity to the rat PTPase, although highly conserved residues within the mammalian enzymes are invariant in the yeast protein. The yeast PTP1 is physicallt linked to the 5'-end of a heat shock gene SSB1. This yeast PTP1 gene was expressed in Escherichia coli and obtained in a highly purified form by a single affinity chromatography step. The recombinant yeast PTPase hydrolyzed phosphotyrosine containing substrates approximately 1000 times faster than a phosphoserine containing substrate. Gene disruption of yeast PTP1 has no visible effect on vegetative growth.     

Analysis of mRNA for class I HLA on human gametogenic cells

We have studied mRNA expression for Class I HLA (human leukocyte antigen) on male germ cells by amplification of gene fragments in PCR techique and by Northern hybridization. RNA was extracted from fractionated gametogenic cells (isolated from testis) and reversely transcribed. Then, cDNA was amplified for specific HLA sequence (1151 bp) representing whole-length coding sequence (HLA, -A, -B, -C). The specificity of this product was confirmed in “nested” PCR of 400 bp gene fragment coding for alpha 2 domain, alpha 3 domain, and the transmembrane portion of Class I HLA. The results indicate minimal expression of classical Class I HLA on gametogenic cells. Northern hybridization with 669 bp cDNA fragment (spanning for alpha 3 domain, transmembrane, cytoplasmic, and 3′ untraslated region) resulted in a low intensity signal from gametogenic cell fractions and confirmed our findings obtained by PCR. The minimal expression of classical HLA antigens may create a neutral cover for the male reproductive system, thereby preventing an immunological response during germ cell differentiation. © 1994 Wiley-Liss, Inc.     

人脑内-含有ACP样结构域新基因的发现

为寻找脑内新基因,以正常成人全脑cDNA为模板,采用锚定PCR方法进行扩增,将经琼脂糖DNA电泳 鉴定获得的一约1200bp大小的特异性条带回收,并克隆入Teasy载体.用310 Genetic Analyzer进行自动测序. 所得序列进行生物信息学分析BLAST相似性分析结果证明所得序列为新序列,读框分析表明,该序列中存在 一完整编码区,编码含357个氨基酸的蛋白质.ProDom软件分析发现其含有酰基携带蛋白(ACP)样结构域. 随后,经3'RACE法克隆到该基因的全长cDNA,其全长为2024bp,染色体定位在14q11.2,含有16个外显子, 15个内含子,该基因已登录到GenBank.经设计编码区引物,从Teasy载体扩增出编码区后再克隆入pGEX-4T1 表达载体,经异丙基硫代-D-乳糖苷(IPTG)化学诱导表达.其编码区克隆人pGEX-4T1表达载体后,转入 JM109宿主菌,经IPTG诱导已得到表达.点杂交及RNA印迹表明,该基因在正常成人脑内广泛高表达.     

Xylose utilisation: cloning and characterisation of the Xylose reductase from Candida tenuis

Xylose reductases catalyse the initial reaction in the xylose utilisation pathway, the NAD(P)H+H+ dependent reduction of xylose to xylitol. In this work, the xylose reductase gene from Candida tenuis CBS 4435 was cloned and successfully expressed in E. coli. From the purified and partially sequenced protein primers were deduced for PCR. The fragment obtained was used for Southern blot analysis and screening of a subgenomic library. The clone containing the open reading frame was sequenced; the gene consisted of 969 nucleotides coding for a 322 amino acids protein with a molecular mass of 36 kDa. Putative regulatory signals were identified with the help of a Saccharomyces cerevisiae regulatory sequence database. In order to express the xylose reductase in E. coli, the gene was placed under positive and negative control. At low temperatures, the xylose reductase was expressed in soluble and active form up to about 10% of the soluble protein; with rising temperatures formation of visible inclusion bodies occurred. In refolding experiments we were able to recover the major portion of xylose reductase activity from the pellet fraction.