Isolation of a human T-lymphotropic virus type I strain from Australian aboriginals.

A human T-lymphotropic virus type I (HTLV-I) strain was isolated in a CD4+ T-lymphocyte culture established from a healthy seropositive Australian Aboriginal. This isolate, identified as HTLV-IMSHR-1, was detected by immunofluorescence with monoclonal antibodies, by the presence of gag-encoded protein p24 in the culture supernatant, and by cocultivation leading to infection and transformation of lymphocytes from an HTLV-I-negative donor. By using the polymerase chain reaction technique, the env gene and segments of the pol and pX regions of the proviral genome of HTLV-I(MSHR-1) were amplified and sequenced. Comparison with the envelope sequences of prototype strains revealed up to 7% divergence at the nucleotide level and 3.1 to 4.3% divergence in the predicted amino acid sequence. Phylogenetic analysis showed that the Australian and Melanesian isolates are related. Differential reactivity with monoclonal antibodies suggests that gag protein p19 of HTLV-I(MSHR-1) is also divergent. The potential for antigenic divergence between the prototype HTLV-I isolates and the Austro-Melanesian variants requires further investigation, because it would have implications for serodiagnosis and vaccine development.     

Isolation and molecular characterization of a serotype 9 human rotavirus strain.

A human rotavirus strain, designated AU32, that belongs to serotype 9 was isolated and was compared by RNA-RNA hybridization with recently established two serotype 9 strains (WI61 and F45) as well as other prototype human strains. These three strains exhibited a very high degree of homology with one another and shared a high degree of homology with strains belonging to the Wa genogroup but not with strains belonging to either the DS-1 or AU-1 genogroup. These results suggest that genetic constellation of the serotype 9 strains is similar to that of the commonest human rotavirus despite the recent recognition of this serotype.     

Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning.

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. Upon infection, this virus gives rise to two species of unintegrated linear viral DNA, which are 8.3 and 5.5 kilobase pairs long and represent the helper virus (REV-A) and the oncogenic component (REV-T), respectively. Restriction endonuclease cleavage maps of these two DNA components indicate that REV-T DNA has a large portion of the genome deleted with respect to REV-A DNA and a substitution about 0.8 to 1.5 kilobase pairs long that is unrelated to REV-A DNA. These additional sequences comprise the putative transforming region of REV-T (rel). A chicken spleen cell line transformed by REV-T produced virus which upon infection gives rise to three species of unintegrated linear viral DNA (8.3, 5.5, and 3,3 kilobase pairs). We isolated the proviruses of the 8.3- and 3.3-kilobase pair species from this cell line by cloning in the phage vector Charon 4A. Restriction enzyme mapping showed that the two proviral clones are proviruses of REV-A and a variant of REV-T, respectively. A subclone of the variant REV-T provirus specific for the rel sequences of REV-T was used as a hybridization probe to demonstrate that the rel sequences are different from the putative transforming sequences of Schmidt-Ruppin Rous sarcoma virus strain A, avain myelocytomatosis virus, avian myeloblastosis virus, avian erythroblastosis virus, Abelson murine leukemia virus, and Friend erythroleukemia virus. In addition, the rel-specific hybridization probe was used to identify a specific set of sequences which are present in uninfected avian DNAs digested with several restriction enzymes. The corresponding cell sequences are not arranged like rel in REV-T.     

Ground squirrel hepatitis virus DNA: molecular cloning and comparison with hepatitis B virus DNA

Ground squirrel hepatitis virus (GSHV) shares many ultrastructural antigenic, molecular, and biological features with hepatitis B virus (HBV) of humans, indicating that they are members of the same virus group. Both viruses contain small circular DNA molecules which are partially single stranded. Here, we ligated an endonuclease EcoRI digest of GSHV DNA with EcoRI-cleaved plasmid vector pBR322 and cloned recombinant plasmids in Escherichia coli C600. Two cloned recombinants were characterized. One (pGS2) was found to contain only part of the GSHV genome, and the other (pGS11) was found to contain the entire viral DNA. A restriction endonuclease cleavage map of the GSHV insert in pGS11 and the locations of certain physical features of the virion DNA were determined. The relative positions of the single-stranded region, the unique 5' end of the short DNA strand, and the unique nick in the long DNA strand in GSHV DNA were found to be the same as those previously described for HBV DNA. Hybridization with an HBV [32P]DNA probe containing the apparent coding sequence for the major polypeptide of HBV surface antigen and a probe containing the putative coding sequence for the major polypeptide of the HBV core revealed specific homology with different restriction fragments of GSHV DNA. The two homologous regions had approximately the same locations relative to the single-stranded region, the 5' end of the short strand, and the nick in the long strand in the two viral DNAs. These results suggest that in both viruses the genes for the major HBV surface antigen and core polypeptides have the same locations relative to unique physical features of the viral DNAs.     

Mobilization of a Sym plasmid from a fast-growing cowpea Rhizobium strain.

A large Sym plasmid from a fast-growing cowpea Rhizobium species was made mobilizable by cointegration with plasmid pSUP1011, which carries the oriT region of RP4. This mobilizable Sym plasmid was transferred to a number of Rhizobium strains, in which nodulation and nitrogen fixation functions for symbiosis with plants of the cowpea group were expressed.     

Virion DNA of ground squirrel hepatitis virus: structural analysis and molecular cloning.

The structure of the encapsidated DNA genome of ground squirrel hepatitis virus (GSHV) has been examined by restriction endonuclease cleavage, nucleic acid hybridization, and molecular cloning. GSHV virion DNA is a relaxed circular molecule of approximately 3,200 bases in length; most molecules harbor an extensive single-stranded region which is largely confined to one-half of the genome. The full-length viral DNA strand is covalently bound to protein. The single-stranded region can be repaired in vitro by the action of the endogenous virion polymerase, exogenously added DNA polymerase from avian myeloblastosis virus, or both. Restriction enzyme cleavage of viral DNA from different isolates demonstrated that multiple variants of GSHV exist in nature. The genomes of two such strains have been cloned in Escherichia coli, and their physical maps have been determined. Nucleic acid hybridization studies revealed that the strains share sequence homology with the DNA of human hepatitis B virus. Regions homologous to the coding regions for the surface and core antigens of human hepatitis B virus have been localized on the GSHV chromosome. Molecular cloning experiments have also led to the identification of a region of the viral genome which is altered in a procaryotic host.     

Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma

A novel plasminogen-binding protein has been isolated from human plasma utilizing plasminogen-Sepharose affinity chromatography. This protein copurified with alpha 2 antiplasmin when the plasminogen affinity column was eluted with high concentrations of epsilon-aminocaproic acid (greater than 20 mM). Analysis by sodium dodecyl sulfate suggests this protein has an apparent Mr of 60,000. The amino-terminal amino acid sequence showed no similarity to other protein sequences. Based on the amino-terminal amino acid sequence, oligonucleotide probes were designed for polymerase chain reaction primers, and an approximately 1,800 base pair cDNA was isolated that encodes this Mr 60,000 protein. The deduced amino acid sequence reveals a primary translation product of 423 amino acids that is very similar to carboxypeptidase A and B and consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide, and a 309-amino acid catalytic domain. This protein shows 44 and 40% similarity to rat procarboxypeptidase B and human mast cell procarboxypeptidase A, respectively. The residues critical for catalysis and zinc and substrate binding of carboxypeptidase A and B are conserved in the Mr 60,000 plasminogen-binding protein. The presence of aspartic acid at position 257 of the catalytic domain suggests that this protein is a basic carboxypeptidase. When activated by trypsin, it hydrolyzes carboxypeptidase B substrates, hippuryl-Arg and hippuryl-Lys, but not carboxypeptidase A substrates, and it is inhibited by the specific carboxypeptidase B inhibitor (DL-5-guanidinoethyl)mercaptosuccinic acid. We propose that the Mr 60,000 plasminogen-binding protein isolated here is a novel human plasma carboxypeptidase B and that it be designated pCPB.     

Replication of mouse hepatitis virus: negative-stranded RNA and replicative form RNA are of genome length

There are seven virus-specific mRNA species in mouse hepatitis virus-infected cells (Lai et al., J. Virol. 39:823-834, 1981). In this study, we examined virus-specific negative-stranded RNA to determine whether there are corresponding multiple negative-stranded RNAs. Intracellular RNA from mouse hepatitis virus-infected cells was separated by agarose gel electrophoresis, transferred to nitrocellulose membranes, and hybridized to positive-stranded genomic 60S [32P]RNA. Only a single RNA species of genomic size was detected under these conditions. This RNA was negative stranded. No negative-stranded subgenomic RNA was detected. We also studied double-stranded replicative-form RNA in the infected cells. Only one replicative-form of genomic size was detected. When the double-stranded RNA isolated without RNase treatment was analyzed, again only one RNA species of genomic size was detectable. Furthermore, most of the virus-specific mRNAs could be released from this RNA species upon heating. These results suggest that all of the mouse hepatitis virus-specific RNAs are transcribed from a single species of negative-stranded RNA template of genomic size.     

The influenza virus haemagglutinin gene: cloning and characterisation of a double-stranded DNA copy.

A protocol has been developed for the synthesis of a double-stranded DNA (dsDNA) copy of the influenza virus RNA genome segment which codes for the major surface antigen, haemagglutinin (HA). This dsDNA copy was inserted, after digestion with S1 nuclease and poly (dC) tailing with terminal transferase, into poly(dG)-tailed, PstI-cut, pBR322 DNA, and used to transform E. coli RR1. Tetracycline-resistant bacterial colonies were screened for the presence of plasmid containing the copied HA gene by testing their ability to hybridise to a specific, 32P-labelled, single-stranded DNA probe. Four cloned hybrid plasmids, containing DNA complementary to the HA gene of the influenza strain 29C (a laboratory derivative of influenza A/NT/60/68 (1)) were analysed by restriction enzyme mapping. Each contained a dsDNA insert equivalent to a full length copy of the HA gene. The nucleotide sequence of a selected restriction fragment from the DNA inserted in one of these cloned plasmids (C89) was determined. The amino acid sequence deduced from these data agreed with the amino acid sequence determined for the corresponding region of HA from the influenza strain A/Mem/102/72, another member of the Hong Kong subtype, identifying the inserted dsDNA of C89 as an authentic copy of the influenza HA gene.     

Complete coding sequence and molecular analysis of hepatitis A virus from a chimpanzee with fulminant hepatitis

Background Hepatitis A virus (HAV) infects both humans and non‐human primates, in experimentally infected chimpanzees is typically milder than in humans. In 1982, Abe and Shikata reported a first case of a chimpanzee with fulminant hepatitis caused by spontaneous HAV infection, and the underlying mechanisms of the disease remain unknown. Methods To characterize denoted CFH‐HAV, we conducted cloning and near full‐length sequence analysis. Results Phylogenetic analyses of VP1‐2A and complete sequence comparison between various genotypes and the sample sequence showed clustering in genotype IB. Based on BLAST analysis, the sequence was most closely related to the wild‐type (HM175/WT) isolate. Amino acid and nucleic acid similarities were 99.8% and 94.41%, respectively. Conclusions The chimpanzee may have been infected with human HAV genotype IB. The substitutions in VP2, VP4, 2B, 2C, and 3D, which may enhance virus proliferation, contributed to disease severity culminating in fulminant hepatic failure.     

赭曲霉毒素A降解菌株的筛选及脱毒酰胺水解酶的基因克隆和表达

【背景】赭曲霉毒素A (ochratoxin A, OTA)是一种可以致癌的真菌毒素,其污染严重影响食品安全,危害人类健康。生物降解法去除OTA污染是近些年的研究热点,发掘高效的OTA降解脱毒酶资源具有重要的意义。【目的】筛选高效的OTA降解菌株并从中克隆降解基因,为生物脱毒方法的开发提供基因和酶资源。【方法】利用OTA为唯一碳源的筛选培养基从土壤中筛选纯化OTA降解菌株,通过16S rRNA基因序列分析确定其分类地位,利用高效液相色谱(high performance liquid chromatography, HPLC)分析其降解产物。通过同源序列比对的方法克隆降解基因并与载体pET-29a(+)相连,然后在大肠杆菌BL21(DE3)中表达。利用Ni²⁺亲和层析对表达产物进行纯化,研究其对OTA的降解活性和酶学特征。【结果】筛选到一株高效的OTA降解菌株,在12 h内能够完全降解1 μg/mL的OTA;初步鉴定该菌株属于Niastella,编号为JX-6;菌株JX-6通过酰胺键断裂途径降解OTA生成无毒的OTα;从菌株JX-6中鉴定了一个OTA酰胺水解酶,命名为NcOTase;NcOTase与已报道的OTA酰胺水解酶序列相似性较低,仅为31%–53%;纯化的NcOTase具有OTA水解活性,比酶活为60.3 U/mg,活性显著高于大部分已报道的OTA降解酶。【结论】NcOTase是一个高效的OTA降解脱毒酶,在去除食品和饲料中OTA污染方面具有很好的应用前景。     

A cytosolic form of aminopeptidase P from Drosophila melanogaster: molecular cloning and characterization

Using a functional genomic approach, we have identified and characterized a cytosolic form of aminopeptidase P from Drosophila melanogaster. This study represents the first characterization of an insect aminopeptidase P. The complete sequence of a 12.5 kbp genomic clone from D. melanogaster showed the presence of a 1,839 bp ORF, encoding a protein of 613 amino acids with a calculated molecular mass of 68.5 kDa. The deduced amino acid sequence was 48% identical and 66% similar to rat and human cytosolic aminopeptidase P. Amino acids important for catalytic activity and the metal binding ligands were found to be conserved between Drosophila AP-P and its mammalian homologues. The recombinant enzyme expressed in Escherichia coli hydrolyzed the amino terminal Xaa-Pro bond of substance P and bradykinin, revealing its functional identity. Further enzyme characterization showed the enzyme to be a manganese-dependent metallopeptidase. Immunoblot analysis showed that DAP-P is located exclusively in the cytosol and is temporally regulated during Drosophila development. In the adult fly, the protein could be detected in gut, testis and ovary, with a high level of expression in brain.     

Isolation and characterization of microplasminogen. A low molecular weight form of plasminogen

A functionally active human microplasminogen without kringle structures was produced by incubation of plasminogen with urokinase-free plasmin at an alkaline pH. The microplasminogen was purified by affinity chromatography on lysine- and soybean trypsin inhibitor-Sepharose and by chromofocusing. Human plasminogen is specifically cleaved at Arg529-Lys530 by plasmin to form microplasminogen, which consists of a single polypeptide of 261 residues from the COOH-terminal portion of native plasminogen. It has an Mr of 28,617, calculated from the sequence, which is consistent with the molecular weight determined by sodium dodecyl sulfate gel electrophoresis. Microplasminogen is a slightly basic protein and is eluted from a chromofocusing column at pH 8.3. It can be activated by urokinase and streptokinase to a catalytically active microplasmin. The specific amidolytic activity of microplasmin is about three times higher than Lys77-plasmin on a weight basis and is about the same on a molar basis. The activation of microplasminogen by streptokinase is slower than that of either Glu-plasminogen or Lys77-plasminogen. On the other hand, the activation of microplasminogen by urokinase is faster than that of either of the latter. The Arg560-Val561 bond is cleaved during activation of both microplasminogen and native plasminogen.