A Random DNA Sequencing,Computer-Based Approach for the Generation of a Gene Map of Molluscum Contagiosum Virus

The genome of Molluscum contagiosum virus (MCV) has a high G+C content, which largely differs from those of vaccinia virus (VAC) and other characterized poxviruses. This has precluded the use of DNA hybridization for the identification of MCV genes and the further establishment of the virus genetic map. To circumvent this problem, we have partially sequenced clones containing virus restriction endonuclease fragments, which were derived by either single or double-digestion of genomic DNA from the subtype I of MCV. The DNA sequences were translated and used to search protein data bases. This analysis resulted in the finding of high-scoring matches to data base entries, including forty-five VAC genes. In addition, MCV-specific sequences that encoded protein domains of known function (i.e. DNA J domain) were found. The locations of MCV clones were inferred from the presumed colinearity of both MCV and VAC genomes, and further confirmed by PCR technology. The data presented here led to the construction of a partial genetic map of MCVI, which revealed that the order and orientation of a large number of MCV genes were equivalent to those of their VAC homologues. The conserved gene arrangement was apparently disrupted in the terminal regions, where MCV sequences showing homologies with the VAC counterparts were not found.     

Identification and typing of molluscum contagiosum virus in clinical specimens by polymerase chain reaction

A polymerase chain reaction (PCR) which enables the detection of molluscum contagiosum virus (MCV) genomes in either fresh or formalin-fixed clinical specimens is described. The primers used were designed to amplify a 167 bp region of the 3.8kbp HindIII fragment K of the MCV 1 genome. The ability of this PCR to detect three common MCV types (1, 1v and 2) in clini-cal specimens was confirmed using frozen extracts from 75 molluscum lesions, and digests of single sections of 11 formalin-fixed, paraf-fin-embedded lesions; all of which had been previously typed by Southern hybridisation. In addition, 2 specimens previously negative by hybridisation were shown to be positive for MCV DNA by PCR. Confirmation of the identity of the PCR products and distinction between the two major MCV types (MCV 1/1v versus MCV 2) was achieved by comparison of the results of cleavage with the restriction endonucleases HhaI and SacI. Sequencing of the PCR products revealed complete homology between MCV 1 and 1v, but minor nucleotide variations between MCV 1/1v and MCV 2 were identified. As well as providing a highly sensitive means of diagnosis, the technique may also prove useful for investigations into the pathogenesis, epidemiology and natural history of molluscum contagiosum infection. J. Med. Virol. 53:205–211, 1997. © 1997 Wiley-Liss, Inc.     

Analysis of the genome of molluscum contagiosum virus by restriction endonuclease analysis and molecular cloning

Virions of molluscum contagiosum virus (MCV), a member of the poxviridae, were isolated directly from lesions of individual patients and characterized by restriction enzyme analysis. The comparative analysis of the cleavage patterns and Southern blot hybridization of 14 independently isolated virus samples revealed that MCV isolates can be classified into two different types. The majority of MCV isolated from clinically typical skin lesions (13 of 14) showed similar DNA cleavage patterns and were termed MCV type 1, whereas one isolate derived from a vaginal lesion showed a completely different DNA cleavage pattern and therefore was termed MCV type 2. For detailed investigation of the viral genome, a defined gene library of MCV DNA sequences was established. The Bam HI DNA fragments of the viral genome of MCV type 1 prototype isolate 1/80 was inserted into the bacterial plasmid vector pAT153. With the exception of terminal fragments (fragments A and B) of the viral genome, all other DNA fragments were cloned. All cloned Bam HI DNA fragments were individually identified by digestion of the recombinant plasmid DNA with different restriction enzymes and screened by hybridization of plasmid DNA to viral DNA.     

Strategy for identifying the gene encoding the DNA polymerase of molluscum contagiosum virus type 1

Molluscum contagiosum virus (MCV) is a member of the family Poxviridae and pathogenic to humans. MCV causes benign epidermal tumors mainly in children and young adults and is a common pathogen in immunecompromised individuals. The viral DNA polymerase is the essential enzyme involved in the replication of the genome of DNA viruses. The identification and characterization of the gene encoding the DNA polymerase of molluscum contagiosum virus type 1 (MCV-1) was carried out by PCR technology and nucleotide sequence analysis. Computer-aided analysis of known amino acid sequences of DNA polymerases from two members of the poxvirus family revealed a high amino acid sequence homology of about 49.7% as detected between the DNA polymerases of vaccinia virus (genus Orthopoxvirus) and fowlpoxvirus (genus Avipoxvirus). Specific oligonucleotide primers were designed and synthesized according to the distinct conserved regions of amino acid sequences of the DNA polymerases in which the codon usage of the MCV-1 genome was considered. Using this technology a 228 bp DNA fragment was amplified and used as hybridization probe for identifying the corresponding gene of the MCV-1 genome. It was found that the PCR product was able to hybridize to theBamHI MCV-1 DNA fragment G (9.2 kbp, 0.284 to 0.332 map units). The nucleotide sequence of this particular region of the MCV-1 genome (7267 bp) between map coordinates 0.284 and 0.315 was determined. The analysis of the DNA sequences revealed the presence of 22 open reading frames (ORFs-1 to-22). ORF-13 (3012 bp; nucleotide positions 6624 to 3612) codes for a putative protein of a predicted size of 115 kDa (1004 aa) which shows 40.1% identity and 35% similarity to the amino acid sequences of the DNA polymerases of vaccinia, variola, and fowlpoxvirus. In addition significant homologies (30% to 55%) were found between the amino acid sequences of the ORFs 3,-5,-9, and-14 and the amino acid sequences of the E6R, E8R, E10R, and a 7.3 kDa protein of vaccinia and variola virus, respectively. Comparative analysis of the genomic positions of the loci of the detected viral genes including the DNA polymerases of MCV-1, vaccinia, and variola virus revealed a similar gene organization and arrangement.     

Genomic sequences homologous to the protein kinase region of the bifunctional herpes simplex virus type 2 protein ICP10

The large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) consists of two functional domains. The amino (N)-terminal domain at residues 1–411 has serine/threonine-specific kinase activity (PK domain) and is encoded by a DNA fragment with transforming potential ( 15,17). The remaining region is required for ribonucleotide reductase activity (RR domain) (14,15). Computer-assisted comparison of the ICP10 sequence to the EMBL database 21 has revealed sequences within the RR domain that are common to all RR1 proteins. Motifs homologous to the catalytic domains of all PKs were identified in the PK region (15). However, based on this database all other sequences were unique. Secondary structure analysis of the PK and RR junction region of ICP10 identified twist angle variations with helical periodicity characteristic of enhancer elements. Sequences homologous to a segment of the PK domain were amplified and cloned from human DNA using the polymerase chain reaction (PCR), suggesting that the PK domain may have originated from a cellular gene.     

丙型肝炎病毒核心蛋白DNA免疫初步研究

目的　观察HCV核心蛋白基因的DNA免疫效果。方法　将HCV核心蛋白基因插入真核表达载体pcDNA3.1( ) ,构建重组质粒pcDNA3.1 c。在证明该重组质粒可在哺乳动物COS7细胞中表达的基础上 ,用重组质粒 10 0 μg免疫小鼠 ,同时设立空白质粒组和PBS组两组对照 ,初次免疫后 4周、8周各进行一次加强免疫。小鼠体液免疫反应和T淋巴细胞增殖检测分别采用间接免疫荧光法和MTT法。结果　pcDNA3.1 c可在COS7细胞内表达HCV核心抗原 ,接种于Balb c小鼠能有效诱导体液和细胞免疫应答。结论　重组质粒pcDNA3.1 c对于丙型肝炎防治具有潜在价值     

小鼠蛋白激酶CK2β亚基的克隆、表达及活性测定

通过反转录PCR从NIH 3T3小鼠成纤维细胞中获得小鼠蛋白激酶CK2β亚基编码区cDNA；构建其表达质粒并经测序证明其编码小鼠蛋白激酶CK2β亚基，但与两种已报道的小鼠CK2β亚基cDNA编码区序列分别存在一个碱基差异，与大鼠、猪、兔和人CK2β亚基cDNA的编码区相应碱基序列则一致。将其在表达菌BL21(DE3)中诱导表达，出现一26kDa分子量蛋白过度表达，表达蛋白占菌体总蛋白的31．7％，但大多数以不溶形式存在。Western blot鉴定表明：过度表达产物能与抗人CK2β亚基抗体发生特异性免疫反应。当CK2α和β亚基以1：1摩尔比混合时，构成性的CK2全酶显示出最大活性，直接表明CK2β亚基对CK2α有激活作用。这些结果有力地证明了克隆表达的重组蛋白是小鼠蛋白激酶CK2β亚基。     

The mitochondrial DNA of the yeast Hansenula petersonii: genome organization and mosaic genes

Summary The mitochondrial genomes of yeasts are circular DNA molecules that vary greatly in size in different species. The mitochondrial DNA of the yeast H. petersonii is about 42 kbp in length, about one half the size of the corresponding genome in S. cerevisiae. Sequences homologous to protein-encoding genes from S. cerevisiae have been identified and localized on this genome by hybridization with DNA from petite mutants. The comparison between the mitochondrial genomes of H. petersonii and S. cerevisiae showed differences in the overall genome organization, but both include genes with mosaic organization. In fact, sequences homologous to the first intron of the S. cerevisiae cob short gene are found in (or adjacent to) the cob and cox1 genes present in the genome of H. petersonii. Moreover, an intron homologous to that present in the 21S rRNA gene of S. cerevisiae seems to have been conserved in the large ribosomal RNA gene of H. petersonii, in a similar position.     

Nucleotide sequence of the gene encoding the matrix protein of mumps virus (Miyahara strain)

The nucleotide sequence of the gene encoding the matrix (M) protein of mumps virus (MuV), Miyahara strain, has been determined from several overlapping cDNA clones. The M protein mRNA is 1248 nucleotides in length, exclusive of the poly(A) tail, and codes for a protein of 375 amino acids (Mr41,556). Comparison of the deduced amino acid sequence of the M protein of the Miyahara strain with that of the SBL-1 strain revealed that the M proteins of both strains are highly conserved. A significantly lower rate of nucleotide differences conducive to amino acid differences in the M gene compared with other genes appeared to indicate the importance of the conserved primary structure of the M protein for its function.Requests for reprints should be addressed to Kiyoshi Tanabayashi, Department of Measles Virus, National Institute of Health, 4-7-1 Gakuen, Musashimurayama, Tokyo 190-12, Japan.     

DNA vaccination using coexpression of cytokine genes with a bacterial gene encoding a 60-kDa heat shock protein

Coexpression of cytokine genes together with antigen-encoding genes in DNA vaccination vectors can increase humoral and cellular immune responses and may steer them in a Th1 or Th2 direction. In this study, the modulatory effect of interleukin (IL)-2, IL-4, and interferon (IFN)-γ coexpressed with the 60-kDa heat shock protein (Hsp60) of Yersinia enterocolitica O:8 (Y-Hsp60) was studied. DNA vaccination with y-hsp60 evoked specific humoral and cellular immune responses as well as reduction of the splenic bacterial load upon challenge with Y. enterocolitica in a mouse infection model. Coexpression of IL-2 or IFN-γ enhanced Y. enterocolitica-specific total IgG (P < 0.05) and IgG2a antibody responses. Coexpression of IFN-γ also improved the proliferative T cell responses upon stimulation with Y-Hsp60. A reduction of the splenic bacterial load as compared with the plasmid encoding Y-Hsp60 only was found for the IFN-γ coexpressing vector. Thus, coexpression of cytokine genes such as IFN-γ in DNA vaccination vectors might improve immunity and help to overcome the side effects of standard adjuvants. Received: 15 May 2000     

Construction of human cell lines which contain and express the adenovirus DNA binding protein gene by cotransformation with the HSV-1 tk gene

We have introduced the DNA binding protein (DBP) gene of human adenovirus type 5 (Ad5) into high molecular weight DNA of permissive human cells by cotransformation of tk- cells with the cloned DBP and HSV-1 thymidine kinase genes. 110 tk+ cell lines were isolated after selection in HAT medium. The amount and arrangement of adenovirus sequences in the tk+ cell lines were analyzed by restriction endonuclease digestion and filter hybridization. Twelve of the 110 lines carry at least a segment of the DBP gene while only three of these contain the entire DBP gene at approximately one copy per cell. Cytoplasmic, polyadenylated DBP mRNA is made in all three cell lines though the amount is very low compared to that present in infected HeLa cells. The cell line U13-2 which contains approximately 1/30 the steady-state level of DBP mRNA found in infected HeLa cells produces a few percent of the amount of DBP made during the peak period of DBP synthesis in infected cells. The other two lines contain lower levels of DBP mRNA and do not synthesize detectable levels of the protein. When these DBP-tk+ cell lines are infected with adenovirus mutants containing temperature-sensitive (ts) mutations in the DBP gene, only U13-2 permits some viral DNA replication (and hence late gene expression) at the nonpermissive temperature, indicating that sufficient quantities of DBP from the integrated gene are produced to allow complementation of the ts mutation in this cell line. However, growth of these ts mutants (as measured by virus production) is only partially complemented in U13-2 at the nonpermissive temperature.     

DNA mediated immunization with encoding the nucleoprotein gene of porcine transmissible gastroenteritis virus

The immune response to a naked plasmid DNA encoding the nucleoprotein (N protein) of porcine transmissible gastroenteritis virus (TGEV) was investigated in this study. A complementary DNA of the entire N gene was amplified by RT-PCR, and inserted into a mammalian expression vector (pcDNA3.1) to construct a recombinant plasmid (pcDNA/N). To evaluate the immunogenicity of the construct, BALB/c mice were intramuscularly immunized with different doses (50, 100 and 200 μg/mouse) of pcDNA/N twice at a 5-week interval. An optimal antibody response was achieved with 100 μg of pcDNA/N. The response lasted at least 11 weeks after primary immunization. By western blotting analysis, the antibodies specifically recognized a 47 kDa protein corresponding to the viral N protein, but they did not reveal neutralizing activity against infectious TGEV in vitro. Immunoglobulin G2a was predominant among these antibodies, which was indicative of Th1 type cell activation in pcDNA/N immunized mice. Moreover, spleen cells from these mice showed stronger immune responses than those from live vaccine or parental vector immunized mice. These results suggest that the construct can elicit both humoral and cell-mediated immune (CMI) responses against TGEV N protein in mice.     

Complete sequence of human cardiac α-myosin heavy chain gene and amino acid comparison to other myosins based on structural and functional differences

We have obtained the 5820 nucleotide sequence encoding all 1939 amino acids of the human cardiac α-myosin heavy chain (α-MHC

1 The sequence has been deposited in the DDBJ base. (Accession no. D00943)

), as established by dideoxy sequencing of cloned cDNA, genomic DNA and polymerase chain reaction (PCR) amplification products. This sequence represents overlapping fragments of the entire coding sequence. Amino acid sequence comparison of the human cardiac α-MHC with the published human cardiac β-MHC have demonstrated that there are, at least, 7 isoform-specific divergent regions, including functionally important binding protein-related sites such as ATP, actin and myosin light chain. It has been reported that in the rat, there are 8 isoform-specific divergent regions. The 7th divergent area (residue area 1633-1657, which is thought to mediate thick filament formation) in the light meromyosin region in the rat is not apparent in the human. The amino acid compositions of cardiac α- and β-MHCs in the human and the rat, and human embryonic skeletal muscle and chicken gizzard smooth muscles were compared. Amino acid sequences in cardiac α- and β-MHCs in the human and the rat are well conserved. In the head portion, the amino acid composition divergence of human cardiac α-MHC is ranked between rat cardiac α-MHC and human cardiac β- or rat cardiac β-MHC; human skeletal muscle MHC is the most divergent of the myosin isoform examined. These data predict that human cardiac α-MHC may have undergone evolutionary changes toward obtaining the biochemical and physiological properties of cardiac β-MHC.     

Sequence analysis of the cystic fibrosis gene in patients with disseminated bronchiectatic lung disease

Summary The diagnosis of classical cystic fibrosis (CF) is easily made by clinical assessment alone, but may be missed or delayed in cases with an atypical clinical course. In a recent major study the age at diagnosis varied between 2 months and 47 years. For diagnostic purposes we have investigated the cystic fibrosis transmembrane regulator (CFTR) gene in 10 adult patients (age 18 to 45 years) with chronic obstructive pulmonary disease since childhood or adolescence and bronchiectases disseminated through both lungs. Only one subject (a 29-year-old male) had exocrine pancreatic insufficiency (PI); all others were pancreatic-sufficient (PS). The first nucleotide (ATP)-binding fold of the CFTR was analyzed by direct sequencing of polymerase chain reaction (PCR)-amplified genomic DNA in these cases. Two patients with different phenotypes (one PI, one PS) were found to be homozygous for the common F508 mutation of the CFTR gene, which proved the diagnosis of cystic fibrosis in their cases and allowed genetic counselling. The PS patient had normal sweat tests and had not previously been recognized as having CF. Four other patients were heterozygous for F508, with no other mutation in exons 10 or 11 of the gene, and four patients had normal sequences of these exons. Because only about 70% of all CF chromosomes carry F508, the unexpectedly high frequency (4/8=50%) of heterozygosity for F508 among the non-F508/F508 patients with bronchiectases suggests that some of these might also have unrecognized CF with rare genotypes and mutations in any of the 22 exons not sequenced. About 90 different mutations have already been detected in coding regions of the CFTR gene and a very broad and so far not fully recognized spectrum of clinical phenotypes may be caused by mutations of this gene. Screening for the most frequent CFTR gene mutations, which is practicable using recent technology, may provide significant new diagnostic information in patients with CF-like pulmonary phenotypes, especially if they have normal or borderline sweat tests and no pancreatic insufficiency.Abbreviations PCR polymerase chain reaction - CF cystic fibrosis - CFTR cystic fibrosis transmembrane conductance regulator - COPD chronic obstructive pulmonary disease - PI pancreatic insufficiency - PS pancreatic sufficiency     

Identification of the gene encoding the DNA (cytosine-5) methyltransferase of lymphocystis disease virus

The gene encoding the DNA (cytosine-5) methyltransferase (m5C-MTase) of lymphocystis disease virus (flounder isolate, LCDV-1) has been identified by polymerase chain reaction (PCR) using oligonucleotide primers synthesized corresponding to different regions of the m5C-MTase gene of frog virus 3 (FV3). A DNA fragment of 487 bp was amplified using oligonucleotide primers L3 and R4 which correspond to the nucleotide positions 87 to 109 and 530 to 550 of the m5C-MTase gene of FV3, respectively. The DNA nucleotide sequence of the PCR product was determined by direct cycle sequencing. The alignment of the deduced amino acid sequence derived from the PCR product and the m5C-MTase protein of FV3 revealed a homology of 55.4% identity and 29.1% similarity. The amino acid sequence which was found to be significantly homologous to the amino acid sequence deduced from the nucleotide sequence of the PCR product was located at the amino acid position 37 to 175 of the m5C-MTase of FV3 indicating the specificity of the amplified PCR product. The DNA nucleotide sequence of the LCDV-1 genome corresponding to the 5 and 3 termini of the m5C-MTase gene was determined by primer walking. The locus of the m5C-MTase gene of LCDV-1 was identified within the EcoRI DNA fragment G of LCDV-1 (7.9 kbp; 0.947 to 0.034 map units). The m5C-MTase gene of LCDV-1 comprises 684 nucleotides coding for a putative protein of 228 amino acid residues. A high degree of amino acid sequence homology (53.3% identity and 25.8% similarity) was detected between the m5C-MTases of LCDV-1 and FV3.