A genomic map of infectious laryngotracheitis virus and the sequence and organization of genes present in the unique short and flanking regions

We present a genomic map of infectious laryngotracheitis virus (ILT) and an 18,912 bp sequence containing the entire unique short region and a portion of the flanking short repeats. In determining the genomic map, an 856 bp region repeated as many as 13 times was identified within the short repeats. The unique short sequence contains nine potential open reading frames (ORFs). Six of these ORFs show homology to other known herpesvirus unique short genes. Using the herpes simplex virus nomenclature, these genes are the US2, protein kinase, and glycoproteins G, D, I, and E (ORF 1, 2, 4, 6, 7, and 8, respectively). Interestingly, an open reading frame with homology to HSV-1 UL47 (ORF 3) is found in the unique short. One very large open reading frame (ORF 5) is present and contains a threonine-rich, degenerate repeat sequence. This gene appears to be unique to ILT among sequenced herpesviruses. Two ORFs were identified within the short repeat (SR) region. SRORF 1 is homologous to a gene (SORF3) found in the unique short region in both MDV and HVT, and appears to be specific to avian herpesviruses. SRORF 2 has homology to HSV US10.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data-base and have been assigned the accession number U28832.     

尿道致病性大肠杆菌新基因R049特征的研究

目的 明确从国内分离的尿道致病性大肠杆菌(UPEC) 132中发现的新基因R049的特征及其表达蛋白在菌体内的定位。方法 提取UPEC132染色体DNA,鸟枪法构建文库,高通量焦磷酸法测序,Newbler软件拼接,进行生物信息学分析,确定R049相关片段的特征。提取UPEC132的内膜和外膜蛋白,与其全菌裂解物一起进行SDS-PAGE,用R049重组蛋白抗血清进行Western blot,确定R049表达蛋白的菌体内位置。结果 成功构建UPEC 132染色体文库,获取R049-contig169022 bp,与UPEC536染色体第233074至451502位碱基序列同源性较高,其中含有R049基因的20773 bp片段替代相当于UPEC536染色体上PAIⅢ536的位置,G+C含量为46.97％,两端具有正向重复序列,并与插入元件整合酶基因和thrW tRNA基因相邻,包含25个ORF,命名为R049 -GI。R049基因位于R049-GI的第13个ORF。SDS-PAGE和Western blot分析显示R049基因表达相对分子质量为47.0× 103蛋白是UPEC132的外膜蛋白。结论 R049基因编码细菌的外膜蛋白,是UPEC132通过基因水平转移获得的染色体基因组岛的组成部分。     

cDNA cloning of a human homologue of the Caenorhabditis elegans cell fate-determining gene mab-21: expression, chromosomal localization and analysis of a highly polymorphic (CAG)n trinucleotide repeat

The two most consistent features of the diseases caused by trinucleotide repeat expansion-neuropsychiatric symptoms and the phenomenon of genetic anticipation-may be present in forms of dementia, hereditary ataxia, Parkinsonism, bipolar affective disorder, schizophrenia and autism. To identify candidate genes for these disorders, we have screened human brain cDNA libraries for the presence of gene fragments containing polymorphic trinucleotide repeats. Here we report the cDNA cloning of CAGR1, originally detected in a retinal cDNA library. The 2743 bp cDNA contains a 1077 bp open reading frame encoding 359 amino acids. This amino acid sequence is homologous (56% amino acid identify and 81% amino acid conservation) to the Caenorhabditis elegans cell fate-determining protein mab-21. CAGR1 is expressed in several human tissues, most prominently in the cerebellum, as a message of approximately 3.0 kb. The gene was mapped to 13q13, just telomeric to D13S220. A 5'-untranslated CAG trinucleotide repeat is highly polymorphic, with repeat length ranging from six to 31 triplets and a heterozygosity of 87-88% in 684 chromosomes from several human populations. One allele from an individual with an atypical movement disorder and bipolar affective disorder type II contains 46 triplets, 15 triplets longer than any other allele detected. Though insufficient data are available to link the long repeat to this clinical phenotype, an expansion mutation of the CAGR1 repeat can be considered a candidate for the etiology of disorders with anticipation or developmental abnormalities, and particularly any such disorders linked to chromosome 13.      

Molecular genetic analysis of autosomal dominant cerebellar ataxia with retinal degeneration (ADCA type II) caused by CAG triplet repeat expansion

Autosomal dominant cerebellar ataxia with retinal degeneration (ADCAII) was previously mapped by linkage analysis studies to chromosome 3p12- p21.1 (SCA7). Positional cloning efforts have recently identified a novel gene, SCA7 , containing a translated CAG repeat, expanded in SCA7 patients. We cloned the SCA7 gene from a yeast artificial chromosome (YAC) clone contig spanning the SCA7 candidate region. Using a combination of genomic sequencing and cosmid-based exon trapping, two expressed sequence tags were identified. Sequencing of the corresponding cDNA clones and RT-PCR analysis identified the full- length SCA7 cDNA. Together, our sequence data defined the intron/exon boundaries of the first two coding exons of the SCA7 gene, with the first exon containing the expanded CAG repeat. Further, sequence comparison with the published SCA7 cDNA identified one additional putative exon in the 5'-UTR region of the SCA7 gene. The SCA7 gene was mapped on the YAC contig in the 2.5 cM interval between D3S1600 and D3S1287. In one extended Belgian SCA7 pedigree the expanded alleles ranged from 38 to at least 55 repeats with allele lengths being inversely correlated with onset age of ADCAII symptoms. The SCA7 repeats increased in length in successive generations. Normal alleles had from four to 18 repeats, with 10 repeats being the most common allele.      

Molecular cloning, complete nucleotide sequence, and gene structure of the provirus genome of a retrovirus produced in a human lymphoblastoid cell line

We found and characterized a type D retrovirus produced in a human lymphoblastoid cell line of B-cell lineage. The amino acid sequence of the N-terminal region of the purified major structural protein (PVTRSQGQVSSNTTGRASPHPDTHTIPE) revealed no high homology with any of the known retroviral amino acid sequences. We have cloned cDNA and the proviral genome integrated in the retrovirus-producing cells, and determined the complete nucleotide sequence and gene structures of the genome. The provirus genome is 8785 bp long and has the structure of LTR-gag-prt-pol-eny-LTR. The nucleotide sequences of the long terminal repeat (LTR) region and a part of the pol gene were closely related to the available sequences of squirrel monkey retrovirus (SMRV), and we designated this virus SMRVHLB' abbreviated as SMRV-H. The primer (tRNA(Lys)1,2)-binding sequence of SMRV-H (TGGCGCCCAGGACGTGGGGCTCGA) has a GG insertion, which is different from that of SMRV. The transmembrane protein of the 3' terminal region of env gene contains an amino acid sequence of an immunosuppressive peptide (EVVLQNRRGLDLLTAEQGGICLALQERCCFYANKS), in which R is unique in SMRV-H. The core sequence of the glucocorticoid regulatory element is found upstream of the two 42-bp imperfect repeats in the LTR. Sequences partially homologous to those of the rat IgE-binding protein gene are in gag and pol genes.     

EcoRI Restriction Fragment-Length Polymorphism of the Human Immunoglobulin Variable Lambda 8 (IGLV8) Subgroup Reveals a Gene Family

The human immunoglobulin lambda locus (IGL) maps on chromosome 22q11.1–q11.2 and directs the synthesis of lambda-type Ig light chains. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes and the J-C cluster plus the joining segments and the constant genes. Recently the variable lambda gene clusters were mapped by the contig methodology which located all the known functional v-lambda genes and pseudogenes. The 30 functional v-lambda genes described so far were subgrouped into ten families (VλI to VλX), but RFLP studies have estimated that the germline repertoire contains about 70 genes. Based on sequence comparisons, we defined specific oligonucleotide primers for the unique IGLV8S1 genedescribed. The cloned 244 bp product obtained from genomic DNA with these primers was sequenced and used as probe in Southern hybridization EcoRI RFLP analysis of Brazilian people. We detected the IGLV8S1 gene in a 3.7 kb EcoRI restriction fragment present in all the individuals analyzed, in agreement with the physical map of the IGL locus. Moreover, we detected an 8.0 kb EcoRI monomorphic fragment and a 6.0 kb EcoRI polymorphic fragment. These data suggest that the IGLV8 subgroup is a gene family.     

The major cysteine proteinase (cruzipain) from Trypanosoma cruzi is encoded by multiple polymorphic tandemly organized genes located on different chromosomes

We demonstrate that cruzipain, the major cysteine proteinase of Trypanosoma cruzi epimastigotes, is encoded by a large number of tandemly arranged genes. Restriction enzyme analysis of 20 clones containing complete repeat units of the gene, as well as sequencing of 2 of these clones, and comparison with previously published partial sequences, indicated that the sequence is conserved among the repeat units, although polymorphisms clearly exist. The repeat units contain an intergenic region of 528 bp and coding regions for pre- and pro-enzyme, a central domain and a C-terminal extension. The predicted amino acid sequences of these regions indicated a sequence identity of 30, 60, 70 and 36%, respectively, when the T. cruzi sequence was compared with the sequence of a similar cysteine proteinase from Trypanosoma brucei [14]. Studies by pulsed field gel electrophoresis, complemented with restriction analysis, indicated that the clusters are located on 2 4 different chromosomes in several parasite isolates.     

Genome sequence of <Emphasis Type="Italic">Leucania seperata</Emphasis> nucleopolyhedrovirus

The nucleotide sequence of the Leucania seperata (Ls) Nucleopolyhedrovirus (LsNPV) genome has been determined and analyzed. The circular dsDNA genome contains 168041 bp, making it the largest NPV sequenced to date. The genome has a G + C content of 48.6% and encodes 169 predicted open reading frames (ORFs), one unique repeat region, and eight homologous repeat regions that are divided into two groups. Of the genome, 82.8% encodes predicted ORFs including five dispersal ORFs that have a large overlaps (range in 149 ∼ 390 bp) with their adjacent ORFs, respectively such as expression factor 10, 11, 5, 2 (lef-10, lef-11, lef-5, lef-2), and telokin-like protein-20 (tlp-20); 4.4% is in repeat regions; the remaining 12.8% of the genome comprises nonrepeat intergenic regions. LsNPV encodes homologues of 133 ORFs identified previously in other baculoviruses. Other than 10 ‘baculovirus repeat ORFs’ (bro) and two ‘inhibitor of apoptosis’ (iap) genes, no duplicated ORFs were found. LsNPV lacks a homologue of the ubiquitin gene, which has been found in all fully sequenced baculoviruses. Iap3 and p49, two genes were proven to be inhibitors of apoptosis by experiment, and are found in the LsNPV genome. It is not found in other baculoviruses that two kinds of inhibitors of apoptosis present in a baculovirus genome. The GenBank accession number of the LsNPV genome sequence is AY394490.     

Nucleotide Sequence of a 5892 Base Pairs Fragment of the LsmNPV Genome and Phylogenetic Analysis of LsMNPV

A 5892 bp fragment of Leucania separata multiple nuclear polyhederosis virus (LsMNPV) containing gp37, 39 k genes and another four ORFs was sequenced in this article. According to regulatory elements on 5 un-coding sequences, the ORF4 and ORF1 are probably two novel baculovirus genes, and the ORF4 probably also is a new early-late gene. The possible functions about GP37 and 39 K proteins were explored. The homology of GP37 protein among LsMNPV and 8 other insect virus was compared. The evolutional position of LsMNPV is also estimated based on the homology of gp37 genes. The structure and its homology of several genes in LsMNPV prove that LsMNPV is far related with other baculovirus and has an exceptional genome organization.     

肝癌组织差异表达基因cDNA序列的筛选与鉴定

目的：筛选并鉴定肝癌组织特异表达基因。方法：通过菌落原位杂交技术筛选用抑制消减杂交法构建肝癌与癌旁肝组织差异表达基因消减cDNA文库，用PCR方法进一步筛选出有插入片段的阳性克隆，将阳性克隆进行DNA测序和同源性比较分析，用Northern印迹方法对新的cDNA序列进行初步鉴定。结果：从消减文库中随机挑取的100个白色克隆中筛选出13个阳性克隆，DNA测序获得11个不同的cDNA序列；同源性比较分析表明，6个cDNA片段与在基因高度同源，5个cDNA片段为新的序列。其长度大于300bp的3个新序列，Norther印迹证实它都来源于肝癌组织。结论：用抑制消减杂交方法构建的肝癌差异表达基因消减cDNA文库富含肝癌特异表达基因，经验证的3个新的cDNA序列可能为肝癌特异的基因序列。     

Cloning,molecular characterization,and expression analysis of the unc45 myosin chaperone b(<Emphasis Type="Italic">unc45b</Emphasis>)gene of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis>)

Unc45 myosin chaperone b(unc45b)gene is a molecular chaperone that mediates the folding, assembly and accumulation of thick-filament myosin in the formation of sarcomere, which plays an important role in the development of striated muscle and the stability of sarcomere. In this study, the complete cDNA sequence of unc45b gene of grass carp was obtained by rapid amplification of cDNA ends (RACE), and the characteristics of the unc45b protein predicted from gene sequence was analyzed by bioinformatics methods. The differential expression pattern in tissues was also detected by quantitative real-time PCR. The results showed that the full-length of unc45b gene of grass carp is 3163 bp, which contains a 60 bp 5′UTR, a 298 bp 3′UTR, and a 2865 bp open reading frame (ORF) encoding a 934 amino acid peptide. The deduced unc45b protein exhibits a homology of 92, 86, 86 % with the protein of zebrafish (Danio rerio), channel catfish (Ietalurus punctatus) and tilapia (Oreochromis niloticus) respectively, and the protein contains UCS myosin head binding domain and TPR peptide repeat domain. The protein is a hydrophilic and non-secretory protein with a molecular mass and isoeletronic point of 103,699.8 and 7.39 Da. The structural elements of the protein includes α-helixes and loops, and the unc45b gene highly expresses in skeletal muscle and heart in grass carp. This study laid a foundation for further research in explaining the myofibril accumulation in crisped grass carp.     

Genomic and proteomic characterization of a thermophilic Geobacillus bacteriophage GBSV1

Phages are present wherever life is found, and play roles in many biogeochemical and ecological processes. The thermophilic bacteriophages, however, have not been well studied. In this study, phage GBSV1 was obtained from a thermophilic bacterium Geobacillus sp. 6k51 isolated from a hot spring. GBSV1 contains a double-stranded linear DNA of 34,683 bp, which encodes 54 putative open reading frames (ORFs). Thirty three of these 54 ORFs exhibit sequence similarities to genes from 7 species of Geobacillus or Bacillus bacteria, as well as of bacteriophages infecting these bacteria. Twenty-two ORFs have been functionally annotated based on both their sequence similarities to known genes and predicted Pfam protein domains. Five structural proteins of the purified GBSV1 virion have been identified by proteomic analyses. Surprisingly, 7 of the GBSV1 ORFs share sequence similarities with genes from bacteria relevant to human diseases. This is the first report that genes of human disease-inducing bacteria are found in a thermophilic phage. It is suggested that thermophilic phages may be the potential evolutionary link between thermophiles and human pathogens. The characterization of GBSV1 may possibly lead to new insights into virus–host interactions and to a better understanding of gene transfers and evolution of life on earth in general.     

Sequence and organization of the Mamestra configurata nucleopolyhedrovirus genome

The nucleotide sequence of the genome of the nucleopolyhedrovirus (NPV) from Mamestra configurata (MacoNPV, isolate 90/2), a group II NPV, was determined and analyzed. The MacoNPV DNA genome consists of 155,060 bp and has an overall G+C content of 41.7%. Computer-assisted analysis predicted 169 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. BLAST searches and comparisons with completely sequenced baculoviruses indicated that there were 66 ORFs conserved among the nine baculoviruses compared and an additional 17 ORFs were conserved among the NPVs. The gene content and gene arrangement in MacoNPV were most similar to those of SeMNPV, including two putative odv-e66 and p26 gene homologues. However, in contrast to SeMNPV, 8 ORFs with homology to baculovirus repeat ORFs (bro) and single copies of enhancin and conotoxin-like protein ORFs were found in MacoNPV. The MacoNPV genome contained four homologous regions, each with 10 to 17 repeated sequences. Each repeat was 60 to 86 nucleotides in length and contained an approximately 43-bp-long imperfect palindrome. There were 13 ORFs unique to MacoNPV, ranging from a small ORF of 196 bp to larger ORFs of up to 1047 bp, and many of these contained typical early and late baculovirus consensus promoters.     

Nucleotide sequence of the left-terminus of infectious laryngotracheitis virus (Gallid herpesvirus 1) SA-2 strain

Summary.  The nucleotide sequence of 10.6 kilobase pairs (kbp) at the left-terminus of infectious laryngotracheitis virus (ILTV) SA-2 vaccine strain was determined. Several features were elucidated, including, 102 base pair (bp) inverted repeats separated by 750 bp of unique sequence which contains an NF-1 binding site indicating that the terminal may be a site for an origin of replication. Other direct repeats were also found in this region. To the right of the inverted repeat region, a 2130 bp region was found to contain small open reading frames (ORFs) of less than 100 aa. Another potential ORF was found to the right of the region containing the small ORFs which consisted of two 184 bp direct repeats inserted into the reading frame, which would truncate the putative product. Only one copy of this repeat was found in the corresponding homologue of the wild type strain SA-0. Six other ORFs were found, which shared little or no identity to homologues of other alphaherpesviruses, suggesting that these putative genes are unique to ILTV. Received December 19, 1996 Accepted April 9, 1997     

Shotgun sequencing of the negative-sense RNA genome of the rhabdovirus Maize mosaic virus

The maize-infecting nucleorhabdovirus, Maize mosaic virus (MMV), was sequenced to near completion using the random shotgun approach. Sequences of 102 clones from a cDNA library constructed from randomly-primed viral RNA were compiled into a 12,133 nucleotide (nt) contig containing six open reading frames. The contig consisted of 97 sequences averaging 660 bp in length. The average sequence coverage was six-fold, and 93% of the contig had sequence reads covering both strands. The remaining sequence was derived from single (5%) or multiple (2%) reads on the same strand. Three of the six ORFs showed significant similarities to the deduced protein sequences of the nucleocapsid, glycoprotein and polymerase sequences of other rhabdoviruses. The predicted gene order of the MMV genome was 3'-N-P-3-M-G-L-5'. Shotgun sequencing of the MMV genome took approximately 127 h and cost 0.38 dollars per nt (including labor), whereas the primer walking approach for sequencing the 13,782-nt MFSV genome [Tsai, C.-W., Redinbaugh, M.G., Willie, K.J., Reed, S., Goodin, M., Hogenhout, S. A., 2005. Complete genome sequence and in planta subcellular localization of maize fine streak virus proteins. J. Virol. 79, 5304-5314] took about 217 h and cost 0.50 dollars per nt. Thus, the shotgun approach gave good depth of coverage for the viral genome sequence while being significantly faster and less expensive than the primer walking method. This technique will facilitate the sequencing of multiple rhabdovirus genomes.