The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus.

The complete nucleotide sequence of RNA beta from the type strain of barley stripe mosaic virus (BSMV) has been determined. The sequence is 3289 nucleotides in length and contains four open reading frames (ORFs) which code for proteins of Mr 22,147 (ORF1), Mr 58,098 (ORF2), Mr 17,378 (ORF3), and Mr 14,119 (ORF4). The predicted N-terminal amino acid sequence of the polypeptide encoded by the ORF nearest the 5'-end of the RNA (ORF1) is identical (after the initiator methionine) to the published N-terminal amino acid sequence of BSMV coat protein for 29 of the first 30 amino acids. ORF2 occupies the central portion of the coding region of RNA beta and ORF3 is located at the 3'-end. The ORF4 sequence overlaps the 3'-region of ORF2 and the 5'-region of ORF3 and differs in codon usage from the other three RNA beta ORFs. The coding region of RNA beta is followed by a poly(A) tract and a 238 nucleotide tRNA-like structure which are common to all three BSMV genomic RNAs.     

一株辛德毕斯病毒的基因组序列测定及分析

目的：对引进的一株辛德毕斯病毒的基因组序列进行测定,阐明其与已报道毒株序列的关系。方法：对辛德毕斯病毒基因组编码区进行分段RT-PCR扩增,对非编码区采用RACE法进行扩增,将扩增产物直接进行测序,应用DNAStar软件将测序结果拼接得到基因组序列,采用MEGA3.1软件对9株辛德毕斯病毒基因组序列进行系统进化发生树的构建。结果与结论：此株辛德毕斯病毒基因组共11663nt,编码3745个氨基酸残基,其中5＇端的2/3基因组编码4种非结构蛋白NSp1、NSp2、NSp3和NSp4,3＇端的1/3基因组编码5种结构蛋白E1、E2、E3、6K和C;结构基因和非结构基因之间有48nt的连接区为非翻译区;病毒基因组5＇末端和3＇末端分别有59、318nt的非编码区;序列同源性分析结果表明,此株病毒与S.A.AR86株的同源性最高,两者核苷酸序列的同源性为99.7%,氨基酸序列的同源性为99.6%,而与本室保存的另一辛德毕斯病毒MEI株的遗传进化关系稍远,系统进化发生树处于不同分支上。     

The nucleotide sequence of cDNA coding for the structural proteins of foot-and-mouth disease virus

The complete nucleotide sequence of cDNA coding for the structural capsid polypeptides of foot-and-mouth disease virus (FMDV) (strain A(10)61) has been determined. Portions of the flanking sequence coding for the nonstructural proteins p20a and p52 are also provided. The three larger structural polypeptides VP1, VP2 and VP3 have unmodified Mrs of 23248, 24649 and 24213, respectively. The size of the smaller polypeptide, VP4, can only be estimated at 7360 because the 5'-limit of its coding region is not yet known with certainty. The sequence data for VP1 (the major immunising antigen) and the amino-terminal quarter of p52 are compared with the data of Kurz et al. (Nucl. Acids Res. 9 (1981) 1919-1931) for a different serotype (O1K). This shows that variation is much greater in the region coding for VP1 than in that coding for p52. This is reflected in the level of amino acid sequence variation predicted for the two proteins. Analysis of relative codon usage reveals a strong bias in favour of C and G over U and A in the third base position. The dinucleotide frequencies show a bias against A-U and U-A, and for A-C and C-A.     

An mRNA from human brain encodes an isoform of the B subunit of the vacuolar H(+)-ATPase

The B subunit (approximately 60 kDa) of the vacuolar H(+)-ATPase is one of the two major subunits comprising the hydrophilic catalytic complex of the enzyme. Using left and catalytic complex of the enzyme. Using left and right primers which bind two highly conserved sequences of the B subunit, an 836-base pair fragment was amplified from human brain cDNA by the polymerase chain reaction. The amplified fragment was used to probe a Northern blot and to screen a brain cDNA library. A single RNA band, 3.2 kilobases (kb) in length, was detected on Northern blots. A positive cDNA clone containing a 2.5-kb insert was isolated and sequenced. It included a long 3'-untranslated region (greater than 1.2 kb) and was missing a minor portion of the 5'-end of the coding region. The coding region of the brain cDNA sequence was 77% identical at the nucleotide level and 90% identical at the amino acid level to the previously reported sequence for the B subunit of the vacuolar H(+)-ATPase from human kidney (Sudhof, T. C., Fried, V. A., Stone, D. K., Johnston, P. A., and Xie, X.-S. (1989) Proc. Natl. Acad. Sci, U. S. A. 86, 6067-6071). Within the coding region of the brain cDNA, which is 6 amino acid residues shorter at the 3'-end than the kidney sequence, an 11% difference in the GC content was calculated. The 3'-noncoding sequence of the brain cDNA was completely unrelated to that of kidney and was three times longer. We conclude that the B subunit cDNAs from human kidney and brain represent different isoforms. This is the first demonstration of an isoform of a vacuolar H(+)-ATPase subunit.     

Comparative sequences of two type 1 dengue virus strains possessing different growth characteristics in vitro

The complete genome sequences of two dengue-1 virus strains having different growth characteristics (Mochizuki and A88) were compared with other published strains. The sequence analysis indicated several unique amino acid changes throughout the coding region of Mochizuki strain, mostly in envelope (E) protein. A unique amino acid, Ile-69 for Mochizuki strain at E protein resulted in the loss of an Asn-67-linked glycosylation site. A Thr substitution for Ala-114 at C protein and amino acid changes found in E, non-structural NS3, NS4a, and NS5 proteins were unique for A88 strain. These substitutions might be correlated to their different growth characteristics in vitro.     

南方菜豆花叶病毒外壳蛋白基因序列的分析

用重组DNA技术及序列分析法测定了南方菜豆花叶病毒RNA基因组3′端1,000个碱基的序列,以及由此序列推导出的整个外壳蛋白的氨基酸顺序,它与巳报导的基本上一致。介绍了用DNA的寡核苷酸水解混合物作为起始引物,以3′端不含PolyA尾巴且不能加上PolyA的病毒RNA作为模板合成互补DNA,及进一步无性繁殖此cDNA的方法。     

Cloning and nucleotide sequence of a carbomycin-resistance gene from Streptomyces thermotolerans

Two plasmids (pOJ158 and pOJ159) containing DNA fragments from the carbomycin(Cb)-producing strain Streptomyces thermotolerans were identified in Streptomyces griseofuscus based on their ability to confer resistance to Cb. The Cb-resistance determinants on pOJ158 and pOJ159 were designated carA and carB, respectively. In S. griseofuscus, pOJ159 also confers resistance to spiramycin, rosaramicin, lincomycin, and vernamycin B, but not to tylosin; in Streptomyces lividans, pOJ159 additionally confers resistance to erythromycin and oleandomycin. The carB gene was localized on pOJ159 to a 1.25-kb region whose nucleotide sequence was determined. The sequence has a G + C content of 68% and contains the coding sequence for carB and portions of the 5' and 3' untranslated regions. A comparison of the amino acid sequence of the protein encoded by carB (as deduced from the nucleotide sequence) with the deduced amino acid sequence of the RNA methylase from Streptomyces erythraeus (encoded by ermE) revealed extensive homology, suggesting that carB also encodes an RNA methylase. The region 5' to the coding sequence does not contain a small ORF or regions of complementarity that are commonly associated with translationally regulated macrolide-lincosamide-streptogramin B resistance genes. The 3' untranslated region contains an inverted repeat sequence that potentially can form a stable RNA stem-loop structure with a calculated delta G of -70 kcal.     

Molecular cloning and the complete nucleotide sequence of the creatine kinase-M cDNA from chicken.

The nucleotide sequence of creatine kinase-M (CK-M) cDNA clones has been determined. It includes the entire coding region of 381 amino acids in addition to 5' and 3' untranslated regions. A comparison with a partial sequence from rat CK-M reveals 84% nucleotide sequence homology in the coding region but divergence in the 3' untranslated region. The amino acid sequence is 94% conserved between chicken and rat. Hybridization to RNA immobilized on filters indicates homology between the CK-M 3' untranslated region and additional muscle specific RNA species. The coding region hybridizes only to CK-M RNA.     

Construction of a rice immature seeds cDNA library and molecular cloning of oryzacystatin cDNA

Total RNA was extracted from rice immature seeds harvested 2 weeks after flowering; then mRNA was purified. cDNA with NotI and SaiI cohesive ends was synthesized and inserted into λgt22A. After packaged in vitno, the cDNA library was constructed with 1.5×106pfu. A 21-mer oligodeoxynucleotide was synthesized according to the 5'-end conserved coding sequence of oryzacystatin (a thiol proteinase inhibitor) and labeled as a probe. From 2.1 × 104 pfu, 9 positive dones have been isolated, 8 of which contain the entire coding region of oryzacystatin. λOC1 has the longest cDNA insert, which contains an open reading frame of 309 bp coding sequence, 84 bp 5'-end non-coding region and a poly(A) signal AATAAA at the 3'-end followed by 31 Nt of poly(A). The coding sequence is the same compared with oryzacystatin genomic DNA sequence, while there are some obvious differences such as insertion and variation in the non-coding region, especially lots of nonsucoessive insertion in the 3' region after poly(A) signal.     

The complete nucleotide sequence of RNa1 of barley mild mosaic virus (asl1) and attempts at bacterial expression of the P3 protein

The complete nucleotide sequence of RNA1 of an Aschersleben isolate of barley mild mosaic virus (BaMMV) was determined. It consists of 7263 nucleotides (nt) excluding the 3' poly (A) tail. The 5' and 3' nontranslated regions (NTR) are 148 and 338 nt in length, respectively, and flank a single large open reading frame coding for a precursor polypeptide with a calculated molecular mass of 256 kDa. Sequence comparison revealed a 96% amino acid (aa) identity to RNA1 translation products of Japanese and French BaMMV isolates. Conserved nucleotide motifs in the 3' sense and 5' complementary sense NTR of the two genomic RNAs were identified that may represent the polymerase recognition sites. A range of constructs containing various parts of the coding region of the P3 nonstructural protein was prepared for expression in Escherichia coli . A short stretch of 35 aa in the C-proximal region of P3 appeared to be highly toxic to the bacterium.     

Structure and gene organization in the transformed Hind III-G fragment of Ad12

The nucleotide sequence of the transforming Hind III-G fragment of Ad12 DNA which encompasses the left 6.8% of the genome has been determined. The fragment was 2320 nucleotides long, and contained a GC cluster at positions 126-155 and a region extremely rich in AT at positions 1098-1142 (number from the leftmost end). Possible coding regions for the two transforming gene products were assigned. The predicted coding region for T antigen g is positions 502-1069 and positions 1144-1373, which are joined by splicing (266 amino acid residues, 30 kd), and that for T antigen f is positions 1845-2126 (94 amino acid residues, 10 kd). The sequence of the Hind III-G fragment was compared with that of the transforming DNA fragment of Ad5 which encompasses the left 8.0% of the genome (2809 nucleotides). There are several discrete regions with significant sequence homology. The comparison suggests that the regions in the left two thirds of the Ad5 and Ad12 transforming DNA fragments (map units 0-4.7% in Ad5 and 0-4.4% in Ad12) bear some resemblance in their gene organizations, and code for proteins containing structurally homologous regions.     

水稻条叶枯病毒(RStV)基因组组分4的克隆与序列分析

利用RTPCR技术合成并扩增了水稻条叶枯病毒(RStV)中国云南分离物基因组组分4的全长cDNA,将PCR产物克隆在载体pCRII上,并进行全序列测定,所得核苷酸序列及推测的氨基酸序列与日本分离物T进行比较。结果表明,在核苷酸水平,两分离物的vORF、vcORF及基因间非编码区序列的同源性分别为94.9%、94.1%、86.1%,5’端非编码区序列相同,而3’非编码区同源性为96.1%,仅有两个核苷酸不同;在氨基酸水平,vORF及vcORF编码蛋白的同源性分别为99.4%和98.3%。可见,编码区的大小及其氨基酸序列和两末端序列都是很保守的。因此,中国云南分离物Y与日本分离物T可能有很近的亲缘关系。