BK virus DNA sequence: extent of homology with simian virus 40 DNA.

The primary nucleotide sequence of three regions of BK virus (BKV) variant (MM) DNA has been determined. The region between map positions 0.715 and 0.900 includes the initiation points and partial coding sequences of the putative VP2 and VP3 proteins of BKV(MM), the amino acid sequences of which show over 80% homology with those of VP2 and VP3 of simian virus 40. The sequence of a potential leader protein X, 66 amino acids long for BKV(MM) and 62 long for simian virus 40, is also deduced. The regions between 0.595 and 0.398 and 0.310 and 0.175 include the coding sequence for the entire small t antigen and most of the large T antigen of BKV(MM). The DNA sequence within these regions comprises over 50% of the complete BKV(MM) genome and shows a 70% sequence homology with the corresponding regions of simian virus 40 DNA. This high degree of homology is at variance with the reported homology values of 11--20% estimated by hybridization measurements of heteroduplex analyses. Possible explanations for the discrepancy are discussed.     

BK virus DNA: cleavage map and sequence analysis.

A detailed physical map of the BK virus (MM strain) genome has been constructed with respect to the cleavage sites of 11 different restriction enzymes. The enzymes cut BKV(MM) DNA at 61 specific sites whose locations have been determined. Preliminary nucleotide sequence was carried out in the region from 0.70-0.75 map positions on BKV(MM) DNA. An 80% homology was found at 0.714-0.744 map positions on BKV(MM) DNA with 0.722-0.752 map positions on simian virus 40 DNA. This region of simian virus 40 DNA codes for the synthesis of the leader sequence of late mRNA.     

Nucleotide sequence of the mRNA encoding the pre-alpha-subunit of mouse thyrotropin.

We have constructed and cloned in bacteria recombinant DNA molecules containing DNA sequences coding for the precursor of the alpha subunit of thyrotropin (pre-TSH-alpha). Double-stranded DNA complementary to total poly(A)+RNA derived from a mouse pituitary thyrotropic tumor was prepared enzymatically, inserted into the Pst I site of the plasmid pBR322 by using poly(dC).poly(dG) homopolymeric extensions, and cloned in Escherichia coli chi 1776. Cloned cDNAs encoding pre-TSH-alpha were identified by their hybridization to pre-TSH-alpha mRNA as determined by cell-free translations of hybrid-selected and hybrid-arrested RNA. The nucleotide sequences of two cDNAs (510 and 480 base pairs) were determined with chemical methods and corresponded to much of the region coding for the alpha subunit and the 3' untranslated region of pre-TSH-alpha mRNA. The sequence of the 5' end of the mRNA was determined from cDNA synthesized by using total mRNA as template and a restriction enzyme DNA fragment as primer. Together these sequences represented greater than 90% of the coding and noncoding regions of full-length pre-TSH-alpha mRNA, which was determined to be 800 bases long. The amino acid sequence of the pre-TSH-alpha deduced from the nucleotide sequence showed a NH2-terminal leader sequence of 24 amino acids followed by the 96-amino-acid sequence of the apoprotein of TSH-alpha. There is greater than 90% homology in the amino acid sequences among the murine, ruminant, and porcine alpha subunits and 75-80% homology among the murine, equine, and human alpha subunits. Several regions of the sequence remain absolutely conserved among all species, suggesting that these particular regions are essential for the biological function of the subunit. The successful cloning of the alpha subunit of TSH will permit further studies of the organization of the genes coding for the glycoprotein hormone subunits and the regulation of their expression.     

Nucleotide sequence of cloned cDNAs encoding human preproparathyroid hormone.

We have cloned cDNA copies of human preproparathyroid hormone in Escherichia coli after insertion of double-stranded DNA into the Pst I site of plasmid pBR322 using the poly(dG) . poly(dC) homopolymer extension technique. Recombinant plasmids coding for preproparathyroid hormone were identified by filter hybridization assay using as a probe 32P-labeled bovine preproparathyroid cDNA. Nucleotide sequence analysis of five recombinant plasmids permitted the assignment of 74 nucleotides of the 5' noncoding region, the entire coding region of 345 nucleotides, and the entire 3' noncoding region of 348 nucleotides of the mRNA. The coding sequence predicts the previously unknown "pre" amino acid sequence and clarifies the hormone's amino acid sequence, which has been disrupted. The 5' noncoding region contains an AUG codon followed by a UGA stop codon before the authentic initiator codon. The 3' noncoding region is 120 nucleotides longer than in bovine preproparathyroid mRNA and contains two A-A-U-A-A-A sequences, potential signals for polyadenylation.     

Nucleotide sequence of tobacco mosaic virus RNA.

Oligonucleotide primers have been used to generate a cDNA library covering the entire tobacco mosaic virus (TMV) RNA sequence. Analysis of these clones has enabled us to complete the viral RNA sequence and to study its variability within a viral population. The positive strand coding sequence starts 69 nucleotides from the 5' end with a reading frame for a protein of Mr 125,941 and terminates with UAG. Readthrough of this terminator would give rise to a protein of Mr 183,253. Overlapping the terminal five codons of this readthrough reading frame is a second reading frame coding for a protein of Mr 29,987. This gene terminates two nucleotides before the initiator codon of the coat protein gene. Potential signal sequences responsible for the capping and synthesis of the coat protein and Mr 29,987 protein mRNAs have been identified. Similar sequences within these reading frames may be used in the expression of sets of proteins that share COOH-terminal sequences.     

Nucleotide sequence preservation of human mitochondrial DNA.

Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mp11; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both between- and within-individual differences were identified. Between-individual differences were identified in approximately 1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation of mammalian mitochondrial DNA sequences in single organisms.     

Nucleotide sequence and predicted amino acid sequence of a protein encoded in a small herpes simplex virus DNA fragment capable of trans-inducing alpha genes.

The five alpha genes of herpes simplex virus 1 are the first set of genes to be expressed after infection. Previous studies have shown that alpha genes resident in eukaryotic cells are induced by infection with herpes simplex virus 1 or 2 but not by other herpesviruses and indicate that the alpha trans-inducing factor was a structural component of the virion. This factor induces genes linked to a bona fide promoter and containing at the 5' end a small sequence derived from the promoter-regulatory domains of alpha genes. We report the sequence of a small DNA fragment shown previously to be capable of expressing the alpha trans-inducing factor in transient expression systems. The only gene encoded in its entirety in this fragment is predicted to specify a 479 amino acid protein with a Mr of 53,053. The precise termini of the 1.74-kilobase mRNA specifying this protein were determined in our 5' and 3' S1 nuclease protection studies.     

Expression of simian virus 40 small t antigen in Escherichia coli and purification of the antigen

A simian virus 40 (SV 40) DNA fragment encoding small t antigen was cloned in expression vector pUC8 for the purification of the antigen. The SV40 Hind III B fragment was inserted into the Hind III site of pUC8. A plasmid having the lacZ' and small t antigen genes in the same orientation was designated as pSVt. pSVt encodes the entire small t antigen and an extra 18 amino acids at the amino terminus of the antigen. E. coli transformed with pSVt produced hybrid small t antigen (22 kDa) which comprised about 6% of the total protein. The hybrid small t antigen reacted in immunoblot analysis with SV40-induced tumor-bearing hamster serum. Hybrid small t antigen was extracted from E. coli, and purified by preparative SDS-PAGE. The antigen was extracted from the gel using formic acid solution with high-yield. The gel-purified antigen showed the same antigenic reactivity as crude antigen.     

Nucleotide sequence of v-rel: the oncogene of reticuloendotheliosis virus.

The nucleotide sequence of v-rel, the oncogene carried by reticuloendotheliosis virus (REV), has been determined. The defective transforming genome REV arose through the insertion of v-rel (1,415 nucleotides) into the env gene of the helper virus REV-A. The predicted rel protein (503 amino acids) employs the REV-A env initiator and terminates within the p20E region of env. Because there are no natural antisera that detect the REV transforming protein, this nucleotide sequence provides the first step toward its isolation and characterization. The predicted protein is clearly distinct from all other reported transforming proteins but may be very distantly related to members of the src family.     

Nucleotide sequence of the Salmonella typhimurium origin of DNA replication.

Construction of deletion derivative plasmids and cloning of restriction fragments from plasmids containing the Salmonella typhimurium origin of replication (ori) were used to locate the functional origin to within a DNA fragment of 296 base pairs between the genes uncB and asn. The nucleotide sequence of the S. typhimurium ori region was determined and compared with the Escherichia coli ori sequence. In the 296-base pair fragment, 85.8% of the bases are conserved between the two species. A nearly equal number of transition and transversion type differences, with no insertions or deletions, occurs between the two bacterial origins, such that the relatively high percentage (adenine plus thymine) of 59.5% is conserved. The 296-base pair fragment contains 14 GATC sequences, all of which are conserved. The high frequency of occurrence of GATC, which is the site of methylation under control of the dam gene, may explain in part why the bacterial ori region appears to be so highly conserved. A large number of secondary structures are possible. One such structure, with a "cloverleaf," is favored by ori nucleotide sequence comparisons and leads to potential novel macromolecular interactions.     

新型乙型肝炎病毒核心区核酸疫苗的免疫原性研究

目的观察新型乙型肝炎病毒(HBV)核心抗原(HBcAg)核酸疫苗的免疫原性。方法应用新型人体应用载体质粒pSW389l构建HBcAg核酸疫苗(pSW3891／HBc)，对照组和实验组Balb／c小鼠分别以基因枪法免疫对照载体质粒(PSW3891)和HBcAg核酸疫苗，采用酶联免疫吸附试验检测抗HBc，乳酸脱氢酶(LDH)释放测定法检测小鼠HBcAg特异性cTL杀伤活性。结果HBcAg核酸疫苗可在体外293T细胞中高效表达，免疫小鼠后可产生高滴度抗HBc(1：97200)，免疫鼠脾细胞HBc特异性CTL杀伤活性达73．25％。结论新型HBcAg核酸疫苗在Balb／c小鼠实验中表现出良好的体液和细胞免疫原性。     

Nucleotide sequence of the genetic loci encoding subunits of Bradyrhizobium japonicum uptake hydrogenase.

An indispensable part of the hydrogen-recycling system in Bradyrhizobium japonicum is the uptake hydrogenase, which is composed of 34.5- and 65.9-kDa subunits. The gene encoding the large subunit is located on a 5.9-kilobase fragment of the H2-uptake-complementing cosmid pHU52 [Zuber, M., Harker, A.R., Sultana, M.A. & Evans, H.J. (1986) Proc. Natl. Acad. Sci. USA 83, 7668-7672]. We have now determined that the structural genes for both subunits are present on this fragment. Two open reading frames are present that correspond in size and deduced amino acid sequence to the hydrogenase subunits, except that the small-subunit coding region contains a leader peptide of 46 amino acids. The two genes are separated by a 32-nucleotide intergenic region and likely constitute an operon. Comparison of the deduced amino acid sequences of the B. japonicum genes with those from Desulfovibrio gigas, Desulfovibrio baculatus, and Rhodobacter capsulatus indicates significant sequence identity.     

Nucleotide sequence and genomic organization of feline immunodeficiency virus.

An infectious molecular clone of the Petaluma strain of feline immunodeficiency virus (FIV) was isolated from a recombinant bacteriophage library containing genomic DNA prepared from FIV-infected Crandall feline kidney (CRFK) cells. The integrated provirus has a total length of 9472 base pairs. Three long open reading frames corresponding to GAG, POL, and ENV gene coding frames are evident. In addition, an open reading frame overlaps the 3' end of POL, in the region that encodes viral infectivity factor in the primate viruses. Several short open reading frames are present in the intergenic region between POL and ENV and within ENV, which may serve as exons for production of TAT and REV equivalents in FIV. Alignment of the predicted amino acid sequences of the FIV proteins with those of other lentiviruses indicates that FIV did not arise recently from any other characterized lentivirus.     

Occurrence of BK virus DNA in DNA obtained from certain human tumors.

DNA sequences from BK virus have been detected by measurements of reassociation kinetics in DNA preparations isolated from five of 12 human tumor tissues and three of four human tumor cell lines; no DNA sequences from BK virus could be found using the same assay in DNA samples obtained from nontumor tissues removed from various patients. Whether or not this association is trivial, indicative of infection by BK virus, or correlative with the tumor state must await the testing of additional samples of normal and tumor tissues as well as a definition of the physical state of the genome of BK virus.     

Nucleotide sequence of microvariant RNA: another small replicating molecule.

Microvariant RNA, a small self-replicating molecule (114 nucleotides long), has been isolated from Qbeta replicase reactions incubated in the absence of exogenous template. Its complete nucleotide sequence has been determined. Comparison with MDV-1 RNA, a somewhat larger endogenous Qbeta replicase product (220 nucleotides long) that had previously been characterized, revealed no significant sequence similarity. Since Qbeta replicase can mediate the synthesis of both of these disparate RNA molecules, primary sequence cannot be the sole determining factor in the processes of enzyme recognition and replication. This implies that the key is to be found in the secondary or tertiary structures. The availability of two different replicating molecules of defined sequence should aid in identifying these critical structural features.     

Nucleotide sequence of the cDNA encoding the precursor of the beta subunit of rat lutropin.

We have determined the nucleotide sequences of cDNAs encoding the precursor of the beta subunit of rat lutropin, a polypeptide hormone that regulates gonadal function, including the development of gametes and the production of steroid sex hormones. The cDNAs were prepared from poly(A)+ RNA derived from the pituitary glands of rats 4 weeks after ovariectomy and were cloned in bacterial plasmids. Bacterial colonies containing transfected plasmids were screened by hybridization with a 32P-labeled cDNA encoding the beta subunit of human chorionic gonadotropin, a protein that is related in structure to lutropin. Several recombinant plasmids were detected that by nucleotide sequence analyses contained coding sequences for the precursor of the beta subunit of lutropin. Complete determination of the nucleotide sequences of these cDNAs, as well as of cDNA reverse-transcribed from pituitary poly(A)+ RNA by using a synthetic pentadecanucleotide as a primer of RNA, provided the entire 141-codon sequence of the precursor of the beta subunit of rat lutropin. The precursor consists of a 20 amino acid leader (signal) peptide and an apoprotein of 121 amino acids. The amino acid sequence of the rat lutropin beta subunit shows similarity to the beta subunits of the ovine/bovine, porcine, and human lutropins (81, 86, and 74% of amino acids identical, respectively). Blot hybridization of pituitary RNAs separated by electrophoresis on agarose gels showed that the mRNA encoding the lutropin beta subunit consists of approximately 700 bases. The availability of cDNAs for both the alpha and beta subunits of lutropin will facilitate studies of the regulation of lutropin expression.     

Nucleotide sequence of the simian virus 40 small-t gene

The nucleotide sequence of the segment of simian virus 40 DNA between standard map positions 0.53 and 0.65, i.e., approximately half of the restriction fragment Hind A, is reported. This segment is located near the beginning of the early region and is transcribed counterclockwise. There is a potential initiating ATG signal at 13 nucleotides from the Hind C-Hind A junction in the strand with the same polarity as the early mRNA. From this signal on, an open reading frame is present which would allow the synthesis of a polypeptide of 174 amino acids until a TAA termination codon is reached at nucleotide 602 (map position 0.547). This polypeptide, revealed by the DNA sequence, corresponds almost certainly to small-t antigen. Correlation of the deduced amino acid sequence with the NH(2)-terminal sequences of small-t and large-T (tumor) antigens of simian virus 40, as established by Paucha et al. [Paucha, E., Mellor, A., Harvey, R., Smith, A. E., Hewick, R. M. & Waterfield, M. D. (1978) [Proc. Natl. Acad. Sci. USA 75, 2165-2169], strongly argues that both proteins are indeed initiated at the ATG triplet. Because the DNA region between 0.547 and 0.534 is blocked for translation in all three reading frames by multiple termination condons, we conclude that the large-T antigen must be coded for by two noncontiguous DNA segments: the segment from 0.65 to around 0.60, which small-t and large-T antigens share, and another segment starting at some point after position 0.534 and continuing counterclockwise until it terminates at map position 0.174. Small-t antigen is methionine-rich and has a remarkably high number of cysteine residues clustered mainly in its COOH-terminal half. It is rich in both basic and acidic residues, the former being slightly in excess.