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The complete sequences of African horsesickness virus serotype 4 (vaccine strain) RNA segment 2 and 6 which encode outer capsid protein.

The complete sequences of RNA segment 2 and segment 6 of African horsesickness virus serotype 4 (AHSV-4) vaccine strain were determined from cDNA clones inserted into pBR 322. The RNAs of segment 2 and 6 are 3229, 1566 bp long respectively and both contain an open reading frame encoding proteins VP2 and VP5 of 1060, 505 amino acid residues. The estimated molecular weight of VP2 was 124,178 dalton and that of VP5 was 56,793 dalton. Their noncoding end sequences were 5'GTTTAA . . . and . . . ACATAC3' (segment 2), 5'GTTTAT . . . and . . . ACTTAC3' (segment 6). They were different from orbivirus characteristic terminal sequences, which were 5'GTTAAA . . . and . . . ACTTAC3'. The comparison of both sequences of AHSV-4 segment 2 and 6 with those of segment 2 and 5 of bluetongue virus (BTV) serotype 10 revealed 53% nucleotide similarity and 23% amino acid similarity (segment 2), and 58% nucleotide similarity and 46% amino acid similarity (segment 6). In the same way, the comparison of both sequences of the vaccine strain with those of the virulent strain segment 2 and segment 6 of AHSV-4 revealed 91% nucleotide and 96% amino acid similarity (segment 2), and 98% nucleotide and 98% amino acid similarity (segment 6).

African Horse Sickness Virus↗

Differential processing of osteopontin transcripts in rat kidney- and osteoblast-derived cell lines.

Using immunoprecipitation and tryptic peptide microsequencing we confirmed the identity of normal rat kidney (NRK) cell-secreted 69-kDa major phosphoprotein as osteopontin (OP). We then immunoselected a 1.4-kilobase pair (kb) OP cDNA from a lambda gt11 library prepared from Kirsten sarcoma virus-transformed NRK (KNRK) cellular mRNA, using rabbit anti-69-kDa OP serum. Sequence analysis of this cDNA revealed the presence of a 52-nucleotide-long insert in the 5'-noncoding region, which was absent in OP cDNA cloned from the cDNA library of ROS 17/2.8 rat osteosarcoma cells. The insert sequence is flanked by putative intron splice junctions and is located 15-nucleotide upstream of the translational initiation site. An insert-specific 30-mer oligonucleotide probe hybridized to a single 1.5-kb RNA species from both NRK and KNRK cells, but not from ROS 17/2.8 cells. However, Southern analysis showed the presence of this insert sequence in the genomic DNA of both NRK and ROS 17/2.8 cells. Furthermore, PCR amplification of the insert-containing region using genomic DNAs from both NRK and ROS 17/2.8 cells gave products of identical size and sequence. Since OP is a single copy gene, these data provide strong evidence for differential cell type-specific processing of OP transcripts. In addition, we demonstrate that, in contrast to most transformed cells, levels of OP expression are significantly reduced in KNRK cells as compared to NRK cells.

Amino Acid Sequence↗

Complete sequences of the glycoproteins and M RNA of Punta Toro phlebovirus compared to those of Rift Valley fever virus.

The complete sequence of Punta Toro virus (Phlebovirus, Bunyaviridae) middle size (M), RNA has been determined. The RNA is 4330 nucleotides long (mol wt 1.46 X 10(6), base composition: 26.7% A, 33.6% U, 18.5% G, 21.2% C) and has 3'- and 5'-terminal sequences that, depending on the arrangement, are complementary for some 15 residues. The viral RNA codes in its viral-complementary sequence for a single primary gene product (the viral glycoprotein precursor) that is comprised of 1313 amino acids (146,376 Da) and is abundant in cysteine residues but has few potential asparagine-linked glycosylation sites. The 5'-noncoding region of the Punta Toro M viral-complementary RNA is short (16 nucleotides); the 3'-noncoding sequence is much longer (372 nucleotides). The latter is rich in short stretches of adenylate residues, like the 3'-noncoding regions of the Punta Toro S mRNA species (T. Ihara, H. Akashi, and D. H. L. Bishop, 1984, Virology 136, 293-306). No other large open reading frame has been identified in either the viral, or viral-complementary, M RNA sequences. Limited amino-terminal sequence analyses of the two viral glycoproteins have indicated the gene order and potential cleavage sites in the glycoprotein precursor. The data suggest the existence of a 30 X 10(3)-Da polypeptide (designated NSM) in the glycoprotein precursor that precedes the G1 protein (i.e., gene product order: NSM-G1-G2). Examination of the sequence of the Punta Toro M gene product reveals the presence of multiple hydrophobic sequences including a 19-amino acid, carboxy-proximal, hydrophobic region (G2). This hydrophobic sequence is followed by a 13-amino acid-terminal sequence rich in charged amino acids. The size and constitution of the carboxy-terminal region is consistent with a transmembranal and anchor function for the glycoprotein in the viral envelope. Other regions of the glycoprotein precursor contain sequences of amino acids with a predominantly hydrophobic character (23, 50, and 20 amino acids in length). Their functions are unknown. The amino terminus of the G1 protein is located near the end of the 23-amino acid-long hydrophobic sequence of the presumptive precursor, the hydrophobic 50-amino acid sequence lies within G1, and the amino terminus of G2 is located in the middle of the 20-amino acid-long hydrophobic sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

Mouse primase p49 subunit molecular cloning indicates conserved and divergent regions.

Primase is a specialized RNA polymerase that synthesizes RNA primers for initiation of DNA synthesis. A full cDNA clone of the p49 subunit of mouse primase, a heterodimeric enzyme, has been isolated using a primase p49-specific polyclonal antibody to screen a lambda gt11 mouse cDNA expression library. The cDNA indicated the subunit is a 417-amino acid polypeptide with a calculated molecular mass of 49,295 daltons. The p49 mRNA is approximately 1500 nucleotides long with a 5'-untranslated region of 74 nucleotides and a 3'-untranslated region of 200 nucleotides. Comparison with a similar sized primase subunit from yeast showed highly conserved amino acid sequences in the N-terminal halves of the polypeptides and included a potential metal-binding domain suggesting the functional importance of this region for DNA binding. In contrast, the 3' portion of the cDNA has rapidly diverged in nucleotide sequence, as primase mRNA can be detected in mouse and rat cells with a 3' probe (including coding and noncoding) but not in RNA from hamster or human cells. A full-length cDNA probe detected mRNA from hamster and human cell lines, indicating a conserved 5' portion and divergent 3' region of the expressed gene. The rapid divergence may be related to the species-specific protein interactions found for the DNA polymerase alpha-primase complex. The mRNA is detected in proliferating but not in quiescent cells consistent with its function in DNA replication.

Amino Acid Sequence↗

Replicase gene of coxsackievirus B3.

A cDNA copy covering two-thirds of the coxsackievirus B3 genome was cloned in the PstI site of the pBR322 vector. A nucleotide sequence containing the gene for the viral replicase and the 3' noncoding region of the coxsackievirus B3 genome was determined. The predicted amino acid sequence of the coxsackievirus B3 replicase was shown to be remarkably similar to that of the poliovirus 1 replicase. The 3' noncoding region, in contrast, was only weakly homologous to the poliovirus 1 sequence but showed a close relationship to the sequence of swine vesicular disease virus, a variant of coxsackievirus B5. A 13-nucleotide-long segment located near the polyadenylic acid junction is conserved in several members of the enterovirus group and may thus serve an important function during replication of viral RNA.

Base Sequence↗

Human adenylate kinase deficiency associated with hemolytic anemia. A single base substitution affecting solubility and catalytic activity of the cytosolic adenylate kinase.

Adenylate kinase deficiency in the erythrocyte is a rare genetic disorder associated with hemolytic anemia. To determine the molecular basis of this disorder, we first cloned the normal gene encoding human cytosolic adenylate kinase (AK1) and determined the structure. The gene was 12 kilobase pairs long and was split into 7 exons. The structures of 5'- and 3'-flanking regions were determined by primer extension and RNA blot analysis. The results showed that two species of mRNA with 0.9 and 2.5 kilobases, which differed at the 3'-end portion, were generated by the AK1 gene. Alu sequences were found in the largest intron (intron 5) and in the noncoding region of exon 7. Next, both alleles of the AK1 gene were cloned from DNA of a patient bearing the adenylate kinase deficiency and their nucleotide sequences determined. A transition (C----T) was found in exon 6 on an allele, which resulted in an Arg to Trp (CGG----TGG) substitution at the 128th residue of AK1. Since chicken AK1 is highly homologous to human AK1 with respect to the amino acid sequence, we introduced an Arg to Trp substitution to chicken AK1 at the same position by oligodeoxynucleotide-directed mutagenesis. The mutant chicken AK1 expressed in Escherichia coli showed a reduced catalytic activity as well as a decreased solubility and a change in affinity to phosphocellulose. Thus it was considered that the observed C----T transition was a cause of the decreased AK1 activity of the patient's erythrocyte. Analysis on phosphocellulose chromatography of erythrocyte AK1 of the patient and parents revealed that the patient's mutant allele was derived from the mother.

Adenylate Kinase↗

Arthropod hemocyanins. Molecular cloning and sequencing of cDNAs encoding the tarantula hemocyanin subunits a and e.

cDNA clones comprising the entire coding region of two out of the seven heterogeneous subunits of hemocyanin from the tarantula, Eurypelma californicum, were isolated from four cDNA libraries constructed from total RNA from the heart tissue of single spiders. Hybridization was first carried out using a tarantula hemocyanin subunit e partial cDNA, and several positive clones were isolated, including one containing a 2.2-kilobase full-length cDNA (lambda M1). The cDNA comprises an open reading frame for 623 amino acids, 34 nucleotides of the 5'noncoding region, and 286 nucleotides of the 3'-noncoding region. To select for other hemocyanin subunits, two 17-mer oligonucleotide mixtures, corresponding to the conserved regions in the copper A and copper B oxygen-binding site of chelicerate hemocyanins, were used as probes. Among the positive clones obtained, full-length cDNAs coding for subunit a were identified. The cDNA sequence determined from clone lambda K1 provides an open reading frame coding for 630 amino acids and includes the 5'- and 3'-noncoding regions. Northern blot analysis revealed single transcripts for subunits a and e, each 2.3 kilobases long. The cDNAs for subunits a and e were both found to lack any leader peptide sequence. This supports the idea that the mature protein accumulates in the cytoplasm and is released by cell rupture.

Amino Acid Sequence↗

Sequence analysis and genome organisation of poinsettia mosaic virus (PnMV) reveal closer relationship to marafiviruses than to tymoviruses.

Sequence comparison and genome organisation of poinsettia mosaic virus (PnMV), a putative member of the tymoviruses, revealed a closer relationship to marafiviruses. The complete nucleotide sequence of PnMV was determined. The 6099-nt RNA genome encodes a putative 221-kDa polyprotein that lacks a stop codon between the replicase and the coat protein genes, as in most tymovirus RNAs. The genomic RNA has a poly(A) tail at its 3'-terminus in contrast to the tRNA-like structure found in the RNA of most tymoviruses, and no homology was observed to the conserved noncoding region of the tymoviral 3'-termini. The tymobox of PnMV, a 16-nt region of the subgenomic RNA (sgRNA) promoter shared by most tymoviruses, differs in 3 nt from the RNA sequence of tymoviruses but is identical to the sequence of marafiviruses. At least three sgRNAs were found in PnMV-infected Euphorbia pulcherrima and in isolated PnMV particles; one that is 650 nt long encodes the 21.4-kDa coat protein, and the others are about 3.5 and 1.7 kb and contain the 5'- and the 3'-terminal parts of genomic RNA, respectively. Like tymoviruses, PnMV particles sediment as top and bottom components. The particles of the top component contain the sgRNA (650 nt) encoding the coat protein, and those of bottom component contain both genomic and sgRNAs.

Amino Acid Sequence↗

Nucleotide sequences of the 3' terminal region of onion yellow dwarf virus isolates from Allium plants in Japan.

The 2032 nucleotide sequence of the 3' terminal region of onion yellow dwarf virus (OYDV) isolated from Allium wakegi, bearing the genes for viral coat protein (CP) and a truncated RNA-dependent RNA polymerase, has been determined. Respective homologies of the nucleotide sequence in the corresponding region and the deduced amino acid sequence of CP with the equivalents of leek yellow stripe virus (LYSV) from garlic were 68.0 and 59.3%. Variation in the nucleotide sequence is concentrated in the boundary region between the putative RNA-dependent RNA polymerase gene and the CP gene as well as in the 3' noncoding region. These sequence divergencies, including the deletion of 79 nucleotides, resulted both in alterations to the amino acid sequence and the absence of 28 amino acid residues in the amino terminal region of OYDV CP in comparison with LYSV CP. In addition, the length of the 3' noncoding sequence of OYDV was one-third that of LYSV. Comparison of the 3' terminal 1197 nucleotides sequence of OYDV with sequences of the respective cDNAs cloned by RT-PCR directly from the total RNA of infected Allium plants that included two varieties of A. fistulosum, "Wakenegi" and "Shimonita-negi", and A. chinense, showed 90.7% overall identities, even though they have long been cultivated in locally restricted area in Japan. These findings appear to suggest that a single strain of OYDV invaded Japanese Allium plants long ago and spread throughout them.

Allium↗

Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes.

We have conducted a comprehensive search for conserved elements in vertebrate genomes, using genome-wide multiple alignments of five vertebrate species (human, mouse, rat, chicken, and Fugu rubripes). Parallel searches have been performed with multiple alignments of four insect species (three species of Drosophila and Anopheles gambiae), two species of Caenorhabditis, and seven species of Saccharomyces. Conserved elements were identified with a computer program called phastCons, which is based on a two-state phylogenetic hidden Markov model (phylo-HMM). PhastCons works by fitting a phylo-HMM to the data by maximum likelihood, subject to constraints designed to calibrate the model across species groups, and then predicting conserved elements based on this model. The predicted elements cover roughly 3%-8% of the human genome (depending on the details of the calibration procedure) and substantially higher fractions of the more compact Drosophila melanogaster (37%-53%), Caenorhabditis elegans (18%-37%), and Saccharaomyces cerevisiae (47%-68%) genomes. From yeasts to vertebrates, in order of increasing genome size and general biological complexity, increasing fractions of conserved bases are found to lie outside of the exons of known protein-coding genes. In all groups, the most highly conserved elements (HCEs), by log-odds score, are hundreds or thousands of bases long. These elements share certain properties with ultraconserved elements, but they tend to be longer and less perfectly conserved, and they overlap genes of somewhat different functional categories. In vertebrates, HCEs are associated with the 3' UTRs of regulatory genes, stable gene deserts, and megabase-sized regions rich in moderately conserved noncoding sequences. Noncoding HCEs also show strong statistical evidence of an enrichment for RNA secondary structure.

3' Untranslated Regions↗

Secretory pancreatic stone protein messenger RNA. Nucleotide sequence and expression in chronic calcifying pancreatitis.

The pancreatic stone protein and its secretory form (PSP-S) are inhibitors of CaCO3 crystal growth, possibly involved in the stabilization of pancreatic juice. We have established the structure of PSP-S mRNA and monitored its expression in chronic calcifying pancreatitis (CCP). A cDNA encoding pre-PSP-S has been cloned from a human pancreatic cDNA library. Its nucleotide sequence revealed that it comprised all but the 5' end of PSP-S mRNA, which was obtained by sequencing the first exon of the PSP-S gene. The complete mRNA sequence is 775 nucleotides long, including 5'- and 3'- noncoding regions of 80 and 197 nucleotides, respectively, attached to a poly(A) tail of approximately 125 nucleotides. It encodes a preprotein of 166 amino acids, including a prepeptide of 22 amino acids. No overall sequence homology was found between PSP-S and other pancreatic proteins. Some homology with several serine proteases was observed in the COOH-terminal region, however. The mRNA levels of PSP-S, trypsinogen, chymotrypsinogen, and colipase in CCP and control pancreas were compared. PSP-S mRNA was three times lower in CCP than in control, whereas the others were not altered. It was concluded that PSP-S gene expression is specifically reduced in CCP patients.

Adolescent↗

Global eradication of poliomyelitis: reinvent the wheel or use existing options effectively?

The development of serum and nasopharyngeal antibody response, and the magnitude and temporal pattern of fecal shedding of vaccine and revertant polioviruses was examined in infants previously immunized with one or more doses of oral poliovirus vaccine (OPV), enhanced-potency inactivated poliovirus vaccine (EIPV), or both. The fecal samples were collected at different intervals after the last OPV dose. The nucleic acid sequences of the purified RNA obtained from all virus isolates were examined in the 5' noncoding region by dideoxysequencing to determine whether the viruses shed represent revertants (vaccine), nonrevertants, or both. The frequency and duration of vaccine virus-shedding appeared to be similar in both immunization schedules. Revertant virus shedding was not demonstrated 30 days after immunization with OPV alone. However, shedding of revertants was detected for as long as 60 days in some subjects previously immunized with EIPV. The duration of shedding of revertant virus differed with different serotypes and different immunization regimens. Prior immunization with one or more doses of OPV reduced the length of shedding of revertant virus. Significantly, however, prior immunization with one or more doses of EIPV was not associated with reduced shedding of revertant virus types. The nature of serum immune response as determined by ELISA or neutralizing antibody appeared to be similar after either immunization schedule, although the antibody titers were quantitatively higher after two doses of IPV than observed after a similar schedule with OPV. Highest antibody activity was detected in subjects immunized with a combination of IPV followed by OPV. ELISA antibody activity in the nasopharynx was regularly detected after either form of immunization.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Viral↗

Complete nucleotide sequence of alfalfa mosaic virus RNA3.

A full-length cDNA clone of alfalfa mosaic virus (AMV) RNA3 was prepared and sequenced. The 2,037 base sequence contains two open reading frames of 903 and 666 nucleotides that code for a 32,400 dalton protein (32.4K protein) and the 24,380 dalton coat protein, respectively. A 5'-noncoding sequence of 240 bases preceeding the 32.4K protein contains homologous regions that may have a function in its translation. The intercistronic junction is 49 bases long, the last 36 bases representing the 5'-end of the subgenomic RNA4. The remaining 179 bases comprise the 3'-terminal noncoding sequence.

Base Sequence↗

Isolation of cDNAs coding for three different forms of liver microsomal cytochrome P-450 from polychlorinated biphenyl-treated beagle dogs.

Three different cDNA clones, namely DM1-1, Dah1, and Dah2, encoding hepatic cytochrome P-450, were isolated from a cDNA library in lambda gt11 constructed from liver RNA of polychlorinated biphenyl-treated beagle dogs. DM1-1 was 1857 base pairs (bp) long and encoded a polypeptide of 457 residues. Dah1 was 2394 bp long and contained an entire coding region for 524 amino acid residues. In addition, Dah2 was 1623 bp long and had an open reading frame consisting of 503 amino acid residues, although it lacked the translational initiation codon. Judging from the similarity of the nucleotide and amino acid sequences, forms of cytochrome P-450 encoded by DM1-1, Dah1, and Dah2 were judged to belong to the P450IIC, P450IA1, and P450IA2 subfamilies, respectively. Northern blot analysis of RNA from various tissues, using the specific 3' noncoding regions of Dah1 and Dah2 as probes, indicated that mRNAs for P-450(Dah1) and P-450(Dah2) were not detectable in tissues from untreated dogs, except for P-450(Dah2) in livers. Polychlorinated biphenyl induced both mRNAs in liver, kidney, and lung, especially in the kidney.

Amino Acid Sequence↗

Different expression of rat aldolase A mRNA in the skeletal muscle and ascites hepatoma cells.

Rat aldolase A mRNAs from the skeletal muscle and ascites hepatoma cells are compared by RNA blot hybridization. The hepatoma AH60C and AH130 mRNA are about 1600 bases long, some 50 bases longer than the skeletal muscle transcript. Aldolase A enzymes encoded by these mRNAs, however, are indistinguishable in electrophoresis on cellulose polyacetate strips and two-dimensional polyacrylamide gel. In addition, comparison of the thermal stability of hybrids between the mRNAs from the skeletal muscle and hepatoma cells and a cDNA probe (pRAAM83) made from the aldolase A mRNA of the skeletal muscle indicate that the mRNAs have extremely high sequence homology, at least, within the most part of coding region in addition to the 3' noncoding region. These findings indicate that these mRNA may be transcribed by the use of different promoter sites and the common structural gene.

Animals↗

The structure and nucleotide sequence of the 5' end of the human c-myc oncogene.

We have established the structure and nucleotide sequence of the 5' end of the human c-myc oncogene, using a cloned genomic fragment isolated from a fetal liver library (clone lambda MC41) and cloned cDNA from the human leukemic cell line K562. The human c-myc oncogene consists of three exons and two introns. Primer extension of the human c-myc mRNA of three different cell lines and S1 nuclease protection experiments served to establish the position of two transcription initiation sites. The splicing site of the first exon-intron boundary was determined by comparative analysis of the sequences of the genomic and cDNA clones. The first exon contains termination codons in all three reading frames and no translation initiation signals, confirming our previous observation that the c-myc mRNA has a long 5' noncoding sequence. This first exon also was found to be utilized in the formation of c-myc mRNAs in a variety of human cell lines.

Base Sequence↗

Retroviral insertions 90 kilobases proximal to the Evi-1 myeloid transforming gene activate transcription from the normal promoter.

The inappropriate production of the Evi-1 zinc finger protein occurs in retrovirus-induced murine myeloid leukemias and human acute myelogenous leukemias. In murine leukemias, expression of the Evi-1 gene is associated with retroviral insertions either in the Evi-1 locus, which is immediately 5' of the coding region of the gene, or in the genetically linked Cb-1/fim-3 locus. In these studies, we demonstrate by chromosomal walking and pulse field electrophoresis that the Cb-1/fim-3 locus is located 90 kb 5' of the Evi-1 locus. Primary structure analysis of Evi-1 cDNA clones from a Cb-1/fim-3 rearranged cell line (DA-3) demonstrates that transcription initiates 5' of the Evi-1 locus and that the first noncoding exon of the gene is 681 bp larger than previously defined. S1 nuclease protection studies reveal multiple transcription initiation sites within this region. Comparable transcriptional initiation sites were identified in RNA from kidney and ovary, in which the gene is normally expressed, suggesting that retroviral insertions in the Cb-1/fim-3 locus activate transcription from the normal promoter. In one myeloid cell line (DA-3), a single long terminal repeat (LTR) is present in the Cb-1/fim-3 locus. No stable transcripts were detectable from this LTR. In cells with retroviral insertions in the Cb-1/fim-3 locus, one allele of the Evi-1 locus becomes hypermethylated in the 5' region of the gene. Together, these results are most consistent with an LTR-mediated, long-range cis activation of Evi-1 gene expression.

Alleles↗

Complete nucleotide sequence of an Indian strain of Japanese encephalitis virus: sequence comparison with other strains and phylogenetic analysis.

The RNA genome of an Indian strain of Japanese encephalitis virus (JEV), GP78, was reverse transcribed and the cDNA fragments were cloned in bacterial plasmids. Nucleotide sequencing of the cDNA clones covering the entire genome of the virus established that the GP78 genome was 10,976 nucleotides long. An open reading frame of 10,296 bases, capable of coding for a 3,432 amino acid polyprotein, was flanked by 95- and 585-base long 5'- and 3'-non-coding regions, respectively. When compared with the nucleotide sequence of the JaOArS982 strain, the JEV GP78 genome had a number of nucleotide substitutions that were scattered throughout the genome except for the 5'-noncoding region, the sequence of which was fully conserved. Comparison of the complete genome sequences of different JEV isolates showed a 1.3-4.1% nucleotide sequence divergence among them, which resulted in 0.6-1.8% amino acid sequence divergence. Analysis based on the complete genome sequences of different JEV isolates showed that the GP78 isolate from India was phylogenetically closer to the Chinese SA14 isolate.

Adult↗