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Biomedical subjects

Y Furuichi

Publications and source records attributed to Y Furuichi.

At least 181 records · Page 10Linked to original sources
PubMed1987

cDNA probes of individual genes of human rotavirus distinguish viral subgroups and serotypes.

The use of cDNA probes for detection of rotaviruses has been investigated using plasmids containing inserts specific for each of the eleven genes of human rotavirus strain Wa. In a dot-blot detection system in which radioactive DNA probes were hybridized to viral RNA extracted from cultivatable rotavirus strains, cDNAs of genes 7, 8, 10 and 11, were found to be the most reliable probes for detecting a range of rotavirus strains. Unexpectedly, rotaviruses could be distinguished with respect to subgroup and subtype specificities when cDNAs of genes 6 and 9, which encode the immunologically relevant proteins VP6 (group-specific antigen) and VP7 (type-specific antigen), were used as probe, even though the nucleic acid sequences of these genes are known to have a high degree of sequence homology.

Child, Preschool↗
PubMed1987

Molecular cloning and sequence of a cDNA coding for bovine lipoprotein lipase.

Lipoprotein lipase (LPL; triacylglycero-protein acylhydrolase, EC 3.1.1.34) was purified from bovine milk. Synthetic oligonucleotides were prepared, based on the amino acid sequences of three peptides obtained from partial digestion of purified LPL, and were used as probes to isolate cDNA clones for LPL mRNA from a bovine mammary gland. One of the clones, pLPL-49R2, contains an insert cDNA (49R2) of about 3.2 kilobases (kb) that hybridizes to all three probes and encodes a polypeptide that includes the NH2-terminal sequence of bovine LPL reported recently [Ben-Avram, C. M., Ben-Zeev, O., Lee, T. D., Hagga, K., Shively, J. E., Goers, J., Pedersen, M. E., Reeve, J. R. & Schotz, M. C. (1986) Proc. Natl. Acad. Sci. USA 83, 4185-4189]. Complete nucleotide sequence analysis revealed that cDNA insert 49R2 contains the entire coding region for LPL as well as a 3' untranslated region of about 1.6 kb. The predicted amino acid sequence indicates that bovine LPL is a hydrophilic protein consisting of 450 amino acids (Mr 50,548) in its unglycosylated form. Blot hybridization analysis of poly(A)+ mRNA from bovine mammary gland demonstrated that there are at least three sizes of LPL mRNAs--3.2, 2.5, and 1.7 kb--with the 2.5-kb mRNA being the most abundant. Restriction endonuclease mapping of other cDNA clones suggested that the variation in mRNA size results from differential utilization of polyadenylylation signals during mRNA processing.

Amino Acid Sequence↗
PubMed1986

Conserved structures among the nucleocapsid proteins of the paramyxoviridae: complete nucleotide sequence of human parainfluenza virus type 3 NP mRNA.

The nucleotide sequence of the mRNA coding for the nucleocapsid protein (NP) of the paramyxovirus, human parainfluenza virus type 3 (PIV-3), has been determined. The NP mRNA was found to contain 1642 bases, excluding poly(A), and encode a protein of 515 amino acids, with a molecular weight of 57,823. Amino acid residues 1 through 420 of PIV-3 NP protein showed extensive sequence homology with the corresponding amino acids of Sendai virus nucleocapsid protein. There was virtually no homology between the last 95 amino acids. Comparison of the NP proteins of PIV-3, Sendai virus, measles virus, and canine distemper virus revealed, from amino acid residues 160 through 390, some conserved areas between the corresponding proteins of these paramyxoviruses. The 5' terminal sequence of PIV-3 NP mRNA (5'-AGGATTAAAG-3') was similar to the conserved sequence (formula; see text) found at the 5' termini of Sendai virus mRNAs. Both PIV-3 NP and Sendai virus mRNAs had a common 3' terminal tetranucleotide (5'-TAAG-3') preceding the poly (A) tail.

Amino Acid Sequence↗
PubMed1985

Sequence of a cloned 523-bp cDNA for thymosin beta 4.

The sequence of a 523-bp cDNA, isolated from a clone bank prepared from partially purified rat spleen mRNA coding for thymosin beta 4, was described. The 3' sequence extended through the poly(A) segment and the 5' sequence included 36 bp preceding the translated sequence. The putative amino acid sequence coded by this segment possesses some of the features of a signal peptide.

Amino Acid Sequence↗
PubMed1985

Diagnosis of rotavirus infection with cloned cDNA copies of viral genome segments.

The diagnostic potential of cloned cDNA copies of human rotavirus (strain WA) genome segments for the detection of rotavirus in clinical specimens has been determined. A hybridization assay in which a mixture of 32P-labeled cDNAs representing the 11 rotavirus segments was used as a probe compared favorably with three frequently used diagnostic tests for rotavirus in terms of both specificity and sensitivity. Significantly, clinical isolates could be readily distinguished when cloned cDNA copies of individual genome segments were used independently as a probe. In assays in which genome RNA from rotaviruses of known subgroups and serotypes were tested, cloned probes that encode nonstructural viral proteins hybridized efficiently to genome RNAs of all strains, whereas cloned probes corresponding to genome segments 6 and 9 exhibited the potential for differentiating strains of different subgroups and serotypes. Cloned cDNA copies of rotavirus genome segments therefore offer considerable potential for improved general diagnosis of rotavirus in clinical specimens, as well as for epidemiological studies in which virus isolates can be distinguished on the basis of nucleotide sequence homology of individual genome segments.

Cloning, Molecular↗
PubMed1985

Synthesis in Escherichia coli of the reovirus nonstructural protein sigma NS.

The coding region of reovirus type 3 genomic segment S3, encoding the nonstructural protein sigma NS, was placed under the control of the bacteriophage lambda pL promoter in the Escherichia coli expression plasmid pRC23 (J.C. Lacal, E. Santos, V. Notario, M. Barbacid, S. Yamazaki, H.-F. Kung, C. Seamans, S. McAndrew, and R. Crowl, Proc. Natl. Acad. Sci. USA 81:5305-5309). Derepression of the pL promoter led to the synthesis of a protein of the same molecular weight as sigma NS produced in reovirus-infected L cells. The expressed protein was indistinguishable from authentic sigma NS by peptide mapping with Staphylococcus aureus V8 protease and by immunoblot analysis. Most importantly, the purified protein had nucleic acid-binding properties similar to that previously shown for sigma NS obtained from infected cells. Binding of single-stranded RNAs by recombinant sigma NS protein was inhibited by GTP.

Carrier Proteins↗
PubMed1984

Cloning and sequence analysis of cDNA for rat spleen thymosin beta 4.

Molecular cloning of a cDNA has established the sequence of the translated portion of the mRNA for rat spleen thymosin beta 4. The presence of a methionyl initiator codon immediately preceding the codon for the first seryl residue of mature thymosin beta 4 is consistent with previous results indicating the absence of a signal peptide in the product translated in vitro from rat spleen mRNA. The cDNA sequence analysis also established the presence of two terminator codons immediately following the codon for the COOH-terminal seryl residue. Thymosin beta 4 is thus synthesized as a 5100-dalton peptide containing 44 amino acid residues. Removal of the initiator methionyl residue and acetylation of the NH2-terminal serine residue would yield mature thymosin beta 4 containing 43 amino acids. The absence of a signal peptide makes it unlikely that thymosin beta 4 is a secreted peptide.

Amino Acid Sequence↗
PubMed1984

Comparative sequence analysis of rotavirus genomic segment 6--the gene specifying viral subgroups 1 and 2.

Cloned DNA copies of rotavirus genomic segment 6 from simian 11 (subgroup 1) and human strain Wa (subgroup 2) rotaviruses have been used to determine the nucleotide sequences of the gene that determines viral subgroup specificity. Both genomic segments are 1,356 nucleotides in length and possess 5'- and 3'-terminal untranslated regions of 23 and 142 nucleotides, respectively. The inferred amino acid sequence reveals VP6 to be a polypeptide of 397 amino acids in which more than 90% of the amino acid sequence is conserved between the two viruses. There are 34 amino acid changes between the subgroup 1 and 2 polypeptides, most clustered in three regions of the molecule at residues 39 through 62, 80 through 122, and 281 through 315.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequence of human rotavirus genome segment 10, an RNA encoding a glycosylated virus protein.

The complete nucleotide sequence of human rotavirus (Wa strain) genome segment 10 was determined by using a cloned DNA copy. The sequence data indicated that segment 10 is A + T rich (65%) and consists of 750 base pairs. The positive strand of segment 10 contains a single open reading frame that extends 175 codons from the first AUG triplet (residues 42 through 44). The amino acid sequence of the segment 10 product was deduced from the nucleotide sequence. There are two distinct glycosylation sites at the N-terminal hydrophobic region, consistent with previous findings that this protein exists in a glycosylated form. The apparent molecular weight (20,000) of the unglycosylated, precursor polypeptide is in good agreement with the one calculated from the predicted amino acid sequence. Structural analysis of the positive strand (mRNA from segment 10) showed that it could form, like mRNA from segment 11, a stable panhandle structure involving the 5' and 3'-terminal regions. The nucleotide sequence of segment 10 from simian rotavirus, recently determined by Both et al. (J. Virol. 48:335-339, 1983) was found to be highly homologous to, and to share several important features with, segment 10 of human rotavirus.

Amino Acid Sequence↗
PubMed1984

Nucleotide sequence of the gene encoding the serotype-specific antigen of human (Wa) rotavirus: comparison with the homologous genes from simian SA11 and UK bovine rotaviruses.

The nucleotide sequence of human (Wa) rotavirus genome segment 9, which encodes the serotype-specific antigen VP7, has been determined. Comparison of the deduced amino acid sequence of Wa VP7 protein to the sequences of simian SA11 and UK bovine VP7 proteins shows that the majority of the amino acid differences are clustered between amino acid residues 37 through 49, 65 through 75, 87 through 105, 122 through 126, 146 through 149, 178 through 181, and 208 through 242. A hydrophilicity profile of the three proteins reveals correlations between hydrophilic peaks, potentially antigenic determinants, and certain clusters of amino acid changes.

Amino Acid Sequence↗
PubMed1984

Reovirus type 3 genome segment S4: nucleotide sequence of the gene encoding a major virion surface protein.

A full-length cDNA copy of reovirus double-stranded RNA genome segment S4 which codes for a major virion structural polypeptide, sigma 3, has been completely sequenced. The 1,196-nucleotide cDNA contains a single long open reading frame in the plus strand extending 1,095 nucleotides from the 5'-proximal A-T-G to a single stop codon. This corresponds to translation of 92% of the S4 gene. The inferred sigma 3 polypeptide is hydrophilic and consists of 365 amino acids, totalling 41,164 daltons.

Amino Acid Sequence↗
PubMed1983

Nucleotide sequence of reovirus genome segment S3, encoding non-structural protein sigma NS.

This report describes the complete nucleotide sequence of human reovirus (Dearing strain) genome segment S3. Previous studies indicated that this RNA encodes the major non-structural viral polypeptide sigma NS, a protein that binds ssRNAs (Huisman & Joklik, Virology 70, 411-424, 1976) and has a poly(C)-dependent poly(G) polymerase activity (Gomatos et al., J. Virol. 39, 115-124, 1981). The genome segment consists of 1,198 nucleotides and possesses an open reading frame that extends 366 codons from the first AUG triplet (residues 28-30). There is no significant sequence homology between the plus strand of genome segment S3 and that of genome segment S2 determined previously (Cashdollar et al., PNAS 79, 7644-7648, 1982). However, S3 RNA has significant dyad symmetry and regions that can potentially hybridize (delta G = -26 KCal/mole) with S2 RNA. From the predicted amino acid sequence a possible secondary structure for sigma NS protein was determined. Structural features of reovirus RNA and sigma NS are discussed in relation to their role(s) in viral genome assembly.

Base Sequence↗
PubMed1983

Molecular cloning of double-stranded RNA virus genomes.

Genome double-stranded RNAs isolated from purified human reovirus (serotype 3) and rotavirus (Wa strain) were modified at the 3' termini by addition of oligo(C) approximately 15 with T4 RNA ligase. These RNAs were transcribed into cDNA by oligo(dG)-primed reverse transcriptase and cloned after insertion into pBR322 at the Pst I site. Hybridization of plasmid-transformed Escherichia coli RR1 colonies with 32P-labeled viral genome RNAs demonstrated the presence of DNA clones representative of each of the 10 reovirus RNAs and 10 of the 11 constituent segments of the rotavirus genome. Analyses of the size and terminal nucleotide sequences of insert DNAs indicated that some clones contained a full-length copy of the virus genome segment. The complete nucleotide sequence of rotavirus genome segment 11 double-stranded RNA was obtained by using this procedure. It provides a general method for cloning double-stranded RNAs and also nonpolyadenylylated single-stranded RNAs.

Amino Acid Sequence↗
PubMed1983

Capped and conserved terminal structures in human rotavirus genome double-stranded RNA segments.

Both 3'- and 5'-terminal structures of human rotavirus genome double-stranded RNA segments were determined. RNAs were labeled at the 3'-termini with [32P]pCp by incubation with RNA ligase and at the 5'-termini with [32P]phosphate by polynucleotide kinase or, in the case of 5' caps, with 3H by chemical modification with [3H]NaBH4. Examination of radiolabeled termini released by digestion with several base-specific RNases revealed that rotavirus RNA segments are base paired end-to-end and contain the same terminal structures: (formula; see text)

Base Sequence↗
PubMed1982

A unique class of compound, guanosine-nucleoside tetraphosphate G(5')pppp(5')N, synthesized during the in vitro transcription of cytoplasmic polyhedrosis virus of Bombyx mori. Structural determination and mechanism of formation.

Two structurally different classes of oligonucleotides accumulate in vitro in cytoplasmic polyhedrosis virus (CPV) transcription mixtures in molar excess as compared to the completed RNA products. The first class consists of oligonucleotides which correspond to the 5'-terminal sequence of the virus mRNAs (referred to as initiator oligonucleotides). The major species of initiator oligonucleotides are (p)ppApG and (p)ppApGpN together with smaller amounts of homologous capped structures (Furuichi, Y. (1981) J. Biol. Chem. 256, 483-493). In addition to initiator oligonucleotides, CPV transcription mixtures yielded a second new class of compounds which were radiolabeled by [alpha-32P]GTP and resistant to phosphatase digestion. Their structures were identified as G(5')pppp(5')A, G(5')pppp(5')C, G(5')pppp(5')G, and G(5')pppp(5')U. With the exception of G(5')pppp(5')G, these compounds have not been observed previously. The mechanism of synthesis of these unique compounds was elucidated as pppG + pppN leads to GppppN + PPi. The reaction resembles, in principle, a guanylylation reaction which occurs during cap formation in CPV and other eukaryotic mRNA syntheses. It is likely that these compounds are formed in a similar way by a condensation reaction involving a viral guanylyltransferase-pG intermediate complex and ribonucleoside triphosphate. When the amounts of G(5')pppp(5')N were measured, it was found that G(5')pppp(5')N reached maximum concentrations (0.4 to 0.7 microM) shortly after the onset of RNA synthesis (1 h) and these levels were maintained or diminished gradually. By contrast, mRNA and (p)ppApG were continuously synthesized. The relative molar ratios of total G(5')pppp(5')N and (p)ppApG versus mRNA were comparable (74:24:1 and 30:27:1 during 1 to 4 h transcription, respectively). The results imply that these unusual compounds G(5')pppp(5')N as well as initiator oligonucleotides may be produced reiteratively during initiation when RNA chain elongation and capping are uncoupled.

Animals↗
PubMed1982

Priming of reovirus transcription by GppppG and formation of CpG(5')GpC.

Compounds of general structure N(5')pn(5')N were used by the reovirus-associated RNA polymerase as primers for template-directed synthesis of virus-specific oligonucleotides and RNA. Structural requirements for activity included a guanosine residue and at least four phosphates--i.e., G(5')pppp(5')N. Gp4G incubated with viral cores in the presence of CTP yielded Gp4GpC and CpGp4GpC. In a complete transcription mixture, Gp4G was also incorporated into the 5' termini of full-length transcripts in the unmethylated forms Gp4GpC and CpGp4GpC, in contrast to viral mRNAs that contain 5'-terminal m7GpppGmpC formed de novo. Gp5G, Gp6G, and Gp4A but not Gp2G, Gp3G, and Ap4A also primed reovirus transcription and inhibited RNA methylation.

Dinucleoside Phosphates↗