The complete nucleotide sequence of apple mosaic virus RNA-3

Summary The complete nucleotide sequence of apple mosaic ilarvirus (ApMV) RNA-3 has been determined from cloned viral cDNAs. The 5 terminus of RNA-3 was determined by direct RNA sequencing, while the 3 end was determined by polyadenylation of genomic RNA and sub-cloning using oligo dT. ApMV RNA-3 is 2056 bases in length and encodes at least two open reading frames. It is similar in size and genome organization to the RNA-3 of other members of theBromoviridae, which includes ilarviruses. The CP gene is in the 3 half of the molecule, and another large open reading frame is upstream of the CP gene and can potentially encode a protein of 32 400 daltons. This peptide is the same size and shows limited sequence homology to an open reading frame located at the 5 end of RNA 3 in tobacco streak and prune dwarf ilarviruses and alfalfa mosaic virus, which is postulated to be the viral movement protein. The nucleic acid sequence was not homologous to tobacco streak virus, prune dwarf virus, alfalfa mosaic virus or other members of theBromoviridae. The 5-non-coding region of ApMV RNA-3 contains a 15 base palindromic sequence which encloses a sequence resembling the ICR-2 regions of eukaryotic tRNA gene promoters.     

Phylogenetic justification for splitting theRymovirus genus of the taxonomic familyPotyviridae

Summary ThePotyviridae family has been divided into four genera on the basis of vector transmission, as follows:Potyvirus genus (aphid),Rymovirus genus (mite),Bymovirus genus (fungus) andIpomovirus genus (whitefly). However recent sequence comparisons of the coat protein and 3 NCR regions of the potyviruses have demonstrated that the rymoviruses appear to be a group of two unrelated clusters namely Ryegrass Mosaic Virus (RGMV), Agropyron Mosaic Virus (AgMV) and Hordeum Mosaic Virus (HoMV) in one group and Wheat Streak Mosaic Virus (WSMV) and Brome Streak Mosaic Virus (BrSMV) in the second group. We therefore propose that RGMV, AgMV and HoMV remain in the genusRymovirus and WSMV and BrSMV form a separate genus, possibly theWhestrevirus genus.     

Stereochemical aspects of the interaction of myosin and actomyosin with nucleotides

Summary The five possible analogues of ATP and the three possible analogues of ADP which contain single non-bridging sulphur atoms instead of oxygen in the polyphosphate structure have been used as probes of the interaction of nucleotides with myosin and actomyosin. Evidence is presented for the requirement of an , , -tridentate complex of magnesium and ATP as the substrate for myosin. Of the four possible tridentate MgATP diastereomers, the exo isomer (nomenclature of Cornelius & Cleland, 1978) appears to be the actual substrate.Abbreviations ATP Adenosine-5-O-triphosphate - ATP(-S) Adenosine-5-O-(1-thiotriphosphate) - ATP(-S) Adenosine-5-O-(2-thiotriphosphate) - ATP(-S) Adenosine-5-O-(3-thiotriphosphate) - ADP Adenosine-5-O-diphosphate - ADP(-S) Adenosine-5-O-(1-thiodiphosphate) - ADP(-S) Adenosine-5-O-(2-thiodiphosphate) - Enzyme Myosin ATPase (EC 3.6.1.3)     

Attenuated Lapinized Chinese Strain of Classical Swine Fever Virus: Complete Nucleotide Sequence and Character of 3′-Noncoding Region

The complete nucleotide sequence including precise 5- and 3-terminal non-coding regions (NCRs) of the attenuated lapinized Chinese strain (HCLV) of Classical Swine Fever Virus (CSFV) was determined from overlapping cDNA clones constructed by separated RT-PCR and rapid amplification of cDNA ends (RACE) methods. The genomic RNA of the HCLV strain consists of 12,310 nucleotides (nts) including 374nts and 242nts in the 5- and 3-NCRs, respectively. It contains one large open reading frame (ORF) encoding a polyprotein of 3,898 amino acids with a calculated molecular weight of 437.6kDa. There is one notable insertion of 12 continuous nts, CTTTTTTCTTTT in the 3-NCR of HCLV genomic cDNA when compared with its parental virulent Shimen strain. Sequence alignment of partial 3-NCR reveals two groups of CSFV vaccine strains carrying similar T-rich insertions at different positions in this region. Computer-predicted secondary structures suggest that T-rich insertion greatly change the structures and thus decrease the promoter functions of 3-NCRs during the replications of these two groups of CSFV vaccine strains.     

Repetitive sequences in the ITS1 region of ribosomal DNA in congeneric microphallid species (Trematoda: Digenea)

In searching for species-specific DNA sequences of microphallid species (Digenea, Trematoda) we examined the ribosomal internal transcribed spacer regions (ITS) of three closely related species (Levinseniella group) hosted by mud snails (first intermediate host) and marine crustaceans (second intermediate host). In the ITS1 region we found consistent patterns of repeating sequences of 130 bp. Within each main repeat there was a varying number of subrepeats specific for each of the species. All repeats including subrepeats were identified by a similar starting sequence: 5-CCTGTGG-3. As this sequence has close resemblance to the chi sequence 5-GCTGGTGG-3 found in phage we speculate if it serves the same function as a recombination hotspot. Alternatively but less likely, it could be an inactive, mutational relic of a sequence that once served this purpose.     

Nucleotide sequence and determination of the extremities of the 26S ribosomal RNA gene in wheat mitochondria: evidence for sequence rearrangements in the ribosomal genes of higher plants

Summary The nucleotide sequence of the wheat mitochondrial 26S ribosomal RNA gene and flanking regions was determined and compared with mitochondrial 26S rRNA genes from maize and Oenothera. All three genes exhibit a high degree of homology except within two variable regions. When the plant mitochondrial 26S rRNA genes are compared with Escherichia coli 23S rRNA and chloroplast 23S and 4.5S rRNA genes, a third variable region is apparent close to the 3 end of the gene. The 5 and 3 ends of the wheat mitochondrial gene were determined by S1 nuclease mapping. Computer analysis of the wheat mitochondrial gene revealed several small sequences present either in the 5 region of the 26S rRNA gene or in the 18S rRNA gene.     

Regulation of the trehalose-6-phosphate synthase complex in Saccharomyces

Summary Trehalose-6-phosphate synthase is another example of an enzyme of carbohydrate metabolism, in Saccharomyces, which could be regulated by interconversion of forms. Deactivation was mediated both in vivo and in vitro by a cyclic AMP-dependent protein kinase. Reversibility of this process was obtained by a phosphatase treatment leading to an increase in activity. The phosphorylated, less active form of the enzyme proved to be more susceptible to activation by ATP.Mg. Mutants with well defined lesions in the cyclic AMP-dependent protein kinase system were used to corroborate our findings of a possible regulatory mechanism of trehalose-6-phosphate synthase activity by interconversion of forms.Abbreviations PMSF phenyl-methyl-sulfonyl fluoride - G-6-P glucose-6-phosphate - UDPG uridine-5-diphosphoglucose - PEP phosphoenol pyruvate - NAD⁺ -nicotinamine adenine dinueleotide - ATP adenonise 5-triphosphate - cAMP adenosine 2:3-cyclic monophosphate - MOPS 3 (N-morpholino) propanesulfonic acid     

A defective proviral DNA with a 2.6-kb deletion of human immunodeficiency virus type 1 (HIV-1) in a persistently HIV-1 infected cell clone

A cell line (H2-5) producing defective doughnut-shaped particles of human immunodeficiency virus type 1 (HIV-1) was found to contain proviral DNA with a large deletion of 2558 bases, corresponding to the 3 half ofpol gene, the entirevif andvpr genes, and the 5 terminal of thetat gene.     

Roles of the polypyrimidine tract and 3′ noncoding region of hepatitis C virus RNA in the internal ribosome entry site-mediated translation

Summary. Hepatitis C virus (HCV) genome contains a 3noncoding region (3NCR) consisting of a variable region, a polypyrimidine tract (polyU/UC) and the X region. To examine the roles of 3NCR and polyU/UC tract in the internal ribosome entry site (IRES)-mediated translation process, a variety of 3NCRs containing different lengths of polyU/UC tract were obtained from HCV infected patients and cloned respectively to the downstream of the firefly luciferase coding gene linked to HCV 5NCR and 30 nucleotides of core gene (containing IRES element). The results of in vitro translation in rabbit reticulocyte lysate (RRL) and cell transfection assay in mammalian cells showed that the IRES-mediated translation efficiency could be enhanced by the full-length of 3NCR of HCV RNA. However, contradictory results were observed when the role of polyU/UC tract in the IRES-mediated translation was studied. While the IRES-mediated translation efficiency was inhibited by the presence of polyU/UC tract in in vitro translation experiments, transfection of these expression cassettes into hepatic cell line showed that polyU/UC tract enhanced IRES-mediated translation efficiency in vivo. Cellular-fraction complement experiments showed that cellular factors were required for the enhancement by the polyU/UC tract. Further antibody blocking assay and UV cross-linking assay suggested the correlation of IRES-mediated translation with host factors, including the La protein. The data above also indicated that the modulations of the IRES-mediated translation by the HCV 3NCR and the polyU/UC tract were in a length-independent manner.These authors contributed equally to this work.     

Specific Interaction of a 25-kilodalton Cellular Protein,a 40S Ribosomal Subunit Protein,with the Internal Ribosome Entry Site of Hepatitis C Virus Genome

Translation initiation of hepatitis C virus (HCV) RNA is controlled by an internal ribosome entry site (IRES) contained in 5 noncoding region (NCR) and in several nucleotides of the coding region. The ability of a 25-kilodalton cellular protein (p25) to bind the HCV 5 NCR is correlated with the efficiency of translation initiation of HCV RNA, indicating that this protein plays a critical role in HCV translation (S. Fukushi, C. Kurihara, N. Ishiyama, F. B. Hoshino, A. Oya, and K. Katayama, J Virol 71, 1662–1666, 1997). We have extended the study for identification of the IRES region required for p25 binding. For this purpose, we have performed UV cross-linking competition analyses using 5- or 3- deleted mutants of the HCV 5 NCR as competitor RNAs for binding of p25 to wild-type HCV 5 NCR. Competitor RNAs lacking nucleotides (nt) 47–74 or nt 279–331 did not inhibit p25 binding to the HCV IRES, indicating that these regions are necessary for interaction of the p25 and HCV IRES. Since p25 binding was not observed in the IRES elements of encephalomyocarditis virus and poliovirus in UV cross-linking competition analyses, the p25 binding may be specific for the HCV IRES. p25 bound to the HCV IRES was detected when a purified 40S ribosomal subunit was used for UV cross-linking experiment, indicating that p25 is one of 40S ribosomal subunit proteins. These results reveal an unique interaction between the 40S ribosomal subunit and HCV IRES to contribute to translation initiation of the HCV genome.     

Dissociation of acetylcholine- and cyclic GMP-induced currents inXenopu oocytes

InXenopus follicular oocytes, activation of muscarinic receptors evokes a slow potassium current (H-response); a similar current is evoked by intracellular injection of cyclic guanosine 3,5-monophosphate, cGMP (Dascal et al. 1984). We have tested the hypothesis that cGMP may be the second messenger that mediates the opening of K channel by acetylcholine (ACh). ACh elevated the intracellular level of cGMP with a time course similar to that of the development of the muscarinic H-response; maximal increase in cGMP concentration above the control was about 0.2 pmole/oocyte. The amount of injected cGMP that produced a detectable K current (threshold dose) varied between 0.5 and 3 pmole/oocyte. At low doses of cGMP, the slope of log dose-log response curve was about 2.5, suggesting involvement of a biochemical process with a positive cooperativity of at least 3. Higher doses of cGMP evoked, in addition to the outward current, an irregular, rapidly developing, long-lasting inward current, that never reached amplitudes comparable to those of ACh-evoked Cl currents. The K current elicited by cGMP was insensitive to elevation or depletion of external Ca. It was potentiated by isobutylmethylxanthine (IBMX). ACh strongly inhibited the cGMP-evoked K current when applied at the plateau of the latter. 4-Phorbol 12,13-dibutyrate (PDBu) (1 M) rapidly and completely inhibited the cGMP response. It is concluded, that most of the results presented in this report contradict the hyothesis that cGMP is the intracellular mediator of ACh-induced changes in membrane conductance in the oocytes.Abbreviations ACh acetylcholine - cAMP cyclic adenosine 3,5-monophosphate - cGMP cyclic guanosine 3,5-monophosphate - EGTA ethylenediaminetetraacetic acid - Hepes N-2-hydroxyethyl-piperazinc-N-2-hydroxypropanesulphonic acid - IBMX 3-isobutyl-l-methylxanthine - IP₃ inositol 1,4,5-trisphosphate - PDBu 4-phorbol 12,13-dibutyrate     

Characterization of the hog cholera virus 5′ terminus

Hog cholera virus (HoCV) 5 terminus of the ALD and GPE(–) strains were analyzed by using rapid amplification of cDNA end method (5RACE). An additional nine nucleotides were found at the 5 termini of genomic RNA in the ALD and GPE(–) strains of HoCV. These nine nucleotides were also conserved in BVDV and were suggested to form a hairpin structure at the 5 terminus by computer-assisted analysis. It seems possible that the secondary structure and/or the 5 terminus sequence has a significant role in the HoCV virus genome.     

Determination of the 5′ and 3′ terminal noncoding sequences of the bi-segmented genome of the avibirnavirus infectious bursal disease virus

Summary Terminal sequences of the bi-segmented dsRNA genome of 3 different strains of infectious bursal disease virus (IBDV) were analyzed by the rapid amplification of cDNA 5 ends (5RACE) procedure. Both segments are 85% homologous in a 32-nucleotide sequence comprising the 5 end, whereas the 3 end has a conserved pentamer. Comparison to published terminal sequences of other IBDV strains revealed high conservation between the two segments but more serotype-specific nucleotide changes (5 on segment A and 3 on segment B) in the 5 noncoding region compared to the 3 noncoding region (none on segment A and 1 on segment B).     

Complete Genomic Sequence of Viral Hemorrhagic Septicemia Virus,a Fish Rhabdovirus

The complete nucleotide sequence of the fish rhabdovirus viral hemorrhagic septicemia virus (VHSV) has been determined. The genome comprises 11158 bases and contains six long open reading frames encoding the nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G, nonstructural viral protein NV, and polymerase L. Genes are arranged in the order 3-N-P-M-G-NV-L-5. The exact 3 and 5 ends were determined after RNA-oligonucleotide ligation or RACE. They show inverse complementarity as in other rhabdovirus genomes. Nucleotide and deduced amino acid sequences exhibit significant homology to corresponding sequences in the related fish rhabdovirus infectious hematopoietic necrosis virus.     

Ca2+-Mg2+-ATPase activity of human red blood cells in healthy and diabetic volunteers

Summary The present investigation was dedicated to support biochemical interpretations of well-known long-term microvascular complications in diabetes. Provided the hypothetical correlation between erythrocyte membrane rigidity and increased intracellular calcium content holds true, a reduced Ca²⁺-Mg²⁺-ATPase activity in diabetic subjects could represent the underlying biochemical mechanism. Thus, we have compared basal and calmodulin-activated ATPase activity in healthy and diabetic volunteers. We could demonstrate a significant reduction of basal and stimulated enzyme activity in diabetic subjects. Furthermore, partial purification of calmodulin from erythrocytes of diabetic patients and healthy subjects yielded experimental evidence that reduced enzyme activity in diabetic volunteers is due to an altered basal activity as well as to a reduced stimulation by calmodulin.Abbreviations ATP Adenosine 5-triphosphate - ATPase Adenosine 5-triphosphatase - DEAE Diethylaminoethyl - EDTA Ethylenediamine tetraacetate - EGTA Ethyleneglycol-bis-(-aminoethyl ether)N,N-tetraacetic acid - P_i Inorganic phosphate - Tris Tris(hydroxymethyl)aminomethane     

Metabolic studies on vaccinia-virus-infected oligodendrocytes in brain cell cultures

Twelve-day-old cultures of dissociated newborn mouse brain were infected with neurotropic vaccinia virus strain WR. Using the indirect immunofluorescence staining technique the total destruction of galactocerebroside (GL) or myelin basic protein (MBP)-positive oligodendrocytes could be detected after 72 h of infection. The activity of the oligodendrocyte-specific enzymes, cerebroside sulfotransferase (CST) and 23-cyolionucleotide 3-phosphohydrolase (CNP), was 27% and 17% respectively of the activity in noninfected controls. This reduction was not a result of viral-induced inhibition of host protein synthesis. In cultures treated with puromycin GC- and MBP-positive oligodendrocytes were detectable at a time at which no CST or CNP activity could be detected.     

Evolution of a Common Structural Core in the Internal Ribosome Entry Sites of Picornavirus

The translational control involving internal ribosome binding occurs in poliovirus (PV), human rhinoviruses (HRV), encephalomyocarditis virus (EMCV), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Internal ribosome binding utilizes cis-acting genetic elements of approximately 450 nucleotides (nt) termed the internal ribosome entry sites (IRES) found in these picornaviral 5-untranslated region (5UTR). Although these IRES elements are quite different in their primary sequence, a similar folding structure with a conserved 3 structural core exists in the IRES. Phylogenetic analysis and RNA folding of the 5 UTR of picornaviruses, including PV types 1–3, coxsackievirus types A and B, swine vesicular disease virus, echoviruses, enteroviruses (human and bovine), HRV, HAV, EMCV, mengovirus, Theilers murine encephalomyelitis viruses, FMDV, and equine rhinoviruses, indicates that the predicted conserved structural core is indeed a general structural feature for all members of the picornavirus family. The evolution of a common structural core likely occurred by the gradual addition or deletion of structural domains and elements to preserve a similar tertiary structure that facilitates the utilization of the IRES in specific host-cell environments.     

The complete nucleotide sequences of the coat protein cistron and the 3′ non-coding region of a newly-identified potyvirus infecting sweetpotato,as compared to those of sweetpotato feathery mottle virus

Summary Complementary DNA representing 728 nucleotides of the 3 end of the genomic RNA of sweetpotato virus G (SPV-G), a newly-identified potyvirus infecting sweetpotato, was cloned and sequenced. This sequence was combined with that previously determined for the 5 terminal part of the coat protein cistron of the virus. The whole sequence contained a single open reading frame (ORF) of 1065 nucleotides, with the capacity to encode a coat protein of 355 amino acids, significantly larger than that of other potyviruses. The ORF was followed by an untranslated region of 222 nucleotides and a poly (A) tail. The coat protein of SPV-G was only distantly related to that of known potyviruses, with the exception of sweetpotato feathery mottle virus (SPFMV). Indeed, sequence identity in the C-terminal three quarters of the coat protein (more than 80%) and in the 3 untranslated region (more than 70%) indicate that SPV-G should be considered as closely related to, though distinct from SPFMV. This subset relationship is similar to that previously reported for members of the bean yellow mosaic virus subgroup or the bean common mosaic virus subgroup.     

Molecular localization of human IgG anti-F(ab′)2 reactivity with variable- and constant-region λ light-chain epitopes

Human IgG antibodies reacting with antigenic determinants on F(ab)₂ fragments represent generic antiidiotypic antibodies present in the serum of normal individuals. Additionally, the titers of these antibodies in the sera of patients with systemic lupus erythematosus (SLE) are inversely related to disease activity. Because these autoantibodies recognize predominantly light chain-related epitopes, especially type, we synthesized constant (C)- and variable (V)-related overlapping 7-mer peptides on polypropylene pins to determine anti-F(ab)₂-reactive epitopes on human light chains. ELISA reactivity of affinity-purified anti-F(ab)₂ antibodies obtained from normal individuals and from patients with SLE, as well as murine anti-human light-chain monoclonal antibodies specific for C and V subgroup-related determinants, was tested using the overlapping 7-mers of human light-chain sequence. The patterns of reactivity against C-related peptides were similar in both normal and SLE-derived anti-F(ab)₂ antibodies. However, reactivity profiles against V-related peptides were distinctively different between the normal and the SLE-associated anti-F(ab)₂ autoantibodies. A decrease in reactivity among the SLE IgG anti-F(ab)₂ antibodies was noted for particular amino acid V complementarity-determining region (CDR) residues, including glycine at positions 27 and 54, alanine at 16 and 37, and tyrosine at 28 and 91. This different pattern of reactivity from normal may indicate that in SLE there is a failure of antiidiotypic control mechanisms, as reflected by a defect in production of antibody to immunodominant V CDR residues.     

Pseudogenes and short repeated sequences in the rice chloroplast genome

Summary The rice chloroplast genome has been derived from a tobacco-like ancestral form by three major inversions. In the rice genome we have found six pseudogenes, trnG, trnI, 3-rps 12a, trnT, trnE and trnfM/G, all located near inversion endpoints, as well as four short repeated sequences. A comparison of rice, wheat and tobacco sequences indicated that similar pseudogenes are present in wheat but not in tobacco, suggesting that the creation of these pseudogenes occurred before the divergence of rice and wheat. The region downstream of rbcL is a variable region and contains rpl23 in rice and wheat and another 3-rps 12b further downstream in rice. This 3-rps 12b shows a higher homology to the functional rps 12 than 3-rps 12a, which suggests that it appeared more recently. The involvement of these pseudogenes in genome inversions and the creation of the pseudogenes and short repeated sequences are discussed.