The long 3′ non-translated regions of Blackcurrant reversion virus RNAs are highly conserved between virus isolates representing different phenotypes and geographic origins

Summary. Blackcurrant reversion virus (BRV) belongs in the subgroup c of nepoviruses. The 3 NTRs of RNAs 1 and 2 of BRV are 1360 and 1363 nucleotides long, respectively, and highly similar (94.8%). In this study we have compared the sequences of the 3 NTRs of ten BRV isolates, originating from different geographic regions or hosts. All deduced sequences were 94.1–98.8% identical with each other, and with the previously deduced 3 NTR sequences of RNAs1 and 2 of the type isolate. The proceeding 480 nucleotides of the CP coding region were 86.9–97.9% identical between the same isolates.     

Sequence determination of the extreme 5′ end of equine arteritis virus leader region

The extreme 5 end of the leader sequence of four equine arteritis virus (EAV) strains was obtained by using rapid amplification of cDNA end method (5 RACE), and sequenced. Seventeen more nucleotides were added upstream of the 5 end of the EAV published genomic sequence. A common feature among the analyzed EAV isolates was the presence of an AUG start codon within the added sequence and the appearance of an intraleader open reading frame (ORF) of 111 nucleotides which was predicted to encode a peptide of 37 amino acids. The role of this putative intraleader ORF has yet to be determined.     

Nucleotide sequence comparison of the 3′-terminal regions of severe,mild, and non-papaya infecting strains of papaya ringspot virus

Summary The 3-terminal 2,561 nucleotide residues of the severe HA strain of papaya ringspot virus (PRSV) was determined. Comparison with the published sequence of the mild strain PRSV HA 5-1 showed that they shared a 99.4% identity in their 3-terminal 2,235 residues. There were ten residues different at the NIb gene, resulting in five amino acid changes, and two residues different in the coat protein gene, resulting in two amino acid changes. The 3-untranslated regions were identical, but HA contained two more nucleotides (AG) at the 3 extreme. Comparison with the published non-papaya infecting type W strain PRSV-W revealed that they shared a 97.9% identity in their 3-terminal 2,235 residues. There were 40 nucleotides different in the coding region, which resulted in four amino acid changes in the NIb gene and six in the CP gene, and seven nucleotides different in the 3-untranslated region.     

On the variability of the 3′ terminal sequence of the turnip mosaic virus genome

Summary The sequence of the 3-terminal 1223 nucleotides (nts) of a Japanese isolate of turnip mosaic virus (TuMV-Jap) RNA has been determined. The sequence reveals a single open reading frame (ORF) which terminates at a position 212 nts upstream of the 3 poly(A)-tract. Determination of the N-terminal amino acids of TuMV-Jap coat protein (CP) mapped the CP cistron within this ORF and revealed a Glu-Ala dipeptide sequence as the putative cleavage site by which the CP is released from the viral polyprotein. The predicted amino acid sequence of the TuMV-Jap CP shows 97.2% identity with that of a Canadian isolate of TuMV (TuMV-Can) and 99% with a second, Chinese, isolate (TuMV-Chi). However, the 3-terminal non-translated region (NTR) of TuMV-Jap RNA is significantly shorter (212 nts) than the 3-NTR of TuMV-Can RNA (668 nts), but of equal length as the 3-NTR of the TuMV-Chi isolate which also measures 212 nts. The 3-NTRs of both the TuMV-Jap and TuMV-Chi RNAs show homology with the first 201 nucleotides of the TuMV-Can RNA 3-NTR. A search in the EMBL nucleotide sequence database revealed that the 467 nt-long unique extension of the 3-NTR of TuMV-Can RNA has 89.8% homology to a part of the chloroplast ribosomal protein 12 gene (rsp 12-gene). Irrespective of the origin of this extra sequence in the reported TuMV-Can sequence, which may have been introduced by a genuine RNA recombination event, it is concluded that the standard TuMV genome has a CP gene of 864 nts and an conserved 3-NTR of approximately 212 nucleotides in length.     

Molecular evidence that the whitefly-transmitted sweetpotato mild mottle virus belongs to a distinct genus of thePotyviridae

Summary Complementary DNA representing 2 108 nucleotides at the 3 end of the genomic RNA of the whitefly-transmitted sweetpotato mild mottle virus (SPMMV) was cloned after PCR. Sequence analysis revealed an open reading frame of 1 797 nucleotides which codes for a protein of 599 amino acids, followed by a 3 non-coding region of 311 nucleotides. Alignment of the deduced amino acid sequence with corresponding sequences of other members of thePotyviridae demonstrated that part of the presumptive RNA-dependent RNA polymerase and the coat protein coding regions of SPMMV are found at the 3 end of its genome, in that order. Alignment of the amino acid sequence of the core of SPMMV coat protein with those of selected members of thePotyviridae showed limited identity, thus demonstrating — with phylogenetic analysis — that SPMMV belongs to a distinct genus of the familyPotyviridae.     

RNA binding properties of core protein of the flavivirus Kunjin

Summary Kunjin virus (KUN) C is a typical flavivirus core protein which is truncated in vivo to a mature form of 105 residues enriched in lysine and arginine. In order to study the possible association of KUN C with RNA in vitro, we prepared several recombinant C proteins with specific deletions, each fused at the amino-terminus to glutathione-S-transferase (GST) and expressed inE. coli. They were reacted with KUN RNA probes transcribed in vitro from cDNA representing the 5 untranslated region (5 UTR, 93 of 96 nucleotides), the 3 UTR (624 nucleotides), and the 5 UTR plus most of the C coding region (t core, 440 nucleotides). Fusion protein C107 (incorporating mature C) bound strongly to all KUN RNA probes with apparent specificity, being completely resistant to inhibition by 800 mM NaCl, and to competition by a large excess of tRNA. In reactions with labelled KUN RNA probes putative binding sites were identified in the isolated amino-terminal (32 residues) and carboxyterminal (26 residues) basic amino acid domains; this binding was strongly competed by unlabelled KUN UTR probes but weakly or not at all by tRNA. These small domains probably acted co-operatively in binding of mature C to KUN RNA probes. The KUN RNA-core protein binding reactions are similar to those reported with other viral coat or capsid proteins and viral RNAs.     

3′-Terminal sequences of influenza C virion RNA

Summary The 3-terminal nucleotides of the genome segments of influenza C/Taylor/47, C/Bavaria/79 and C/Johannesburg/1/66 were identified by two RNA sequencing techniques. These comprised 11 nucleotides (3 UCGUCUUCGUC) which were found to be conserved among the genome segments of each virus.With 3 Figures     

Genetic Analysis of Pestiviruses at the 3′ End of the Genome

Specific PCR primers were selected for each pestivirus genotype which flanked the 3-part of the NS5B gene and more than three quarters of the 3-UTR. PCR products were sequenced in both directions using an automatic sequencing device and analyzed by computer package program DNASTAR. A comparative analysis of the 3 untranslated region (3-UTR) of 82 viruses, representing the four genotypes of the Pestivirus genus, provided a similar phylogenetic grouping as other genomic regions. Intertypic recombination was not observed, but Border disease virus (BDV) and Bovine viral diarrhoea virus type 1 (BVDV I) showed great intragenotypic variability. In most pestiviruses the stop codon is TGA, but BDV isolates were found to have either a TAG or a TAA stop codon. Various deletions and insertions were observed in the 3-UTR region. Viruses of the BVDV Ib group contained a characteristic deletion of 41 nucleotides. Compared to the 5-UTR, the 3-UTR was less conserved. The first 50–60 nucleotides were particularly variable, whilst the most conserved part was found at the 3 end of the studied region. All 82 viruses contained AT-rich stretches, the positions and sizes of which differed between the genotypes.     

Full-genome Nucleotide Sequence of a Hepatitis C Virus Variant (Isolate Name VAT96) Representing a New Subtype within the Genotype 2 (Arbitrarily 2k)

Hepatitis C virus (HCV), a single-stranded RNA virus of the family Flaviviridae, has a wide range of genetic heterogeneity: 6–11 genotypes (or 6 clades) have been known and each genotype comprises multiple subtypes. Here we report the entire nucleotide sequence of an HCV isolate from a patient in Moldova with chronic hepatitis (isolate name VAT96). The genetic organization of VAT96 was, from 5 to 3 ends, 5UTR (341 nt), polyprotein ORF (9099 nt), 3UTR (38 nt except for the poly-U and poly-pyrimidine stretch), and X-tail (98 nt). Comparison of the polyprotein amino acid sequence of VAT96 with those of known full-genome isolates assigned VAT96 to the genotype 2 (or clade 2), and further phylogenetic analysis based on a 447-nt sequence that covers part of the C and E1 regions suggested that VAT96 represents a new subtype within the genotype 2, arbitrarily designated 2k VAT96 was unique in that it possessed a U residue prior to GCC at the 5 end of its genome while all the other full-genome HCV sequences start with GCC or ACC. In addition, the polyprotein ORF of HCV-VAT96, like HCV-BEBE1 of 2c, encoded several additional amino acids in excess, compared to 2a and 2b sequences. Despite these characteristics that may be unique to VAT96, the 98-nt sequence of the X-tail of VAT96 was highly homologous to those of other isolates with different genotypes so far reported.     

The nucleotide sequence of the coat protein genes and 3′ non-coding regions of two resistance-breaking tobamoviruses in pepper shows that they are different viruses

Summary The nucleotide sequence of the coat protein genes and 3 non-coding regions of two different resistance-breaking tobamoviruses in pepper have been determined. The deduced coat protein of an Italian isolate of pepper mild mottle virus (PMMV-I) consists of 156 amino acids and its 3 non-coding region is 198 nucleotides long. They have been found to be very similar in sequence and structure to those previously reported for a Spanish isolate (PMMV-S). In contrast, a Dutch isolate termed P 11 codes for a coat protein of 160 amino acids and its 3 non-coding region is 291 nucleotides long, which may have arisen by duplication. The nucleotide and the predicted coat protein amino acid sequence analysis show that this isolate should be considered as a new virus within the tobamovirus group. The term paprika mild mottle virus (PaMMV) is proposed.     

Phosphorylation-regulated low-conductance Cl− channels in a human pancreatic duct cell line

A low-conductance Cl^– channel has been identified in the apical membrane of the human pancreatic duct cell Capan-1 using patch-clamp techniques. Cell-attached channels were activated by the vasoactive intestinal polypeptide (VIP, 0.1 mol/l), dibutyryl-adenosine 3,5-cyclic monophosphate (db-cAMP, 1 mmol/l), 8-bromo adenosine 3,5-cyclic monophosphate (8-BrcAMP, 1 mmol/l), 3-isobutyl-1-methyl-xanthine (IBMX, 100 mol/l) and forskolin (10 mol/l). No channel activity was observed in non-stimulated control cells. In both cell-attached and excised inside-out patches, the channel had a linear current/voltage relationship and a unitary conductance of 9 pS at 23°C and 12 pS at 37°C. Its opening probability was not voltage dependent although pronounced flickering was induced at negative potentials. Anionic substitution led to the selectivity sequence Cl^–>I^–>HCO₃ ^–>gluconate. In insideout excised patches, the channel activity declined spontaneously within a few minutes. Reactivation of silent excised channels was achieved by adding protein kinase A (PKA, in the presence of ATP, cAMP and Mg²⁺). Conversely, active channels were silenced in the presence of alkaline phosphatase. The PKA-activated Cl^– channel was 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS, 100 mol/l) and 4-acetamido-4-isothiocyanatostilbene-2, 2-disulphonic acid (SITS, 100 mol/l) insensitive, but was blocked by diphenylamine-2-carboxylic acid (DPC, 100 mol/l). These results demonstrate that the apical low-conductance Cl^– channel in Capan-1 is regulated on-cell by VIP receptors via cAMP and off-cell by PKA and phosphatases. They provide evidence that this channel is closely related to the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel.     

Cloning of the 3′-terminal region encoding movement and coat proteins of a Korean isolate of odontoglossum ringspot virus

Summary The 3-terminal 1855 nucleotides (nts) of a Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) were cloned and sequenced. The sequence contained two open reading frames, which encode the cell-to-cell movement protein (MP) and coat protein genes, and are 912 nts and 477 nts long, respectively. The MP gene contained a conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA locating between 1117 nts and 1292 nts from the 3-end. The 3 untranslated region (UTR) of the virus comprises 414 nts, includes nine pseudoknots and a tRNA-like structure domain containing aminoacyl acceptor arm and the anticodon hairpin loop coding for histidine.The nucleotide sequence data for ORSV-Cy reported in this paper will appear in the EMBL, GenBANK and DDBJ nucleotide databases under the accession numbers X78966 and X80053.     

Zur Bedeutung des cyclischen Adenosin-3′:5′-monophosphat als „second messenger“ bei der Säuresekretion des Magens

Zusammenfassung Ob bei einer bestimmten Hormonwirkung cyclisches Adenosin-3:5-monophosphat (cAMP) als intracellulärer Vermittler fungiert, läßt sich nach den von Sutherland entwickelten Kriterien beurteilen; dabei sind die qualitativen, quantitativen und zeitlichen Beziehungen zwischen dem Hormoneffekt auf den intracellulären cAMP-Spiegel und die jeweilige physiologische Antwort zu prüfen. Im Falle der Histamin- bzw. Pentagastrin-stimulierten gastralen Säuresekretion des Frosches (Necturus maculosus) und der Ratte erfüllt das cAMP die Bedingungen eines second messenger. Dagegen ließ sich beim Hund und Menschen eine derartige Funktion des cAMP bisher nicht verifizieren; ob bei diesen Species cyclisches Guanosin-3:5-monophosphat anstelle des cAMP die intracelluläre Vermittlerrolle übernimmt, werden erst noch systematische Untersuchungen erweisen müssen.Die in diesem Zusammenhang zitierten eigenen Arbeiten wurden von der Deutschen Forschungsgemeinschaft, Bad Godesberg, unterstützt.     

Predicted stem-loop structures and variation in nucleotide sequence of 3′ noncoding regions among animal calicivirus genomes

Caliciviruses are nonenveloped with a polyadenylated genome of approximately 7.6 kb and a single capsid protein. The RNA Fold computer program was used to analyze 3-terminal noncoding sequences of five feline calicivirus (FCV), rabbit hemorrhagic disease virus (RHDV), and two San Miguel sea lion virus (SMSV) isolates. The FCV 3-terminal sequences are 40–46 nucleotides in length and 72–91% similar. The FCV sequences were predicted to contain two possible duplex structures and one stem-loop structure with free energies of –2.1 to –18.2 kcal/mole. The RHDV genomic 3-terminal RNA sequences are 54 nucleotides in length and share 49% sequence similarity to homologous regions of the FCV genome. The RHDV sequence was predicted to form two duplex structures in the 3-terminal noncoding region with a single stem-loop structure, resembling that of FCV. In contrast, the SMSV 1 and 4 genomic 3-terminal noncoding sequences were 185 and 182 nucleotides in length, respectively. Ten possible duplex structures were predicted with an average structural free energy of –35 kcal/mole. Sequence similarity between the two SMSV isolates was 75%. Furthermore, extensive cloverleaflike structures are predicted in the 3 noncoding region of the SMSV genome, in contrast to the predicted single stem-loop structures of FCV or RHDV.Disclaimer: No endorsements are herein implied. Brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standards of the products, and the use of the names by the USDA implies no approval of the products to the exclusion of others that may also be suitable.     

Sequence of the 3′-terminal region of a Zimbabwe isolate of cowpea aphid-borne mosaic virus (CABMV)

Summary The 3-terminal 1221 nucleotides of a Zimbabwe isolate of cowpea aphid-borne mosaic potyvirus (CABMV) genome have been sequenced. The sequence comprises an open reading frame (ORF) of 990 nucleotides and a 3 non-coding-region of 231 nucleotides followed by a poly-A. The ORF has high similarity to NIb and coat proteins (CP) of potyviruses. A potential CP Q/S cleavage site was identified, yielding a CP of 30.5 kDa containing 275 amino acids. The CABMV sequence is closely related to that of South African passiflora virus (SAPV) which should therefore be regarded as a strain of CABMV.The sequence described in this paper has been deposited in the EMBL database under accession number X82873.     

DNA fingerprinting involving fluorescence-labeled termini of any enzymatically generated fragments of DNA

Summary We have developed a new fluorescence-based method for DNA fingerprinting that does not require a fluorescent linker or a synthetic oligonucleotide primer, both of which are normally used for labeling of DNA. Cosmid DNAs are digested with appropriate restriction enzymes and the 3 termini of DNA fragments are labeled with the corresponding, fluorescent dye-conjugated dideoxynucleotide triphosphate terminator (dye-ddNTP) by the Klenow fragment of DNA polymerase I fromEscherichia coli, which has 35 exonuclease and replacement activities as well as its main 53 polymerase activity. Samples are separated on a DNA-sequencing gel and data are analyzed by application of both the Version 0.3.8a mapper program (Applied Biosystem Inc., Foster City, CA) and our Overlap I program that facilitate rapid analysis of the frequency of overlapping of cosmid DNAs. Using this method we have determined the overlap frequency of DNA fragments of each cosmid clone from the mouse MHC class I gene cluster.     

Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence

Summary Recently, a parvo-like virus was newly isolated from silkworm larvae and the two viral DNAs (VD1 and VD2) with different electro-mobilities were identified. We cloned the viral DNAs in a plasmid pUC119 and demonstrated that these two DNAs were not a bimorphic molecules though they shared a common terminal sequence of 53 nucleotides. In addition, the sequence at the 5 terminus of each strand of the viral DNA was located in inverted form at its 3 terminus. On the other hand, the nucleotide sequences of VD1 and VD2 were different from that of the Bombyx densovirus (Ina isolate) DNA.     

A Matrix Associated Region Localizes the Human SOCS-1 Gene to Chromosome 16p13.13

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3 end of the human PRM1 PRM2 TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3 of the spermatid-expressed PRM1 PRM2 TNP2 domain at position 16p13.13.     

Bean yellow mosaic,clover yellow vein,and pea mosaic are distinct potyviruses: evidence from coat protein gene sequences and molecular hybridization involving the 3′ non-coding regions

Summary The sequences of the 3 1019 nucleotides of the genome of an atypical strain of bean yellow mosaic virus (BYMV-S) and of the 3 1018 nucleotides of the clover yellow vein virus (CYVV-B) genome have been determined. These sequences contain the complete coding region of the viral coat protein followed by a 3 non-coding region of 173 and 178 nucleotides for BYMV-S and CYVV-B, respectively. When the deduced amino acid sequences of the coat protein coding regions were compared, a sequence identity of 77% was found between the two viruses, and optimal alignment of the 3 untranslated regions of BYMV-S and CYVV-B gave a 65% identity. However, the degree of homology of the amino acid sequences of coat proteins of BYMV-S with the published sequences for three other strains of BYMV ranged from 88% to 94%, while the sequence homology of the 3 untranslated regions between the four strains of BYMV ranged between 86% and 95%. Amplified DNA probes corresponding to the 3 non-coding regions of BYMV-S and CYVV-B showed strong hybridization only with the strains of their respective viruses and not with strains of other potyviruses, including pea mosaic virus (PMV). The relatively low sequence identities between the BYMV-S and CYVV-B coat proteins and their 3 non-coding regions, together with the hybridization results, indicate that BYMV, CYVV, and PMV are distinct potyviruses.     

Komplement- und Antikörper-Titer bei Patienten mit chronischer Bronchitis

Zusammenfassung Bei insgesamt 76 Patienten mit chronischer Bronchitis wurden Komplement(C)- und Antikörper(Ak)Titer (gegen die jeweiligen aus Sputumproben gezüchteten Keime) bestimmt. Die C-Titer (CH50/ml) lagen bei 57 Patienten (= 75%) im Normbereich, bei 17 Patienten (= 22,4%) oberhalb und bei 2 Patienten (= 2,6%) unterhalb der Grenzwerte bei Kontrollpersonen. Dabei tendierten bei höheren Ak-Titern die C-Titer zu niedrigeren Werten. Es wird vermutet, daß kontinuierliche Immunreaktionen den relativen Abfall der C-Titer bei gleichzeitig hohen Ak-Titern bedingen.

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Summary In 76 patients with chronic bronchitis total complement (C) activity and antibody titer (versus correspondent microorganisms cultured from sputum samples) were determined. 57 patients (= 75%) were found to have normal C titers (CH50/ml), in 17 cases (= 22,4%) the C titers were elevated above and in 2 cases (= 2,6%) reduced under limiting values of control persons. The C titers tended to lower levels when antibody titers increased. It is supposed that continuous immune reactions cause the relative decrease of complement activity when antibody titers are increased.

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Mit finanzieller Beihilfe der Europäischen Gemeinschaft für Kohle und Stahl durchgeführte Forschungsarbeit.