Cloning of the potato virus Y genes encoding the capsid protein CP and the nuclear inclusion protein NIb

Summary DNA was synthesized complementary to the RNA genome of potato virus Y (PVY; strain GO 16) and cloned into vectors. The size of PVY-specificEco RI-restricted cDNA ranged from 0.3 to approximately 22kb. Two of the cDNA clones each of which contained some 4kb of cDNA sequence starting from the 3-polyadenylated terminus were characterized by sequence analysis. Presence of a single open reading frame suggests that PVY-specific proteins are synthesized as a polyprotein precursor. As with other sequenced potyvirus RNAs the gene for the PVY capsid protein CP is located next to the 3-untranslated region followed by the genes for the putative RNA polymerase (nuclear inclusion protein NIb) and the virus-specific protease (nuclear inclusion protein NIa). The 3-trailing sequence of the PVY strains cloned is highly homologous to the corresponding region of pepper mottle virus (PeMV) and suggests that PeMV is not a distinct member of the potyvirus group, but another strain of PVY.     

Isolation of the missing 5′-end of the encoding region of the bovine leukemia virus cell receptor gene

Summary The missing 5-end of the encoding region of the bovine leukemia virus (BLV) cell receptor gene (BLVRcp1/5) was isolated from a lambda gt11 cDNA library using the³²P-labeledEcoRI-SamI fragment corresponding to the 5-end of a 2.3 kbp cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor gene (BLVRcp1). The nucleotide and amino acid sequence analysis of the BLVRcp1/5 cDNA revealed that the 1058 bpEcoRI fragment at its 5-end contained a new 114 amino acid long sequence, and at its 3-end contained a completely identical 88 amino acid overlapping region with the 5-end of the BLVRcp1 cDNA. The combined sequences of both cDNAs represent the whole encoding region of the BLV cell receptor gene. The longest open reading frame of the BLV cell receptor gene encodes a protein containing 843 amino acids with a calculated molecular mass of 94.2 kDa which concurs with experimentally detected native BLV receptor protein. Search for homology has shown that about 250 bp of the BLV cell receptor gene is highly homologous to Venter's tag sequences of an unidentified gene from the human brain library.     

Isolation and partial characterization of a full-length cDNA clone for 3α-hydroxysteroid dehydrogenase: A potential target enzyme for nonsteroidal anti-inflammatory drugs

Homogeneous 3-hydroxysteroid dehydrogenase (3-HSD; EC 1.1.1.50) of rat liver cytosol is a monomeric (MR 34000) NAD(P)⁺ dependent oxidoreductase which displays 9-, 11- & 15-hydroxyprostaglandin dehydrogenase activity. The enzyme is potently inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting that 3-HSD may be a target enzyme for NSAIDs. A monospecific, polyclonal anti-sera raised against the purified enzyme was used to screen a gt11 expression library and oligonucleotide probes complementary to the 5 and 3 ends of immunopositive clones were used to isolate a 2.1 kb full-length cDNA. Digestion of the full-length cDNA with Eco RI generated two fragments of 1.1 and 1.0 kb in length. Both fragments were subcloned into pGEM3 and partially sequenced. The 1.1. kb fragment contains the C-terminus of 3-HSD which was confirmed by an in-frame stop codon and comparison of the predicted amino acid sequence to peptide sequence obtained from two endo lys-C peptides of 3-HSD. The 1.0 kb fragment is 5 to the 1.1 kb fragment and is sufficient in length to contain the remainder of the entire open reading frame for 3-HSD. Dideoxysequencing reveals significant sequence homology with bovine lung prostaglandin PGF₂ synthase. These findings support the role 3-HSD in inflammation and suggest that hydroxysteroid dehydrogenases, hydroxyprostaglandin dehydrogenases and prostaglandin F₂ synthase may be members of a common gene family.     

Proteins are attached to the ends of a linear plasmid in the filamentous fungus Ascobolus immersus

Summary In seven out of eleven wild strains of the Ascomycete Ascobolus immersus plasmid DNA was found. There was great variability with respect to size and number of the plasmids in the strains concerned. For a further analysis two plasmids originating from one wild strain were submitted to restriction analysis and electron microscopy. Both turned out to be linear having different molecular weights (pAIl = 7.9 kb, pAI2 = 5.6 kb). Denaturation of pAI2 and subsequent renaturation revealed the presence of inverted repeats (0.7 kb) at both ends. After treatment with proteinase K and 5 and 3 specific exonucleases it became evident that the 5 ends of pAI2 are linked with proteins. In this respect it is similar in structure to other linear genetic elements such as the linear plasmids found in Zea mays and the genomes of adenoviruses.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Rhinovirus detection using probes from the 5′ and 3′ end of the genome

Summary This study investigated the abilities of cDNA probes from the 5 and 3 ends of the genome of human rhinoviruses (HRV-) 14, 9, and 1B to detect RNA from 59 rhinovirus serotypes. The results show that probes from the 5 end of the genomes of HRV-14, 9, and 1B detected a large number of serotypes but the detection rate was variable and depended on the degree of homology with the particular probe. In contrast, all the 3 end probes were specific for the homologous virus. However, along HRV-9 probe detected a large number of serotypes.It was concluded that such cDNA probes would not detect all serotypes with equal efficiency. Synthetic oligonucleotides corresponding to short but highly conserved regions in the 5 non coding region may overcome this problem.     

Comparison of expression of the endo-β-1,3-1,4-glucanase gene from Bacillus subtilis in Saccharomyces cerevisiae from the CYC1 and ADH1 promoters

Summary The endo--1,3-1,4-glucanase gene from B. subtilis was placed under yeast promoter control in a number of different yeast expression vectors. The hybrid plasmids were transformed into S. cerevisiae where they directed the synthesis of varying amounts of active enzyme. The presence of B. subtilis DNA sequences 5 to the initiation codon for the B. subtilis -glucanase gene reduced expression of the gene in yeast. A 1,000-fold increase in the yield of -glucanase was obtained using the ADH1 promoter compared with the CYC1 promoter.     

Mapping of sporulation-specific functions in the yeast syntaxin gene<Emphasis Type="Italic"> SSO1</Emphasis>

The yeast Saccharomyces cerevisiae has two closely related plasma membrane syntaxins, Sso1p and Sso2p, which together provide an essential function in vegetative cells. However, Sso1p is also specifically needed during sporulation; and this function cannot be provided by Sso2p. We used fusions between SSO1 and SSO2 to map the sporulation-specific function of SSO1. We found that the two N-terminal -helices Ha and Hb of Sso1p are important for sporulation, since it is reduced 8-fold for fusions where Ha and Hb are derived from Sso2p. In contrast, the C-terminal half of Sso1p does not seem to be specifically required for sporulation. Surprisingly, we further found that the 3 untranslated region (3UTR) of SSO1 is essential for sporulation. Western blots failed to reveal a preferential expression of Sso1p in sporulating cells, indicating that effects on gene expression are unlikely to explain why the SSO1 3UTR is needed for sporulation.Communicated by S. Hohmann     

Messenger RNA 3′-end formation of a DNA fragment from the human c-myc 3′-end region in Saccharomyces cerevisiae

We have tested the functioning of the human c-myc polyadenylation signal in Saccharomyces cerevisiae. A DNA fragment containing the two AATAAA polyadenylation signals of the c-myc gene was inserted into a plasmid designed for the in-vivo testing of polyadenylation signals in yeast. The c-myc fragment had a partial capacity for directing mRNA 3-end formation in yeast. The 3-endpoints were 50–100 bp distant from the mRNA 3-ends mapped in humans. This human DNA fragment is therefore unspecifically functional in yeast, indicating that other sequence elements than the human polyadenylation signal, AATAAA, are necessary for 3-end formation.     

Acute paw oedema formation induced by ATP: Re-evaluation of the mechanisms involved

ATP-induced inflammation was investigated using subplantar injection in the mouse hind paw. The order of efficacy of purinoceptor agonists for inducing paw oedema (30 nmol per paw) was ATP=,-methylene ATP=2-methylthio ATP > adenosine > UTP > ADP > AMP. Diadenosine polyphosphates effectively induced paw oedema formation with an order of efficacy of: P¹, P⁴-di(adenosine-5)tetraphosphate =P¹,P⁵-di(adenosine-5)-pentaphosphate= P¹,P⁶-di(adenosine-5) hexaphosphate ATP=P¹,P³-di(adenosine-5)triphosphate > P¹,P²-di(adenosine-5)pyrophosphate. Systemic administration of P₂-purinoceptor antagonists (30–100 mol/kg), suramin, 4,4-diisothiocyanatostilbene-2,2-disulphonate, pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid and cibacron blue, reduced the intensity of ATP-induced oedema. At 30 mol/kg 8-(p-sulfophenyl)theophylline (non-selective adenosine receptor antagonist), 3,7-dimethyl-1,1-propargyl-xanthine (adenosine A₂ receptor antagonist), triprolidine (histamine H₁ receptor antagonist), ranitidine (histamine H₂ receptor antagonist) and ketanserin (5-hydroxytryptamine 5-HT₂ receptor antagonist), but neither 8-cyclopentyl-1,3-dipropylxanthine (adenosine A₁ receptor antagonist), nor indomethacin (cyclooxygenase inhibitor) inhibited the ATP-induced swelling. Topical (100 nmol per paw), but not systemic (100 mol/kg) administration of N^G-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor) reduced the intensity of the ATP-induced paw oedema. These results show that ATP can induce an inflammatory oedematous reaction and contribute to our understanding of the underlying mechanisms.accepted by G. Bowen     

3′-Deoxy-3′-fluoroinosine as a potent antileishmanial agent

We studied the antileishmanial activity of 3-deoxy-3-fluoroinosine (3-FI) againstLeishmania tropica andL. donovani. In in vitro cultivation, the EC₅₀ values (the concentration of drug necessary to inhibit the growth rate of cells to 50% of the control value) obtained for 3-FI against the promastigotes ofL. tropica andL. donovani were 2.3×10^–7 and 1.0×10^–6 M, respectively. It was less toxic toward mouse mammary-tumor FM3A cells, a model host; the EC₅₀ value was 1.9×10^–4 M. Leishmania promastigote metabolized 3-FI to 3-deoxy-3-fluoroadenosine 5-triphosphate (3-FATP) but FM3A cells did not. 3-FI was effective againstL. donovani amastigotes in J774.1 cells in an in vitro cultivation system under conditions similar to those used in the in vivo assay. 3-FI (50 mg/kg, given i.v.)showed a cytotoxic effect against the amastigotes ofL. donovani in mice.     

Cyclische Nucleotide als intracelluläre Überträgerstoffe bei Hormonwirkungen

Zusammenfassung Hormone dienen als extracelluläre Informationsüberträger zwischen ihrem Bildungsort, einer endokrinen Drüse, und den Zellen, deren Funktion sie regulieren. Durch die Reaktion des Hormons mit den an der Zellmembran gelegenen Receptoren wird die Aktivität der mit diesen eng verknüpften Adenyl-Cyclase beeinflußt. Die meisten Hormone erhöhen in ihrem Zielorgan die Aktivität dieses Enzyms und führen hierdurch zu einem raschen Anstieg der intracellulären Konzentration von Adenosin-3:5-monophosphat (Ado-3:5-P). Dieses cyclische Nucleotid wird durch eine spezifische Phosphodiesterase zu Adenosin-5-monophosphat abgebaut. Auch die Aktivität dieses Enzyms bestimmt die intracelluläre Ado-3:5-P-Konzentration, die im Vergleich zu der anderer Nucleotide sehr gering ist.Ado-3:5-P beeinflußt als zweiter, intracellulärer Überträgerstoff die Aktivität zahlreicher Schlüsselenzyme. Die Ado-3:5-P-Konzentration bestimmt hierdurch das Gleichgewicht verschiedener Stoffwechselwege zueinander und damit die Reaktion einer Zelle auf eine hormonale Stimulierung. An einer Reihe von Enzymen wird die durch Ado-3:5-P bedingte Aktivitäts-Änderung durch einen gleichartigen Mechanismus bewirkt. Das cyclische Nucleotid stimuliert Proteinkinasen, die eine Phosphatgruppe des ATP auf verschiedene Proteine übertragen und hierdurch deren Eigenschaften verändern können. So steigt bei Phosphorylierung durch eine Ado-3:5-P-stimulierbare Proteinkinase die Aktivität der Triglyceridlipase und der Glykogen-Phosphorylase-b-kinase an, dagegen nimmt die Aktivität der Glykogen-Synthetase ab; durch Phosphorylierung von Histonen kann deren Repressorcigenschaft vermindert und die Synthese von Enzymen gesteigert werden.In manchen tierischen Geweben wurde auch eine spezifisch durch Guanosin-3:5-monophosphat (Guo-3:5-P) stimulierbare Proteinkinase nachgewiesen. Dieses cyclische Nucleotid kommt wie Ado-3:5-P in allen Säugerorganen vor. Die Bildung von Guo-3:5-P aus GTP wird durch die Guanyl-Cyclase katalysiert, ein Ferment, das im Gegensatz zur Adenyl-Cyclase zum großen Teil nicht an die Zellmembranen gebunden ist. Die Konzentration von Guo-3:5-P in verschiedenen Geweben, im Blutplasma und im Urin wird durch Hormone beeinflußt. Es ist noch nicht bekannt, welche hormonalen Regulationen durch Guo-3:5-P vermittelt werden; dagegen ist bei vielen, rasch einsetzenden Hormonwirkungen die Beteiligung von Ado-3:5-P nachgewiesen worden.

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Abkürzungen Ado-3:5-P Adenosin-3:5-monophosphat - dAdo-3:5-P Desoxy-adenosin-3:5-monophosphat - Guo-3:5-P Guanosin-3 : 5-monophosphate - Nuc-3:5-P Nucleosid-3:5-monophosphat - NTP Nucleosidtriphosphat - NMP Nuclcosid-5-monophosphat - dATP Desoxyadenosintriphosphat - P_i anorganisches Phosphat - PP_i anorganisches Pyrophosphat - DNS Desoxyribonucleinsäure - RNS Ribonucleinsäure - r-RNS ribosomale RNS - m-RNS Boten-RNS - Glykogen-Synthetase UDP-Glucose--1,4-glucan--4-glucosyltransferase - ICSH interstitial-cell-stimulating hormone     

Sequence of the coding regions from the 3.0 kb and 3.9 kb mRNA

Summary Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA clones covering the genome region from the 3 end of the pelomer gene to the start of the integral membrane protein gene. The nucleotide sequence of this area was determined using clone pTG11 and a previously reported cDNA clone pTG22. Three open reading frames (ORFs) were identified encoding putative polypeptides of relative molecular masses (M_r) 6,600, 27,600, and 9,200. The sequence encoding the M_r 9,200 polypeptide was found to be present on the unique 5 region of the 3.0 kb mRNA species whereas the other two ORFs mapped on the 3.9 kb mRNA species. Differences between the ORFs from this strain of TGEV and those from a previously reported avirulent strain of TGEV were compared.     

Bleomycin-kanamycin resistance as a marker of the presence of transposon Tn5 in clinical strains ofEscherichia coli

The aminoglycoside modifying enzyme aminoglycoside 3- phosphotransferase II (APH(3)II) is encoded for on transposon Tn5 by theaphA gene, in the same operon as theble gene determining bleomycin resistance. To document this linkage 82 kanamycin-resistantEscherichia coli strains of clinical origin were studied; all 18 isolates presenting bleomycin-kanamycin resistance were shown by an enzymatic assay to produce APH(3)II, and the presence of Tn5 was demonstrated by gene hybridization. Similarly, bleomycin-kanamycin resistance was shown to be linked to APH(3)II production inSalmonella spp. The epidemiology of strains with Tn5-encoded APH(3)II may thus be studied, at least inEscherichia coli, by a simple diffusion test using bleomycin and kanamycin discs.     

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.     

Cyclic nucleotide changes induced in human leukocytes by a product of axenically grownEntamoeba histolytica that inhibits human monocyte locomotion

Pulse exposure of human mononuclear phagocytes to the monocyte locomotion-inhibitory factor produced byEntamoeba histolytica (i.e., the 369- to 765-Da chromatographic fraction obtained from the supernatant fluid of axenically grownE. histolytica) led to a swift increase in the intracellular concentration of adenosine 3:5 cyclic monophosphate (cAMP). A weaker response was observed in human polymorphonuclear leukocytes, the locomotion of which, however, is not inhibited by this amebic factor. The same chromatographic fraction obtained from the axenic medium control lacked this effect, at least upon mononuclear phagocytes. On the other hand, both the monocyte locomotion-inhibitory factor and the axenic medium control, possibly through shared culture medium components, induced comparable increases in guanosine 3:5 cyclic monophosphate (cGMP) in human mononuclear phagocytes and in polymorphonuclear leukocytes, thus suggesting that the latter nucleotide is not critical for the leukotactic inhibitory phenomenon. Our results suggest that like other leukotactic inhibitors, the monocyte locomotion-inhibitory factor produced byE. histolytica operates through modulations of intracellular cAMP.Abbreviations MP Mononuclear phagocytes (monocytes) - MLIF monocyte locomotion-inhibitory factor - PMN polymorphonuclear leukocytes - cAMP adenosine 3:5 cyclic monophosphate - cGMP guanosine 3:5 cyclic monophosphate - PBS phosphate-buffered (pH 7.4) normal saline - AMC axenic medium control Gey's-A; Gey's medium with 2% albumin - ZAS zymosan-activated serum - hpf high-power field (400x)     

Evolutionarily recent transfer of a group I mitochondrial intron to telomere regions in Saccharomyces cerevisiae

Summary The junctions between X and Y subtelomeric repeats in Saccharomyces cerevisiae usually contain a stretch of telomere sequences, (G_1–3T)_n. Two of three cloned X-Y junctions from strain YP1 have a replacement of about 200 bp of X, the internal telomere sequence, and 49 bp of Y by a 292 bp sequence. The first 227 bp of this insertion sequence are 100% identical to the fourth intron of cytochrome b. The rest of the insertion has homology to an unknown dispersed nuclear sequence. Recombination among subtelomeric regions can explain the nuclear distribution of this sequence and why telomeres can trap and maintain sequences that would otherwise be lost.     

A Defective RNA Associated with Bamboo Mosaic Virus and the Possible Common Mechanisms for RNA Recombination in Potexviruses

A naturally occurring 1.1 kb RNA was isolated from purified virions of bamboo mosaic potexvirus isolate S (BaMV-S). This RNA is a defective RNA (D RNA) derived from a single internal deletion of the BaMV genome. A cDNA clone representing the complete nucleotide sequence of the BaMV-S D RNA was generated and its nucleotide sequence was determined. The BaMV D cDNA is 1015 nts in length [excluding the poly(A) tail] and consists of two regions corresponding to 867 nts of the 5 terminus and 148 nts of the 3 terminus of the BaMV genomic RNA. BaMV D cDNA contains a single open reading frame (ORF) encoding a putative 29.7 kDa protein comprised of a fusion of the first 258 amino acids of BaMV ORF 1 and the last 2 amino acids of coat protein. The coding capacity of D RNA was verified by in vitro translation of native BaMV-S D RNA and of 1.1 kb RNA transcribed in vitro from the full-length D cDNA. BaMV D RNA can be reproducibly generated by serial passages of BaMV-S in Nicotiana benthamiana and is the first D RNA in the potexvirus group shown to be generated de novo. Alignments of sequences surrounding the 5 and 3 junction borders of reported potexvirus D RNAs reveal a 65.2–84.6% sequence identity, suggesting that common mechanisms for viral RNA recombination are involved in the generation of potexvirus D RNAs.     

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.     

Homologous maturase-like proteins are encoded within the group I introns in different mitochondrial genes specifying Yarrowia lipolytica cytochrome c oxidase subunit 3 and Saccharomyces cerevisiae apocytochrome b

A mitochondrial cox3 gene in the alkane yeast, Yarrowia lipolytica, encodes a subunit-3 protein of cytochrome c oxidase, and contains a 1044 base-pair-long intron, as compared with the corresponding intronless gene in Saccharomyces cerevisiae. The intron belongs to a group I intron as determined by the cDNA sequence for the splicing sites as well as the predicted RNA secondary structure. Remarkably, this intron could code for a protein of 206 amino-acid residues which showed 63% similarity with an RNA maturase encoded by the second intron of the mitochondrial apocytochrome b gene in S. cerevisiae. Both introns occurred within the conserved exon sequence, 5-TT(G/C)AGGTGC-3, suggesting the possible transposition of a common ancestral intron.     

The VP3 gene of human group C rotavirus

The complete nucleotide sequence of genome segment 4 from the human group C rotavirus (Bristol strain) was determined. Comparison of the nucleotide sequences of the genome termini with the consensus 5 and 3 terminal non-coding sequences of the human group C rotavirus genome revealed characteristic 5 and 3 sequence motifs. Human group C rotavirus genome segment 4 is 2, 166bp long and encodes a single open reading frame of 2,082 nucleotides (693 amino acids) starting at nucleotide 55 and terminating at nucleotide 2,136 giving a 3 untranslated region of 30 nucleotides. Alignment with the porcine group C VP3 equivalent gene showed the human gene is one amino acid longer, and that the proteins have 84.1% amino acid sequence identity. A conserved potential nucleotide binding motif shared with the porcine VP3 sequence was identified. Analogy with the group A rotaviruses suggested that the genome segment 4 encodes the group C rotavirus guanylyltransferase.