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.     

Nucleotide Sequences of Double-Stranded RNA Segments from a Hypovirulent Strain of the White Root Rot Fungus Rosellinia necatrix: Possibility of the First Member of the Reoviridae from Fungus

Twelve double-stranded (ds) RNA segments were detected from a hypovirulent strain W370 of the white root rot fungus Rosellinia necatrix. The estimated molecular weights ranged from 0.41×10⁶ to 2.95×10⁶. Full length cDNA clones for eight segments were obtained. Northern blot analysis suggested that each segment was genetically unique. The nucleotide sequences of eight full length dsRNA segments were determined. One long open reading frame was found in each segment. Conserved sequences at the 5-end (5-ACAAUUU-3) and at the 3-end (5-UGCAGAC-3) were identified in all eight segments. Segment-specific panhandle structures, formed by inverted terminal repeats, were also found in all segments. Comparative analyses of the predicted translational products of eight dsRNA segments showed that the deduced amino acid sequence partially matched those of the Reoviridae family members: Colorado tick fever virus, Nilaparvata lugens reovirus, and rice black streaked dwarf virus. The results suggested that W370 dsRNA is derived from a new member of the family Reoviridae detected in fungus.     

Two polymorphicAvaI andHhaI sites in a differentially methylated region of the human H19 gene

Summary The H19 gene is paternally imprinted both in the human and mouse (Bartolomeiet al., 1991; Zhang and Tycko, 1992), although its expression pattern seems somewhat different between the two species (Jinno,et al., 1995). DNA-methylation is a promising candidate for a parent-of-origin mark of the gene, and a paternal allele-specific methylation imprint was recently identified at the mouse H19 locus (Tremblayet al., 1995). We found a 50% methylated region in the human H19 gene (Jinno, unpublished data). A search for polymorphisms in this region revealed two novelAvaI andHhaI RFLPs, which contribute to the detection of allele-specific methylation at the human H19 locus.PCR primers for the AvaI-site PANL2 5-GAGCCTGCCAAGCAGAGCG-3 - PANR2 5-CACATAAGTAGGCGTGACTTGA-3 PCR primers for the HhaI-site ASMA 5-CAATGAGGTGTCCCAGTTCCA-3 - PANR2 5-CACATAAGTAGGCGTGACTTGA-3     

Organization of ATPA coding and 3′ flanking sequences associated with cytoplasmic male sterility in Phaseolus vulgaris L.

Summary A region of the mitochondrial genome associated with cytoplasmic male sterility (CMS) in Phaseolus vulgaris was flanked by two different repeated sequences designated x and y. The DNA sequence of the CMS-unique region and a portion of each flanking repeat was determined. Repeat x contained a complete coding copy of the F1 ATPase subunit A (atp A) gene, as well as an open reading frame (orf) predicting a protein of 209 amino acids. The TGA termination codon of the atpA gene and the ATG initiation codon of orf209 were overlapping. These reading frames were oriented with their 3 ends proximal to the CMS-unique region. The CMS-unique region of 3736 nucleotides contained numerous orfs. The longest of these predicted proteins being of 239, 98 and 97 amino acids. The 3 coding and 3 flanking regions of orf98 were derived from an internal region of the higher plant chloroplast tRNA alanine intron. The region of repeat y immediately adjacent to the CMS-unique region contained the 111 carboxy-terminal coding residues of the apocytochrome b (cob) gene. This segment was oriented with its 5 end proximal to the CMS-unique region, but cob gene sequences were not fused to an initiation codon within the unique region.     

Identification and characterization of U1 small nuclear RNA genes from two crustacean isopod species

Four different units containing three variants of the U1 snRNA gene have been identified in the genome of Asellus aquaticus and only one unit has been identified in the genome of Proasellus coxalis. All four identified U1 snRNA genes can be folded according to the proper secondary structure and possess the functionally useful conserved sequences. Moreover, in the 3 flanking regions, all genes present both the 3 box, a conserved sequence required for 3 processing of mature snRNA, and a polyadenylation signal which is unusual for these genes. The PCR products were used as probes in fluorescent in-situ hybridization (FISH) experiments to locate them on chromosomes of A. aquaticus and P. coxalis.     

Structure of the cloned Locusta migratoria mitochondrial genome: restriction mapping and sequence of its ND-1 (URF-1) gene

Summary We have cloned the entire mitochondrial genome of Locusta migratoria in four fragments and characterised by restriction mapping. In addition, we have sequenced a 1,095 kb region containing the ND-1 (URF-1) gene. The inferred primary structure of the protein is highly homologous to its Drosophila counterpart (68%). The gene is flanked at the 5 end by the tRNA _CUN ^leu gene, interrupted by the sequence TTG. The 3 end is flanked by the tRNA _ser ^UCN gene, followed by a sequence homologous to the 3 end of D. yakuba cytochrome b. The relative position of the genes is conserved between Locusta and Drosophila, thus indicating conservation of mitochondrial gene order in insects.     

Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement

Summary Nucleotide sequence analysis of a 17043 basepair (bp) region of the Chlamydomonas reinhardtii plastome indicates the presence of three open reading frames (ORFs) similar to RNA polymerase subunit genes. Two, termed rpoB1 and rpoB2, are homologous to the 5-and 3-halves of the Escherichia coli beta subunit gene, respectively. A third, termed rpoC2, is similar to the 3-half of the bacterial beta' subunit gene. These genes exhibit several unusual features: (1) all three represent chimeric structures in which RNA polymerase gene sequences are juxtaposed in-frame with long sequences of unknown identity; (2) unlike their counterparts in plants and eubacteria, rpoB1 and rpoB2 are separated from rpoC2 by a long (7 kilobase-pair, kbp) region containing genes unrelated to RNA polymerase; (3) DNA homologous to the 5 half of rpoC (termed rpoC1 in other species) is not present at the 5 end of rpoC2 and could not be detected in C. reinhardtii chloroplast DNA. RNA expression could not be detected for any of the RNA polymerase genes, suggesting that they are pseudogenes or genes expressed at stages of the C. reinhardtii life-cycle not investigated. The three genes are flanked by GC-rich repeat elements. We suggest that repeat DNA-mediated chloroplast recombination events may have contributed to their unusual arrangement.     

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     

Nucleotide sequence of the coat protein genes of <Emphasis Type="Italic">Alstroemeria mosaic virus</Emphasis> and Amazon lily mosaic virus,a tentative species of genus <Emphasis Type="Italic">Potyvirus</Emphasis>

Summary. The nucleotide sequences of the 3 terminal region of the genomes of Alstroemeria mosaic virus (AlsMV) and the Amazon lily mosaic virus (ALiMV) have been determined. These sequences contain the complete coding region of the viral coat protein (CP) gene followed by a 3-untranslated region (3-UTR). AlsMV and ALiMV share 74.9% identity in the amino acid sequence of the CP, and 55.6% identity in the nucleotide sequence of the 3-UTR. Phylogenetic analysis of these CP genes and 3-UTRs in relation to those of 79 potyvirus species revealed that AlsMV and ALiMV should be assigned to the Potato virus Y (PVY) subgroup. AlsMV and ALiMV were concluded to have arisen independently within the PVY subgroup.     

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.     

A broad bean mitochondrial atp6 gene with an unusually simple,non-conserved 5′ region

Summary A nucleotide sequence of broad bean mitochondrial DNA (mtDNA) that contains an atp6 gene of 876 ntp is presented. Relative to other plant atp6 genes, this broad bean gene comprises a 90 ntp non-conserved 5 region, a 759 ntp highly conserved central region and a 27 ntp non-conserved 3 region. The non-conserved, 5 region of the broad bean atp6 gene differs from the corresponding regions of most other plant atp6 genes in that it contains only one potential translation initiation codon and, following this codon, a 63 ntp segment that predicts an amino acid sequence with a predominance of alternating leucines.     

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.     

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.     

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     

Suppression of a mitochondrial point mutation in a tRNA gene can cast light on the mechanisms of 3′ end-processing

We used a genetic approach to study the nuclear factors involved in the biogenesis of mitochondrial tRNAs. A point mutation in the mitochondrial tRNA^Asp gene of Saccharomyces cerevisiae had previously been shown to result in a temperature-sensitive respiratory-deficient phenotype as a result of the absence of 3 end-processing of the tRNA^Asp. Analysis of mitochondrial revertants has shown that all revertants sequenced have a G-A compensatory change at position 53, which restores the hydrogen-bond with the mutated nucleotide. We then searched for nuclear suppressors to identify the nuclear gene(s) involved in mitochondrial tRNA 3 end-processing. One such suppressor mutation was further characterized: it restores tRNA^Asp maturation and growth at 36°C on glycerol medium in heterozygous diploids, but leads to a defective growth phenotype in haploids.     

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.     

Selection and characterization of pyrG mutants of Penicillium chrysogenum lacking orotidine-5′-phosphate decarboxylase and complementation by the pyr4 gene of Neurospora crassaa

Summary Pyrimidine auxotrophs of Penicillium chrysogenum have been isolated at a high frequency among mutants resistant to 5-fluoroorotic acid (5.2 mM). Some of the pyrimidine auxotrophs (e.g. strain pyrG1) showed no reversion. A radiometric assay based on the conversion of (6-¹⁴C)orotidine 5-monophosphate (OMP) into (6-¹⁴C)uridine 5-monophosphate (UMP) was developed to determine OMP-decarboxylase activity. One of the pyrimidine auxotrophs (P. chrysogenum pyrGl) was studied in detail. It was deficient in OMP-decarboxylase activity, whereas the parental strain (P. chrysogenum Wis. 54-1255) showed a normal enzyme activity. A five-fold higher OMP-decarboxylase activity was found in a P. chrysogenum pyrGI clone transformed with plasmids containing the Neurospora crassa pyr4 gene (which codes for the same enzyme).Abbreviations OMP orotidine 5-monophosphate - UMP uridine 5-monophosphate