Cloning and characterization of a repetitive sequence fromPneumocystis carinii

Four repetitive sequence clones measuring 10.9–23.4 kb in length were isolated from the genomic library ofPneumocystis carinii. Restriction enzymes mapping and cross-hybridization studies revealed that these clones are interrelated and that they derive from the common repeat unit, which is specific forP. carinii. Dot-blot analysis suggested that the copy number of the repeat sequence is about 100, assuming that the genome size is 1.5×10⁷ bp. Interestingly, the repetition unit extended over at least 23.4 kb and included long, 5.2-kb inverted repeats, for example, A-B-A-C, in which A is the inversion of A.     

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.     

Long,nearly identical untranslated sequences at the 3′ terminal regions of the genomic RNAs of cherry leafroll virus (walnut strain)

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3 UTR) immediately 5 of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3 UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.The nucleotide sequence reported in this paper has been submitted to the EMBL data library and assigned the accession number Z34265.     

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.     

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.     

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.     

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.     

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.     

Messenger RNA intron in the nuclear 18s ribosomal RNA gene of deuteromycetes

Introns within messenger RNA genes have characteristic border sequences and a conserved region near the 3 end of the intron. All are involved in splicing to produce the mature mRNA. Introns in ribosomal RNA genes have less well-defined borders and contain no internal conservation. We report here mRNA-type introns located near the 3 end of the 18s rRNA genes of the deuteromycetes Phialophora americana and Cenococcum geophilum. Inserted sequences of various sizes have also been located at the same point in several other deuteromycete species.     

The family of mouse phosphoglycerate kinase genes and pseudogenes

The mammalian genome contains two genes encodingphosphoglycerate kinase; the pgk-1gene is X-linked and is expressed in all cells except sperm, while the pgk-2gene is expressed exclusively in sperm cells. The mouse genome contains no pseudogenes derived from pgk-2.On the other hand, the genomes of Balb/c and C3H/He strain mice contain six other regions with sequences homologous to those of pgk-1cDNA. These pgk-related sequences are likely derived from the pgk-1gene by retroposition because all are located on autosomal chromosomes and because none appear to be interrupted by introns. Two of the presumed pseudogenes contain sequences homologous to all regions of the pgk-1cDNA while the other four genomic regions were truncated at the 5, 3, or both ends. One of the truncated pseudogenes was sequenced. Its pgk-related sequence was not flanked by direct repeats, suggesting that loss of the 5 and/or 3 ends of this retrogene may have occurred following its integration into the genome. Our evidence suggests that pgk-1-derived retroposons arose initially more than 100 million years ago and have continued to arise until so recently that some are unique to different mouse strains.     

The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5 termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3–5 exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.     

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.     

Complete nucleotide sequence of the RNA 1 of a grapevine isolate of <Emphasis Type="Italic">Arabis mosaic virus</Emphasis>

Summary. The complete nucleotide sequence of the genomic RNA 1 of the grapevine isolate NW (Neustadt an der Weinstrasse) of Arabis mosaic virus (ArMV) was determined. It is 7334 nucleotides long excluding the poly(A) tail, and contains one long open reading frame encoding a polypeptide of 2284 amino acids. The 5 and 3 non-coding regions were 227 and 252 nucleotides long respectively, and showed stretches of high identity with the corresponding 5 and 3 non-coding regions of ArMV-NW RNA 2. The analysis of the amino acid sequence of the polyprotein encoded by the RNA 1 of the ArMV-NW showed that the conserved amino acid motifs, characteristic for the viral protease co-factor, the NTP-binding protein, the cystein protease, and the RdRp core domains, were all present. Amino acid sequence comparisons between the polyproteins encoded by the RNAs 1 of ArMV-NW and other nepoviruses showed 75% identity with the GFLV-F13, and up to 36% with other nepoviruses.     

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.     

Linear mitochondrial plasmid inBrassica has terminal protein

Summary A linear 11.3 kb plasmid occurs in preparations of mitochondrial DNA from certainBrassica species and cultivars. Evidence that protein is associated with the ends of the plasmid was obtained in this study. Plasmid DNA ran as a distinct band during electrophoresis only if it had been treated with proteinase K. The hybridization patterns of clones from different regions of the plasmid to restriction digests of the protein-DNA complex revealed that the protein is associated with the end fragments of the plasmid. Exonuclease treatments indicated that the 5 ends of the plasmid DNA molecule are blocked but that the 3 ends are free.     

Comparative sequence analysis of four complete primary structures of plum pox virus strains

The complete nucleotide sequence of plum pox virus (PPV) strain SK 68 was determined from a series of overlapping cDNA clones. The exact 5 terminus was determined by direct RNA sequencing. The RNA sequence was 9786 nucleotides in length, excluding a 3 terminal poly(A) sequence. The large open reading frame starts at nucleotide position 147 and is terminated at position 9568. Comparison of cistrons from other plum pox virus strains with those predicted for the SK 68 strain indicated the same genomic organizations. Comparison of sequences leads to the following conclusions: (1) The genetic organization of all four PPV strains is identical, containing one large polyprotein gene and two noncoding regions at the 5 and 3 ends; (2) pairwise comparison of the genomic sequence of PPV SK 68 with other PPV strains shows 11% alteration. Sequence differences among strains are spread in a uniform manner upon the genome, except for the P1, HC-pro, and two noncoding regions, which are more conserved (with a 4% and 6.6% change). The stability of the noncoding regions is probably linked to their role in replication. The sequence variation has little effect on the amino acid sequence of the corresponding polypeptides, as changes occur preferentially in the third position of the reading frame triplets, except in the case of the 5 end of the coat protein gene (2.7% average difference in amino acid level, while in the case of coat protein it is 7.7%). The sequence analysis of the coat protein region of the four complete and one partial sequence indicates that the Hungarian plum pox virus strain diverges at the larger extent, similar to the El Amar strain, from which only less than half of the sequence is available.     

Comparison of the capsid protein cistron from serologically distinct strains of sweetpotato feathery mottle virus (SPFMV)

Summary Complementary DNA clones corresponding to the 3 terminus of sweetpotato feathery mottle virus (SPFMV) strains RC and C were synthesized and sequenced. An open reading frame followed by a 3 terminal non-coding region of 222 nucleotides and a terminal polyadenylation track was present in clones from both strains. Putative N-terminal capsid protein cleavage sites were identified for both strains 945 nucleotides 5 of the first stop codon. Sequence comparisons of these strains show 98% nucleic acid identity in the last 351 nucleotides of the capsid protein cistron and 100% in the corresponding amino acids. This relatively short homologous sequence element near the C terminus is responsible for the wide spectrum hybridization among SPFMV strains using in vitro transcribed antiviral RNA probes (riboprobes). The sequence similarity in the remaining N terminal 645 nucleotides is only 62% and 65% for their predicted amino acids. A tendency of decreasing nucleotide mismatches in the alignment from 5 to 3 end of both capsid protein cistrons was detected. Although the alignment of the predicted amino acid sequence of the SPFMVRC capsid protein with those of other potyviruses showed significant homology, hybridization with riboprobes from both the 5 and 3 regions of the capsid protein cistron of SPFMV was virus-specific.     

Molecular Characterization of an Isolate of Citrus Tristeza Virus that Causes Severe Symptoms in Sweet Orange

The complete sequence (19,249 nucleotides) of the genome of citrus tristeza virus (CTV) isolate SY568 was determined. The genome organization is identical to that of the previously determined CTV-T36 and CTV-VT isolates. Sequence comparisons revealed that CTV-SY568, a severe stem-pitting isolate from California, has more than 87% overall sequence identity with CTV-VT, a seedling yellows isolate from Israel. Although SY568 has an overall sequence identity of 81% with CTV-T36, a quick decline isolate from Florida, the sequence identity in the 3 half of the genome is over 90% while the sequence identity in the 5 half of the genome is as low as 56%. Based on the sequence alignments of these three isolates, sequences in the 3 half of the genome are generally well conserved, while the sequences in the 5 half are relatively divergent. Sequence data of independent overlapping clones from the CTV-SY568 genome revealed two regions with highly divergent sequences. In open reading frame 1b (RNA dependent RNA polymerase), there were 118 nucleotide differences that lead to 16 amino acid changes. In the open reading frame of the divergent coat protein gene, 5 amino acid changes result from 48 nucleotide differences. Most differences occurred in the third position of the codons, and resulted in silent amino acid substitutions. RNase protection assays demonstrated that most of the clones obtained are representative of the major RNA species of this isolate. Northern analysis indicated that CTV-SY568 accumulated more viral RNA including genomic and certain subgenomic RNAs than isolates VT or T36 in sweet orange.     

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.