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PubMed2003

Identification and characterization of phytoplasmal genes, employing a novel method of isolating phytoplasmal genomic DNA.

Phytoplasmas are unculturable, insect-transmissible plant pathogens belonging to the class Mollicutes. To be transmitted, the phytoplasmas replicate in the insect body and are delivered to the insect's salivary glands, from where they are injected into the recipient plant. Because phytoplasmas cannot be cultured, any attempt to recover phytoplasmal DNA from infected plants or insects has resulted in preparations with a large background of host DNA. Thus, studies of the phytoplasmal genome have been greatly hampered, and aside from the rRNA genes, only a few genes have hitherto been isolated and characterized. We developed a unique method to obtain host-free phytoplasmal genomic DNA from the insect vector's saliva, and we demonstrated the feasibility of this method by isolating and characterizing 78 new putative phytoplasmal open reading frames and their deduced proteins. Based on the newly accumulated information on phytoplasmal genes, preliminary characteristics of the phytoplasmal genome are discussed.

Animals↗

PubMed2005

Structure and genome organization of AFV2, a novel archaeal lipothrixvirus with unusual terminal and core structures.

A novel filamentous virus, AFV2, from the hyperthermophilic archaeal genus Acidianus shows structural similarity to lipothrixviruses but differs from them in its unusual terminal and core structures. The double-stranded DNA genome contains 31,787 bp and carries eight open reading frames homologous to those of other lipothrixviruses, a single tRNA(Lys) gene containing a 12-bp archaeal intron, and a 1,008-bp repeat-rich region near the center of the genome.

Genome, Viral↗

PubMed2006

Analysis of the human cytomegalovirus genomic region from UL146 through UL147A reveals sequence hypervariability, genotypic stability, and overlapping transcripts.

BACKGROUND: Although the sequence of the human cytomegalovirus (HCMV) genome is generally conserved among unrelated clinical strains, some open reading frames (ORFs) are highly variable. UL146 and UL147, which encode CXC chemokine homologues are among these variable ORFs. RESULTS: The region of the HCMV genome from UL146 through UL147A was analyzed in clinical strains for sequence variability, genotypic stability, and transcriptional expression. The UL146 sequences in clinical strains from two geographically distant sites were assigned to 12 sequence groups that differ by over 60% at the amino acid level. The same groups were generated by sequences from the UL146-UL147 intergenic region and the UL147 ORF. In contrast to the high level of sequence variability among unrelated clinical strains, the sequences of UL146 through UL147A from isolates of the same strain were highly stable after repeated passage both in vitro and in vivo. Riboprobes homologous to these ORFs detected multiple overlapping transcripts differing in temporal expression. UL146 sequences are present only on the largest transcript, which also contains all of the downstream ORFs including UL148 and UL132. The sizes and hybridization patterns of the transcripts are consistent with a common 3'-terminus downstream of the UL132 ORF. Early-late expression of the transcripts associated with UL146 and UL147 is compatible with the potential role of CXC chemokines in pathogenesis associated with viral replication. CONCLUSION: Clinical isolates from two different geographic sites cluster in the same groups based on the hypervariability of the UL146, UL147, or the intergenic sequences, which provides strong evidence for linkage and no evidence for interstrain recombination within this region. The sequence of individual strains was absolutely stable in vitro and in vivo, which indicates that sequence drift is not a mechanism for the observed sequence hypervariability. There is also no evidence of transcriptional splicing, although multiple overlapping transcripts extending into the adjacent UL148 and UL132 open reading frames were detected using gene-specific probes.

Amino Acid Sequence↗

PubMed2002

DicodonUse: the programme for dicodon bias visualization in prokaryotes.

The DicodonUse programme is aimed at a fast and simple assessment of genes present in prokaryotic nucleotide sequences. It identifies open reading frames that are not genes, and it distinguishes the genes that inherently belong to the genome in question from the genes that were inserted into the genome in the course of evolution. The programme is based on frequencies of dicodons used by the organism.

Bacteria↗

PubMed1991

Characterization of mRNAs and coding potential of the PET54 gene from Saccharomyces cerevisiae.

The nuclear PET54 gene in yeast controls expression of two mitochondrial genes: COX1 at the level of pre-mRNA splicing and COX3 at the level of mRNA translation. Two size classes (1.6 and 1.1 kb) of transcripts that contain the PET54 coding region are produced in vivo. Relative to the majority of yeast mRNAs analyzed so far, the 5' untranslated leader region of the 1.6 kb transcript is unusually long (254 bases), while that for the major 1.1 kb transcript is unusually short (1 base). The majority of each class of PET54 mRNA was associated with polysomes in vivo. The possibility that two polypeptides are produced in vivo from the 1.1 kb PET54 mRNA was raised by the work of Sedman et al. [J. Virol. 64: 453-457, 1990], which showed that translation initiation at a downstream AUG occurs with increased efficiency when the upstream AUG is located very close to the 5' end of the mRNA. However, two sensitive assays for production of a second polypeptide, which is predicted to be 22 kD, were employed and no second polypeptide was detected. Furthermore, a nonsense mutation introduced near the beginning of the PET54 open reading frame abolished both COX1 and COX3 gene expression. These results indicate that the PET54 gene encodes predominantly a single functional polypeptide that is employed for expression of both the COX1 and COX3 genes of mitochondrial DNA.

Base Sequence↗

PubMed1995

Nucleotide sequence analysis of a full-length human T-cell leukemia virus type I from adult T-cell leukemia cells: a prematurely terminated PX open reading frame II.

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically associated with adult T-cell leukemia/lymphoma (ATL). The prototypic HTLV-1, ATK, is the only full-length provirus cloned from uncultured leukemic cells and completely sequenced prior to this study. We have determined the complete nucleotide sequence of another full-length HTLV-1 provirus cloned directly from leukemic cells. A premature termination codon was found in the second open reading frame (orf II) of the pX region. Our finding indicates that open reading frame II of the HTLV-1 pX region is not required for outgrowth of ATL leukemic clones in vivo.

Amino Acid Sequence↗

PubMed1999

Comparisons of defective HTLV-I proviruses predict the mode of origin and coding potential of internally deleted genomes.

Cell lines infected with a variety of HTLV-I isolates were examined for the presence of defective proviruses that contain deletions spanning the gag, pol, and env genes. Internally deleted proviruses were identified by Southern blotting and by PCR amplification with 5' and 3' primers complementary to gag and tax sequences, respectively. PCR products representing eight defective proviruses from seven different cell lines were subsequently cloned and sequenced. The objectives of this study were twofold: first, we sought to determine whether nucleotide sequences surrounding sites of deletion shared common features that might reveal the mechanisms by which the defective genomes originated. Second, we asked whether deleted proviruses encode Gag fusion proteins with related C-terminal residues derived from open reading frames in the pX region. While most of the defective proviruses had incurred a single, large deletion, two of them displayed a more complex pattern of multiple rearrangements. Alignments of bases flanking the 5' and 3' deletion endpoints within each provirus showed tracts of sequence identity consistent with a mechanism involving aberrant intramolecular strand-transfer events during replication. We suggest that the amount or activity of HTLV-I polymerase in virions may contribute both to the poor infectivity of the virus and to the high deletion frequency. Two of the eight proviruses that were examined encoded a gag gene joined to an extended open reading frame; the other six had very short open reading frames (one to six amino acids) derived from pX or env regions joined to gag that showed no apparent amino acid sequence similarity.

Amino Acid Sequence↗

PubMed1991

Nucleotide sequence of the RAD57 gene of Saccharomyces cerevisiae.

We have determined the nucleotide (nt) sequence of the RAD57 gene of Saccharomyces cerevisiae. RAD57 contains an open reading frame of 1380 bp. The deduced amino acid sequence of 460 residues contains a potential nt-binding sequence and shows significant similarity to the preliminary sequence of RAD51.

Adenosine Triphosphatases↗

PubMed1997

Nucleotide sequence of the genes encoding the matrix and small hydrophobic proteins of pneumonia virus of mice.

The nucleotide sequences of the genes encoding the matrix (M) protein and the small hydrophobic (SH) protein of pneumonia virus of mice (PVM) are described. The matrix protein gene contains a large open reading frame encoding polypeptide of 257 residues which shows considerable (38.6-42.1%) amino acid identity with the matrix proteins of other pneumoviruses. The matrix gene also contains a second, smaller, open reading frame (ORF) as seen with the equivalent genes of other pneumoviruses. The PVM second open reading frame is capable of encoding a polypeptide of 46 residues and shows no significant similarity with the proteins encoded by the equivalent open reading frames of the other pneumoviruses. The gene adjacent to that encoding the matrix protein encodes a small, 92 residue, polypeptide which has a central hydrophobic domain and is structurally similar to the small hydrophobic protein of respiratory syncytial virus.

Amino Acid Sequence↗

PubMed1998

Complete nucleotide sequence of the genomic RNA of tobacco mosaic virus strain Cg.

Tobacco mosaic virus (TMV)-Cg is a crucifer-infecting tobamovirus that was isolated from field-grown garlic. We determined the complete nucleotide sequence of the genomic RNA of TMV-Cg. The genomic RNA of TMV-Cg consists of 6303 nucleotides and encodes four large open reading frames, organized basically in the same way as that of other tobamoviruses. The nucleotide and deduced amino acid sequences are very similar to those of the other crucifer-infecting tobamoviruses that have been sequenced so far.

Amino Acid Sequence↗

PubMed1994

RNP1, a new ribonucleoprotein gene of the yeast Saccharomyces cerevisiae.

A previously unidentified ribonucleoprotein (RNP) gene of yeast has been cloned and sequenced. The gene, named RNP1, was found adjacent to a previously sequenced gene encoding the second gene for ribosomal protein L4. RNP1 contains two RNA Recognition Motifs (RRM), [alternatively known as RNA binding Domains (RBD)], but unlike most RNP genes does not contain any auxiliary simple sequence domains. The first RRM (RRM1) most resembles RRM domains found in the hnRNP A/B class of RNP proteins. The second RRM (RRM2) most resembles a RRM so far seen only in the single RRM of the yeast SSB1 gene. Two null mutants of RNP1 that were created, a frameshift disruption and a complete deletion of the gene, were viable, demonstrating that the gene is not essential for cell growth. Two double null mutants of yeast RNP genes that were created (delta RNP1/delta SSB1 and delta SSB1/delta NPL3) were also viable. A fragment identical in size to the RRM1 domain could be amplified by PCR from the DNA of fungi, plants, and animals, using primers matching the ends of this domain, indicating that the structure of RRM1 is conserved. Another potential open reading frame on the same cloned fragment of DNA encodes a gene product whose structure resembles that of a seven-transmembrane-segment membrane receptor protein.

Amino Acid Sequence↗

PubMed1994

Cloning of the early promoters of Pseudomonas aeruginosa bacteriophage D3: sequence of the immunity region of D3.

The early promoters of bacteriophage D3 of Pseudomonas aeruginosa were cloned and physically mapped to the right 25% of the phage genome. The promoters were cloned into promoter selection vector pQF26, and their relative strengths, the direction of transcription, and whether they were directly regulated by repressor were determined. A 3.3-kb fragment of the genome containing the immunity region was sequenced and analyzed (GenBank accession number: L22692). The promoter activity associated with this region was determined to be bidirectional and repressible, indicating that this region contains operator-promoter complexes. Sequence and functional analyses suggest that this region is analogous to the immunity region of coliphage lambda. Two strong promoters, one of which was repressible, were found to be located adjacent to the immunity region. Clear-plaque mutant phage D3c contains insertion element IS222, which causes it to behave as a repressor-negative (c1) variant. The site of insertion of IS222 was sequenced and determined to lie within the c1 gene open reading frame. This phage shows remarkable similarity in genomic organization to coliphage lambda and its relatives.

Amino Acid Sequence↗

PubMed1991

Characterization of variable regions in the envelope and S3 open reading frame of equine infectious anemia virus.

The polymerase chain reaction was used to amplify and clone parts of the envelope gene and overlapping S3 open reading frame, thought to encode rev, of the virulent in vivo-derived Th-1 isolate of equine infectious anemia virus (EIAV). The results indicated that EIAV consists of a heterogeneous mixture of genotypes present at the first febrile cycle after initial infection. We showed that the Th-1 isolate apparently contains nondefective genotypes as well as types which have transmembrane protein truncations or are rev deficient. Furthermore, we could confirm the presence of a hypervariable region in the gp90 envelope glycoprotein. Taken together with earlier data on the heterogeneity of the regulatory motifs present in the long terminal repeat sequences of viruses from the same in vivo isolate (S. Carpenter, S. Alexandersen, M. J. Long, S. Perryman, and B. Chesebro, J. Virol. 65:1605-1610, 1991), our findings indicate that EIAV uses a complex system of diversity in biological phenotypes together with variation in regulatory and antigenic makeup to evade host response and to cause persistent infection and recurrent chronic disease.

Amino Acid Sequence↗

PubMed2002

RAPD isolation of a Y chromosome specific ORF in a dioecious plant, Silene latifolia.

Silene latifolia is a dioecious plant and has heteromorphic sex chromosomes: the X and Y chromosomes. The Y chromosome is the largest, and its genetic control seems to be most strict among dioecious plants. To identify the putative sex-determination elements on the Y chromosome, random amplified polymorphic DNA (RAPD) analysis was used to screen for Y chromosome specific DNA fragments, and 31 clones were successfully produced. Genomic Southern hybridization and FISH (fluorescence in situ hybridization) analyses revealed that one of the clones, #2-2, is a Y chromosome specific fragment that has a single copy on the Y chromosome. Sequence tagged site (STS)-PCR analysis also succeeded in amplifying one fragment in males and no fragments in females. Cloning and sequencing of the #2-2 flanking region using inverse PCR revealed an open reading frame (ORF) corresponding to 285 amino acids in length (ORF285), but no expression of the ORF285 gene was identified. ORF285 may be a clue to the origin of dioecy.

Amino Acid Sequence↗

PubMed2003

DNA sequencing of a plasmid with virulence from marine fish pathogen Vibrio anguillarum.

DNA sequence of a plasmid pEIB1 associated with virulence from the marine fish pathogen Vibrio anguillarum was determined using the methods of restriction endonuclease digestion, subcloning, and primer walking. The whole length of obtained pEIB1 DNA sequence was 66 164 bp, and the overall G+C content of DNA sequence is 42.7%. This sequence encodes 44 open reading frames containing the genes of DNA replication, biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes.

Animals↗

PubMed1994

Nucleotide sequence of RNA 1, the largest genomic segment of rice stripe virus, the prototype of the tenuiviruses.

The complete nucleotide sequence of RNA 1, the largest genomic segment of rice stripe virus (RSV), was determined using two sets of overlapping cDNA clones. RNA segment 1 comprises 8970 nucleotides and on the viral complementary sequence has a single long open reading frame coding for a protein of 2919 amino acids with an estimated M(r) of 336860. Amino acid sequence comparisons of the putative protein indicated strong homology (30% amino acid identity over about 1500 residues) with the L protein of the genus Phlebovirus of the Bunyaviridae, but no detectable similarity with other members of the Bunyaviridae. However, weak similarity was detected with the L protein of Tacaribe arenavirus. The highly homologous sequence domain includes the conserved motifs of the putative RNA-dependent RNA polymerase. The data presented here, along with previous work clearly show significant similarities in genome organization, structure and expression between RSV and members of the genus Phlebovirus of the Bunyaviridae. Taken together, we propose that tenuiviruses should be included in the Bunyaviridae under the genus Tenuivirus.

Amino Acid Sequence↗

PubMed1993

Identification and analysis of the transport/capsid assembly protein (tp/cap) gene of human herpesvirus-6 (HHV6).

The transport/capsid assembly protein (tp/cap) gene of human herpesvirus 6 (HHV6) strain U1102 has been identified and localized on the restriction enzyme map of the viral genome, to the EcoRI-Q fragment. The complete DNA sequence of the tp/cap gene was determined. The tp/cap gene encodes a protein product of 726 amino acids and has the strongest similarity with the homologous gene (HCMV UL56) from HCMV. Upstream of the tp/cap open reading frame is the gene for the major DNA binding protein (mdbp) and downstream is the glycoprotein B (gB) gene. This gene block arrangement is common to all herpesviruses.

Amino Acid Sequence↗

PubMed2005

Nucleotide sequence analyses of genomic RNAs of Peanut stunt virus Mi, the type strain representative of a novel PSV subgroup from China.

The complete nucleotide sequence of Peanut stunt virus strain Mi (PSV-Mi) from China was determined and compared to other viruses of the genus Cucumovirus. The tripartite genome of PSV-Mi encoded five open reading frames (ORFs) typical of cucumoviruses. Distance analyses of four ORFs indicated that PSV-Mi differed sufficiently in nucleotide sequence from other PSV strains of subgroups I and II to warrant establishment of a third subgroup of PSV.

Arachis↗