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PubMed2004

Molecular cloning and characterization of canine ICOS.

Inducible costimulatory receptor (ICOS) is one recently identified member of the CD28 family of costimulatory molecules. Evidence suggests ICOS functions as a critical immune regulator and, to evaluate these effects, we employed the canine model system that has been used to develop strategies currently in clinical use for hematopoietic stem cell transplantation. To investigate the effects of blocking the ICOS pathway in the canine hematopoietic cell transplantation model, we tested existing murine and human reagents and cloned the full length of the open reading frame of canine ICOS cDNA to allow the development of reagents specific for the canine ICOS. Canine ICOS contains a major open reading frame of 624 nucleotides, encoding a protein of 208 amino acids, and localizes to chromosome 37. Canine ICOS shares 79% sequence identity with human ICOS, 70% with mouse, and 69% with rat. Canine ICOS expression is limited to stimulated PBMC.

Amino Acid Sequence↗

PubMed1998

Strawberry vein banding virus--definitive member of the genus Caulimovirus.

The complete DNA sequence (7876 nucleotides) of strawberry vein banding virus (SVBV) has been determined. Seven open reading frames are detected that potentially code for proteins of calculated weight 37.8; 18.3; 16.6; 56.0; 81.1; 59.0 and 12.6 kDa, respectively. Their position on the viral genome is the same as that of the corresponding proteins on the cauliflower mosaic virus (CaMV) genome. Phylogenetic analysis based on the amino acid sequence of this protein shows a closer relationship of SVBV with CaMV, figwort mosaic virus and carnation etched ring virus than with other caulimoviruses.

Base Sequence↗

PubMed2001

Actinomyces naeslundii fimbrial protein Orf977 shows similarity to a streptococcal adhesin.

The nucleotide sequence of the chromosomal DNA, upstream of Actinomyces naeslundii T14V fimbrial gene fimA, was determined. One open reading frame (orf977) encoding 977 amino acids was found, preceded by a gene homologous to elongation factor TU. Database searches revealed that Orf977 was homologous to CshA, a Streptococcus gordonii protein involved in cell adhesion. Previous studies had already determined two genes in the type 2 fimbrial gene cluster of A. naeslundii T14V: the structural subunit fimA, and orf365 with unknown function, followed by ribosomal genes. This study completes the type 2 fimbrial gene cluster sequence.

Actinomyces↗

PubMed1993

Cloning and characterization of the C. elegans histidyl-tRNA synthetase gene.

In this paper, we report the cloning and sequencing of the C. elegans histidyl-tRNA synthetase gene. The complete genomic sequence, and most of the cDNA sequence, of this gene is now determined. The gene size including flanking and coding regions is 2230 nucleotides long. Three small introns (45-50 bp long) are found to interrupt the open reading frame. The open reading frame translates to 523 amino acids. This putative protein sequence shows extensive homology with the human and yeast histidyl-tRNA the histidyl-tRNA synthetase gene is a single copy gene. Hence, it is very likely that it encodes both the cytoplasmic and the mitochondrial histidyl-tRNA synthetases. It is likely to be trans-spliced since it contains a trans-splice site in its 5' untranslated region.

Amino Acid Sequence↗

PubMed1995

A novel human c-sis mRNA species is transcribed from a promoter in c-sis intron 1 and contains the code for an alternative PDGF B-like protein.

The human platelet-derived growth factor (PDGF) B chain precursor is usually translated from a 3.5 kb c-sis/PDGF B gene transcript. The first exon of the c-sis/gene contains the code for the signal peptide of the PDGF B chain precursor, preceded by a 1 kb long untranslated sequence with potent translation inhibitory activity. In this paper we show that a novel 2.6 kb c-sis mRNA present in the human choriocarcinoma cell line JEG-3 initiates at an alternative exon 1, which we refer to as exon 1a. The 90 bp long exon 1a is located in the center of the first intron of the gene. It coincides with a very pronounced DNase-I-hypersensitive site and is preceded by a functional promoter. Of the three ATG codons present in exon 1a, the third one perfectly matches the criteria of a consensus start codon. It initiates an open reading frame that is continuous with the code for the PDGF B chain precursor but lacks the code for a signal peptide. We conclude that this novel 2.6 kb c-sis mRNA species lacks the strong translation inhibitory potential of the regular exon 1 and contains the code for a PDGF B-like protein that may be targeted to the cell nucleus.

Alternative Splicing↗

PubMed1995

BS a novel LINE-like element in Drosophila melanogaster.

Transposable elements with long terminal inverted repeats are rare and only one family of elements of this sort has been identified in the genome of Drosophila melanogaster. An insertion associated with the HSBS mutation of the achaete-scute complex has been reported to be a second element of this type. We have determined the complete sequence of this insertion and have shown that it is in fact two copies of a new LINE-like transposable element, that we have called BS, inserted in opposite orientation 337 bp apart. Like other elements of this type, BS has two open reading frames that appear to encode a gag-like polypeptide and a reverse transcriptase. There are few complete BS elements in the five strains of D.melanogaster that we have tested and they appear to transpose infrequently. The events that may have lead to the double BS insertion are discussed in terms of the supposed mechanism of transposition of LINE-like elements.

Amino Acid Sequence↗

PubMed1998

A race-specific insertion of transposable element IS801 in Pseudomonas syringae pv. phaseolicola.

The isolation and cloning of a random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) band specific for Pseudomonas syringae pv. phaseolicola race 1 allowed us to design a pair of primers that amplify 1.2-kb race 1-specific and 2.7-kb race 2-specific fragments, providing a rapid method for the identification of races by standard PCR methods. Restriction analysis revealed identical endonuclease sites in both fragments but the race 2 fragment contains a 1.5-kb insertion, identified as transposable element IS801 by sequence comparison. One complete and one partial open reading frame (ORF), each with a high probability of encoding a protein, have been identified in the 1.2-kb fragment common to both race 1 and race 2 sequences. As IS801 disrupts the partial ORF in the race 2 fragment, the complete sequence of this ORF has been obtained as well as its promoter region. The possibility that it may encode an avirulence gene is discussed as well as the role of transposable elements in pathogen evolution.

Base Sequence↗

PubMed1992

Complete sequence of maize stripe virus RNA4 and mapping of its subgenomic RNAs.

The complete nucleotide sequence of maize stripe virus RNA4 was determined and found to consist of 2227 nucleotides containing two significant open reading frames. One, in the 5' end of the viral RNA, encodes the major non-capsid protein of M(r) 19,815. The other is located in the 5' end of the viral complementary RNA and could encode a protein of M(r) 31,900. This protein has not been identified previously and has been designated NS4, a non-structural protein. RNA-RNA hybridization detected subgenomic RNAs encoding these proteins, a characteristic of RNA possessing an ambisense gene organization.

Amino Acid Sequence↗

PubMed2002

Nucleotide sequences of segments 1, 3 and 4 of the genome of Bombyx mori cypovirus 1 encoding putative capsid proteins VP1, VP3 and VP4, respectively.

The complete nucleotide sequences of genomic segments S1, S3 and S4 from Bombyx mori cypovirus 1 (BmCPV-1) have been determined. The segments consisted of 4190, 3846 and 3262 nucleotides encoding putative proteins of 1333, 1239 and 1058 amino acids with molecular masses of approximately 148, 140 and 120 kDa (p148, p140 and p120, respectively). All segments possess a single open reading frame. Homology searches showed that all three proteins have homologies to proteins of Rice ragged stunt virus, a member of the genus Oryzavirus within the family REOVIRIDAE: Partial homologies of p140 to structural proteins in other viruses were also found. The predicted molecular masses and the homologies with structural proteins in other viruses lead us to suggest that S1, S3 and S4 encode the capsid proteins VP1, VP3, and VP4, respectively, of BmCPV-1.

Amino Acid Sequence↗

PubMed1998

A cytoplasmic male sterility-associated mitochondrial peptide in common bean is post-translationally regulated.

Cytoplasmic male sterility in the common bean plant is associated with a dominant mitochondrial mutation designated pvs-or f 239 (for Phaseolus vulgaris sterility sequence open reading frame 239). The sequence is transcribed in both vegetative and reproductive tissues, but the translation product, ORF239, is present only in reproductive tissues. We present evidence to support a model of post-translational regulation of ORF239 expression based on the following observations. In organello translation experiments using purified mitochondria from young seedlings demonstrated accumulation of ORF239 only when a protease inhibitor was included. Proteolytic activity against ORF239 was observed in mitochondrial extracts fractionating with the mitochondrial inner membrane. The DNA sequence encoding a serine-type protease, similar to the lon protease gene of Escherichia coli, was cloned from the Arabidopsis genome. The expression product of this sequence demonstrated proteolytic activity against ORF239 in vitro, with features resembling the activity detected in mitochondrial inner membrane preparations. Antibodies generated against the overexpressed Lon homolog reduced proteolytic activity against ORF239 when added to mitochondrial extracts. Our data suggest that ORF239 was undetected in vegetative tissue due to rapid turnover by at least one mitochondrial protease that acts against ORF239 post-translationally.

Amino Acid Sequence↗

PubMed1996

Characterization and sequence analysis of a stable cryptic plasmid from Enterococcus faecium 226 and development of a stable cloning vector.

A small cryptic plasmid, pMBB1, isolated from Enterococcus faecium 226 was characterized. The plasmid contained an extremely stable replicon which has limited homology to the lactococcal plasmid pCI305. Sequence analysis of the replicon detected one open reading frame of 822 bp capable of encoding a 32-kDa protein. No detectable single-stranded intermediates were found for the replicon, suggesting that pMBB1 may be included in the same family as pCI305, although pCI305 exhibits a more narrow host range. A small stably maintained vector able to replicate in a variety of lactic acid bacteria, containing a large multiple cloning region, was constructed by using the pMBB1 replicon.

Amino Acid Sequence↗

PubMed2001

Nucleotide sequence and analysis of conjugative plasmid pVT745.

The complete nucleotide sequence and genetic map of pVT745 are presented. The 25-kb plasmid was isolated from Actinobacillus actinomycetemcomitans, a periodontal pathogen. Two-thirds of the plasmid encode functions related to conjugation, replication, and replicon stability. Among potential gene products with a high degree of similarity to known proteins are those associated with plasmid conjugation. It was shown that pVT745 derivatives not only mobilized a coresident nontransmissible plasmid, pMMB67, but also mediated their own conjugative transfer to different A. actinomycetemcomitans strains. However, transfer of pVT745 derivatives from A. actinomycetemcomitans to Escherichia coli JM109 by conjugation was successful only when an E. coli origin of replication was present on the pVT745 construct. Surprisingly, 16 open reading frames encode products of unknown function. The plasmid contains a conserved replication region which belongs to the HAP (Haemophilus-Actinobacillus-Pasteurella) theta replicon family. However, its host range appears to be rather narrow compared to other members of this family. Sequences homologous to pVT745 have previously been detected in the chromosomes of numerous A. actinomycetemcomitans strains. The nature and origin of these homologs are discussed based on information derived from the nucleotide sequence.

Aggregatibacter actinomycetemcomitans↗

PubMed2003

Characterization of endogenous retroviruses in sheep.

Endogenous retrovirus (ERV) sequences have been found in all mammals. In vitro and in vivo experiments revealed ERV activation and cross-species infection in several species. Sheep (Ovis aries) are used for various biotechnological purposes; however, they have not yet been comprehensively screened for ERV sequences. Therefore, the aim of the study was to classify the ERV sequences in the ovine genome (OERV) by analyzing the retroviral pro-pol sequences. Three OERV beta families and nine OERV gamma families were revealed. Novel open reading frames (ORF) in the amplified proviral fragment were found in one OERV beta family and two OERV gamma families. Hybrid OERV produced by putative recombination events were not detected. Quantitative analysis of the OERV sequences in the ovine genome revealed no relevant variations in the endogenous retroviral loads of different breeds. Expression analysis of different tissues from fetal and pregnant sheep detected mRNA from both gammaretrovirus families, showing ORF fragments. Thus, the release of retroviruses from sheep cells cannot be excluded.

Animals↗

PubMed1995

[Synthesis and antigenic activity of peptides from the ORF3 protein sequence of hepatitis E virus].

The hepatitis E virus (HEV) is an infection agent (detected recently) responsible for an enterically transmitted non-A, non-B hepatitis [1,2]. Hepatitis E is a big problem in many developing countries, including the Central Asian areas of the former Soviet Union [2]. By cloning followed by sequence analysis of the HEV genome, three open reading frames (ORF) have been identified, among them ORF3 encoding a protein containing 123 amino acid residues, the function of this protein being unknown. Recently [3], one of the immunodominant regions of ORF3 protein was revealed between the 91st and 123rd amino acid residue. The purpose of the present study was a more precise localization of epitopes in the C-terminal portion of HEV ORF3 protein by using synthetic peptides.

Amino Acid Sequence↗

PubMed2006

Molecular cloning and characterization of a C-type lectin from Ancylostoma ceylanicum: evidence for a role in hookworm reproductive physiology.

Lectins comprise a family of related proteins that mediate essential cell functions through binding to carbohydrates. Within this protein family, C-type lectins are defined by the requirement of calcium for optimal biologic activity. Using reverse transcription PCR, a cDNA corresponding to a putative C-type lectin has been amplified from the hookworm parasite Ancylostoma ceylanicum. The 550 nucleotide open reading frame of the A. ceylanicum C-type Lectin-1 (AceCTL-1) cDNA corresponds to a 167 amino acid mature protein (18,706 Da) preceded by a 17 amino acid secretory signal sequence. The recombinant protein (rAceCTL-1) was expressed in Drosophila S2 cells and purified using a combination of affinity chromatography and reverse phase HPLC. Using in vitro carbohydrate binding studies, it was determined that rAceCTL-1 binds N-acetyl-d-glucosamine, a common component of eukaryotic egg cell membranes. Using a polyclonal IgG raised against the recombinant protein, the native AceCTL-1 was identified in sperm and soluble protein extracts of adult male A. ceylanicum by immunoblot. Probing of adult hookworm sections with the polyclonal IgG demonstrated localization to the testes in males, as well as the spermatheca and developing embryos in females, consistent with its role as a sperm protein. Together, these data strongly suggest that AceCTL-1 is a male gender-specific C-type lectin with a function in hookworm reproductive physiology.

Amino Acid Sequence↗

PubMed1992

Nucleotide sequence of 9.2 kb left of CRY1 on yeast chromosome III from strain AB972: evidence for a Ty insertion and functional analysis of open reading frame YCR28.

We report the 9210 bp sequence from a segment of yeast chromosome III cloned from strain AB972 in lambda PM3270. Analysis of this sequence and its comparison with the one derived from the corresponding segment of strain XJ24-24A revealed that the AB972 region contains a duplication of about 2 kb and a Ty element, which are not found in XJ24-24A and cause a quite significant rearrangement of the whole region. We performed functional analysis of YCR28, the largest open reading frame we found in both AB972 and XJ24-24A. YCR28 encodes a putative protein of 512 amino acids with some similarities to yeast allontoate permease. Its disruption does not cause any detectable phenotype on rich medium or on allantoate medium, while we observed a strain-dependent effect on sensitivity to amino acid balance and to 3-aminotriazole, when cells were grown in synthetic medium.

Amino Acid Sequence↗

PubMed1994

Characterization of cDNAs species encoding the Tat protein of caprine arthritis encephalitis virus.

Two distinct species of caprine arthritis encephalitis virus (CAEV) tat cDNAs were isolated early after infection of a Himalayan tahr cell line. Sequence analyses predicted that one cDNA (pCEV/e1) represented a polycistronic transcript that encodes Tat and Rev as well as an N-terminally truncated transmembrane protein and a protein, designated X, whose function is unknown; whereas the other cDNA (pCEV/f1) encodes Tat and the env gene products. pCEV/e1 trans-activated a CAEV LTR-chloramphenicol acetyltransferase reporter gene in goat synovial membrane cells. This activity was shown to be encoded by the Tat open reading frame by analysis of a deletion mutant. Because the pCAEV/f1 insert was unstable in plasmid form, its Tat activity could not be convincingly demonstrated. The target sequences for Tat within the CAEV LTR were localized to the U3 region which, when placed in either orientation upstream of heterologous promoters, was able to confer responsiveness to Tat.

Amino Acid Sequence↗

PubMed1999

Complete genomic sequence of the lytic bacteriophage DT1 of Streptococcus thermophilus.

Streptococcus thermophilus lytic bacteriophage DT1, isolated from a mozzarella whey, was characterized at the microbiological and molecular levels. Phage DT1 had an isometric head of 60 nm and a noncontractile tail of 260 x 8 nm, two major structural proteins of 26 and 32 kDa, and a linear double-stranded DNA genome with cohesive ends at its extremities. The host range of phage DT1 was limited to 5 of the 21 S. thermophilus strains tested. Using S. thermophilus SMQ-301 as a host, phage DT1 had a burst size of 276 +/- 36 and a latent period of 25 min. The genome of phage DT1 contained 34,820 bp with a GC content of 39.1%. Forty-six open reading frames (ORFs) of more than 40 codons were found and putative functions were assigned to 20 ORFs, mostly in the late region of phage DT1. Comparative genomic analysis of DT1 with the completely sequenced S. thermophilus temperate phage O1205 revealed two large homologous regions interspersed by two heterologous segments. The homologous regions consisted of the early replication genes, the late morphogenesis genes, and the lysis cassette. The divergent segments contained the DNA packaging machinery, the major structural proteins, and remnants of a lysogeny module.

Amino Acid Sequence↗