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PubMed1991

Molecular cloning of the Candida maltosa ADE1 gene.

The structural gene (ADE1) encoding phosphoribosyl-aminoimidazole-succinocarboxamide synthetase (SAICAR synthetase; EC 6.3.2.6) in Candida maltosa has been isolated by functional complementation of an ade1 strain of Saccharomyces cerevisiae. The gene was localized on a 2.5-kb BamHI DNA fragment. Nucleotide sequence analysis of the cloned gene has revealed an open reading frame encoding a protein (SAICAR synthetase) with an Mr of 32,751. The codon bias index, 0.68, indicates that the ADE1 gene is a moderately highly expressed gene. The cloned gene shows 63.5% nt identity and 65.2% deduced amino acid identity with the S. cerevisiae ADE1 gene which encodes the same enzymatic activity. The gene may be used as a convenient genetic marker for construction of a new host-vector system for C. maltosa.

Amino Acid Sequence↗

PubMed2001

A small upstream open reading frame causes inhibition of human major vault protein expression from a ubiquitous mRNA splice variant.

Overexpression of the major vault protein (MVP) has been linked to a multidrug resistance (MDR) phenotype. We describe a ubiquitously expressed MVP mRNA splice variant (long (L)-MVP) differing from the regular isoform (short (S)-MVP) within the 5'-leader. Only L-MVP mRNA contains a small upstream open reading frame which was proven to inhibit in vitro and in vivo MVP expression in cis. L-MVP represented an almost constant portion of total MVP mRNA in diverse normal tissues, but was more variable in malignant cell types. MDR sublines with altered MVP expression displayed changed S-MVP/L-MVP ratios as compared to their drug-sensitive counterparts. Our results suggest alternative splicing as one mechanism for regulation of MVP expression.

5' Untranslated Regions↗

PubMed1998

A novel Bacillus natto plasmid pLS32 capable of replication in Bacillus subtilis.

Plasmid pLS32 is a relatively large (approximately 70 kbp), cryptic, low copy-number plasmid present in Bacillus natto. We isolated and analyzed the replication region of the plasmid in B. subtilis, and the following results were obtained: the replication region contained an open reading frame encoding a 287-amino acid protein (RepN), whose amino acid sequence was partially homologous with those of the Rep proteins encoded on plasmids pAD1 and pLJ1 isolated from Enterococcus faecalis and Lactobacillus helveticus, respectively; the replication origin (oriN) was located in the repN-coding region; the copy number of a pLS32 derivative, pBET131, was 2 to 3 per chromosome; replication of pBET131 required poll. These features show that pLS32 is a novel plasmid capable of replication in B. subtilis.

Amino Acid Sequence↗

PubMed1997

Sequence of a melibiose transporter gene of Enterobacter cloacae.

We cloned a fragment of the chromosomal DNA of Enterobacter cloacae, which enabled a melibiose-negative Escherichia coli mutant lacking melB to grow on melibiose as the sole source of carbon. Transformed cells harboring the hybrid plasmid carrying the cloned DNA showed melibiose transport activity. The nucleotide sequence of the DNA region was determined. One complete open reading frame (ORF) and a part of another ORF were found in the region, and the amino acid sequences were deduced. The complete ORF was found to encode a melibiose transporter which consisted of 425 amino acid residues. Hydropathy analysis revealed that there are about 12 hydrophobic domains in this transporter. The incomplete ORF which exists in the upstream region of the transporter gene seemed to encode an alpha-galactosidase.

Amino Acid Sequence↗

PubMed2004

PIG-V involved in transferring the second mannose in glycosylphosphatidylinositol.

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors many proteins to the eukaryotic cell surface. The biosynthetic pathway of GPI is mediated by sequential additions of sugars and other components to phosphatidylinositol. Four mannoses in the GPI are transferred from dolichol-phosphate-mannose (Dol-P-Man) and are linked through different glycosidic linkages. Therefore, four Dol-P-Man-dependent mannosyltransferases, GPI-MT-I, -MT-II, -MT-III, and -MT-IV for the first, second, third, and fourth mannoses, respectively, are required for generation of GPI. GPI-MT-I (PIG-M), GPI-MT-III (PIG-B), and GPI-MT-IV (SMP3) were previously reported, but GPI-MT-II remains to be identified. Here we report the cloning of PIG-V involved in transferring the second mannose in the GPI anchor. Human PIG-V encodes a 493-amino acid, endoplasmic reticulum (ER) resident protein with eight putative transmembrane regions. Saccharomyces cerevisiae protein encoded in open reading frame YBR004c, which we termed GPI18, has 25% amino acid identity to human PIG-V. Viability of the yeast gpi18 deletion mutant was restored by human PIG-V cDNA. PIG-V has two functionally important conserved regions facing the ER lumen. Taken together, we suggest that PIG-V is the second mannosyltransferase in GPI anchor biosynthesis.

Amino Acid Sequence↗

PubMed2002

Upstream open reading frames regulate translation of Mona/Gads adapter mRNA in the megakaryocytic lineage.

Mona, also called Gads, is a molecular adapter that plays a key role in T-cell and platelet signalling by linking the adaptors Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (Slp-76) and linker for activation of T cells (LAT) upon T-cell receptor and collagen receptor activation. Platelets express a specific form of Mona mRNA, called 1B, which is transcribed from a megakaryocyte-specific promoter. Mona 1B mRNA differ from 1A transcripts found in T cells and some myeloid cells only by the 5'UTR. We report here that 1B mRNA expressing cells do not express detectable amounts of Mona protein, in contrast to 1A expressing cells, and we show that 1B 5'UTR contains upstream open reading frames (uORFs). Mutating the corresponding uAUG restored efficient Mona translation, or that of an unrelated ORF. This suggested that Mona protein expression in 1B mRNA expressing cells is tightly controlled at the translational level. Accordingly, Mona protein was not detected in resting platelets. Strikingly, platelet activation by thrombin resulted in the rapid induction of Mona protein expression, suggesting that translation inhibition of 1B mRNA may be relieved in activated platelets.

5' Untranslated Regions↗

PubMed2004

GENIUS II: a high-throughput database system for linking ORFs in complete genomes to known protein three-dimensional structures.

GENIUS II is an automated database system in which open reading frames (ORFs) in complete genomes are assigned to known protein three-dimensional (3D) structures. The system uses the multiple intermediate sequence search method in which query and target sequences are linked by intermediate sequences gathered by PSI-BLAST search. By applying the system to 129 complete genomes, 43.8% on average of the ORFs in the genomes were assigned to known 3D structures and the results are available for free at GENIUS II web site.

Algorithms↗

PubMed1994

Sequence and organization of the lactococcal prolate-headed bIL67 phage genome.

bIL67 is a broad-host-range prolate-headed phage that is active against Lactococcus cells. The complete phage genome sequence of 22195 bp was established. Thirty-seven open reading frames (ORFs) organized in two clusters were identified. Functions were assigned to the putative products of six of the ORFs on the basis of comparison of the deduced amino acid sequences to known proteins, analysis of structural features of the proteins and search for conserved motifs. These were a DNA polymerase, a protein involved in recombination, a lysin, a terminase subunit, a structural protein and a holin.

Amino Acid Sequence↗

PubMed2005

Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice.

A metagenomic (community genomic) library consisting of 5,760 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B from DNA extracted from the large-bowel microbiota of BALB/c mice. DNA inserts detected in 61 randomly chosen clones averaged 55 kbp (range, 8 to 150 kbp) in size. A functional screen of the library for beta-glucanase activity was conducted using lichenin agar plates and Congo red solution. Three clones with beta-glucanase activity were detected. The inserts of these three clones were sequenced and annotated. Open reading frames (ORF) that encoded putative proteins with identity to glucanolytic enzymes (lichenases and laminarinases) were detected by reference to databases. Other putative genes were detected, some of which might have a role in environmental sensing, nutrient acquisition, or coaggregation. The insert DNA from two clones probably originated from uncultivated bacteria because the ORF had low sequence identity with database entries, but the genes associated with the remaining clone resembled sequences reported in Bacteroides species.

Amino Acid Sequence↗

PubMed1993

Identification of an early-stage gene of Chlamydia psittaci 6BC.

Chlamydiae are parasitic bacteria characterized by a temporally regulated developmental cycle. In the early stage of the cycle, metabolically inert elementary bodies reorganize to dividing reticulate bodies, a process about which little is known. The purpose of this investigation was to identify and clone chlamydial genes that are expressed preferentially during the early stage of the developmental cycle of Chlamydia psittaci 6BC. Several potential early genes were cloned with highly radioactive, host-free-generated RNA probes to screen a genomic library. One clone appeared to encode a gene that was particularly well expressed at 1 h postinfection. In further characterization, we found that it encodes two complete open reading frames and one partial open reading frame of 370 codons. The partial open reading frame, designated gltX, is very similar to bacterial glutamyl-tRNA synthetases and was demonstrated to be transcribed in vivo at 24 h postinfection by primer extension analysis. A lysine-rich open reading frame (LRO) of 117 codons was found upstream and divergent from gltX. The LRO lacks homology to known proteins, and we were unable to demonstrate that it is transcribed in vivo. The third open reading frame, of 182 codons, was found to be convergent with and partially overlap the LRO. It was confirmed to be preferentially expressed within the first 1.5 h of infection by Northern (RNA) blot analysis and was designated the early upstream open reading frame (EUO). Like the LRO, the EUO is not homologous to known proteins. A major potential transcription start site of the EUO was identified by primer extension analysis. However, the sequence upstream of the site does not closely resemble the consensus recognition sequences of bacterial sigma factors even though it is AT rich. The EUO is the first chlamydial gene specific to the early stage to be cloned and sequenced.

Amino Acid Sequence↗

PubMed1991

Biologic properties and nucleotide sequence analysis of human papillomavirus type 51.

Human papillomaviruses (HPVs) may be grouped according to the site from which they are isolated and the disease with which they are associated. We recently identified and cloned HPV type 51 (HPV-51) from a low-grade precancerous lesion (G. Nuovo, E. DeVilliers, R. Levine, S. Silverstein, and C. Crum. J. Virol. 62:1452-1455, 1988). Molecular epidemiologic analysis of cervical lesions, including condylomata and low- and high-grade precancers, revealed that HPV-51 was present in about 5% of the samples we examined. We have now determined the complete nucleotide sequence of this virus and compared it with other sequenced HPVs. Our analysis reveals that the 7,808-bp genome is composed of eight open reading frames which are encoded on the same strand and that this virus is most closely related to HPV-31. Sequence comparisons place this virus in the group of high-risk viruses (those with an increased risk of progressing to malignancy) along with HPV-16, -18, -31, and -33. Morphologic transformation experiments demonstrated that HPV-51 had transformation potential and that transformed cells contained RNAs homologous to E6 and E7.

Amino Acid Sequence↗

PubMed1993

Evidence for a functional subclass of the RTVL-H family of human endogenous retrovirus-like sequences.

We report the discovery of a subgroup of RTVL-H human endogenous retroviral elements, designated RTVL-Hp, that is intact in the pol region which is deleted in the vast majority of RTVL-H elements. As a consequence, RTVL-Hp elements contain critical functional domains in their pol region that other RTVL-H elements lack. We estimate that the haploid genomes of humans, apes, and Old World monkeys contain 50 to 100 copies of RTVL-Hp elements and 800 to 1,000 deleted sequences. The major amplification of deleted elements appears to have occurred after the divergence of Old World and New World monkeys, since we have obtained evidence that a few intact RTVL-Hp elements, but no deleted forms, are present in marmoset DNA. Using the polymerase chain reaction coupled with a direct screen for open reading frames, we have isolated fragments from four RTVL-Hp elements amplified from human DNA that contain an open reading frame throughout a region of pol that is disrupted by diagnostic mutations in all other RTVL-H sequences that we had previously analyzed. Northern (RNA) hybridization analysis shows that unit-length RTVL-Hp transcripts are expressed in the human teratocarcinoma cell line Tera-1. Together, the results presented here suggest that a small functional subfamily of RTVL-H elements is present in the human genome.

Amino Acid Sequence↗

PubMed2003

Transcriptional and post-transcriptional mechanisms regulating the rat pituitary vasopressin V1b receptor gene.

The number of V1b vasopressin receptors (V1bR) in the anterior pituitary plays an important role during adaptation of the hypothalamic-pituitary-adrenal axis to stress in rats. Regulation of V1bR expression involves transcriptional and translational mechanisms. One of the elements mediating transcriptional activation of the rat V1bR gene is a long stretch of GAGA repeats (GAGA box) in the promoter located near the transcription start point capable of binding a protein complex of 127 kDa present in pituitary nuclear extracts. There is a lack of correlation between changes in V1bR mRNA and the number of VP binding sites, suggesting that V1bR expression depends on the efficiency of V1b R mRNA translation into protein. Two mechanisms by which the 5' untranslated region (5'-UTR) of the rat V1bR mRNA can mediate either inhibition or activation of V1bR mRNA translation have been identified. First, upstream open reading frames (ORF) present in the 5'-UTR repress translation of the major ORF encoding the V1b receptor, and secondly, an internal ribosome entry site (IRES) activates V1bR translation. Stimulation of IRES activity through protein kinase C-mediated pathways results in V1bR mRNA translation increasing V1bR protein levels. The existence of multiple loci of regulation for the V1bR at transcriptional and translational levels provides a mechanism to facilitate plasticity of regulation of the number of pituitary vasopressin receptors according to physiological demand.

5' Untranslated Regions↗

PubMed2002

[cDNA cloning and sequence analysis of genome segment S7 of rice black-streaked dwarf virus].

Genome segments 7 of zhejiang and Hebei isolates of rice black-streaked dwarf virus (RBSDV) were amplified and sequenced. Segment 7 of Zhejiang isolate was consisted of 2193 nts (EMBL accession no. AJ297427) in length and that of Hebei isolate was 2190 nts (AJ297428). Both segments contained two non-overlapping open reading frames (ORFs), which encoded two polypeptides with molecular weights of 41 kD and 36 kD. These two segments shared 99% nucleotide identity, 100% and 94.4% amino acid identities of ORF1 and ORF2, shared 93.5% and 93.8% identities at nucleotide level, 98.1% (ORF1) and 96.5%/97.8% (ORF2) at amino acid level with S7 of Japanese RBSDV, and shared 85.1% and 85.3% identities at nucleotide level, 92.3% (ORF1), 85.5%/86.8% (ORF2) at amino acid level with S6 of Italian MRDV.

Amino Acid Sequence↗

PubMed2004

Expression of 17beta-hydroxysteroid dehydrogenease in testis of the ascidian Ciona intestinalis [corrected].

Ascidians have been employed as model organisms in investigating spermatogenesis. 17beta-hydroxysteroid dehydrogenase (HSD) is a steroidogenic enzyme essential for invertebrate spermatogenesis. A homologue of HSD was found in the EST database of Ciona intestinalis and cloned. Sequence analysis showed significant homology to zebra fish, sea urchin and human 17beta-HSD. The gene has an open reading frame (ORF) of 918 nucleotides coding for a polypeptide of 306 amino acids and a calculated mass of 35-kDa. Immunoblotting with an antibody raised against HSD recognized a 35-kDa protein purified from the C. intestinalis testis. The HSD protein was localized in steroidogenic cells in the Ciona testis. These results suggest that C. intestinalis 17beta-HSD is equivalent to the enzyme of vertebrate Leydig cells and that 17beta-HSD could be a phylogenetic marker for organisms producing steroids.

17-Hydroxysteroid Dehydrogenases↗

PubMed1995

The human ribosomal protein S7-encoding gene: isolation, structure and localization in 2p25.

We have identified a gene encoding the human ribosomal protein (r-protein) S7. The S7 gene contains seven exons and six introns spanning about 6 kb. Organization of the gene is similar to that of Xenopus laevis S8, the only homologous intron-containing gene isolated so far. An mRNA transcribed from this gene has an open reading frame (ORF) of 582 nucleotides (nt), which encodes a protein of 194 amino acids (22.1 kDa). The transcription start point (tsp) was mapped by a primer extension assay to a C residue within a pyrimidine-rich tract. Human S7 (hS7) is identical to rat S7 (rS7) and exhibits significant similarity with the X. laevis, insect and plant homologs. We have used fluorescence in situ hybridization (FISH) to localize S7 to chromosome 2p25.

Amino Acid Sequence↗

PubMed1998

Generation of Saccharomyces cerevisiae deletants and basic phenotypic analysis of eight novel genes from the left arm of chromosome XIV.

The disruption of eight novel genes was realized in two genetic backgrounds. Among these open reading frames, NO333, NO348 and NO364 presented homologies with other proteins of yeast or other organisms, whereas NO320, NO325, NO339, NO384 and NO388 showed no similarity with any protein. Tetrad analysis of heterozygous deletant strains revealed that NO348, NO364 and NO388 are essential genes for vegetative growth, whereas NO320, NO325, NO333, NO339 and NO384 are non-essential. Basic phenotypic analyses of the non-lethal deletant strains as suggested in the six-pack B0 programme did not reveal any significant differences between parental and mutant strains.

Base Sequence↗

PubMed1995

Sequence analysis of the right end of chromosome XV in Saccharomyces cerevisiae: an insight into the structural and functional significance of sub-telomeric repeat sequences.

Approximately 3.9 kb of DNA, centromere proximal to the previously sequenced Y' element at the right end of chromosome XV in Saccharomyces cerevisiae strain YP1, has been sequenced. A number of the known sub-telomeric repeat sequences were identified, including Y', core X and STRs A, B. C and D. Several of these repeat elements contain potentially functional sequences. In addition, two other members of repeated gene families were identified. The first of these shows 61% and 60% DNA sequence identity to Enolases 1 and 2 respectively. The Enolase-like sequence appears to be species specific, with three copies being found in all strains of S. cerevisiae studied. The location of the three copies is the same for all strains. The second repeated sequence has homology with known open reading frames on chromosomes III, V and XI. There are five or six copies of this sequence in all S. cerevisiae and S. paradoxus strains studied and three in S. bayanus strains. The analysis of this region and comparison to sub-telomeric regions on other chromosomes gives some indication as to the potential functional and structural significance of sub-telomeric repeat sequences. In addition, these findings are consistent with the idea that sub-telomeric regions may be targets for unusual recombination events.

Amino Acid Sequence↗