Identification of six open reading frames in the Salmonella enterica subsp. enterica ser. Typhi viaB locus involved in Vi antigen production.
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Antigens, Bacterial↗
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The complete sequence of a 6146 bp fragment of Saccharomyces cerevisiae chromosome III contains two new open reading frames.
As part of the EEC project to sequence the entire chromosome III of Saccharomyces cerevisiae we have sequenced a total of 11,040 bp from near the right end of the chromosome. A new protein kinase gene was found at one extremity of the sequenced region (Wilson et al., 1992), while the previously sequenced actin binding protein gene, ABP1, (Drubin et al., 1990) was found at the other extremity. We present here the sequence of the region between these two genes which has the potential to code for two new open reading frames (ORFs).
Amino Acid Sequence↗
The TSM1 gene of Saccharomyces cerevisiae overlaps the MAT locus.
We have cloned the region from MAT to THR4 on chromosome III of Saccharomyces cerevisiae. Although the region is only 15 kb, the two loci are genetically separated by 22 cM. This is in sharp contrast to the very low level of recombination (2 cM in 22 kb) that is observed in the adjacent CRY1-MAT interval, and suggests that there may be a "hot spot" for recombination in the MAT-THR4 region. The DNA sequence of the first 4.4 kb distal to MAT reveals an open reading frame that we have identified as the essential gene, TSM1. Surprisingly, the TSM1 open reading frame of 1,410 amino acids extends into the MAT locus, such that the 3'-end of the MAT alpha 1 transcript ends 15 bp from the 3'-end of the TSM1 open reading frame.
Alleles↗
Reconstitution of transcription from the human U6 small nuclear RNA promoter with eight recombinant polypeptides and a partially purified RNA polymerase III complex.
The human U6 small nuclear (sn) RNA core promoter consists of a proximal sequence element, which recruits the multisubunit factor SNAP(c), and a TATA box, which recruits the TATA box-binding protein, TBP. In addition to SNAP(c) and TBP, transcription from the human U6 promoter requires two well defined factors. The first is hB", a human homologue of the B" subunit of yeast TFIIIB generally required for transcription of RNA polymerase III genes, and the second is hBRFU, one of two human homologues of the yeast TFIIIB subunit BRF specifically required for transcription of U6-type RNA polymerase III promoters. Here, we have partially purified and characterized a RNA polymerase III complex that can direct transcription from the human U6 promoter when combined with recombinant SNAP(c), recombinant TBP, recombinant hB", and recombinant hBRFU. These results open the way to reconstitution of U6 transcription from entirely defined components.
Amino Acid Sequence↗
Cloning and characterization of a complete open reading frame of the hepatitis C virus genome in only two cDNA fragments.
The synthesis of long cDNA molecules encoding the complete genome of RNA viruses has recently been demonstrated; this major improvement has numerous practical applications such as construction of infectious cDNA clones or study of sequence variability at the level of a single RNA molecule. Using hepatitis C virus (HCV) as a model, we established an RT-PCR technique for amplification of cDNA fragments with a length of about 5 kb. The RT reaction was carried out with a Moloney murine leukaemia virus reverse transcriptase lacking detectable RNase H activity. For PCR reactions an enzyme mix containing Taq and Pwo DNA polymerases was used. Hot start and addition of 5% DMSO were also important to efficiently achieve long PCR products. About 10(6) HCV genome equivalents/ml in serum were needed in order to amplify the HCV genome in only two cDNA fragments covering about 98% of the complete genome. Analysis of the HCV quasi-species is also possible by this method as shown by sequencing of the hypervariable region 1 (HVR1) after cloning of cDNAs. The integrity of the long cDNA clones was proven by (1) restriction analyses, (2) partial sequencing and (3) expression of respective gene products. In vitro transcribed cDNAs were translated in rabbit reticulocyte lysate. Structural and nonstructural HCV proteins were identified by immunoprecipitation using patient serum. These results suggest that the two cDNA clones encode a complete and functional open reading frame of HCV.
Amino Acid Sequence↗
Antisense RNA mediates transcriptional processing in an archaebacterium, indicating a novel kind of RNase activity.
Strains of the extremely halophilic archaebacterium Halobacterium salinarium that are lysogenic for the phage phi H produce an antisense RNA transcript complementary to the first 151 nucleotides (nt) of the early lytic phage transcript T1. This is the first case of antisense control of gene expression in an archaebacterium. We show through transformation of H. salinarium that the antisense RNA functions in trans, rendering the early lytic phage transcript T1 susceptible to specific cleavage by an unidentified RNase of unique endonucleolytic activity. The single-stranded ends of RNA are cut off at the ends of the 151 nt RNA duplex, removing the ribosomal binding sites from the first open reading frame of transcript T1 but without concomitant digestion of the products.
Amino Acid Sequence↗
Plasmids from two morphologically distinct cyanobacterial strains share a novel replication origin.
A 2.9-kbp replication origin from a plasmid endogenous to the filamentous cyanobacterium Fremyella diplosiphon UTEX 481 was genetically characterized and sequenced. Deletion analysis of the 2.9-kbp DNA fragment delimited the minimum region necessary for replication in F. diplosiphon Fd33 to approximately 2.5 kbp. DNA sequence analysis revealed that the F. diplosiphon plasmid replication origin is structurally very similar to and shares significant identity with the 1.75-kbp replication origin reported for plasmid pDU1, isolated from the morphologically distinct cyanobacterium Nostoc sp. strain PCC 7524. Each cyanobacterial plasmid replication origin includes a large open reading frame that predicts a conserved protein of unknown function; the predicted proteins of the replication origins are of similar sizes and 30% identical in amino acid sequence. Each cyanobacterial plasmid replication origin also possesses a region of dyad symmetry approximately 300 bp upstream of the conserved open reading frame.
Amino Acid Sequence↗
xln23 from Streptomyces chattanoogensis UAH23 encodes a putative enzyme with separate xylanase and arabinofuranosidase catalytic domains.
The xylanase gene xysA of Streptomyces halstedii JM8 was used to isolate a DNA fragment from a gene library of Pstl-digested chromosomal DNA of the lignocellulolytic actinomycete Streptomyces chattanoogensis CECT-3336. Nucleotide sequence analysis revealed a gene (xln23) encoding a bifunctional multimodular enzyme bearing two independent xylanase and alpha-L-arabinofuranosidase domains separated by a Ser/Gly-rich linker. The N terminus of the predicted protein showed high homology to family F xylanases. The C terminus was homologous to amino acid sequences found in enzymes included in the glycosyl hydrolase family 62 and, in particular, to those of alpha-L-arabinofuranosidase AbsB from Streptomyces lividans. PCR and RT-PCR experiments showed that the nucleotide sequences corresponding to each domain are arranged as expected on the chromosomal DNA and that they are cotranscribed. To our knowledge, this is the first description of xylanase and arabinofuranosidase domains in a same open reading frame.
Amino Acid Sequence↗
Analysis of the DNA sequence of a 34,038 bp region on the left arm of yeast chromosome XV.
We report the DNA sequence of a 34,038 bp segment of Saccharomyces cerevisiae chromosome XV. Subsequent analysis revealed 20 open reading frames (ORFs) longer than 300 bp and two tRNA genes. Five ORFs correspond to genes previously identified in S. cerevisiae, including RPLA2, PRE6, MSE1, IFM1 and SCM2 (TAT2, TAP2, LTG3). Two putative proteins share considerable homology with other proteins in the current data libraries. ORF O2145 shows 41.2% identity with the glycophospholipid-anchored surface glycoprotein Gas1p of S. cerevisiae and ORF O2197 has 53.2% identity to chromosome segregation protein Dis3p of Schizosaccharomyces pombe.
Amino Acid Transport Systems↗
Sequence of a 17.1 kb DNA fragment from chromosome X of Saccharomyces cerevisiae includes the mitochondrial ribosomal protein L8.
We have sequenced a continuous segment of 17,137 bp on chromosome X. Sequence analysis of this stretch revealed 14 open reading frames (ORFs) at least 100 amino acids long. One gene, encoding the mitochondrial 60S ribosomal protein L8, had already been sequenced. Four ORF products show weak homologies with known protein sequences. The nine remaining ORF products have no homologies with sequences in data banks.
Carboxylesterase↗
Social and biological early life influences on the prevalence of open bite in Brazilian 6-year-olds.
OBJECTIVE: Little is known about the effects of social and biological risk factors for open bite on the primary dentition. The aim of this study was to assess the early-life risk factors affecting anterior open bite. METHODS: A cross-sectional study using a birth cohort was carried out in Pelotas, Brazil. A sample of 400, 6-year-old children was employed. The Foster and Hamilton criteria were used to classify open bite. Data concerning social conditions, and perinatal and childhood health and behaviour were obtained from birth to 12 months of age and during the fifth year of the children's lives. Unconditional bivariate and multiple logistic regression analysis were performed. RESULTS: The prevalence of anterior open bite was 46.3%. Risk factors included: a maternal age of between 30 and 39 years, as compared with children whose mothers were younger; breast-feeding for < 9 months; dental caries experience; pacifier sucking between 12 months and 5 years, as compared to no sucking or a shorter duration of sucking; and the presence of finger-sucking at 6 years of age. CONCLUSION: Open bite in the primary dentition was associated with older mothers, early weaning, dental caries occurrence, long-term use of a pacifier and finger-sucking at 6 years of age. These findings support the common risk approach for intervention to prevent open bite in the primary dentition.
Bottle Feeding↗
A chimeric open reading frame in the 5' flanking region of coxI mitochondrial DNA from cytoplasmic male-sterile wheat.
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Amino Acid Sequence↗
Sequence analysis of a 13.4 kbp fragment from the left arm of chromosome XV reveals a malate dehydrogenase gene, a putative Ser/Thr protein kinase, the ribosomal L25 gene and four new open reading frames.
A 13421 bp fragment located near the left telomere of chromosome XV (cosmid pEOA461) has been sequenced. Seven non-overlapping open reading frames (ORFs) encoding polypeptides longer than 100 residues have been found (AOB859, AOC184, AOE375, AOX142i, AOE423, AOA476 and AOE433). An additional ORF (AOE131) is found within AOA476. Three of them (AOC184, AOA476 and AOE433) show no remarkable identity with proteins deposited in the data banks. ORF AOB859 is quite similar to a hypothetical yeast protein of similar size located in chromosome VI, particularly within the C-terminal half. AOE375 encodes a new member of the glycogen synthase kinase-3 subfamily of Ser/Thr protein kinases. AOX142i is the gene encoding the previously described ribosomal protein L25. AOE423 codes for a protein virtually identical to the MDH2 malate dehydrogenase isozyme. However, our DNA sequence shows a single one-base insertion upstream of the reported initiating codon. This would produce a larger ORF by extending 46 residues the N-terminus of the protein. The existence of this insertion has been confirmed in three different yeast strains, including FY1679.
Amino Acid Sequence↗
Sequence of the CDC10 region at chromosome III of Saccharomyces cerevisiae.
A 4.74 kb DNA fragment from the right arm of chromosome III of Saccharomyces cerevisiae, adjacent to the centromere region was sequenced. Four open reading frames with an ATG initiation codon and larger than 200 bp were found in this fragment. The largest open reading frame of 966 bp was identified as the CDC10 gene.
Base Sequence↗
Cloning and analysis of YMR26, the nuclear gene for a mitochondrial ribosomal protein in Saccharomyces cerevisiae.
The nuclear gene for a mitochondrial ribosomal protein, termed YMR26, of Saccharomyces cerevisiae strain DC-5 was cloned by hybridization with synthetic oligonucleotide mixtures corresponding to the N-terminal amino acid sequence of this protein. The gene was found to occur in a single copy on either chromosome VII or chromosome XV. The nucleotide sequence of the cloned segment containing this gene showed the presence of an open reading frame capable of encoding a basic protein of 18.5 kDa with 158 amino acid residues. The deduced amino acid sequence showed no significant similarity to any known ribosomal proteins of prokaryotic or eukaryotic origin or to any other proteins in the NBRF protein data bank. When the gene was disrupted by insertion of a 2.9 kb restriction fragment containing LEU2, cells became PET- indicating that the gene is essential for yeast mitochondria. Northern blot analysis indicated that the size of the transcript from the YMR26 gene was approximately 530 nucleotides long. The expression level of the YMR26 gene was monitored upon catabolite repression, in strains with various mitochondrial genetic backgrounds and in strains harboring an increased dosage of the YMR26 gene. In rho+ cells, the transcription of the YMR26 gene was more repressed in a medium with glucose than in the presence of either galactose or nonfermentable carbon sources. However, in rho o cells, its transcription appeared not to be repressed even by high concentrations of glucose. The amount of the YMR26 mRNA was increased 10-fold when cells carried the YMR26 gene on a high-copy number plasmid.
Amino Acid Sequence↗
Historical background: Why is it important to improve automated particle selection methods?
A current trend in single-particle electron microscopy is to compute three-dimensional reconstructions with ever-increasing numbers of particles in the data sets. Since manual--or even semi-automated--selection of particles represents a major bottleneck when the data set exceeds several thousand particles, there is growing interest in developing automatic methods for selecting images of individual particles. Except in special cases, however, it has proven difficult to achieve the degree of efficiency and reliability that would make fully automated particle selection a useful tool. The simplest methods such as cross correlation (i.e., matched filtering) do not perform well enough to be used for fully automated particle selection. Geometric properties (area, perimeter-to-area ratio, etc.) and the integrated "mass" of candidate particles are additional factors that could improve automated particle selection if suitable methods of contouring particles could be developed. Another suggestion is that data be always collected as pairs of images, the first taken at low defocus (to capture information at the highest possible resolution) and the second at very high defocus (to improve the visibility of the particle). Finally, it is emphasized that well-annotated, open-access data sets need to be established in order to encourage the further development and validation of methods for automated particle selection.
Algorithms↗
Molecular cloning and sequence analysis of alpha- and beta-globin cDNAs from yellowtail, Seriola quinqueradiata.
Two alpha- and two beta-globin genes from the yellowtail were cloned and their nucleotide sequences determined. The entire open reading frames of alpha-globin A and B genes were 432 and 435 bp long, respectively, whereas the corresponding values of the yellowtail beta-globin A and B genes were both 447 bp. Amino acid identity of the yellowtail alpha- or beta-globin gene compared with those reported in other vertebrates including shark, teleosts and human, ranged from 35.4 to 83.0%. The yellowtail alpha-globin B has a unique inserted amino acid residue in the 48th position, not found in other fish alpha-globin genes. The orientation of alpha- and beta-globin genes open reading frames was head-to-head.
Amino Acid Sequence↗
Cloning of Prevotella intermedia loci demonstrating multiple hemolytic domains.
A gene bank was created from Prevotella intermedia strain 27 chromosomal DNA, and a clone was isolated that conferred the expression of two separate modes of hemolytic activity in recombinant Escherichia coli. The original recombinant hemolytic strain (EB34) contained plasmid, pEB34, with a 5.6-kb insert from Sau 3 AI-digested P. intermedia strain 27 chromosomal DNA cloned into the Bam HI site of pUC18. EB34 and deletion subclones were tested for expression of hemolytic activity in a standard tube assay, measuring lysis of erythrocytes spectrophotometrically as a function of hemoglobin release. Cell suspensions of EB34 demonstrated a dose-dependent hemolytic activity, inhibitable by proteases, and heat treatment but not dependent on calcium ions, and not inhibitable by osmoprotectants. Cell-free lysates also demonstrated a heat inhibitable, dose dependent hemolytic activity. Sub-cloning experiments localized the hemolytic region of the insert to a 3.9-kb fragment under direction of the lac promoter. Sequence analysis of the entire insert revealed the presence of multiple open reading frames (1 to 3) in this region which correlated to different forms of hemolytic expression, such that subclones containing all open reading frames 1 to 3 demonstrated strong hemolytic phenotype on blood plates and in the tube assay. Subclones containing only ORF1 demonstrated hemolysis on plates, but not in the tube assay. Subclones containing only open reading frames 2 and 3, but not ORF1 demonstrated hemolysis in the tube assay but not on plates. Homology searches of DNA and protein databases have not revealed significant homologies with reported hemolysins or proteins in any of the open reading frames.
Amino Acid Sequence↗