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At least 649 records · Page 36Linked to original sources
PubMed1999

Molecular phylogenetic analysis of archaeal intron-containing genes coding for rRNA obtained from a deep-subsurface geothermal water pool.

Molecular phylogenetic analysis of a naturally occurring microbial community in a deep-subsurface geothermal environment indicated that the phylogenetic diversity of the microbial population in the environment was extremely limited and that only hyperthermophilic archaeal members closely related to Pyrobaculum were present. All archaeal ribosomal DNA sequences contained intron-like sequences, some of which had open reading frames with repeated homing-endonuclease motifs. The sequence similarity analysis and the phylogenetic analysis of these homing endonucleases suggested the possible phylogenetic relationship among archaeal rRNA-encoded homing endonucleases.

Archaea↗
PubMed1993

Characterization of a small plasmid from Desulfovibrio desulfuricans and its use for shuttle vector construction.

A 2.3-kb plasmid present in about 20 copies per genome was identified in extracts of Desulfovibrio desulfuricans G100A and designated pBG1. It appears to be unable to replicate in Escherichia coli. Although composite plasmids of pBG1 inserted into pTZ18U are stable in E. coli, few if any pBG1-specific transcripts are detectable. The plasmid sequence reveals several features typical of the origin of replication of non-ColE1 enterobacterial plasmids as well as several potential open reading frames. This small replicon has been shown to support the replication of recombinant plasmids in D. desulfuricans G100A and Desulfovibrio fructosovorans. A conjugable shuttle vector has been constructed.

Base Sequence↗
PubMed1988

Effects of uterine position on rate of sexual development in female Mongolian gerbils.

Examination of the rate of sexual maturation of 79 female gerbils from 32 Caesarean-delivered, foster-reared litters revealed that those females that, as fetuses, occupied uterine positions adjacent to one or two males (1M and 2M females) were less likely than those females occupying uterine positions not adjacent to males (0M females) to exhibit early vaginal opening. Our data further indicated that the uterine environment provided by late-maturing female gerbils biased their fetal daughters to themselves be late maturing to a greater extent than the daughters' uterine positions could explain. Daughters of late-maturing females in 1M and 2M uterine locations were more likely to be late-maturing than were daughters of early-maturing females in similar uterine locations. Because in female Mongolian gerbils age at vaginal opening is a powerful predictor of future reproductive strategy, the present results indicate that in female gerbils both prenatal maternal influence and uterine location are important determinants of future reproductive behaviors.

Animals↗
PubMed1993

Translation of the mRNA of the maize transcriptional activator Opaque-2 is inhibited by upstream open reading frames present in the leader sequence.

The protein encoded by the Opaque-2 (O2) gene is a transcription factor, translated from an mRNA that possesses an unusually long 5' leader sequence containing three upstream open reading frames (uORFs). The efficiency of translation of O2 mRNA has been tested in vivo by a transient assay in which the level of activation of the b32 promoter, a natural target of O2 protein, is measured. We show that uORF-less O2 alleles possess a higher transactivation value than the wild-type allele and that the reduction in transactivation due to the uORFs is a cis-dominant effect. The data presented indicate that both uORF1 and uORF2 are involved in the reducing effect and suggest that both are likely to be translated.

Alleles↗
PubMed1993

Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes.

The avrRpt2 locus from Pseudomonas syringae pv. tomato causes virulent strains of P. syringae to be avirulent on some, but not all, lines of Arabidopsis thaliana and Glycine max (soybean). We determined the DNA sequence of the avrRpt2 locus and identified the avrRpt2 gene as a 768-bp open reading frame encoding a putative 28.2-kDa protein. Deletion analysis and transcription studies provided further evidence that this open reading frame encodes AvrRpt2. We found that the avrRpt2 gene also has avirulence activity in P. syringae pathogens of Phaseolus vulgaris (common bean), suggesting that disease resistance genes specific to avrRpt2 are functionally conserved among diverse plant species. The predicted AvrRpt2 protein is hydrophilic and contains no obvious membrane-spanning domains or export signal sequences, and there was no significant similarity of AvrRpt2 to sequences in the GenBank, EMBL, or Swiss PIR data bases. A comparison of the avrRpt2 DNA sequence to nine other P. syringae avirulence genes revealed a highly conserved sequence, GGAACCNA-N14-CCACNNA, upstream of the translation initiation codon. This motif is located 6 to 8 nucleotides upstream of the transcription start site in all four P. syringae avirulence genes for which a transcription start site has been determined, suggesting a role as a binding site for a novel form of RNA polymerase. Regulation of avrRpt2 was similar to other P. syringae avirulence genes; expression was high in minimal medium and low in rich medium and depended on the hrpRS locus and an additional locus at the opposite end of the hrp cluster of P. syringae pv. tomato.

Amino Acid Sequence↗
PubMed1999

Update in thrombolytic therapy.

Several studies have proven the usefulness of thrombolytic agents in the therapy of ischemic stroke. Data from open and small placebo-controlled trials show a relationship between recanalization and an improved clinical outcome. Three large scaled studies have examine the effect of streptokinase treatment. Because of an increasing mortality the results of these studies were negative, and so streptokinase should not be used to treat acute ischemic stroke. In contrast, three rt-PA studies with together more than 2000 patients showed a significant benefit. Recent meta-analyses including data of all these trials show an odds ratio for death and disability of 0.67 (95 p. 100 CI 0.56; 0.8). The number needed to treat to prevent one death or disability is 11 in a 6 hour time window and 7 in a 3 hour time window. These are impressive numbers that are rarely found in other areas of internal or neurological medicine. The most important risk of thrombolytic therapy is the occurrence of intracerebral hemorrhage, which is reported as between 0-18 p. 100, but which is not associated with increased morbidity or mortality. At present, thrombolytic therapy cannot be recommended for all patients with acute stroke. Careful selection and experiences with this therapy and its risks are necessary.

Clinical Trials as Topic↗
PubMed1994

Identification and characterization of a murine cytomegalovirus gene with homology to the UL25 open reading frame of human cytomegalovirus.

Monoclonal antibody 1B4, previously shown to be protective in vivo and to cross-react with both virally encoded and normal host cell proteins, was used to screen a lambda gt11 cDNA derived from mRNA harvested from mouse embryo fibroblasts 24 hr after infection with murine cytomegalovirus (MCMV). A 700-bp cDNA was identified representing the 5'terminus of a 2460-bp open reading frame (ORF) with significant homology to the human cytomegalovirus UL25 ORF. The UL25 ORF of MCMV potentially encodes an 820 amino acid viral tegument protein with an estimated molecular weight of approximately 90 kDa. Amino acid homology with eukaryotic nucleolins was identified in the acidic N-terminal third of the MCMV UL25 proteins, suggesting that the protein may be involved in transcriptional activation or interactions with chromatin. Northern analysis and S1 nuclease data indicated that the gene is expressed late in infection as an approximately 3-kb transcript and that expression is dependent on viral DNA replication. An epitope recognized by MAb 1B4 was identified using recombinant pGEX plasmids expressing fusion proteins representing the N-terminal region of the MCMV UL25 protein. The identification of the MCMV UL25 ORF as a member of the CMV-specific UL25/UL35 gene family provides an opportunity for the investigation of the role these genes and their products in CMV pathogenesis in an animal model.

Amino Acid Sequence↗
PubMed1991

AUG codons in the RNA leader sequences of the yeast PET genes CBS1 and SCO1 have no influence on translation efficiency.

We report that the major transcription start sites of the yeast PET gene SCO1 are located at positions -149 and -125 relative to the AUG initiation codon of the SCO1 reading frame. The leader sequences of the resulting mRNAs possess a single AUG codon at position -49, which initiates a short open reading frame of three amino acids. The recent finding of a similar situation in the case of the PET gene CBS1 prompted us to address the question as to whether these AUG codons might play some role in the expression of these PET genes. After removal of the upstream AUG codons by site-directed mutagenesis, expression was monitored by use of lacZ fusions and compared to the respective wild-type constructs. Our data show that under all growth conditions tested the leader-contained AUG initiation codons have no significant influence on the expression of both PET genes.

Amino Acid Sequence↗
PubMed2006

Identification of a dehydration and ABA-responsive promoter regulon and isolation of corresponding DNA binding proteins for the group 4 LEA gene CpC2 from C. plantagineum.

The resurrection plant Craterostigma plantagineum (Scrophulariaceae) is used as a model system to investigate the molecular and biochemical basis of desiccation tolerance. Genes which contribute to desiccation tolerance are expressed during dehydration of this plant. One of the dehydration-induced genes is CpC2, a group 4 LEA gene. The CpC2 promoter was analysed and a core promoter region (CPR) was identified which is critical for the responsiveness of the gene to dehydration and the plant hormone ABA. The CPR motif contains two ABA-response elements (ABRE) and a binding site for HDZIP transcription factors. A yeast one-hybrid screen was performed to isolate CPR binding proteins. This resulted in the isolation of a bZIP transcription factor (CpbZIP1) and three highly conserved CpHistone H3 proteins. Two of these CpHistone H3 proteins are constitutively expressed histone H3 variants which are suggested to be involved in gene regulation via histone modification. The CpbZIP1 belongs to the group S of bZIP genes which possess long 5'-UTRs with a putative regulatory function. A second very similar bZIP clone, CpbZIP2, was isolated which contains a conserved small upstream open reading frame (uORF) within the 5'-leader sequence. A possible regulatory role of the uORF is discussed.

Abscisic Acid↗
PubMed1996

Complete nucleotide sequence of the mouse CTLA8 gene.

The gene encoding the mouse cytotoxic T-lymphocyte-associated antigen 8 (CTLA8) has been cloned and its complete nucleotide (nt) sequence determined. Sequence and polymerase chain reaction (PCR) analysis indicated that the published CTLA8 sequence[Rouvier et al., J. Immunol. 150 (1993), 5445-5456] was of rat rather than mouse origin. The mouse CTLA8 gene contains two exons and one intron. The 5'flanking region contains several consensus motifs for binding to transcription factors and the 3' untranslated region (UTR) has AU-rich motifs associated with RNA instability. The putative exon sequences predict that the full-length mouse CTLA8 molecule contains 147 amino acids (aa) and shares 88% aa identity with rat CTLA8 and 57% aa identity to HSV13, an open reading frame (ORF) from herpesvirus saimiri (HVS).

Animals↗
PubMed1994

Maintenance of an open reading frame as an additional level of scrutiny during splice site selection.

Although nonsense mutations have been associated with the skipping of specific constitutively spliced exons in selected genes, notably the fibrillin gene, the basis for this association is unclear. Now, using chimaeric constructs in a model in vivo expression system, premature termination codons are identified as determinants of splice site selection. Nonsense codon recognition prior to RNA splicing necessitates the ability to read the frame of precursor mRNA in the nucleus. We propose that maintenance of an open reading frame can serve as an additional level of scrutiny during exon definition. This process may have pathogenic and evolutionary significance.

Base Sequence↗
PubMed2002

Engineered herpes simplex virus 1 is dependent on IL13Ralpha 2 receptor for cell entry and independent of glycoprotein D receptor interaction.

In the first stage of engineering a herpes simplex virus (HSV)-1 that specifically targets human malignant glioma cells, we constructed a recombinant virus designated R5111 in which we have ablated the binding sites for sulfated proteoglycans in glycoproteins B and C, replaced the amino-terminal 148 aa in glycoprotein C by IL-13 flanked at its amino terminus with a signal peptide, and inserted a second copy of IL-13 after the amino acid 24 of glycoprotein D. In the process, the binding site for HveA, a viral entry receptor, was disrupted. We have also transformed a cell line (J1.1) lacking HSV-1 receptors to express IL13Ralpha2 receptor (J13R cells). We report the following: the R5111 recombinant virus replicates as well as wild-type virus in a variety of cell lines including cell lines derived from brain tumors. R5111 failed to replicate in the parent J1.1 cell line but multiplied to titers similar to those obtained in other cell lines in the J13R cell line. On the basis of the evidence that R5111 can use the IL13Ralpha2 receptor for entry, we conclude that HSV-1 can use receptors other than HveA or nectins, provided it can bind to them. The domains of gD that interact with HveA and nectin receptors are independent of each other. Lastly, the fusogenic activities of the glycoproteins in the viral envelope are not dependent on a set of unique interactions between glycoprotein D and its receptor. The construction of R5111 opens the way for construction of viruses totally dependent on selected receptors for entry or imaging of targeted cells.

Base Sequence↗
PubMed1993

Mapping cell-mediated immunodominant domains of the rubella virus structural proteins using recombinant proteins and synthetic peptides.

Although it is known that rubella-immune individuals have T cells that proliferate in vitro in response to rubella virus (RV), the determinants that evoke this response have not been identified. This study utilized recombinant proteins that express overlapping sequences of the RV structural open reading frame to identify domains of the structural proteins that contain cell-mediated immunodominant sequences. Lysates enriched with RV fusion proteins (RecA-RV-LacZ) were prepared from Escherichia coli transformed with plasmids which contained specific RV cDNA inserts. Approximately 62% of RV-immune individuals gave RV-specific responses to one or more of the RV fusion proteins. Over 10% of immune individuals recognized the capsid sequence C1-C29. Lymphoproliferation data from studies using six overlapping synthetic peptides representing this sequence suggested that as much as 70% of the immune population may recognize this domain. An E1 sequence, E1(202)-E1(283), was recognized by 15% of the RV-immune individuals with the fusion proteins. Five synthetic peptides representing this sequence had an overall response rate of 50%. The sequence C64-C97 failed to evoke any RV-specific responses with the fusion proteins and synthetic peptides representing this sequence were used to verify that the RV fusion proteins and the criteria used to identify RV-specific responses were adequate. These peptides gave a response rate of only 6%. In general, significant responses to specific fusion proteins correlated with high responses (stimulation index > or = 4.0) to representative synthetic peptides. This study suggests that the recombinant proteins were beneficial in identifying cell-mediated immunodominant domains of the RV structural proteins which could be further characterized with synthetic peptides.

Adult↗
PubMed1993

Detection of the outer membrane lipoprotein I and its gene in fluorescent and non-fluorescent pseudomonads: implications for taxonomy and diagnosis.

The open reading frame of the OprI lipoprotein gene from Pseudomonas aeruginosa was amplified by polymerase chain reaction (PCR) starting from purified DNA or colony lysates. A fragment of the expected size (249 bp) was detected in all P. aeruginosa strains from various clinical and geographical origins. The gene could only be amplified in pseudomonads of rRNA group I which are considered to be the authentic genus Pseudomonas. Digestions with HaeIII, PvuII and SphI of the amplified fragments demonstrated a sequence variation in the oprI gene. Colony, dot and Western blots with two monoclonal antibodies (mAbs) against the lipoprotein I confirmed our PCR results. These findings open interesting perspectives for the molecular taxonomy of the genus Pseudomonas and the development of diagnostic tools.

Antibodies, Monoclonal↗
PubMed1996

Sequence of plasmid pGT5 from the archaeon Pyrococcus abyssi: evidence for rolling-circle replication in a hyperthermophile.

The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced. Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence. The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication. Upstream of this open reading frame, we have detected an 11-bp motif identical to the double-stranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism. A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites. A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P. abyssi. These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication. Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.

Amino Acid Sequence↗
PubMed2000

Human cytomegalovirus latency-associated protein pORF94 is dispensable for productive and latent infection.

Human cytomegalovirus latency in bone marrow-derived myeloid progenitors is characterized by the presence of latency-associated transcripts encoded in the ie1/ie2 region of the viral genome. To assess the role of ORF94 (UL126a), a conserved open reading frame on these transcripts, a recombinant virus (RC2710) unable to express this gene was constructed. This virus replicated at wild-type levels and expressed productive as well as latency-associated ie1/ie2 region transcripts. During latency in granulocyte-macrophage progenitors, RC2710 DNA was detected at levels indistinguishable from wild-type virus, latent-phase transcription was present, and RC2710 reactivated when latently infected cells were cocultured with permissive fibroblasts. These data suggest pORF94 is not required for either productive or latent infection as assayed in cultured cells despite being the only known nuclear latency-associated protein.

Base Sequence↗
PubMed2005

Identification and expression of amphioxus beta-microseminoprotein (MSP)-like gene encoding an ancient and rapidly evolving protein in chordates.

The cDNA encoding beta-microseminoprotein-like (beta-MSPL) was identified from the gut cDNA library of amphioxus. It contains a 336 bp open reading frame corresponding to a deduced protein of 111 amino acids and has eight cysteines conserved and located at the same positions as those in the vertebrate beta-MSPs. At amino acid level, it shares 12-20% similarity to the vertebrate beta-MSPs, and seems lacking the signal peptide at the N-terminus. This not only confirms that beta-MSP is a rapidly evolving protein during phylogeny, but also provides further data on the degree of diversity between species of this protein. RT-PCR and Northern blotting show that amphioxus beta-MSPL is expressed in all tissues examined, suggesting that beta-MSPL plays a fundamental role. However, in situ hybridization reveals that positive hybridization signals were present in all blastomeres of the embryos from 4-cell to gastrula stages, while its expression is restricted exclusively to notochord, somites and primitive gut in neurulae and larvae, and disappears in the ectoderm including the neural tube differentiated from the ectoderm. This suggests that beta-MSPL is possibly involved in the differentiation of ectoderm during embryonic development of cephalochordate amphioxus though it is ubiquitously expressed in embryos prior to gastrula stage and in the adult animal.

Amino Acid Sequence↗