Peritoneal leak and chronic pericardial effusion in a CAPD patient.
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Aged↗
Biomedical subjects
C Grimm
Publications and source records attributed to C Grimm.
A common maturation pathway for small nucleolar RNAs.
We have shown that precursors of U3, U8 and U14 small nucleolar RNAs (snoRNAs) are not exported to the cytoplasm after injection into Xenopus oocyte nuclei but are selectively retained and matured in the nucleus, where they function in pre-rRNA processing. Our results demonstrate that Box D, a conserved sequence element found in these and most other snoRNAs, plays a key role in their nuclear retention, 5' cap hypermethylation and stability. Retention of U3 and U8 RNAs in the nucleus is saturable and relies on one or more common factors. Hypermethylation of the 5' caps of U3 RNA occurs efficiently in oocyte nuclear extracts lacking nucleoli, suggesting that precursor snoRNAs are matured in the nucleoplasm before they are localized to the nucleolus. Surprisingly, m7G-capped precursors of spliceosomal small nuclear RNAs (snRNAs) such as pre-U1 and U2, can be hypermethylated in nuclei if the RNAs are complexed with Sm proteins. This raises the possibility that a single nuclear hypermethylase activity may act on both nucleolar and spliceosomal snRNPs.
Animals↗
The hrpRS locus of Pseudomonas syringae pv. phaseolicola constitutes a complex regulatory unit.
The right part of the hrp cluster of Pseudomonas syringae pv. phaseolicola contains two regulatory genes, the previously described hrpS gene and an adjacent locus, hrpR. In this study we determined the sequence of hrpR and analysed the functional organization of the two genes. HrpR and HrpS show high sequence similarities to each other and to other response regulators of the two-component regulatory system. This has recently also been described for the hrpRS system of the closely related pathogen Pseudomonas syringae pv. syringae. The results of our genetic analyses strongly indicate that hrpS expression is regulated by the hrpR gene product. DNA-protein binding studies and site-directed mutagenesis of the hrpR sequence provided further evidence that HrpR activates hrpS transcription by binding to an activator site. This HrpR binding site was mapped in a fragment which is located 378-609 nucleotides upstream of the hrpS transcription start site. The hrpS transcription start site maps 179 nucleotides upstream of the initiation codon ATG, as determined by primer extension analysis, and is preceded by a typical -12/-24 promoter motif.
Amino Acid Sequence↗
In vivo selection of RNA sequences involved in nucleocytoplasmic RNA trafficking.
We used Xenopus oocytes and microinjection techniques to select for RNAs containing nuclear localization signals. A 20 nucleotide long randomized sequence was inserted into a U1-like carrier RNA giving a pool of over 10(12) possible RNA sequences. RNAs that were found in the nucleus 20 hours after nuclear or cytoplasmic injection were amplified and used for the next round of selection and amplification. After 12 rounds, the pools were highly enriched for RNAs that remained in, or were transported into, the nucleus. When individual RNAs of these pools were cloned and sequenced two different types of nuclear localization signals were identified. Some RNAs contain a binding site (whose consensus is AAUUUUUGG) for the Sm proteins common to spliceosomal snRNPs; the 5' caps of these RNAs are hypermethylated during nuclear localization and the RNAs follow the transport pathway of snRNAs. Other molecules could be folded to make long duplexes at their 5' ends; nuclear localization of these RNAs is independent of the 5' caps, and transport from the cytoplasm resembles the pathway for protein import.
Animals↗
A new, pathogen-inducible gene of Arabidopsis is expressed in an ecotype-specific manner.
In this study we describe a novel gene, which was isolated in an attempt to search for specific plant resistance genes of Arabidopsis against isolates of the phytopathogenic bacterium Xanthomonas campestris pv. campestris. The gene was cloned by differential screening of a genomic library of the Xcc 750-resistant ecotype Col-0, using cDNA populations derived from ecotype Col-0 and the Xcc 750-susceptible ecotype Oy-0. The isolated gene, CXc750, is differentially expressed in ecotypes of Arabidopsis thaliana. In addition, although highly expressed in uninfected plants, gene expression increases in response to pathogen attack. CXc750 potentially codes for a small, basic protein of about 10 kDa. The predicted protein product contains a potential signal leader peptide at the amino-terminal end but no ER retention sequence and no further transmembrane domain. This indicates that the gene product is transported to other compartments or out of the cell. The possible function of CXc750 as a member of the plant defense response system is discussed.
Amino Acid Sequence↗
M26 recombinational hotspot and physical conversion tract analysis in the ade6 gene of Schizosaccharomyces pombe.
At the ade6 locus of Schizosaccharomyces pombe flanking markers have been introduced as well as five silent restriction site polymorphisms: four in the 5' upstream region and one in the middle of the gene. The mutations ade6-706, ade6-M26 (both at the 5' end) and ade6-51 (middle of the gene) were used as partners for crosses with the 3' mutation ade6-469. From these three types of crosses, wild-type recombinants were selected and analyzed genetically to assess association with crossing-over and physically to determine conversion tract lengths. The introduced restriction site polymorphisms (five vs. only one) neither influenced the pattern of recombinant types nor the distribution of conversion tracts. The hotspot mutation M26 enhances crossing-over and conversion to the same proportion. M26 not only stimulates conversion at the 5' end, but does this also (to a lower extent) at the 3' end of ade6 at a distance of more than 1 kb. The majority of meiotic conversion tracts are continuous and postmeiotic segregation of polymorphic sites is rare. Conversion tracts are slightly shorter with M26 in comparison with its control 706. The mean minimal length of tracts varies from 670 bp (M26) to 890 bp (706) to 1290 bp (51). It is concluded that M26 acts as an initiation site of recombination or enhances initiation of recombination. M26 does not act by termination of conversion. A region of recombination initiation exists at the 5' end of the ade6 gene also in the absence of the ade6-M26 hotspot mutation.
Crosses, Genetic↗
The low abundance of U7 snRNA is partly determined by its Sm binding site.
In transient expression studies after DNA transfection of HeLa cells, the mouse U7 gene produces only approximately 30% of the RNA produced by a mouse U1b gene. This difference persists even when the transfected genes have all their 5' and 3' flanking sequences exchanged suggesting a post-transcriptional effect. When the special U7 Sm binding site is mutated to a consensus derived from the major snRNAs (Sm-opt), the U7 RNA level increases 4- to 5-fold, whereas no RNA is detected from a U7 gene with a non-functional Sm binding site (Sm-mut). Moreover, U1b genes with the U7 Sm binding site yield reduced RNA levels. The Sm-opt site also alters the cellular behaviour of the corresponding U7 snRNA. It accumulates to a higher level in the nucleus than wild type U7 RNA, and is better immunoprecipitable with anti-Sm antibodies. Injection experiments in Xenopus oocytes indicate that the U7 genes with either Sm-opt or Sm-mut sites produce similar amounts of RNA as wild type U7, but that they differ in opposing ways in the processing of precursors to mature size U7 snRNA and in nuclear accumulation. However, in reconstitution experiments using Xenopus oocytes, we show that U7 Sm-opt RNA, despite its efficient nuclear accumulation, is not active in 3' processing of histone pre-mRNA, whereas wild type U7 RNA is assembled into functional snRNPs, which correctly process histone pre-mRNA substrate. This suggests a functional importance of the special U7 Sm sequence.
Animals↗
[The discovery of individual physiognomy in modern art].
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Anthropometry↗
Synchronized meiosis and recombination in fission yeast: observations with pat1-114 diploid cells.
The mutation pat1-114 has been used to synchronize meiosis in the fission yeast Schizosaccharomyces pombe. We have investigated several aspects of such synchronized meiotic cultures. In both pat1-114 and pat1+ diploids, meiotic landmark events are initiated at the same time after meiosis induction, but synchrony is much more pronounced in the pat1-114-driven meiosis. Commitment to recombination and to meiosis have been timed at 2 h after meiotic induction. Due to a seven-fold reduction of intragenic recombination frequency in the ade6 region of pat1-114 diploids, physical analysis of recombination has not been possible. We have distinguished three factors that influence intragenic recombination frequencies: temperature, azygotic versus zygotic meiosis, and the nature of the pat1 allele. Differences and similarities in the timing of meiotic landmarks in S. cerevisiae and S. pombe are discussed.
Cell Cycle↗
The strong ADH1 promoter stimulates mitotic and meiotic recombination at the ADE6 gene of Schizosaccharomyces pombe.
The effect of the strong promoter from the alcohol dehydrogenase gene on mitotic and meiotic intragenic recombination has been studied at the ade6 locus of the fission yeast Schizosaccharomyces pombe. A 700-bp fragment containing the functional adh1 promoter was used to replace the weak wild-type promoter of the ade6 gene. Analysis of mRNA showed that strains with this ade6::adh1 fusion construct had strongly elevated ade6-specific mRNA levels during vegetative growth as well as in meiosis. These increased levels of mRNA correlated with a 20- to 25-fold stimulation of intragenic recombination in meiosis and a 7-fold increased prototroph formation during vegetative growth. Analysis of flanking marker configurations of prototrophic recombinants indicated that simple conversions as well as conversions associated with crossing over were stimulated in meiosis. The strongest stimulation of recombination was observed when the adh1 promoter was homozygous. Studies with heterologous promoter configurations revealed that the highly transcribed allele was the preferred acceptor of genetic information. The effect of the recombinational hot spot mutation ade6-M26 was also investigated in this system. Its effect was only partly additive to the elevated recombination rate generated by the ade6::adh1 fusion construct.
Alcohol Dehydrogenase↗
The recombinational hot spot mutation ade6-M26 of Schizosaccharomyces pombe stimulates recombination at sites in a nearby interval.
With the help of in vitro constructed intragenic double mutants, we investigated the influence of the recombinational hot spot mutation ade6-M26 on meiotic recombination between two additional ade6 mutations proximal to it. Recombination was stimulated four-fold when M26 was present in a heterozygous condition and ten-fold when homozygous. M26 itself remained unaffected in a substantial number of these events. This indicates that the stimulation can not only be due to a preferred conversion of M26 to wild-type with co-conversion of the second mutation in cis. A model is proposed in which M26 acts as an "entry site" for recombinational enzymes.
Chromosome Mapping↗
The predicted protein product of a pathogenicity locus from Pseudomonas syringae pv. phaseolicola is homologous to a highly conserved domain of several procaryotic regulatory proteins.
A ca. 20-kilobase (kb) region (hrp) that controls the interaction of Pseudomonas syringae pv. phaseolicola with its host (pathogenicity) and nonhost plants (hypersensitive reaction) was previously cloned and partially characterized. In this study we defined the limits and determined the nucleotide sequence of a hrp locus (hrpS), located near the right end of the hrp cluster. The largest open reading frame (ORF302) in hrpS has a coding capacity for a 302-amino-acid polypeptide. The predicted amino acid sequence of the translation product of ORF302 (HrpS) shows significant similarity to several procaryotic regulatory proteins, including the NtrC, NifA, and DctD proteins of Rhizobium spp., the NtrC and NifA proteins of Klebsiella pneumoniae, and the TyrR protein of Escherichia coli. These proteins regulate diverse operons involved in nitrogen fixation, transport and metabolism of amino acids, and transport of C-4 dicarboxylic acids. The HrpS protein appears to be the shortest naturally occurring member of this family of proteins, corresponding for the most part to the highly conserved central domain of these proteins, which contains a putative ATP-binding site. A C-terminal segment analogous to the less-well-conserved domain, involved in DNA binding of NtrC and NifA, is also present in HrpS. These similarities suggest that HrpS is a regulatory protein. In line with this prediction is the finding that a functional hrpS gene is necessary for the activation of another hrp locus during the plant-bacterium interaction.
Amino Acid Sequence↗
Interlaboratory comparison of methane electron capture negative ion mass spectra.
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Mass Spectrometry↗
Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker.
A system is described for gene disruption and replacement in Schizosaccharomyces pombe based on the homologous selectable marker, ura4, the structural gene for orotidine-5'-phosphate decarboxylase. The presence of a single copy of the wild-type gene can rescue a ura4 auxotrophic mutant. Furthermore, ura4- cells can be selected for in the presence of 5-fluoroorotic acid (5-FOA). This allows a convenient means of selecting for both forward and backward mutations. The sequence of a 1.8 kb HindIII fragment which contains the functional gene is reported. It encodes a single open reading frame of 264 amino acids which shows considerable conservation with the orotidine-5'-phosphate (OMP) decarboxylases from other organisms. The ura4 transcript is approximately 850 nucleotides long. It begins 51 bp upstream of the protein coding sequence and is unusual in that transcription termination occurs at or very close to the translational stop codon. To facilitate the use of ura4 in gene disruption experiments we have also constructed a novel strain of S. pombe called ura4-D18, in which the 1.8 kb HindIII fragment has been deleted from the chromosome. Using a combination of this strain and vectors containing ura4 as a selectable marker, we present a general method for targeting recombination events to the chromosomal locus under investigation.
Amino Acid Sequence↗
Observations on integrative transformation in Schizosaccharomyces pombe.
Three different Schizosaccharomyces pombe strains have been transformed with a circular or linearized non-ars plasmid carrying the ura4+ gene as a selectable marker. The first strain shows full homology between the genomic ura4-294 gene (point mutation) and the marker gene on the plasmid. The second strain carries a 600 bp deletion (ura4-D6) that decreases homology between plasmid and chromosome. No homology remains in the third strain which has a complete deletion of the ura4 gene on the chromosome (ura4-D18). When sequence homology exists between transforming DNA and the chromosomal ura4 region, gene conversion is strongly preferred over integration of the circular plasmid. Reduction of the length of homology leads to a decrease of transformation frequencies, and homology dependent as well as a minority of homology independent integrations are observed. In the complete absence of homology two rare types of transformants are encountered: either the circular plasmid replicates autonomously, although it is devoid of an ars sequence, or alternatively the plasmid integrates into the genome at various positions. Transformation with plasmid cut within the coding region of ura4 can lead to tandemly arranged multiple integrations, when no homology exists between the free ends and the chromosome. The integrations occur at the ura4 locus, when homology is retained between plasmid and chromosome, and at various sites in the genome of the strain with a complete deletion of the ura4 gene. The results suggest that homology dependent events (conversion, integration) are strongly preferred in transformation of S. pombe with non-ars plasmids. In addition low frequency integration by illegitimate recombination is observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Chromosome Deletion↗
Influence of calcium ions on proopiomelanocortin mRNA levels in clonal anterior pituitary cells.
The concentration of mRNA encoding proopiomelanocortin (POMC) was measured in AtT-20/D-16v cells, a clonal pituitary tumor cell line. Treatment of the cells with potassium (20 mM) or veratridine (10 microM) for 12, 24 and 48 h caused a time-dependent increase in the levels of POMC mRNA which became significant after 24 h. These effects were not seen in the presence of the sodium channel blocker tetrodotoxin (5 microM). In addition, the calcium channel blocker verapamil (10 microM) completely abolished the responses to either potassium or veratridine, whereas the calcium channel agonist Bay K 8644 (0.1 microM) potentiated the effect of potassium. Furthermore, the calcium channel blockers verapamil (10 microM) and nidefipine (1 microM) significantly decreased not only basal levels of POMC mRNA but also the increase of mRNA levels induced by corticotropin-releasing factor (CRF; 0.1 microM), 8-bromo-cAMP (1 mM) or cholera toxin (100 ng/ml). The drug-induced alterations in the mRNA POMC levels of the cells were, in each case, associated with similar alterations of immunoreactive beta-endorphin in the medium. These results indicate that membrane depolarization to activate sodium channels and calcium channels initiates an entry of calcium ions which triggers POMC gene expression in the AtT-20 cells. Moreover, calcium entry into the cells may exert a tonic stimulatory effect on POMC mRNA under basal conditions and may also contribute to the enhancing effect of CRF or cAMP on POMC mRNA in these cells.
Animals↗
The hypervariable region in the genes coding for entomopathogenic crystal proteins of Bacillus thuringiensis: nucleotide sequence of the kurhd1 gene of subsp. kurstaki HD1.
One of the genes for the entomophatogenic crystal protein of Bacillus thuringiensis (subsp. kurstaki strain HD1) has been cloned in Escherichia coli, and its nucleotide sequence determined completely. The gene is contained within a 4360-bp-long HpaI-PstI DNA restriction fragment and codes for a polypeptide of 1,155 amino acid residues. The protoxin protein has a predicted Mr of 130,625. The E. coli-derived protoxin gene product is biologically active against Heliothis virescens larvae in a biotest assay. Extensive computer comparisons with other published B. thuringiensis subsp. kurstaki strains HD1, HD73, and B. thuringiensis subsp. sotto gene sequences reveal hypervariable regions in the first half of the protoxin coding sequence. These regions are responsible for the biological activity of the protein product of the cloned gene, and may explain the different biological activities of these different protoxins.
Bacillus thuringiensis↗
Identification of a thyroid microsomal antigen by Western blot and immunoprecipitation.
The molecular identity of the thyroid microsomal antigen was investigated by Western blot and immunoprecipitation using sera from eight patients with autoimmune thyroid disease. All sera had high microsomal antibody titers by hemagglutination test and ELISA; only one had thyroglobulin (Tg) antibody. In an immunoprecipitation study using Triton X-100-solubilized 125I-labeled microsomes, all of the sera precipitated a 107K protein. However, this 107K protein was visualized by only three patients' sera by Western blot analysis under reducing conditions. In Western blots run under nonreducing conditions, four sera, including the three sera mentioned above, recognized poorly defined large mol wt proteins. One serum which also had Tg antibody recognized additional bands by both immunoprecipitation and Western blot. Preincubation of serum with 100 micrograms/ml Tg or 100 mU/ml bovine TSH did not alter the binding of antibody to 107K protein in Western blot analysis. The 107K protein was not visualized in Western blots of liver and kidney microsomes using patients' sera. Experiments were performed to characterize the difference between one serum (no. 1) which visualized the 107K band in Western blots and a serum (no. 4) which did not. Reactivity at different dilutions was compared by ELISA and Western blot studies. Serum 1 recognized the 107K protein even at a 1:3200 dilution, which gave a lower optical density value than a 1:200 dilution of serum 4 in ELISA, while serum 4 failed to recognize the 107K band at any dilution. An affinity gel prepared from serum 4 immunoglobulin G linked to agarose [Reacti-Gel (6 X)] removed from Triton X-100-solubilized microsomes the 107K protein which patient 1 serum recognized by Western blot analysis under reducing conditions. The data indicate that serum 4 can recognize the 107K protein under nondenaturing conditions. These data indicate that the 107K protein described here is the microsomal antigen, and that may associate with another protein or maintain a unique conformation due to disulfide bonds present in the native state. In addition, the 107K protein includes at least two antigen epitopes and different patients have different antibodies or different populations of antibodies against these epitopes.
Antigens↗