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At least 379 records · Page 21Linked to original sources
PubMed1995

The sequence of an 11.1 kb fragment on the left arm of Saccharomyces cerevisiae chromosome VII reveals six open reading frames including NSP49, KEM1 and four putative new genes.

We report the sequence of an 11.1 kb fragment located on the left arm of chromosome VII of Saccharomyces cerevisiae. By sequence analysis we have detected six open reading frames (ORFs) longer that 300 bp, which cover 87% of the entire sequence. ORF G1645 is 100% identical to the KEM1 gene, also identified as DST2, XRN1, SEP1 and RAR5, while G1648 is 100% identical to the NSP49 or NUP49 gene. ORF G1642 shares some identity with a hypothetical protein of Caenorhabditis elegans, while the other four ORFs show no significant homology to known proteins.

Amino Acid Sequence↗
PubMed1998

TAT1 encodes a low-affinity histidine transporter in Saccharomyces cerevisiae.

Previous studies have revealed the presence of at least two histidine uptake systems in S. cerevisiae; one with high affinity and the other with low affinity for histidine. The HIP1 gene is known to encode the high affinity permease. The purpose of this study was to identify the gene that encodes the low affinity permease. A mutant strain of S. cerevisiae that is both a histidine auxotroph and a hip1 deletion mutant is unable to grow on low histidine media. This strain was transformed with a yeast cDNA library constructed in a yeast expression vector. Transformants with increased histidine transport were selected by their ability to grow on a low histidine media. Sequencing of the inserts revealed the presence of the HIP1 gene and also the presence of the TAT1 gene. Estimated Km and Vmax values for histidine transport by each system were determined. In a hip1 tat1 double mutant, the level of histidine required for growth increased eight-fold in comparison to the hip1 single mutant. Our results suggest that the TAT1-encoded protein, previously characterized as the high-affinity tyrosine permease, also acts as the low affinity histidine permease.

ATP-Binding Cassette Transporters↗
PubMed1999

Effects of anti-PM-Scl 100 (Rrp6p exonuclease) antibodies on prenucleolar body dynamics at the end of mitosis.

Prenucleolar bodies (PNBs) are transitory structures which serve as building blocks for nucleoli at the transition mitosis/interphase. The assembly of PNBs and their pathway are not clearly understood. To better understand these events, the behavior of the PNB-containing PM-Scl 100 protein was compared with that of other PNB proteins. This nucleolar protein was chosen because its yeast homologue, Rrp6p exonuclease [1], is known to participate in late events in 5.8 S rRNA (ribosomal RNA) processing. There was a heterogeneous distribution of nucleolar proteins in different classes of PNBs. The PM-Scl 100 colocalized predominantly with protein B23. The PM-Scl-100-containing PNBs were translocated at later times to nucleoli as opposed to the fibrillarin-containing PNBs. Microinjections of antibodies directed against PM-Scl 100 during mitosis inhibited targeting of PM-Scl 100 to the nucleolus. However fibrillarin and protein B23 still participated in nucleolar assembly in early G1. We conclude that there are different kinds of PNBs whose translocation to the nucleoli follow ordered kinetics. Interestingly, proteins involved in late steps of processing as PM-Scl 100 are translocated late, suggesting that they are not cotranscriptionally associated with the rRNA precursors.

Amino Acid Sequence↗
PubMed1999

Cloning and mapping of the XRN2 gene to human chromosome 20p11.1-p11.2.

The Dhm1 gene is the mouse homologue of the dhp1(+) gene of Schizosaccharomyces pombe, which is involved in homologous recombination and RNA metabolism, such as RNA synthesis and RNA trafficking, in S. pombe. Complementation analysis showed the Dhm1 gene on a multicopy plasmid can rescue the temperature-sensitivity mutation of dhp1(ts) and the lethality of the dhp1 null mutation. This finding suggests that Dhm1 has a function in mouse similar to that of dhp1(+). The human homologue of this gene, XRN2, has been identified. A 3.6-kb transcript of XRN2 was detected in 16 tissues examined and was more abundant in testis. By radiation hybrid panel mapping, the XRN2 gene was localized to chromosome 20p11.1-p11.2 between markers D20S180 and D20S871.

Adult↗
PubMed2000

Structure and localization of mouse Pmscl1 and Pmscl2 genes.

Sera from some patients with polymyositis-scleoderma overlap syndrome (PM-SCL) recognize two antigenically unrelated proteins, PMSCL1 and PMSCL2. Complete mouse Pmscl1 and Pmscl2 cDNA sequences, chromosomal localizations, exon/intron structure, and promoter region sequences of the mouse Pmscl2 gene are presented. The PMSCL1 gene was found to overlap significantly with cyclin A2 in both human and mouse. As such, it may be deduced that PMSCL1 sequences map to human chromosome 4q27 and the proximal portion of mouse chromosome (Chr) 3 where human and mouse cyclin A2 genes reside. Analysis of human and mouse PMSCL1 cDNA sequences provides evidence that the PMSCL1 protein is 68 amino acids longer than previously thought. A BAC containing mouse Pmscl2 was localized to distal mouse Chr 4 by FISH. This BAC contains the microsatellite D4Mit310. D4Mit310 colocalizes with a number of genes that map to human 1p36. In fact, a STS (G25404) located 54.6 cR from the top of human chromosome 1 was found to contain PMSCL2 sequence upon BLAST search.

5' Untranslated Regions↗
PubMed1993

Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway.

We have identified a family of abundant acidic human keratinocyte proteins with apparent molecular masses ranging between 30,000 and 31,100 (isoelectric focussing sample spot proteins 9109 (epithelial marker stratifin), 9124, 9125, 9126 and 9231 in the master two-dimensional gel database of human keratinocyte proteins) that share peptide sequences with each other, with protein 14-3-3 and with the kinase C inhibitory protein. Immunofluorescence staining of keratinocytes showed that two of these proteins (IEF SSPs 9124 and 9126) localize to the Golgi apparatus, while stratifin is distributed diffusely in the cytoplasm. Significant levels of stratifin, and in smaller amount the sample spot proteins 9124, 9125 and 9126, were detected in the medium of cultured human keratinocytes suggesting that they are partially secreted by these cells. Two-dimensional gel analysis of proteins from cultured human cells and fetal tissues showed that polypeptides comigrating with proteins 9124, 9125 and 9126 are ubiquitous and highly expressed in the brain. Stratifin, however, was present only in cultured epithelial cells and was most abundant in fetal and adult human tissues enriched in stratified squamous keratinising epithelium. We have cloned and sequenced cDNAs coding for members of this family. The complete identity of the sequenced peptides from stratifin with the amino acid sequence translated from the stratifin cDNA clone indicated that this cDNA codes for stratifin. The identity of clones 1054, HS1 and AS1 is less clear as, with few exceptions, none of the individual peptide sequences fits the predicted protein sequences. The polypeptides synthesized by clones 1054 and HS1 in the vaccinia expression system, on the other hand, comigrate with proteins 9126 and 9124, suggesting cell-type-specific expression of members of the protein family. Database searches indicated that clone HS1 corresponds to a human T-cell cDNA 14-3-3 clone, while the high level of similarity of clones 1054 and AS1 with the 14-3-3 beta and eta sequences respectively, suggested that they code for the human equivalent of the two bovine proteins. Microsequence data indicated that IEF SSP 9124 corresponds to the human homolog of bovine 14-3-3 gamma.

14-3-3 Proteins↗
PubMed1995

Electron microscopic visualization of RecT protein and its complexes with DNA.

Electron microscopy has been used to examine Escherichia coli RecT protein alone and in the complexes it forms with DNA substrates, with which it catalyzes strand exchange in vitro. Negative staining has revealed that the 33 kDa RecT protein monomers form open C-shaped and closed O-shaped particles. RecT protein monomers assemble into donut-shaped oligomers containing seven or eight protein monomers and rod-like structures. When bound to single-stranded DNA, RecT forms highly twisted nucleoprotein filaments that are 18 nm in diameter and have a helical pitch of 10 nm. When added to linear duplex DNA in the presence of active RecE protein (exonuclease VIII), filamentous nucleoprotein complexes are formed on the DNA ends and the DNA molecules are frequently cyclized through protein-protein interactions.

Bacterial Proteins↗
PubMed1998

Dissecting histidine interactions of ribonuclease T1 with asparagine and glutamine replacements: analysis of double mutant cycles at one position.

His92 of Ribonuclease T1 combines functional and structural features involving both imidazole nitrogens. To evaluate the use of Asn and Gln substitutions in dissecting the properties of histidines, we analysed the consequences of the His92Gln and His92Asn substitutions on the enzyme's structure, function, and conformational stability by protein engineering and X-ray crystallographic methods. In the X-ray structures of wild-type and His92Gln RNase T1 in complex with 2'-GMP the His92-N epsilon 2 and Gln92-N epsilon 2 atoms are isosterically equivalent. Similarly, the His92N delta 1H...OAsn99 hydrogen bond observed in wild-type is replaced by an equivalent Asn92N delta 2H...OAsn99 in the His92Asn mutant structure. Double mutant cycles at a single position were used to analyse the intermolecular and intramolecular interactions of the exchangeable proton and the individual histidine nitrogens. Urea denaturation measurements as a function of pH revealed that the exchangeable proton of His92, rather than its imidazole ring is contributing about 1 kcal/mol to the conformational stability of RNase T1. The stabilizing and the destabilizing effects of the (His-->Gln) and the (His-->Asn) mutations on urea denaturation of RNase T1 at pH 9.0 suggest that the unprotonated N delta 1 and N epsilon 2 atoms contribute in a compensating way to the conformational stability of RNase T1. A comparative study of the kinetics of all mutants suggests that the protonated His92 imidazole is a strictly co-operative catalytic device.

Amino Acid Substitution↗
PubMed1999

Degradation of FinP antisense RNA from F-like plasmids: the RNA-binding protein, FinO, protects FinP from ribonuclease E.

Transfer of F-like plasmids is regulated by the FinOP system, which controls the expression of traJ, a positive regulator of the transfer operon. F FinP is a 79 base antisense RNA, composed of two stem-loops, complementary to the 5' untranslated leader of traJ mRNA. Binding of FinP to the traJ leader sequesters the traJ ribosome binding site, preventing its translation and repressing plasmid transfer. The FinO protein binds stem-loop II of FinP and traJ mRNA and promotes duplex formation in vitro. FinO stabilizes FinP, increasing its effective concentration in vivo. To determine how FinO protects FinP from decay, the degradation of FinP was examined in a series of ribonuclease-deficient strains. Using Northern blot analysis, full-length FinP was found to be stabilized sevenfold in an RNase E-deficient strain. The major site of RNase E cleavage was mapped on synthetic FinP, to the single-stranded region between stem-loops I and II. A secondary site near the 5' end ( approximately 10 bases) was also observed. A GST-FinO fusion protein protected FinP from RNase E cleavage at both sites in vitro. Two duplexes between FinP and traJ mRNA were detected in an RNase III-deficient strain. The larger duplex resulted from extension of the FinP transcript at its 3' end, suggesting readthrough at the terminator that corresponds to FinP stem-loop II. A point mutant of finP (finP305; C30U) that is unable to repress traJ in the presence of FinO was also characterized. The pattern of RNase E digestion of finP305 RNA differed from FinP, and GST-FinO did not protect finP305 RNA from cleavage in vitro. The half-life of finP305 RNA decreased more than tenfold in vivo, such that the steady-state levels of finP305 RNA, in the presence of FinO, were insufficient to significantly reduce the level of traJ mRNA available for translation, allowing derepressed levels of transfer.

Bacterial Outer Membrane Proteins↗
PubMed1984

Cyclic AMP-dependent regulation of activities of synthetase and phosphodiesterase of 2',5'-oligoadenylate in NIH 3T3 cells.

Treatment of NIH 3T3 cells with adenylate cyclase activator adrenaline (10(-6) M) or cAMP phosphodiesterase inhibitor theophylline (10(-3) M) was shown to lead to intracellular cAMP elevation followed by a 2.0-to 2.5-fold increase in the 2',5'-oligoadenylate synthetase activity. This process was blocked by actinomycin D. The rise in the intracellular cAMP level was also followed by a 3-4-fold decrease in the activity of 2'-phosphodiesterase. Propranolol prevented this inhibition but actinomycin D produced only a negligible effect on the process. Incubation of the cell homogenate with purified catalytic subunit of cAMP-dependent protein kinase and ATP also resulted in a decrease of 2'-phosphodiesterase activity. These results indicate that cAMP is involved in the regulation of enzymes of the 2',5'-oligoadenylate system. The possibility that certain biological functions of cAMP are implemented via 2',5'-oligoadenylate-dependent processes is discussed.

2',5'-Oligoadenylate Synthetase↗
PubMed1994

Molecular analysis of the dhp1+ gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST2 and RAT1 genes of Saccharomyces cerevisiae.

The DST2 gene of Saccharomyces cerevisiae encodes a DNA strand exchange protein, STP beta, which is required for homologous recombination in both mitotic or meiotic cells. We have cloned a DST2-related gene from the fission yeast Schizosaccharomyces pombe and designated it dhp1+. The nucleotide sequence of dhp1+ revealed an open reading frame encoding a protein composed of 991 amino acids. The predicted amino acid sequence was significantly homologous to the S. cerevisiae STP beta, but lacked the carboxy-terminal sequence present in STP beta. Furthermore, dhp1+ shows greater homology to RAT1/HKE1, a gene which is involved in RNA trafficking and processing. Genetic experiments showed that dhp1+ on an S. cerevisiae expression vector could rescue both the defects of the S. cerevisiae DST2 disruptant, slow growth rate and a sporulation defect, and the lethality of the S. cerevisiae rat1ts mutation. This implies the functional similarity of dhp1+ to both DST2 and RAT1. However unlike DST2, dhp1+ is an essential gene for cell growth in S. pombe, suggesting that dhp1+ is not the true homologue of DST2 but rather of RAT1 in S. pombe. The possible roles of dhp1+ in recombination and cell growth in S. pombe are discussed.

Amino Acid Sequence↗
PubMed1995

Identification of functional domains in the Sep1 protein (= Kem1, Xrn1), which is required for transition through meiotic prophase in Saccharomyces cerevisiae.

The Sep1 (also known as Kem1, Xrn1, Rar5, DST2/Stpbeta) protein of Saccharomyces cerevisiae is an Mr 175,000 multifunctional exonuclease with suspected roles in RNA turnover and in the microtubular cytoskeleton as well as in DNA recombination and DNA replication. The most striking phenotype of SEP1 null mutations is quantitative arrest during meiotic prophase at the pachytene stage. We have constructed a set of N- and C-terminal as well as internal deletions of the large SEP1 gene. Analysis of these deletion mutations on plasmids in a host carrying a null allele (sep1 ) revealed that at least 270 amino acids from the C-terminus of the wild-type protein were dispensable for complementing the slow growth and benomyl hypersensitivity of a null mutant. In contrast, any deletion at the N-terminus abrogated complementing activity for these phenotypes. The sequences essential for function correspond remarkably well with the regions of Sep1 that are homologous to its Schizosaccharomyces pombe counterpart Exo2. In addition, these experiments showed that, despite the high intracellular levels of Sep1, over-expression of this protein above these levels is detrimental to the cell. We discuss the potential cellular roles of the Sep1 protein as a microtubule-nucleic acid interface protein linking its suspected function in the microtubular cytoskeleton with its role as a nucleic acid binding protein.

Alleles↗
PubMed1994

Saccharomyces cerevisiae cells lacking the homologous pairing protein p175SEP1 arrest at pachytene during meiotic prophase.

Saccharomyces cerevisiae cells containing null mutations in the SEP1 gene, which encodes the homologous pairing and strand exchange protein p175SEP1, enter pachytene with a delay. They arrest uniformly at this stage of meiotic prophase, probably revealing a checkpoint in the transition from pachytene to meiosis I. At the arrest point, the cells remain largely viable and are cytologically characterized by the duplicated but unseparated spindle pole bodies of equal size and by the persistence of the synaptonemal complex, a cytological marker for pachytene. In addition, fluorescence in situ hybridization revealed that in arrested mutant cells maximal chromatin condensation and normal homolog pairing is achieved, typical for pachytene in wild type. A hallmark of meiosis is the high level of homologous recombination, which was analyzed both genetically and physically. Formation and processing of the double-strand break intermediate in meiotic recombination is achieved prior to arrest. Physical intragenic (conversion) and intergenic (crossover) products are formed just prior to, or directly at, the arrest point. Structural deficits in synaptonemal complex morphology, failure to separate spindle pole bodies, and/or defects in prophase DNA metabolism might be responsible for triggering the observed arrest. The pachytene arrest in sep1 cells is likely to be regulatory, but is clearly different from the RAD9 checkpoint in meiotic prophase, which occurs prior to the pachytene stage.

Chromosomes, Fungal↗
PubMed1990

New detection method for ribonuclease 2 (RNase 2) using immunoblotting with specific antibody.

A ribonuclease (RNase) was isolated from the urine of a 35-year-old male and purified to electrophoretic homogeneity. The enzyme was tentatively designated RNase 2. A rabbit antibody produced by injection of the purified RNase 2 was able to distinguish RNase 2 from another type of RNase coexisting in body fluids. With this antibody it was possible to detect RNase 2 isozymes in human serum and urine without difficulty using isoelectric focusing or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting. Both RNase 2 in serum and urine seemed to exist in multiple forms with regard to their molecular masses and pI values. This technique may prove to be useful in genetic and forensic studies of RNase polymorphism.

Adult↗
PubMed1995

Precise physical mapping of the Escherichia coli rnb gene, encoding ribonuclease II.

Ribonuclease II (encoded by rnb) is one of the two main exonucleases involved in mRNA degradation in Escherichia coli. We report the precise physical mapping of rnb to 29 min on the chromosomal map in the vicinity of pyrF, and clarify the genetic and physical maps of this E. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted for rnb.

Blotting, Southern↗