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C Saavedra

Publications and source records attributed to C Saavedra.

At least 19 recordsLinked to original sources
PubMed1999

The amino acidic substitution of cysteine 167 by serine (C167S) in BstVI restriction endonuclease of Bacillus stearothermophilus V affects its conformation and thermostability.

The restriction endonuclease BstVI from Bacillus stearothermophilus V contains three cysteine residues at positions 134, 167 and 180. Titration of Cys residues with DTNB showed that none of them are involved in disulphide bond formation. Cysteine triplets 134 and 167 were modified by recombinant PCR to introduce a serine residue in each case. The mutated genes were cloned into pGEM-T vector and transformed into E. coli JM109. Even though pGEM-T is not designed for expression, the mutant proteins were efficiently expressed in E. coli. The endonuclease carrying the mutation C134S was purified to homogeneity but appeared to be very unstable. In contrast, the C167S mutant enzyme was stable when pure and was studied biochemically. This mutant enzyme was as stable and resistant to protein-denaturing agents as the wild type enzyme. The activity of both enzymes was not affected by preincubations of 2 h at 80 degrees C. A short preincubation at 95 degrees C caused a complete inactivation of the mutant enzyme while the wild type endonuclease retained 30% of its activity. Moreover, the C167S BstVI was more susceptible to be hydrolyzed by proteinase K and trypsine compared to the wild type endonuclease. These results show that the substitution Cys --> Ser at position 167 affects the configuration and thermostability of BstVI restriction endonuclease.

Amino Acid Substitution↗
PubMed1999

Structural studies of the BstVI restriction-modification proteins by fluorescence spectroscopy.

Structural studies of the proteins of the BstVI restriction-modification system of Bacillus stearothermophilus V were carried out using intrinsic fluorescence techniques. The exposure and environments of their tryptophanyl residues were determined using collisional quenchers. Quenching of BstVI endonuclease by iodide suggested a heterogeneous class of tryptophan residues, while the results obtained with M.BstVI methylase were consistent with a rather exposed tryptophan population. A comparison of the quenching efficiencies at 20 degrees C and 55 or 60 degrees C showed that their structures are more flexible and open at the temperature at which they exhibit maximal activity. The endonuclease reached its active conformation only after 1 h of incubation at 60 degrees C. Fluorescence changes were observed upon Mn2+ and Mg2+ binding, with Kd values in the range 3-5 microM. The binding of S-adenosyl-L-methionine to the methylase produced conformational changes, which were consistent with binding to a single site of Kd 550 and 680 microM at 20 degrees C and 55 degrees C, respectively. Quenching experiments with iodide showed that the presence of S-adenosyl-L-methionine leads to different conformational states at 20 degrees C and 55 degrees C. These results were interpreted in terms of differences in the structural characteristics of these restriction-modification proteins as well as in terms of differences in the conformational states that these enzymes exhibit at 20 degrees C and at the temperature at which they are most active.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1998

Biochemical characterization of tellurite-reducing activities of Bacillus stearothermophilus V.

Bacillus stearothermophilus V is a naturally occurring Gram-positive rod which exhibits resistance to potassium tellurite. Crude extracts of this bacterium catalyse the NADH-dependent, protease-sensitive reduction of K2TeO3 in vitro. Two fractions which showed the ability to reduce potassium tellurite (H1 and H2) were obtained. Fraction H1 behaved as a macroaggregate exhibiting a very high molecular mass that could not be estimated accurately. Upon electrophoresis in polyacrylamide gels in the presence of SDS, however, it was resolved into three distinct bands of 60, 41 and 37.5 kDa. On the other hand, an M(r) of 121 was determined for fraction H2 by means of gel filtration and high-pressure liquid chromatography. In SDS-PAGE a unique protein band of 60 kDa was observed, suggesting that it is actually a dimer. Both fractions showed pH and temperature optima of 7.5 and 57 degrees C, respectively. Concentrations of 2.5 M NaCl or 0.35 mM SDS inhibited fraction H2 almost completely, while fraction H1 retained 20% of its activity under the same conditions. Concentrations of 5 mM EDTA caused the activity of both fractions to increase 2-fold. In addition to reducing tellurite, they were also able to reduce Na2SeO3 and Na2SO3 in vitro.

Edetic Acid↗
PubMed1998

TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus.

The constitutive transport element (CTE) of the type D retroviruses promotes nuclear export of unspliced viral RNAs apparently by recruiting host factor(s) required for export of cellular messenger RNAs. Here, we report the identification of TAP as the cellular factor that specifically binds to wild-type CTE but not to export-deficient CTE mutants. Microinjection experiments performed in Xenopus oocytes demonstrate that TAP directly stimulates CTE-dependent export. Furthermore, TAP overcomes the mRNA export block caused by the presence of saturating amounts of CTE RNA. Thus, TAP, like its yeast homolog Mex67p, is a bona fide mRNA nuclear export mediator. TAP is the second cellular RNA binding protein shown to be directly involved in the export of its target RNA.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Studies on the heterologous expression of BstVI restriction endonuclease in Escherichia coli.

Bacterial restriction and modification systems must be regulated to avoid self-restriction. It is generally accepted that cognate DNA methyltransferases normally protects both, the host's chromosome and extrachromosomal elements from the activity of their endonuclease counterparts. When the bstVIRM genes from Bacillus stearothermophilus V were subcloned into Escherichia coli, several clones exhibiting a r+m- phenotype were originated. The present work was undertaken to analyze the possibility that mechanisms other than DNA methylation could account for the viability of these cells. No evidence was found for an inhibitory agent or endonuclease compartmentation. In vivo experiments showed that lambda phage multiplication was poorly restricted by the heterologous enzyme. The restricting activity against the incoming phase increased however when phage adsortion was performed at higher temperatures. Analogous experiments in which a DNA-repair deficient strain was used as a host for the thermophilic R-M system suggested, to some extent, the participation of the repair machinery in the viability of r+m- clones.

Bacteriophage lambda↗
PubMed1997

The simian retrovirus-1 constitutive transport element, unlike the HIV-1 RRE, uses factors required for cellular mRNA export.

BACKGROUND: A hallmark of retroviral gene expression is that unspliced retroviral genomic RNA is exported to the cytoplasm, whereas endogenous intron-containing cellular RNAs are usually retained in the nucleus. In complex retroviruses, such as human immunodeficiency virus-1 (HIV-1), nuclear export is accomplished by the interaction of a virally encoded protein, Rev, with a cis-acting RNA element, the Rev-responsive element (RRE). In type D retroviruses, such as the simian retrovirus type 1 (SRV-1), however, genomic RNA is exported by cellular factor(s) that interact with a conserved cis-acting RNA element, the constitutive transport element (CTE). RESULTS: We found that the CTE was exported in a specific and saturable fashion from Xenopus oocyte nuclei. When inserted into the intron of an adenovirus-derived pre-mRNA, the CTE did not affect splicing efficiency but promoted the nuclear export of the excised intron lariat that is normally retained within the nucleus. Export of CTE-containing RNAs to the cytoplasm was not affected by the heterogeneous nuclear ribonucleoprotein A1 or an excess of peptides corresponding to the Rev nuclear export signal. Microinjection of saturating amounts of CTE RNA did not affect tRNA export or Rev-mediated export but did inhibit mRNA export. CTE-mediated export was found to be dependent on Ran-mediated GTP hydrolysis. CONCLUSION: The Rev-RRE system and the CTE direct intron-containing RNAs to distinct export pathways. Although previous data have suggested that Rev uses the same export pathway as uracil-rich small nuclear RNAs and 5S ribosomal RNA, the CTE seems to interact with evolutionarily conserved factors that are essential for cellular mRNA export.

Animals↗
PubMed1997

Quantitative trait loci analysis affecting contextual conditioning in mice.

In an extensive backcross of mice between C3H/HeJ (C3H) and C57BL/6J (B6), we sought to map genes that influence learning and memory as measured by performance in a contextual fear-conditioning paradigm. Our results indicate that there are several genetic regions that have a strong influence on performance in this paradigm. The strongest influences map to the proximal and distal ends of chromosome 1 (lod scores of 5.14 and 4.76, respectively). Other chromosomal regions (chromosomes 3, 7, 8, 9 and 18) were also identified as candidates for regions containing genes influencing contextual fear conditioning, with lod scores ranging from 1.8 to 2.7.

Animals↗
PubMed1997

Male-dependent doubly uniparental inheritance of mitochondrial DNA and female-dependent sex-ratio in the mussel Mytilus galloprovincialis.

We have investigated sex ratio and mitochondrial DNA inheritance in pair-matings involving five female and five male individuals of the Mediterranean mussel Mytilus galloprovincialis. The percentage of male progeny varied widely among families and was found to be a characteristic of the female parent and independent of the male to which it was mated. Thus sex-ratio in Mytilus appears to be independent of the nuclear genotype of the sperm. With a few exceptions, doubly uniparental inheritance (DUI) of mtDNA was observed in all families fathered by four of the five males: female and male progeny contained the mother's mtDNA (the F genome), but males contained also the father's paternal mtDNA (the M genome). Two hermaphrodite individuals found among the progeny of these crosses contained the F mitochondrial genome in the female gonad and both the F and M genomes in the male gonad. All four families fathered by the fifth male showed the standard maternal inheritance (SMI) of animal mtDNA: both female and male progeny contained only the maternal mtDNA. These observations illustrate the intimate linkage between sex and mtDNA inheritance in species with DUI and suggest different major roles for each gender. We propose a model according to which development of a male gonad requires the presence in the early germ cells of an agent associated with sperm-derived mitochondria, these mitochondria are endowed with a paternally encoded replicative advantage through which they overcome their original minority in the fertilized egg and this advantage (and, therefore, the chance of an early entrance into the germ line) is countered by a maternally encoded egg factor.

Animals↗
PubMed1997

Phylogenetic evidence for role-reversals of gender-associated mitochondrial DNA in Mytilus (Bivalvia: Mytilidae).

Distinct gender-associated mitochondrial DNA (mtDNA) lineages (i.e., lineages which are transmitted either through males or through females) have been demonstrated in two families of bivalves, the Mytilidae (marine mussels) and the Unionidae (freshwater mussels), which have been separated for more than 400 Myr. The mode of transmission of these M (for male-transmitted) and F (for female-transmitted) molecules has been referred to as doubly uniparental inheritance (DUI), in contrast to standard maternal inheritance (SMI), which is the norm in animals. A previous study suggested that at least three origins of DUI are required to explain the phylogenetic pattern of M and F lineages in freshwater and marine mussels. Here we present phylogenetic evidence based on partial sequences of the cytochrome c oxidase subunit I gene and the 16S RNA gene that indicates the DUI is a dynamic phenomenon. Specifically, we demonstrate that F lineages in three species of Mytilus mussels, M. edulis, M. trossulus, and M. californianus, have spawned separate lineages which are now associated only with males. This process is referred to as "masculinization" of F mtDNA. By extension, we propose that DUI may be a primitive bivalve character and that periodic masculinization events combined with extinction of previously existing M types effectively reset the time of divergence between conspecific gender-associated mtDNA lineages.

Amino Acid Sequence↗
PubMed1996

Regulation of mRNA export in response to stress in Saccharomyces cerevisiae.

The response of eukaryotic cells to heat shock and other forms of stress occurs at both transcriptional and post-transcriptional levels. We used in situ hybridization to determine whether stress affected the subcellular distribution of poly(A)+ RNA in Saccharomyces cerevisiae. Following induction of stress by either heat shock (42 degrees C) or addition of a high concentration of ethanol (10%), the nucleocytoplasmic export of most poly(A)+ RNA was blocked. In situ hybridization indicated that heat-inducible SSA4 and SSA1 mRNAs were exported from nuclei under these same conditions. On the other hand, both GAL1 and URA3 transcripts expressed from the SSA4 promoter accumulated in nuclei following heat shock. Sequences within either the 5' 1600 or the 3' 500 nucleotides of SSA4 mRNA were sufficient to direct GAL1 mRNA to the cytoplasm during stress. The export of SSA4 mRNA following stress required functional nuclear pore complexes, as SSA4 mRNA accumulated in nuclei following heat shock of cells containing temperature-sensitive nucleoporins. However, the selective export of SSA4 mRNA was maintained in heat-shocked cells carrying temperature-sensitive alleles of RNA1, PRP20, or an inducible dominant-negative allele of GSP1, the S. cerevisiae homolog of RAN/TC4. The results reported here suggest that there is selective export of mRNA in yeast.

Adenosine Triphosphatases↗
PubMed1996

Species-specific segregation of gender-associated mitochondrial DNA types in an area where two mussel species (Mytilus edulis and M. trossulus) hybridize.

In each of the mussel species Mytilus edulis and M. trossulus there exist two types of mtDNA, the F type transmitted through females and the M type transmitted through males. Because the two species produce fertile hybrids in nature, F and M types of one may introgress into the other. We present the results from a survey of a population in which extensive hybridization occurs between these two species. Among specimens classified as "pure" M. edulis or "pure" M. trossulus on the basis of allozyme analysis, we observed no animal that carried the F or the M mitotype of the other species. In most animals of mixed nuclear background, an individual's mtDNA came from the species that contributed the majority of the individual's nuclear genes. Most importantly, the two mtDNA types in post-F1 male hybrids were of the same species origin. We interpret this to mean that there are intrinsic barriers to the exchange of mtDNA between these two species. Because such barriers were not noted in other hybridizing species pairs (many being even less interfertile than M. edulis and M. trossulus), their presence in Mytilus could be another feature of the unusual mtDNA system in this genus.

Animals↗
PubMed1995

The GTP-bound form of the yeast Ran/TC4 homologue blocks nuclear protein import and appearance of poly(A)+ RNA in the cytoplasm.

Ran/TC4, a Ras-like GTP-binding protein, and its nucleotide exchanger, RCC1, have been implicated in control of protein movement into the nucleus and cytoplasmic accumulation of mRNA. Saccharomyces cerevisiae contains two homologues of the mammalian Ran/TC4, encoded by the GSP1 and GSP2 genes. We have constructed yeast strains that overproduce either wild-type Gsp1 or a form of Gsp1 with glycine-21 converted to valine (Gsp1-G21V), which we show stabilizes the GTP-bound form. Cells producing Gsp1-G21V have defects in localization of nuclear proteins; nuclear proteins accumulate in the cytoplasm following galactose induction of Gsp1-G21V. Similarly, cells producing Gsp1-G21V retain poly(A)+ RNA in their nuclei. These findings suggest that hydrolysis of GTP by Ran/TC4 is necessary for proper import of proteins into the nucleus and appearance of poly(A)+ RNA in the cytoplasm.

Animals↗
PubMed1995

Male and female mitochondrial DNA lineages in the blue mussel (Mytilus edulis) species group.

In blue mussels of the Mytilus edulis species complex, mitochondrial DNA (mtDNA) inheritance is coupled with gender. Females receive their mother's mtDNA and pass it on to both their daughters and sons. In addition, males receive mtDNA from their father and transmit this male mtDNA to their sons. If this pattern of "doubly uniparental inheritance" is older than the M. edulis species complex, then all members of this group must have two distinct mtDNA lineages: a maternal lineage that is transmitted through females and a paternal lineage that is transmitted through males. To test this hypothesis, we scored mtDNA variation in two taxa in this complex, M. edulis and M. trossulus, by means of restriction fragment profiles of whole-mtDNA genomes and DNA sequence of a region of the cytochrome c oxidase subunit III gene (COIII). The various mitotypes present in these mussels were classified as "male" or "female" based on their gender association and as belonging to M. edulis or M. trossulus based on species-specific allozymes. Both maximum parsimony and neighbor-joining phylogenies based on the COIII sequences grouped female and male mtDNAs into two distinct lineages irrespective of specific origin in accordance with the hypothesis that the origin of these lineages predates the divergence of M. edulis and M. trossulus.

Animals↗
PubMed1994

An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilus.

In animals, mitochondrial DNA (mtDNA) inheritance is predominantly maternal. In a few cases incidental transmission of paternal mtDNA was observed and estimated to account for only 10(-4)-10(-3) of an individual's mtDNA content. In contrast, biparental inheritance is common in mussels of the genus Mytilus. Here we present direct evidence that sex and mtDNA inheritance are coupled in Mytilus. Females inherit mtDNA only from their mother, but they transmit it to both daughters and sons. Males inherit mtDNA from both parents, but they transmit to sons only the mtDNA they inherited from their father. In pair matings, this mtDNA inheritance pattern is associated with a strong sex-ratio bias. These findings establish a newly discovered type of cytoplasmic DNA transmission. We also present evidence that the phenomenon breaks down in interspecific hybrids.

Aging↗
PubMed1994

The expression of the bstVIM gene from Bacillus stearothermophilus V is restricted to vegetative cell growth.

The activity of BstVI DNA methyltransferase was monitored during the sporulative cycle of Bacillus stearothermophilus V. Significant methylase activity was found only in bacteria growing vegetatively. This was confirmed by Northern hybridization, which indicated that the bstVIM gene was not transcribed in cells undergoing sporulation. Supporting evidence came from experiments which demonstrated that the RNA polymerase holoenzyme from these cells did not recognize the promoter elements upstream of the bstVIM gene.

Bacterial Proteins↗