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B Piña

Publications and source records attributed to B Piña.

At least 19 recordsLinked to original sources
PubMed2003

The different (sur)faces of Rap1p.

The DNA-binding protein Rap1p fulfills many different functions in the yeast cell. It targets 5% of the promoters, acting both as a transcriptional activator and as a repressor, depending on the DNA sequence context. In addition, Rap1p is an essential structural component of yeast telomeres, where it contributes to telomeric silencing. Here we review the evidence indicating that Rap1p function is modulated by the precise architecture of the its binding site and its surroundings: long tracts of telomeric repeats for telomeric functions, specific sequences and orientation for maximal transcriptional activation, and specific DNA recognition sequences for complementary factors in other cases. Many of these functions are probably related to chromatin organization around Rap1p DNA binding sites, resulting from the very tight binding of Rap1p to DNA. We propose that Rap1p alters its structure to bind to different versions of its DNA binding sequence. These structural changes may modulate the function of Rap1p domains, providing different interacting surfaces for binding to specific co-operating factors, and thus contributing to the diversity of Rap1p function.

Base Sequence↗
PubMed2001

Alternative mechanisms of transcriptional activation by Rap1p.

Single Rap1p DNA-binding sites are poor activators of transcription of yeast minimal promoters, even when fully occupied in vivo. This low efficiency is due to two independent repression mechanisms as follows: one that requires the presence of histones, and one that requires Hrs1p, a component of the RNA polymerase II mediator complex. Both repression mechanisms were greatly reduced for constructs with tandemly arranged sites. In these constructs, UASrpg sequences (ACACCCATACATTT) activated better than telomere-like sequences (ACACCCACACACCC) in an orientation-dependent manner. Both mutations in the SWI/SNF complex and a deletion of amino acids 597--629 of Rap1p (Tox domain) decreased synergistic effects of contiguous telomeric sites. Conversely, deletion of amino acids 700--798 of Rap1p (Sil domain) made UASrpg and telomeric sites functionally indistinguishable. We propose that the Sil domain masks the main transactivation domain of Rap1p in Rap1p-telomere complexes, where the Tox domain behaves as a secondary activation domain, probably by interacting with chromatin-remodeling complexes. Rap1p DNA-binding sites in ribosomal protein gene promoters are mainly UASrpg-like; their replacement by telomeric sequences in one of these promoters (RPS17B) decreased transcription by two-thirds. The functional differences between UASrpgs and telomeric sequences may thus contribute to the differential expression of Rap1p-regulated promoters in vivo.

Gene Expression Regulation, Fungal↗
PubMed2001

Genetic analysis of the karyotype instability in natural wine yeast strains.

Yeast strains isolated from the wild may show high rates of changes in their karyotypes during vegetative growth. We analysed over 500 karyotypes from mitotic and meiotic derivatives of strain DC5, which has a chromosome rearrangement rate of 8.2 x 10(-3) changes/generation. About 70% of the meiotic derivatives of DC5 had low rearrangement rates, with an average of 5.8 x 10(-4) changes/generation, suggesting that karyotype instability behaved as a dominant phenotype. Diploid derivatives with low karyotype variability in mitosis also had low rates of chromosomal rearrangement during meiosis, suggesting that the two phenotypes may be linked. DC5 and some of its meiotic derivatives (both with high and low karyotype variability) had chromosome XII hypervariable bands. Their distribution among the meiotic products indicates that they are not indicators for genetic instability. To our knowledge, data in this paper are the first to indicate that karyotypically unstable yeast strains may give stable progeny at high rates. Understanding of the relevant mechanism(s) may allow the design of genetic strategies to stabilize karyotypes from natural and/or industrial wine yeasts with unacceptable karyotype rearrangement rates.

Blotting, Southern↗
PubMed2001

Monitoring of endocrine disruptors in surface waters by the yeast recombinant assay.

Endocrine disruptors exert physiological effects at very low concentrations. Surface waters present often a mixture of high concentrations of low-potency disruptors and low amounts of very powerful ones, making their chemical analysis complicated and expensive. We developed a recombinant yeast assay (RYA) for estrogenic compounds using 96-well microtiter plates. This assay is based on three yeast strains, transformed with self-propagating plasmids. One strain contains an expression plasmid for the human estrogen hormone receptor and an appropriate reporter; it detects estrogenic and antiestrogenic activities. The two other yeast strains, one expressing the human progesterone receptor and a second based on the yeast activator Gal4p, served to analyze the nature of antiestrogenic activities. We applied this technique to water samples from two tributaries on the Llobregat river (NE Spain) as well as from four sewage treatment plants discharging on them. Our results indicate that the efficiency of sewage treatment plants for eliminating estrogenic compounds varied notably, being in at least one case completely inefficient. We also observed a prevalence of an inhibitory activity all through the two rivers; this inhibition was hormone specific. These results were consistent to previously obtained chemical analyses of the same samples, although chemical and in vivo analyses showed rather different levels of sensitivity for some compounds. Our findings demonstrate the utility of the yeast recombinant assay for analyzing complex natural samples; at the same time, they stress the necessity of a panel of different yeast systems to adequately describe endocrine-disruptor activities.

Algorithms↗
PubMed2000

Benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides: first phosphodiesterase 7 inhibitors.

The synthesis of a new family of benzyl derivatives of 2,1,3-benzo- and benzothieno[3,2-a]thiadiazine 2,2-dioxides was achieved. The biological data revealed the first heterocyclic family of compounds with PDE 7 inhibitory properties appearing to be a new objective for the treatment of T-cell-dependent disorders. The IC(50) values or percent inhibition values of the compounds against PDE 7 were calculated by testing them against human recombinant PDE 7 expressed in S. cerevisiae. In this expression system the only cyclic nucleotide hydrolyzing activity present in cell extracts corresponded to human PDE 7. Isoenzyme selectivity PDE 7 versus PDE 4 and PDE 3 was also measured. Considering simultaneously inhibition of the three different isoenzymes, monobenzyl derivatives 15 and 23 showed interesting PDE 7 potency (around 10 microM); although not statistically significant, a trend toward selectivity with respect to PDE 3 and PDE 4 was obtained. Benzothiadiazine 16, although less potent at PDE 7 (IC(50) = 25 microM), also showed a trend of selectivity toward PDE 3 and PDE 4. These compounds are considered the best leads for further optimization.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Functional divergence between the half-sites of the DNA-binding sequence for the yeast transcriptional regulator Rap1p.

The yeast transcriptional regulator Rap1p binds to the DNA consensus sequence ACACCCAYACAYYY. We have previously shown that DNA-binding sites in which all four Y (Y=T or C) positions were Ts (UASrpg sequences) synergized more efficiently to activate transcription than sequences in which all Ys were Cs (telomere sequences) [F.-Z. Idrissi, J. Fernández-Larrea and B. Piña (1998) J. Mol. Biol. 284, 925-935]. Here we provide evidence that the DNA consensus sequence for Rap1p behaves as a combination of two ACAYYY half-sites with different functionality, the presence of Ts in the second half-site being the determinant for the transcriptional behaviour of the UASrpg sequences. DNA structure in the different complexes with Rap1p varied from being relatively uniform to appear rather distorted, this also being dependent on the presence of Ts in the second half-site. These distortions did not cause sharp bends or kinks in the DNA molecule. Computer analysis suggests that high-affinity binding of Rap1p to UASrpg sequences requires a rearrangement of the C-terminal Myb domain of the protein. We propose that the structural alterations in Rap1p-DNA complexes, both in the DNA and in the protein, affect the transcription potential of the complex in an allosteric manner. We also propose that the dimeric nature of the Rap1 DNA-binding domain is a key structural feature that explains the disparate functions of its DNA-binding sites in vivo.

Base Sequence↗
PubMed1999

DDP1, a single-stranded nucleic acid-binding protein of Drosophila, associates with pericentric heterochromatin and is functionally homologous to the yeast Scp160p, which is involved in the control of cell ploidy.

The centromeric dodeca-satellite of Drosophila forms altered DNA structures in vitro in which its purine-rich strand (G-strand) forms stable fold-back structures, while the complementary C-strand remains unstructured. In this paper, the purification and characterization of DDP1, a single-stranded DNA-binding protein of high molecular mass (160 kDa) that specifically binds the unstructured dodeca-satellite C-strand, is presented. In polytene chromosomes, DDP1 is found located at the chromocentre associated with the pericentric heterochromatin but its distribution is not constrained to the dodeca-satellite sequences. DDP1 also localizes to heterochromatin in interphase nuclei of larval neuroblasts. During embryo development, DDP1 becomes nuclear after cellularization, when heterochromatin is fully organized, being also associated with the condensed mitotic chromosomes. In addition to its localization at the chromocentre, in polytene chromosomes, DDP1 is also detected at several sites in the euchromatic arms co-localizing with the heterochromatin protein HP1. DDP1 is a multi-KH domain protein homologous to the yeast Scp160 protein that is involved in the control of cell ploidy. Expression of DDP1 complements a Deltascp160 deletion in yeast. These results are discussed in view of the possible contribution of DNA structure to the structural organization of pericentric heterochromatin.

Amino Acid Sequence↗
PubMed1999

Analysis and dynamics of the chromosomal complements of wild sparkling-wine yeast strains.

We isolated Saccharomyces cerevisiae yeast strains that are able to carry out the second fermentation of sparkling wine from spontaneously fermenting musts in El Penedès (Spain) by specifically designed selection protocols. All of them (26 strains) showed one of two very similar mitochondrial DNA (mtDNA) restriction patterns, whereas their karyotypes differed. These strains showed high rates of karyotype instability, which were dependent on both the medium and the strain, during vegetative growth. In all cases, the mtDNA restriction pattern was conserved in strains kept under the same conditions. Analysis of different repetitive sequences in their genomes suggested that ribosomal DNA repeats play an important role in the changes in size observed in chromosome XII, whereas SUC genes or Ty elements did not show amplification or transposition processes that could be related to rearrangements of the chromosomes showing these sequences. Karyotype changes also occurred in monosporidic diploid derivatives. We propose that these changes originated mainly from ectopic recombination between repeated sequences interspersed in the genome. None of the rearranged karyotypes provided a selective advantage strong enough to allow the strains to displace the parental strains. The nature and frequency of these changes suggest that they may play an important role in the establishment and maintenance of the genetic diversity observed in S. cerevisiae wild populations.

Blotting, Southern↗
PubMed1998

Structural and functional heterogeneity of Rap1p complexes with telomeric and UASrpg-like DNA sequences.

Rap1p binds to a variety of related DNA sequences. We studied complexes of Rap1p and its DNA-binding domain with two of these sequences, the UASrpg sequence (5'-ACACCCATACATTT-3') and the Saccharomyces cerevisiae telomeric consensus (5'-ACACCCACACACCC-3'). When cloned in front of a minimal CYC1 promoter, the two sequences differed in their transcriptional potential. Whereas UASrpg or telomeric single binding sites activated transcription with approximately the same strength, adjacent UASrpg sequences showed higher synergistic activity and orientation-dependence than telomeric sequences. We also found different sequence requirements for Rap1p binding in vitro to both sequences, since a single base-pair that severely reduced binding of Rap1p to UASrpg sequences had very little effect on the telomeric sequence. The Rap1p binding domain distorted DNA molecules encompassing the UASrpg sequence or the telomeric-like sequence, as revealed by both KMnO4 hypersensitivity and by hydroxyl radical foot-printing analysis. We propose that Rap1p is able to form structurally and functionally different complexes, depending on the type of DNA sequence the complex is assembled from. This functional and structural heterogeneity may be responsible for the multiple functions that Rap1p binding sites appear to have in vivo.

Base Sequence↗
PubMed1997

Specific deactivation of the mouse mammary tumor virus long terminal repeat promoter upon continuous hormone treatment.

We have studied the transcriptional behavior of the mouse mammary tumor virus long repeat (MMTV-LTR) promoter during a prolonged exposure to glucocorticoids. When integrated into XC-derived cells, MMTV-LTR expression reached its maximum during the first day of dexamethasone treatment, but longer exposure to the hormone resulted in the deactivation of the promoter. In contrast, glucocorticoid-responsive resident genes or MMTV-based transiently transfected plasmids maintained or even increased their mRNA levels during the same period of hormone treatment. An integrated chimeric construct containing the hormone-responsive elements from MMTV-LTR but in different sequence context became also deactivated after a prolonged hormone treatment but with a deactivation kinetics significantly slower than constructs containing the entire, chromatin-positioning MMTV-LTR sequence. The decrease on MMTV-LTR-driven transcription was concomitant with a parallel closure of the MMTV-LTR chromatin and with a decrease in glucocorticoid receptor (GR) concentration in the cell. We concluded that the chromatin-organized MMTV-LTR promoter is particularly sensitive to any decrease on GR levels. We propose that chromatin structure may contribute decisively to the differential expression of MMTV-LTR by two mechanisms: limiting MMTV-LTR accessibility to activating transcription factors and accelerating its shutting down upon a decrease on GR levels.

Animals↗
PubMed1997

ADA1, a novel component of the ADA/GCN5 complex, has broader effects than GCN5, ADA2, or ADA3.

The ADA genes encode factors which are proposed to function as transcriptional coactivators. Here we describe the cloning, sequencing, and initial characterization of a novel ADA gene, ADA1. Similar to the previously isolated ada mutants, ada1 mutants display decreases in transcription from various reporters. Furthermore, ADA1 interacts with the other ADAs in the ADA/GCN5 complex as demonstrated by partial purification of the complex and immunoprecipitation experiments. We estimate that the complex has a molecular mass of approximately 2 MDa. Previously, it had been demonstrated that ada5 mutants displayed more severe phenotypic defects than the other ada mutants (G. A. Marcus, J. Horiuchi, N. Silverman, and L. Guarente, Mol. Cell. Biol. 16:3197-3205, 1996; S. M. Roberts and F. Winston, Mol. Cell. Biol. 16:3206-3213, 1996). ada1 mutants display defects similar to those of ada5 mutants and different from those of the other mutants with respect to promoters affected, inositol auxotrophy, and Spt- phenotypes. Thus, the ADAs can be separated into two classes, suggesting that the ADA/GCN5 complex may have two separate functions. We present a speculative model on the possible roles of the ADA/GCN5 complex.

Acetylation↗
PubMed1996

Molecular polymorphism distribution in phenotypically distinct populations of wine yeast strains.

Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to analyze natural yeast populations from fermenting musts in El Penedès, Spain. Both analyses revealed a considerable degree of polymorphism, indicating heterogeneous natural populations. By specifically designed genetic selection protocols, strains showing potentially interesting phenotypes, such as high tolerance to ethanol and temperature or the ability to grow and to ferment in wine-water-sugar mixtures, were isolated from these natural populations. Genetic analysis showed a strong correlation between the selected phenotypes and mitochondrial DNA polymorphisms. Karyotype analysis revealed several genetically similar yeast lineages in the natural yeast microflora, which we interpret as genetically isolated subpopulations of yeast strains with distinct genetic traits, which may correspond to specific microenvironments. Thus, molecular polymorphism analysis may be useful not only to study the geographical distribution of natural yeast strains but also to identify strains with specific phenotypic properties.

DNA, Fungal↗
PubMed1993

ADA3: a gene, identified by resistance to GAL4-VP16, with properties similar to and different from those of ADA2.

We describe the isolation of a yeast gene, ADA3, mutations in which prevent the toxicity of GAL4-VP16 in vivo. Toxicity was previously proposed to be due to the trapping of general transcription factors required at RNA polymerase II promoters (S. L. Berger, B. Piña, N. Silverman, G. A. Marcus, J. Agapite, J. L. Regier, S. J. Triezenberg, and L. Guarente, Cell 70:251-265, 1992). trans activation by VP16 as well as the acidic activation domain of GCN4 is reduced in the mutant. Other activation domains, such as those of GAL4 and HAP4, are only slightly affected in the mutant. This spectrum is similar to that observed for mutants with lesions in ADA2, a gene proposed to encode a transcriptional adaptor. The ADA3 gene is not absolutely essential for cell growth, but gene disruption mutants grow slowly and are temperature sensitive. Strains doubly disrupted for ada2 and ada3 grow no more slowly than single mutants, providing further evidence that these genes function in the same pathway. Selection of initiation sites by the general transcriptional machinery in vitro is altered in the ada3 mutant, providing a clue that ADA3 could be a novel general transcription factor involved in the response to acidic activators.

Amino Acid Sequence↗
PubMed1992

Genetic isolation of ADA2: a potential transcriptional adaptor required for function of certain acidic activation domains.

We have devised a genetic strategy to isolate the target of acidic activation domains of transcriptional activators based on toxicity in yeast cells of the chimeric activator, GAL4-VP16. Toxicity required the integrity of both the VP16 acidic activation domain and the GAL4 DNA-binding domain, suggesting that inhibition resulted from trapping of general transcription factors at genomic sites. Mutations that break the interaction between GAL4-VP16 and general factors would alleviate toxicity and identify transcriptional adaptors, if adaptors bridged the interaction between activators and general factors. We thus identified ADA1, ADA2, and ADA3. Mutations in ADA2 reduced the activity of GAL4-VP16 and GCN4 in vivo. ada2 mutant extracts exhibited normal basal transcription, but were defective in responding to GAL4-VP16, GCN4, or the dA:dT activator. Strikingly, the mutant extract responded like wild type to GAL4-HAP4. We conclude that ADA2 potentiates the activity of one class of acidic activation domain but not a second class.

Amino Acid Sequence↗
PubMed1992

Transcriptional control by steroid hormones.

Gene regulation by steroid hormones leads to induction or repression of particular sets of genes. These effects are mediated by intracellular hormone receptors that, in the unliganded state, are maintained in an inactive form by unknown mechanisms possibly involving association with other cellular proteins. Induction of the mouse mammary tumor virus (MMTV) requires binding of the hormone receptor to a complex hormone-responsive element (HRE) located between 75 and 190 bp upstream from the start of transcription. The interaction of several receptor molecules with the four receptor binding sites in the HRE is highly cooperative on circular DNA molecules and each individual site is needed for optimal induction. In chromatin the HRE is precisely organized in phased nucleosomes. Following hormone treatment and receptor binding, changes in chromatin structure are detected that correlate with binding of transcription factors, including nuclear factor I, to the MMTV promoter. However, though nuclear factor I acts as a basal transcription factor on the MMTV promoter it does not cooperate with the hormone receptors in terms of binding to free DNA, and mutation of the nuclear factor I binding site does not eliminate hormonal stimulation. This residual induction is mediated by octamer motifs, upstream of the TATA box, that bind the ubiquitous transcription factor OTF-1. Mutation of these octamer motifs does not influence basal transcription in vitro, but completely abolishes the stimulatory effect of progesterone receptor.

Amino Acid Sequence↗
PubMed1992

Histone H1 subtype synthesis in neurons and neuroblasts.

Rat cerebral cortex neurons contain the five histone H1 subtypes H1a-e and the subtype H1 zero present in other mammalian somatic tissues. The four subtypes H1a-d decay exponentially during postnatal development and are partially or totally replaced by H1e that becomes the major H1 subtype in adults. H1 zero accumulates in a period restricted to neuronal terminal differentiation. Here we study the synthesis of the H1 subtypes in cortical neurons and their neuroblasts by in vivo labeling with [14C]lysine. The subtype synthesis pattern of neuroblasts has been determined by labeling gravid rats during the period of proliferation of cortical neurons and synthesis in neurons has been studied by postnatal labeling. The subtype H1a is synthesized in neuroblasts but not in neurons and is therefore rapidly removed from neuronal chromatin. The synthesis of H1b and H1d is much lower in neurons than in neuroblasts so that these subtypes are replaced to a large extent during postnatal development. H1c is synthesized at levels much higher than the other subtypes both in neurons and neuroblasts, but its very high turnover, about one order of magnitude faster than that of H1e in neurons, favors its partial replacement during postnatal development. Comparison of the synthesis rates of H1 zero in newborn and 30-day-old rats shows that the accumulation of H1 zero in differentiating neurons is due to an increased level of synthesis.

Animals↗
PubMed1990

Structural features of a regulatory nucleosome.

DNA sequences from the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR) position nucleosomes both in vivo and in vitro. Here, were present chromatin reconstitution experiments showing that MMTV-LTR sequences from -236 to +204 accommodate two histone octamers in positions compatible with the in vivo data. This positioning is not influenced by the length of the DNA fragment and occurs in linear as well as in closed circular DNA molecules. MMTV-LTR DNA sequences show an intrinsic bendability that closely resembles its wrapping around the histone octamer. We propose that bendability is responsible for the observed rotational nucleosome positioning. Translational nucleosome positioning seems also to be determined by the DNA sequence. These data, along with the results from reconstitution experiments with insertion mutants, support a modular model of nucleosome phasing on MMTV-LTR, where the actual positioning of the histone octamer results from the additive effect of multiple features of the DNA sequence.

Base Sequence↗