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M Remacha

Publications and source records attributed to M Remacha.

At least 19 recordsLinked to original sources
PubMed1997

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.

Base Sequence↗
PubMed1996

Functional domains of chlamydial histone H1-like protein.

Chlamydial trachomatis is one of the few prokaryotic organisms known to contain proteins that bear amino acid similarity to eukaryotic histone H1. It is also appreciated that chlamydial histone-like proteins, designated Hc1 and Hc2, can bind DNA and are presumably involved in the condensation of infectious elementary bodies. However, there is no information on either the orientation of Hc1 and Hc2 or the mechanism of their DNA-protein and protein-protein interactions. Whereas the C-terminal domain of Hc1 between amino acids 63 and 125 shows best alignment with sea-urchin histone H1, and N-terminus between amino acids 1 and 62 is highly conserved among various chlamydial species, suggesting a bifunctional role for this unique protein. In order to delineate the regions responsible for the Hc1 characteristics, we have expressed these two fragments independently in Escherichia coli and studied the binding of double-stranded DNA to either whole Hc1 protein or its two termini. Our results support the role of the carboxyl portion in DNA-protein interaction, a function similar to its eukaryotic counterpart. Although this interaction initiates DNA condensation in the absence of the N-terminal domain, it is not sufficient to produce complete compaction. Intra- or inter-molecular protein-protein interactions may be necessary to achieve such an effect.

Amino Acid Sequence↗
PubMed1996

The sequence of a 17,933 bp segment of Saccharomyces cerevisiae chromosome XIV contains the RHO2, TOP2, MKT1 and END3 genes and five new open reading frames.

We report the DNA sequence of a 17,933 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae. Analysis of the sequence reveals the presence of ten open reading frames (ORFs) larger than 100 codons. Four of these were previously identified as genes RHO2, TOP2, MKT1 and END3. Additionally, the NH2 end coding region of PMS1 is found in the 3' end of the sequence. No significant homology to any known protein has been found for the other five ORFs.

Amino Acid Sequence↗
PubMed1996

The sequence of a 16,691 bp segment of Saccharomyces cerevisiae chromosome IV identifies the DUN1, PMT1, PMT5, SRP14 and DPR1 genes, and five new open reading frames.

As part of the European BIOTECH programme, the nucleotide sequence of a 16691 bp fragment from the left arm of chromosome IV of Saccharomyces cerevisiae has been deduced. Analysis of the sequence reveals the presence of 13 open reading frames (ORFs) larger than 100 codons. five of these were previously identified as genes DUN1, PMT1, PMT5, SRP14 and DPR1. One putative protein, D2371p, contains an ATP-GTP binding site, and shares homology to the ArsA component of an Escherichia coli arsenical pump. No significant homology to any known protein has been found for the other ORFs. D2378p contains a zinc finger domain.

Amino Acid Sequence↗
PubMed1995

Heterologous expression of the highly conserved acidic ribosomal phosphoproteins from Dictyostelium (changed from Dictyosteliumm) discoideum in Saccharomyces cerevisiae.

The genes encoding the acidic ribosomal phosphoproteins DdP1 and DdP2 from Dictyostelium discoideum have been cloned into yeast plasmid vectors under the control of the inducible GAL1 promoter. These constructions have been used to transform S. cerevisiae strains D45 and D67 lacking the equivalent ribosomal components. The D. discoideum genes are properly transcribed when cells are grown in the presence of the inducer galactose and the mRNAs incorporated into polysomes. However, the heterologous ribosomal proteins are not able to rescue the growth deficiency in S. cerevisiae caused by the absence of their own ribosomal proteins. When the heterologous proteins are analyzed using specific antibodies, only protein DdP1 is found in the ribosomes of the transformed S. cerevisiae D67 strain. No other heterologous protein is found in any other transformed strain, suggesting that the heterologous acidic ribosomal components are rapidly degraded when they are not bound to the ribosomes. The results indicate that D. discoideum DdP1 protein is able to interact with the yeast ribosome, though the interaction is functionally inefficient. Protein DdP2, in spite of having a higher sequence similarity to its yeast counterparts, is completely inactive in S. cerevisiae. Since the P proteins from both organisms have extensive amino acid sequence similarity ranging from 60% to 70%, these results warns about establishing a direct relationship between the extent of amino acid sequence similarity and the capacity of heterologous proteins to be functional in host species. Moreover, our data suggest that evolution affected the interaction of the acidic proteins with the ribosome rather than the structural features responsible for their primary functions.

Amino Acid Sequence↗
PubMed1995

Eukaryotic acidic phosphoproteins interact with the ribosome through their amino-terminal domain.

Variable-size fragments of the four yeast acidic ribosomal protein genes rpYP1 alpha, rpYP1 beta, rpYP2 alpha and rpYP2 beta were fused to the LacZ gene in the vector series YEp356-358. The constructs were used to transform wild-type Saccharomyces cerevisiae and several gene-disrupted strains lacking different acidic ribosomal protein genes. The distribution of the chimeric proteins between the cytoplasm and the ribosomes, tested as beta-galactosidase activity, was estimated. Hybrid proteins containing around a minimum of 65-75 amino acids from their amino-terminal domain are able to bind to the ribosomes in the presence of the complete native proteins. Hybrid proteins containing no more than 36 amino terminal amino acids bind to the ribosomes in the absence of a competing native protein. The fused YP1-beta-galactosidase proteins are also able to form a complex with the native YP2 type proteins, promoting their binding to the ribosome. The stability of the hybrid polypeptides seems to be inversely proportional to the size of their P protein fragment. These results indicate that only the amino-terminal domain of the eukaryotic P proteins is needed for the P1-P2 complex formation required for interaction with the ribosome. The highly conserved P protein carboxyl end is not implicated in the binding to the particles and is exposed to the medium.

Cell Compartmentation↗
PubMed1995

Chlamydia trachomatis RNA polymerase alpha subunit: sequence and structural analysis.

We describe the cloning and sequence analysis of the region surrounding the gene for the alpha subunit of RNA polymerase from Chlamydia trachomatis. This region contains genes for proteins in the order SecY, S13, S11, alpha, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins. The incorporation of chlamydial alpha subunit protein into the E. coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among alpha subunits from distantly related genera.

Amino Acid Sequence↗
PubMed1995

Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.

Cell Differentiation↗
PubMed1994

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

Base Sequence↗
PubMed1994

Cloning and characterization of a secY homolog from Chlamydia trachomatis.

Characterization of the genes involved in the process of protein translocation is important in understanding their structure-function relationships. However, little is known about the signals that govern chlamydial gene expression and translocation. We have cloned a 1.7 kb HindIII-PstI fragment containing the secY gene of Chlamydia trachomatis. The complete nucleotide sequence reveals three open reading frames. The amino acid sequence shows highest homology with Escherichia coli proteins L15, SecY and S13, corresponding to the spc-alpha ribosomal protein operons. The product of the C. trachomatis secY gene is composed of 457 amino acids with a calculated molecular mass of 50,195 Daltons. Its amino acid sequence shows 27.4% and 35.7% identity to E. coli and Bacillus subtilis SecY proteins, respectively. The distribution of hydrophobic amino acids in the C. trachomatis secY gene product is suggestive of it being an integral membrane protein with ten transmembrane segments, the second, third and seventh membrane segments sharing > 45% identity with E. coli SecY. Our results suggest that despite evolutionary differences, eubacteria share a similar protein export apparatus.

Amino Acid Sequence↗
PubMed1994

Effect of acidic ribosomal phosphoprotein mRNA 5'-untranslated region on gene expression and protein accumulation.

Constructions were made from genes encoding ribosomal acidic phosphoproteins YP1 beta (L44') and YP2 beta (L45) from Saccharomyces cerevisiae in which different parts of the 5'-untranslated regions were included. The constructs were inserted into centromeric plasmids under the control of the GAL1 promoter and expressed in yeast strains in which the genes coding for each acidic protein family, P1 and P2, had been disrupted. Deletions in the 5' region of the two genes have been found to oppositely affect their expression. Deletion of most of this region strongly stimulates the expression of YP2 beta (L45), increasing the translation efficiency of the mRNA, and generating a 6-fold excess of protein in the cell. A similar deletion in the rpYP1 beta gene represses the expression of the protein, reducing drastically the amount of the mRNA in the cell. The overexpression of rpYP2 beta affects the cell growth by inhibiting protein synthesis at the level of initiation. Reduction of the YP2 beta(L45) overproduction by growing in controlled concentrations of glucose abolishes the inhibitory effect. The excess protein, probably as a high molecular weight complex, apparently interferes with the joining of the 60 S subunit to the initiation complex generating the accumulation of polysome half-mers. In addition, the results indicate the existence of a regulatory mechanism by which each one of the two acidic proteins controls the expression of the other polypeptide. YP1 beta(L44') represses the expression of YP2 beta(L45), while this protein stimulates the expression of YP1 beta(L44').

Gene Expression Regulation, Fungal↗
PubMed1993

The acidic phosphoproteins from Saccharomyces cerevisiae ribosomes. NH2-terminal acetylation is a conserved difference between P1 and P2 proteins.

Isoelectrofocusing gels of acidic ribosomal proteins from most yeast strains reveal the presence of up to 10 bands which are the product of only 4 genes. The proteins have been characterized by NH2-terminal amino acid sequencing, specific antibodies, HPLC, and by taking advantage of acidic protein-defective yeast strains obtained by gene disruption methods. The four most basic proteins coincide with the phosphorylated and dephosphorylated forms of the YP2 proteins, YP2 alpha and YP2 beta, formerly named L44 and L45. Amino-terminal sequencing has shown that these two polypeptides have free amino-terminal ends starting at the first methionine residue. The bands defined earlier as L44' correspond to the phosphorylated and dephosphorylated processed forms of protein YB1 beta lacking the first eight amino acids. The formation of this truncated YP1 beta form seems to be stimulated by salt during protein extraction and is also favored by some modifications at the amino termini of the protein. On the other hand, the previously uncharacterized band, called Ax, corresponds to an NH2-terminal acetylated form of YP1 beta which starts at the serine in the second position of the nucleotide-derived sequence. Finally, the most acidic band is the phosphorylated product of the fourth acidic protein gene. This protein, called YP1 alpha, which is very poorly stained by silver and Coomassie blue, has not been characterized in detail previously. It is also monophosphorylated in the ribosome and, like YP1 beta, is present as an NH2-terminal acetylated form starting at the second serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1993

The activity-controlling phosphorylation site is not the same in the four acidic ribosomal proteins from Saccharomyces cerevisiae.

By using site-directed mutagenesis and chemical analysis of phosphopeptides, a unique phosphorylation site has been shown at serine 73 in the amino acid sequence of the Saccharomyces cerevisiae acidic ribosomal protein YP1 beta (L44'). The mutation in this position prevents in vitro phosphorylation by protein kinases that modify the wild-type polypeptide. The unphosphorylatable mutated protein is unable to bind to the ribosomes and to rescue the growth deficiency of yeast strains in which the corresponding original gene is inactivated by gene disruption. Sequencing of tryptic phosphopeptides has shown that acidic proteins YP1 alpha and YP2 alpha (L44) are also phosphorylated at positions near the carboxyl end. These results contrast with the data indicating that in the highly homologous protein YP2 beta, phosphorylation takes place at serine 19, close to the amino terminus. The results show that phosphorylation is definitely required for the biological activity of these ribosomal proteins. However, the differences in the phosphorylation sites suggest that the effect of this modification is not the same in all of them, confirming the heterologous role of these peculiar ribosomal components. In fact, the different context of the modification sites in the four polypeptides suggests the existence of more than one protein kinase specific for this set of proteins.

Amino Acid Sequence↗
PubMed1993

The complete sequence of a 15,820 bp segment of Saccharomyces cerevisiae chromosome XI contains the UBI2 and MPL1 genes and three new open reading frames.

As part of the EEC yeast genome program, a fragment of 15,820 bp from the right arm of Saccharomyces cerevisiae chromosome XI has been sequenced. This fragment corresponds roughly to the centromere-distal half of cosmid pUKG046 and to a small fragment of cosmid pUKG096, which are located approximately 150 kb from the centromere. It contains four open reading frames (ORFs) which encode potential proteins of more than 100 amino acid residues, as well as the UBI2 gene which carries an intron and does not show up as an ORF in the sequence analysis programs. One of the putative proteins, YKR412, is very rich in serine and has significant homology at the carboxyl end to Nopp140 phosphoprotein. YKR413 has several predicted transmembrane domains. YKR15, which has been recently cloned as the MPL1 gene, encodes a polypeptide that shows homologies to myosin heavy chain and to the cytoskeleton protein Uso1.

Amino Acid Sequence↗
PubMed1992

Stable binding of the eukaryotic acidic phosphoproteins to the ribosome is not an absolute requirement for in vivo protein synthesis.

The genes encoding the four acidic ribosomal phosphoproteins have been inactivated in Saccharomyces cerevisae by recombination with truncated genes carrying different genetic markers. By crossing single haploid disruptants, strains harboring two simultaneously inactivated acidic protein genes were constructed. None of the six possible double disruptions was lethal, but the simultaneous inactivation of either YP1 alpha and YP1 beta(L44') or YP2 alpha(L44) and YP2 beta(L45) caused an important decrease in the cell growth rate. Ribosomes isolated from these slow-growing strains did not contain acidic proteins, not even the two polypeptides whose genes were still intact, although these proteins were present in the cell extracts and they seem to be able to form high-molecular weight protein complexes. Transformation of a slow-growing double transformant with a plasmid containing one of the disrupted genes restored the presence of the acidic proteins in the ribosomes and normal growth rates. The particles of the slow-growing strains were active in an in vitro amino acid polymerizing system, although their activity could be stimulated by the exogenous addition of the missing proteins. These results indicate that in the absence of either YP1 alpha and YP1 beta(L44') or YP2 alpha (L44) and YP2 beta(L45), the remaining acidic proteins are unable to interact with the ribosome in a stable manner, but that a strong interaction of these ribosomal components with the particle is not an absolute requirement for in vivo and in vitro protein synthesis.

Blotting, Southern↗
PubMed1991

Characterization of the yeast acidic ribosomal phosphoproteins using monoclonal antibodies. Proteins L44/L45 and L44' have different functional roles.

In order to characterize the acidic ribosomal proteins immunologically and functionally, a battery of monoclonal antibodies specific for L44, L44' and L45, the three acidic proteins detected in Saccharomyces cerevisiae, were obtained. Eight monoclonal antibodies were obtained specific for L45, three for L44' and one for L44. In addition, two mAbs recognizing only the phosphorylated forms of the three proteins were obtained. The specific immunogenic determinants are located in the middle region of the protein structure and are differently exposed in the ribosomal surface. The common determinants are present in the carboxyl end of the three proteins. An estimation of the acidic proteins by ELISA indicated that, in contrast to L44 and L45, L44' is practically absent from the cell supernatant; this suggests that protein L44' does not intervene in the exchange that has been shown to take place between the acidic proteins in the ribosome and in the cytoplasmic pool. It has also been found that, while IgGs specific for L44 and L45 do not inhibit the ribosome activity, the anti-L44' effectively blocks the polymerizing activity of the particles. These results show for the first time that the different eukaryotic acidic ribosomal proteins play a different functional role.

Animals↗
PubMed1990

The acidic ribosomal proteins as regulators of the eukaryotic ribosomal activity.

The acidic proteins, A-proteins, from the large ribosomal subunit of Saccharomyces cerevisiae grown under different conditions have been quantitatively estimated by ELISA tests using rabbit sera specific for these polypeptides. It has been found that the amount of A-protein present in the ribosome is not constant and depends on the metabolic state of the cell. Ribosomes from exponentially growing cultures have about 40% more of these proteins than those from stationary phase. Similarly, the particles forming part of the polysomes are enriched in A-proteins as compared with the free 80 S ribosomes. The cytoplasmic pool of A-protein is considerably high, containing as a whole as much protein as the total ribosome population. These results are compatible with an exchanging process of the acidic proteins during protein synthesis that can regulate the activity of the ribosome. On the other hand, cells inhibited with different metabolic inhibitors produce a very low yield of ribosomes that contain, however, a surprisingly high amount of acidic proteins while the cytoplasmic pool is considerably reduced, suggesting that under stress conditions the ribosome and the A-protein may aggregate, forming complex structures that are not recovered by the standard preparation methods.

Electrophoresis, Polyacrylamide Gel↗