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O Poch

Publications and source records attributed to O Poch.

At least 19 recordsLinked to original sources
PubMed1997

Who's who among the Saccharomyces cerevisiae actin-related proteins? A classification and nomenclature proposal for a large family.

Inspection of the complete Saccharomyces cerevisiae genome sequence and analysis of the actin-related proteins (ARPs) found therein revealed seven proteins, in addition to the previously designated actin-related proteins Arp1, Arp2 and Arp3, which contained substantial blocks of conservation relative to a chosen sub-set of actins. We have ordered the new ARPs relative to this group of actins and propose to name the more distantly related ARP members, according to their amino acid identity and similarity, Arp4-Arp10. Most of these proteins appear to represent the first example of new classes of ARPs, each of which may have specific localization(s) and cellular function(s). Recently reported ARPs from other species have also been included in the phylogenetic tree derived from the overall alignment of 29 actins and 28 ARPs.

Actins↗
PubMed1997

Sequence comparison of the Ty1 and Ty2 elements of the yeast genome supports the structural model of the tRNAiMet-Ty1 RNA reverse transcription initiation complex.

In the reverse transcription initiation complex of the yeast Ty1 retrotransposon, interaction between the template RNA and primer tRNAiMet is not limited to base pairing of the primer binding site (PBS) with ten nucleotides at the 3' end of tRNAiMet, but three regions named boxes O, 1 and 2.1 interact with the T and D stems and loops of tRNAiMet. Sequence comparison of 33 Ty1 elements and 13 closely related Ty2 elements found in the yeast genome shows that the nucleotide sequence of all elements is highly conserved in the region spanning the PBS and the three boxes. Since the domain of the template RNA encodes a portion of protein TyA, we have calculated its amino acid profile and its nucleotide profile to evaluate the role played by nucleotide sequence conservation in the selection for TyA function and in the maintenance of base pairing interactions for the priming function of Ty1 RNA. Our results show that the nucleotide sequence conservation of Ty1 RNA is constrained not only by selection for Ty1 function but also by maintenance of a given nucleotide sequence able to base pair with the tRNAiMet in the primer-template initiation complex.

Amino Acid Sequence↗
PubMed1997

Conservation of a putative inhibitory domain in the GAL4 family members.

The GAL4 family members are fungal transcriptional activators composed of several functional domains: a characteristic cysteine-rich DNA-binding domain common to all members, a dimerization domain, various transactivation domains generally exhibiting a high acidic content and a highly variable central region supposed to be involved in regulation and in effector recognition. We report here that the central region of the GAL4 family members share eight conserved motifs embedded in a large functional domain of 225 up to 405 residues. This domain may also be present in four proteins belonging to another family of transcriptional activators sharing a C2H2-type zinc finger. Analysis of the biochemical data available on the well-studied GAL4 protein suggests that this domain may be involved in the regulation of the activity of the protein, particularly in an inhibitory function. This hypothesis is further supported by deletion and site-directed mutagenesis experiments on other GAL4 family members. The mean secondary structure prediction performed on the eight motifs strongly suggests that the inhibitory activity may be mediated by hydrophobic interactions linked to the presence of amphipathic alpha-helices.

Amino Acid Sequence↗
PubMed1997

Analysis of a 35.6 kb region on the right arm of Saccharomyces cerevisiae chromosome XV.

We report the sequence of a 35,600 bp fragment covering the PET123 region on the right arm of chromosome XV from Saccharomyces cerevisiae. This region contains 19 possible open reading frames (ORFs) of which 16 are non-overlapping ORFs. Eight ORFs correspond to the SPP2, SMP3, PDR5, NFI1, PUP1, PET123 and MTR10 loci, described previously. Two ORFs correspond to yeast homologues of genes from other organisms: O3530 is a member of the large ribosomal subunit protein L13 family and O3560 (SME1 gene) is a 94-codon ORF and is a homologue of the mammalian SmE spliceosomal core protein. Three ORFs (O3513, O3521, O3548) present significant similarities to proteins of unknown function and three ORFs (O3510, O3536, O3545) lack homology to sequences within the databases screened.

Amino Acid Sequence↗
PubMed1996

Analysis of a 22,956 bp region on the right arm of Saccharomyces cerevisiae chromosome XV.

We present here the sequence analysis of a DNA fragment (cosmid pUOA1258) located on the right arm of chromosome XV. The 22,956 bp sequence reveals 14 open reading frames (ORFs) longer than 300 bp and the 201 bp RPS33 gene. Among the 14 large ORFs, two overlapping frames are likely to be non-expressed and one corresponds to the known GLN4 gene encoding glutaminyl-tRNA synthetase. Two ORFs, O3571 and O3620, encode putative transcriptional regulators with a Zn(2)-Cys(6) DNA binding domain characteristic of members of the GAL4 family. Among the nine remaining ORFs, five (O3568, O3575, O3590, O3615 and O3625) present significant similarity to proteins of unknown function and four (O3580, O3595, O3630 and O3635) lack homology to sequences present in the databases screened.

Amino Acid Sequence↗
PubMed1994

RPK1, an essential yeast protein kinase involved in the regulation of the onset of mitosis, shows homology to mammalian dual-specificity kinases.

We report here the sequence of RPK1 (for Regulatory cell Proliferation Kinase), a new Saccharomyces cerevisiae gene coding for a protein with sequence similarities to serine/threonine protein kinases. The protein sequence of 764 amino acids includes an amino-terminal domain (residues 1-410), which may be involved in regulation of the kinase domain (residues 411-764). The catalytic domain of Rpk1 is not closely related to other known yeast protein kinases but exhibits strong homology to a newly discovered group of mammalian kinases (PYT, TTK, esk) with serine/threonine/tyrosine kinase activity. Null alleles of RPK1 are lethal and thus this gene belongs to the small group of yeast protein kinase genes that are essential for cell growth. In addition, eliminating the expression of RPK1 gives rise to the accumulation of non-viable cells with less than a 1 N DNA content suggesting that cells proceed into mitosis without completion of DNA synthesis. Therefore, the Rpk1 kinase may function in a checkpoint control which couples DNA replication to mitosis. The level of the RPK1 transcript is extremely low and constant throughout the mitotic cycle. However it is regulated during cellular differentiation, being decreased in alpha-factor-treated a cells and increased late in meiosis in a/alpha diploids. Taken together, our results suggest that Rpk1 is involved in a pathway that coordinates cell proliferation and differentiation.

Amino Acid Sequence↗
PubMed1994

Structure and function of poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP) participates in the intricate network of systems developed by the eukaryotic cell to cope with the numerous environmental and endogenous genetoxic agents. Cloning of the PARP gene has allowed the development of genetic and molecular approaches to elucidate the structure and the function of this abundant and highly conserved enzyme. This article summarizes our present knowledge in this field.

Amino Acid Sequence↗
PubMed1994

Rift Valley fever virus L segment: correction of the sequence and possible functional role of newly identified regions conserved in RNA-dependent polymerases.

The sequence of Rift Valley fever virus L segment that we published in a previous paper was erroneous in the 3'-terminal region of the antigenomic RNA molecule. Here, we have shown that the L segment is in fact 6404 nucleotides long and encodes a polypeptide of 237.7K in the viral complementary sense. Sequence comparisons performed between the RNA-dependent RNA polymerases of 22 negative-stranded RNA viruses revealed the existence of two novel regions located at the amino termini of the proteins and conserved only in the polymerases of bunya- and arenaviruses. In the region conserved in all RNA-dependent polymerases, corresponding to the so-called 'polymerase module', we identified a new motif, designated premotif A, common to all RNA-dependent polymerases, as well as amino acids located in the region between motifs preA and A which are strictly conserved for segmented negative-stranded RNA viruses. Using the recently released coordinates of human immunodeficiency virus reverse transcriptase and the alignment between all RNA-dependent polymerases in the 'polymerase module', we have determined the position of the conserved residues in these polymerases and discuss their possible functions in light of the available structural information.

Amino Acid Sequence↗
PubMed1993

Identification of potential active-site residues in the human poly(ADP-ribose) polymerase.

The carboxyl-terminal catalytic domain of the human poly(ADP-ribose) polymerase (PARP) exhibits sequence homology with the NAD(P)(+)-dependent leucine and glutamate dehydrogenases. To clarify the role played by some conserved residues between PARP and NAD(P)(+)-dependent dehydrogenases, point mutations were introduced into the whole enzyme context. Non-conservative mutations of Lys-893 (K893I) and Asp-993 (D993A) completely inactivate human PARP, whereas conservative and nonconservative mutations of Asp-914 (D914E and D914A, respectively) and Lys-953 (K953R and K953I, respectively) partially alter PARP activity. The consequences of conservative substitution of Lys-893 and Asp-993 on the kinetic properties of human poly(ADP-ribose) polymerase enzyme and the polymer it synthesizes suggest that these 2 amino acids are directly involved in the covalent attachment of the first ADP-ribosyl residue from NAD+ onto the acceptor amino acid. In addition, the recent resolution of the three-dimensional structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum (Baker, P.J., Britton, K.L., Engel, P.C., Farrants, G.W., Lilley, K.S., Rice, D.W., and Stillman, T.J. (1992) Proteins 12, 75-86) strongly supports our alignment with leucine and glutamate dehydrogenases and provides an interesting structural framework for the analysis of our results of site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1992

Sequence of the Kluyveromyces lactis beta-galactosidase: comparison with prokaryotic enzymes and secondary structure analysis.

The LAC4 gene encoding the beta-galactosidase (beta Gal) of the yeast, Kluyveromyces lactis, was cloned on a 7.2-kb fragment by complementation of a lacZ-deficient Escherichia coli strain. The nucleotide sequence of the structural gene, with 42 bp and 583 bp of the 5'- and 3'-flanking sequences, respectively, was determined. The deduced amino acid (aa) sequence of the K. lactis beta Gal predicts a 1025-aa polypeptide with a calculated M(r) of 117618 and reveals extended sequence homologies with all the published prokaryotic beta Gal sequences. This suggests that the eukaryotic beta Gal is closely related, evolutionarily and structurally, to the prokaryotic beta Gal's. In addition, sequence similarities were observed between the highly conserved N-terminal two-thirds of the beta Gal and the entire length of the beta-glucuronidase (beta Glu) polypeptides, which suggests that beta Glu is clearly related, structurally and evolutionarily, to the N-terminal two-thirds of the beta Gal. The structural analysis of the beta Gal alignment, performed by mean secondary structure prediction, revealed that most of the invariant residues are located in turn or loop structures. The location of the invariant residues is discussed with respect to their accessibility and their possible involvement in the catalytic process.

Amino Acid Sequence↗
PubMed1990

Expression and site-directed mutagenesis of the catalytic domain of human poly(ADP-ribose)polymerase in Escherichia coli. Lysine 893 is critical for activity.

Bacterially expressed fusion proteins containing the COOH-terminal domain of the human poly(ADP-ribose)polymerase were analyzed by means of a novel assay, the "activity blot," which allows the detection of transferred polypeptides involved in poly(ADP-ribose) synthesis. Deletion analysis demonstrated that the 40-kDa COOH-terminal region of the enzyme is an autonomous catalytic domain exhibiting both the polymerizing and branching activities in the absence of DNA. Site-directed mutagenesis demonstrated that lysine 893 is essential for these catalytic processes. In addition, sequence similarities obtained with the NAD(P)+ amino acid dehydrogenases suggest that (i) lysine 893 may interact with the substrates of poly(ADP-ribose)polymerase and (ii) the COOH-terminal part of the 40-kDa fragment may also contain a Rossman fold structure.

Amino Acid Sequence↗
PubMed1990

Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs.

The aminoacyl-transfer RNA synthetases (aaRS) catalyse the attachment of an amino acid to its cognate transfer RNA molecule in a highly specific two-step reaction. These proteins differ widely in size and oligomeric state, and have limited sequence homology. Out of the 18 known aaRS, only 9 referred to as class I synthetases (GlnRS, TyrRS, MetRS, GluRS, ArgRS, ValRS, IleRS, LeuRS, TrpRS), display two short common consensus sequences ('HIGH' and 'KMSKS') which indicate, as observed in three crystal structures, the presence of a structural domain (the Rossman fold) that binds ATP. We report here the sequence of Escherichia coli ProRS, a dimer of relative molecular mass 127,402, which is homologous to both ThrRS and SerRS. These three latter aaRS share three new sequence motifs with AspRS, AsnRS, LysRS, HisRS and the beta subunit of PheRS. These three motifs (motifs 1, 2 and 3), in a search through the entire data bank, proved to be specific for this set of aaRS (referred to as class II). Class II may also contain AlaRS and GlyRS, because these sequences have a typical motif 3. Surprisingly, this partition of aaRS in two classes is found to be strongly correlated on the functional level with the acylation occurring either on the 2' OH (class I) or 3' OH (class II) of the ribose of the last nucleotide of tRNA.

Amino Acid Sequence↗
PubMed1990

Analysis of the structural diversity of monoclonal antibodies to cyclosporine.

The immunosuppressive cyclic undecapeptide cyclosporine (Cs) represents a useful model for studying the molecular basis of antibody-antigen interactions. The three-dimensional structure of the Cs molecule is known and a large panel of monoclonal antibodies (mAbs) to Cs has been well characterized by cross-reactivity studies with numerous Cs analogs. In the present study, the sequences of the variable regions of seven mAbs to Cs were determined and a striking relationship was found between the expressed variable region genes and the Cs recognition pattern. An analysis of the length and hydrophobic content of the hypervariable regions and sequence similarities suggested that the heavy chain plays a major role in Cs recognition. Different fine specificities were observed for mAbs exhibiting identical light chains, while two antibodies differed by only a single amino acid located in the heavy chain. The presence of a duplication of 12 nucleotides within the heavy chain third hypervariable region of two antibodies suggests the existence of an additional mechanism for creating antibody diversity.

Amino Acid Sequence↗
PubMed1990

An attempt to unify the structure of polymerases.

With the great availability of sequences from RNA- and DNA-dependent RNA and DNA polymerases, it has become possible to delineate a few highly conserved regions for various polymerase types. In this work a DNA polymerase sequence from bacteriophage SPO2 was found to be homologous to the polymerase domain of the Klenow fragment of polymerase I from Escherichia coli, which is known to be closely related to those from Staphylococcus pneumoniae, Thermus aquaticus and bacteriophages T7 and T5. The alignment of the SPO2 polymerase with the other five sequences considerably narrowed the conserved motifs in these proteins. Three of the motifs matched reasonably all the conserved motifs of another DNA polymerase type, characterized by human polymerase alpha. It is also possible to find these three motifs in monomeric DNA-dependent RNA polymerases and two of them in DNA polymerase beta and DNA terminal transferases. These latter two motifs also matched two of the four motifs recently identified in 84 RNA-dependent polymerases. From the known tertiary architecture of the Klenow fragment of E. coli pol I, a spatial arrangement can be implied for these motifs. In addition, numerous biochemical experiments suggesting a role for the motifs in a common function (dNTP binding) also support these inferences. This speculative hypothesis, attempting to unify polymerase structure at least locally, if not globally, under the pol I fold, should provide a useful model to direct mutagenesis experiments to probe template and substrate specificity in polymerases.

Amino Acid Sequence↗
PubMed1990

Sequence comparison of five polymerases (L proteins) of unsegmented negative-strand RNA viruses: theoretical assignment of functional domains.

The large (L) protein subunit of unsegmented negative-strand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagreement with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis.

Amino Acid Sequence↗