Biomedical subjects

C Marck

Publications and source records attributed to C Marck.

At least 19 recordsLinked to original sources

PubMed2000

Genomic exploration of the hemiascomycetous yeasts: 4. The genome of Saccharomyces cerevisiae revisited.

Since its completion more than 4 years ago, the sequence of Saccharomyces cerevisiae has been extensively used and studied. The original sequence has received a few corrections, and the identification of genes has been completed, thanks in particular to transcriptome analyses and to specialized studies on introns, tRNA genes, transposons or multigene families. In order to undertake the extensive comparative sequence analysis of this program, we have entirely revisited the S. cerevisiae sequence using the same criteria for all 16 chromosomes and taking into account publicly available annotations for genes and elements that cannot be predicted. Comparison with the other yeast species of this program indicates the existence of 50 novel genes in segments previously considered as 'intergenic' and suggests extensions for 26 of the previously annotated genes.

Ascomycota↗

PubMed1999

Interaction between yeast RNA polymerase III and transcription factor TFIIIC via ABC10alpha and tau131 subunits.

Yeast TFIIIC mediates transcription of class III genes by promoting the assembly of a stable TFIIIB-DNA complex that is sufficient for RNA polymerase III recruitment and function. Unexpectedly, we found an interaction in vivo and in vitro between the TFIIIB-recruiting subunit of TFIIIC, tau131, and ABC10alpha, a small essential subunit common to the three forms of nuclear RNA polymerases. This interaction was mapped to the C-terminal region of ABC10alpha. A thermosensitive mutation in the C terminus region of ABC10alpha (rpc10-30) was found to be selectively suppressed by overexpression of a mutant form of tau131 (tau131-DeltaTPR2) that lacks the second TPR repeat. Remarkably, the rpc10-30 mutation weakened the ABC10alpha-tau131 interaction, and the suppressive mutation, tau131-DeltaTPR2 increased the interaction between the two proteins in the two-hybrid assay. These results point to the potential importance of a functional contact between TFIIIC and RNA polymerase III.

Alleles↗

PubMed1999

A protein-protein interaction map of yeast RNA polymerase III.

The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein-protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10alpha, and ABC10beta), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.

Binding Sites↗

PubMed1999

Fritless capillary electrochromatography.

The preparation of packed capillaries with stable frits of good quality can be a hurdle to obtain efficient separations in capillary electrochromatography (CEC). Especially with particles smaller than 3 microm, frit preparation is cumbersome. Highly efficient separations using packed capillaries without frits are presented. Under appropriate CEC conditions the particles were retained by electrophoretic attraction towards the anode by a tapered capillary inlet, without the need of a frit at the outlet end. Such fritless capillaries, packed with 1.5 microm nonporous reversed-phase particles, allowed separations with efficiencies of more than 500,000 plates/m. Once the capillaries were conditioned properly, more than 100 separations could be performed with good repeatability. With respect to separation efficiency, fritless capillaries packed with 3 microm particles were comparable with standard CEC capillaries with frits. Examples of separations of steroids, a pesticide and its by-products, and cardiac glycosides under various CEC conditions are shown.

Chromatography, Liquid↗

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↗

PubMed1993

The TFIIIB-assembling subunit of yeast transcription factor TFIIIC has both tetratricopeptide repeats and basic helix-loop-helix motifs.

The multisubunit yeast transcription factor IIIC (TFIIIC; also called tau) can undergo considerable conformational changes upon binding to the A and B blocks of tRNA genes. After binding to DNA encoding tRNA (tDNA), TFIIIC acts as an assembly factor to recruit an initiation factor, TFIIIB, via its tau 131 subunit. We have cloned the gene encoding the tau 131 subunit and named it TFC4. This gene is unique, essential for cell viability, and encodes a M(r) 120,153 protein. Epitope-tagging and mobility-shift assays indicated the presence of a single tau 131 subunit in TFIIIC-tDNA complexes. tau 131 contains two sequence motifs, accounting for nearly one-half of the protein mass, that may provide a molecular explanation for the properties of TFIIIC-tDNA complex. A series of 11 copies of the tetratricopeptide repeat motif may account for the flexibility and interaction properties of TFIIIC. A motif akin to the basic helix-loop-helix motif of MyoD suggests the direct involvement of tau 131 in promoting DNA binding of TFIIIB.

Amino Acid Sequence↗

PubMed1992

Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae.

We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A. In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains. tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes. At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13 degrees C. This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDC55, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A. At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III. Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIIIB. These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase.

Amino Acid Sequence↗

PubMed1991

A new oxygen-regulated operon in Escherichia coli comprises the genes for a putative third cytochrome oxidase and for pH 2.5 acid phosphatase (appA)

The Escherichia coli acid phosphatase gene appA is expressed in response to oxygen deprivation and is positively controlled by the product of appR (katF) which encodes a putative new sigma transcription-initiation factor. However, transcription of appA from its nearest promoter (P1) did not account for total pH 2.5 acid phosphatase expression and was not subject to regulation. The cloned region upstream of appA was extended and analyzed by insertions of transposon TnphoA and by fusions with lacZ. It contains two new genes, appC and appB, which both encode extracytoplasmic proteins. appC and appB are expressed from a promoter (P2) lying just upstream of appC. Both genes are regulated by oxygen, as is appA, and by appR gene product exactly as previously shown for appA. Analysis of the nucleotide sequence and of the origins of transcription have confirmed that the P2-appC-appB- (ORFX)-P1-appA region is organized on the chromosome as an operon transcribed clockwise from P2 and that P1 is a minor promoter for appA alone. Genes appC and appB encode proteins of Mr 58,133 and 42,377, respectively, which have the characteristics of integral membrane proteins. The deduced amino acid sequences of appC and appB show 60% and 57% homology, respectively, with subunits I and II of the E. coli cytochrome d oxidase (encoded by genes cydA and cydB). The notion that the AppC and AppB proteins constitute a new cytochrome oxidase or a new oxygen-detoxifying system is supported by the observation of enhanced sensitivity to oxygen of mutants lacking all three genes, cyo (cytochrome o oxidase), cyd (cytochrome d oxidase) and appB, compared to that of cyo cyd double mutants.

Acid Phosphatase↗

PubMed1990

Nucleotide sequence and transcriptional analysis of the Escherichia coli agp gene encoding periplasmic acid glucose-1-phosphatase.

The nucleotide sequence of the agp gene, which encodes a periplasmic glucose-1-phosphatase, was determined. The deduced amino acid sequence corresponds to a 413-amino-acid-residue polypeptide with a typical hydrophobic signal sequence of 22 amino acids. The mature protein lacks the N-terminal signal peptide and has a calculated Mr of 43,514. Its promoter was defined by primer extension of the mRNA made in vivo. Like many genes under positive control, its -35 promoter region does not match the consensus. The agp gene is both preceded and followed by transcription termination signals, so it appears to be transcribed as a single unit.

Amino Acid Sequence↗

PubMed1990

The complete nucleotide sequence of the Escherichia coli gene appA reveals significant homology between pH 2.5 acid phosphatase and glucose-1-phosphatase.

The whole nucleotide sequence of Escherichia coli gene appA, which encodes periplasmic phosphoanhydride phosphohydrolase (optimum pH, 2.5), and its flanking regions was determined. The AppA protein is significantly homologous to the product of the nearby gene agp, acid glucose-1-phosphatase. Because identical amino acids are distributed over the whole lengths of the proteins, it is likely that appA and agp originate from the same ancestor gene.

Acid Phosphatase↗

PubMed1990

The ABF1 factor is the transcriptional activator of the L2 ribosomal protein genes in Saccharomyces cerevisiae.

The same factor, ABF1, binds to the promoters of the two gene copies (L2A and L2B) coding for the ribosomal protein L2 in Saccharomyces cerevisiae. In vitro binding experiments and in vivo functional analysis showed that the different affinities of the L2A and L2B promoters for the ABF1 factor are responsible for the differential transcriptional activities of the two gene copies. The presence of ABF1-binding sites in front of many housekeeping genes suggests a general role for ABF1 in the regulation of gene activity.

Base Sequence↗

PubMed1989

The two DNA-binding domains of yeast transcription factor tau as observed by scanning transmission electron microscopy.

Yeast transcription factor tau interacts with the intragenic promoter of tRNA genes, binding to both the A and B block elements. Affinity-purified tau factor and tau-tDNA complexes were examined by scanning transmission electron microscopy to analyze the structural features of free and DNA bound factor. The free factor appeared as two tightly associated globular domains of roughly similar size (10 nm in diameter) and mass (approximately 300 kd). A combination of these two domains results in a mass for the factor of 510-670 kd. When tau was allowed to interact with recombinant tRNA(3Leu) genes with variable A block-B block spacing, different structures were observed. With short genes, the two globular domains were not resolved and tau appeared as a large particle covering the A and B block region. On the other hand, with genes having a larger A-B distance (53 or 74 bp), mostly dumb-bell-shaped complexes were formed with individualized factor domains bound separately to the A and B blocks. A smaller proportion of the complexes appeared to consist of a large particle bound at only one site, essentially on the B block. Mapping of the binding domains in the DNA showed a good correlation with the respective positions of the A and B promoter elements. Factor binding did not induce a noticeable DNA bending, although with extended genes apparent DNA shortening and cases of DNA looping were observed. Upon cleavage of the tRNA(3Leu) gene between the A and B blocks after or prior to complex formation, the two factor domains remained attached to the same DNA fragment (mostly the B-DNA fragment). In addition, images of protein-linked, reconstituted full-length genes were also observed. These different conformational states of the tau-tDNA complexes probably reflect the dynamic aspect of the interaction of the factor with its DNA target.

DNA Polymerase III↗

PubMed1988

'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.

DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds.

Amino Acid Sequence↗

PubMed1988

RPA190, the gene coding for the largest subunit of yeast RNA polymerase A.

Yeast RNA polymerases are being extensively studied at the gene level. The entire gene encoding the largest subunit of RNA polymerase A, A190, was isolated and characterized in detail. Southern hybridization and gene disruption experiments showed that the RPA190 gene is unique in the haploid yeast genome and essential for cell viability. Nuclease S1 mapping was used to identify mRNA 5' and 3' termini. RPA190 encodes a polypeptide chain of 186,270 daltons in a large uninterrupted reading frame. A dot matrix comparison of the deduced amino acid sequence of subunit A190 with Escherichia coli beta' and cognate subunits B220 and C160 from yeast RNA polymerases B and C showed a conserved pattern of homology regions (I-VI). A potential DNA-binding site (zinc-binding motif) is conserved in the N-terminal region I. Remarkably, the A190 subunit does not harbor the heptapeptide repeated sequence present in the B220 subunit. The sequence of the A190 subunit diverges from B220 and C160 by the presence of two hydrophilic domains inserted between homology regions I and II, and V and VI. From their codon usage and third base pyrimidine bias, RNA polymerase genes RPA190, RPB220, RPC160, and RPC40 fall among yeast genes expressed at an average level. The RPA190 5'-flanking region contains features present in other polymerase genes that might function in regulation.

Amino Acid Sequence↗

PubMed1986

Fast analysis of DNA and protein sequence on Apple IIe: restriction sites search, alignment of short sequence and dot matrix analysis.

A fast restriction sites search algorithm using a quadruplet look-ahead feature has been written in 6502 assembly language code. The search time, tested on the sequence of pBR322, is 4.1 s/kilobase using a restriction site library including 112 specificities corresponding to a total site length of over 700 bases. The search for a short sequence (less than 36 bases) within a longer one (up to 9999 bases) with a given number of mismatches or gaps allowed has also been written in assembly language. Typical run time for the search of a 12 base sequence with 1, 2 or 3 gaps allowed are 6.2, 9.4 or 13.6 s/kilobase, respectively. The dot matrix analysis needs 7.5 minutes per square kilobase when using a stringency of 15 matched bases out of 25. A 7/21 matrix of two 500 amino acid proteins is obtained in 3 minutes. These three routines are included in DPSA, a general package of programs allowing manipulation and analysis of DNA and protein sequences.

Amino Acid Sequence↗

PubMed1985

A general upstream binding factor for genes of the yeast translational apparatus.

Fractionation of yeast extracts on heparin-agarose revealed the presence of a DNA footprinting activity that interacted specifically with the 5'-upstream region of TEF1 and TEF2 genes coding for the protein synthesis elongation factor EF-1 alpha, and of the ribosomal protein gene RP51A. The protected regions encompassed the conserved sequences 'HOMOL1' (AACATC TA CG T A G CA) or RPG-box (ACCCATACATT TA) previously detected 200-400 bp upstream of most of the yeast ribosomal protein genes examined. Two types of protein-DNA complexes were separated by a gel electrophoresis retardation assay. Complex 1, formed on TEF1, TEF2 and RP51A 5'-flanking region, was correlated with the protection of a 25-bp sequence. Complex 2, formed on TEF2 or RP51A probes at higher protein concentrations, corresponded to an extended footprint of 35-40 bp. The migration characteristics of the protein-DNA complexes and competition experiments indicated that the same component(s) interacted with the three different promoters. It is suggested that this DNA factor(s) is required for activation and coordinated regulation of the whole family of genes coding for the translational apparatus.

Base Sequence↗

PubMed1985

Cloning, nucleotide sequence, and expression of one of two genes coding for yeast elongation factor 1 alpha.

One gene coding for yeast cytoplasmic elongation factor 1 alpha (EF-1 alpha) was isolated by colony hybridization using a cDNA probe prepared from purified EF-1 alpha mRNA. A recombinant plasmid, pLB1, with a 6-kilobase yeast DNA insert, was found by hybrid selection and translation experiments to carry the entire gene. The nucleotide sequence of the gene with its 5'- and 3'-flanking regions was determined. The 5' and 3' ends of EF-1 alpha mRNA were localized by the S1 nuclease mapping technique. The cloned gene, called TEF1, encodes a protein of 458 amino acids (Mr = 50,071) in a single, uninterrupted reading frame. The amino acid sequence shows a strong homology with several domains of Artemia salina EF-1 alpha cytoplasmic factor, as evidenced by diagonal dot matrix analysis. Protein sequence homology is comparatively much lower with the yeast mitochondrial elongation factor. S1 nuclease mapping of the mRNA, hybridization analysis of chromosomal DNA using intragenic or extragenic DNA probes, and gene disruption experiments demonstrated the existence of two genes coding for the cytoplasmic elongation factor EF-1 alpha/haploid genome. The presence of an intact chromosomal TEF1 gene is not essential for growth of haploid yeast cells.

Base Sequence↗

PubMed1983

Z-DNA and other non-B-DNA structures are reversed to B-DNA by interaction with netropsin.

The interaction between the B-form specific ligands netropsin (Nt) and distamycin-3 (Dst-3) and DNA duplexes has been studied under conditions of salt concentration and low water activity that modify the polymer conformation into a non-B DNA form, putatively a Z-like form. Three polymers with strict alternating purine-pyrimidine sequences and GC content from 100-0% have been tested: poly(dG-dC) . poly(dG-dC), poly(dA-dC) . poly(dG-dT) and poly(dA-dT) . poly(dA-dT). The titrations by Nt and Dst-3 were followed by circular dichroism. Although specific binding of Nt to the Z-form of poly(dG-dC) . poly(dG-dC) does not occur, Nt reverses this Z structure to the B-type conformation; Dst-3 is, however, totally inefficient. The presumed non-B or Z-like structure of poly(dA-dC) . poly(dG-dT) is reversed to the B-form upon interaction with Nt; Dst-3 also induces this reversal but at higher ligand ratios. The modified B-structure of poly(dA-dT) . poly(dA-dT) in low water activity is efficiently reversed to the B-form by interaction with both Nt and Dst-3.

Chemical Phenomena↗