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A M de Recondo

Publications and source records attributed to A M de Recondo.

At least 19 recordsLinked to original sources
PubMed1992

Expression of the catalytic subunits of pol alpha and pol delta from fission yeast Schizosaccharomyces pombe.

This paper reports on expression and posttranslational modifications of the catalytic subunits of pol alpha and pol delta from fission yeast Schizosaccharomyces pombe. Okadaic acid treatment of S. pombe spheroplasts in amounts known to inhibit phosphatases 1 and 2A resulted in decreased proteolysis of both pol alpha and pol delta. Computer analysis of pol alpha and pol delta sequences confirmed the presence of consensus motifs for protein phosphorylation. Indirect immunofluorescence microscopy of S. pombe cells showed nuclear location of both proteins in wild type cells. However, whereas cells transformed with a vector expressing pol alpha produced a clear increase of the nuclear signal, no increase was detectable in cells transformed with pol delta. This observation suggests the existence of a mechanism limiting the cell concentration of pol delta in the cell. Constitutive expression of S. pombe pol delta in E. coli was possible only with vectors containing truncated forms of its gene, indicating a toxic effect of pol delta on E. coli growth.

Amino Acid Sequence↗
PubMed1992

DNA polymerase alpha in the fission yeast Schizosaccharomyces pombe: identification and tracing of the catalytic subunit during the cell cycle.

A recombinant protein was obtained in Escherichia coli by subcloning part of the Schizosaccharomyces pombe POL1 gene at the 3'-end of lacZ. Antibodies raised against this protein were used to identify the POL1 gene product in extracts of exponentially growing S. pombe cells. A major 170-kDa protein, whose structure and properties were typical of the catalytic subunit of eukaryotic DNA polymerases alpha (pol alpha), was detected. The same antibodies were used to trace pol alpha and to quantify its level during the S. pombe cell cycle. We found that pol alpha was present at all stages of the cycle and that its cellular pool was subject to limited (three-fold) increase in G1 and S phases, with a decline to the initial level soon after. In addition, we found that a second form of pol alpha with slightly lower molecular weight (165 kDa) existed only during late G1 and S phases. Moreover, absence of initiation or perturbations in the course of DNA replication induced overproduction of the 165-kDa form.

Blotting, Western↗
PubMed1991

Characterization of the POL3 gene product from Schizosaccharomyces pombe indicates inter-species conservation of the catalytic subunit of DNA polymerase delta.

The Schizosaccharomyces pombe POL3 gene was isolated by sequence homology with a region of the Saccharomyces cerevisiae POL3 gene, the only gene sequenced to date encoding the catalytic subunit of eukaryotic DNA polymerase delta. The fission yeast POL3 gene contains a 52 base-pair (bp) intron and encodes a 3600 bp transcript the 5'-end of which is located 32 bp upstream from the initiation codon. The polypeptides predicted from budding and fission yeast POL3 genes share 52% of conserved amino acid residues and have a 60% identical central region. This structural conservation of the catalytic subunit of DNA polymerases delta is probably related to functional constraints. A portion of the most conserved region was used to raise antibodies against an S. pombe polymerase delta/beta-galactosidase fusion protein expressed in Escherichia coli. The purified antibodies recognized a 123,000 Da protein in S. pombe wild-type cell extracts and inhibited an aphidicolin-sensitive DNA polymerase activity that was distinct from DNA polymerase alpha. The antibodies also detected a 140,000 Da protein in extracts from different proliferating mammalian cells, indicating that the catalytic subunits of DNA polymerase delta are highly conserved between yeast and higher eukaryotes.

Amino Acid Sequence↗
PubMed1991

Identification of a gene encoding the predicted ribosomal protein L7b divergently transcribed from POL1 in fission yeast Schizosaccharomyces pombe.

A 0.85 Kb RNA molecule is transcribed in the region upstream from the 5'-end of the S. pombe POL1 gene encoding the catalytic subunit of DNA polymerase alpha. The nucleotide sequence of the DNA region hybridizing with the 0.85 Kb transcript allowed us to identify an open reading frame coding for a predicted peptide which shows 50% identity with the rat ribosomal protein L7 and which is transcribed divergently from POL1. We have named this gene RPL7b because of the existence in S. pombe of a different sequence, named RPL7, which also codes for a putative protein showing homology with the rat ribosomal protein L7. The RPL7b gene includes a 291 bp-long intron containing the sequences necessary for intron excision and RNA splicing in S. pombe. The precise location of the intron was established by amplification and sequencing of a partial cDNA copy of the mRNA, whereas the initiation site of transcription was determined by reverse transcription of the 5' region of the mRNA. The 320 bp separating the starting methionine codons of RPL7b and POL1 genes should contain the signals necessary for their divergent transcription and regulation. The sequence 5'-AAGACAGTCACA-3', whose primary structure is homologous to a conserved block present in the 5'-untranscribed regions of other S. pombe genes of ribosomal proteins, is located about 50 bp upstream the transcription initiation site of RPL7b.

Amino Acid Sequence↗
PubMed1991

The POL1 gene from the fission yeast, Schizosaccharomyces pombe, shows conserved amino acid blocks specific for eukaryotic DNA polymerases alpha.

The POL1 gene of the fission yeast, Schizosaccharomyces pombe, was isolated using a POL1 gene probe from the budding yeast Saccharomyces cerevisiae, cloned and sequenced. This gene is unique and located on chromosome II. It includes a single 91 bp intron and is transcribed into a mRNA of about 4500 nucleotides. The predicted protein coded for by the S. pombe POL1 gene is 1405 amino acid long and its calculated molecular weight is about 160,000 daltons. This peptide contains seven amino acid blocks conserved among several DNA polymerases from different organisms and shares overall 37% and 34% identity with DNA polymerases alpha from S. cerevisiae and human cells, respectively. These results indicate that this gene codes for the S. pombe catalytic subunit of DNA polymerase alpha. The comparisons with human DNA polymerase alpha and with the budding yeast DNA polymerases alpha, delta and epsilon reveal conserved blocks of amino acids which are structurally and/or functionally specific only for eukaryotic alpha-type DNA polymerases.

Amino Acid Sequence↗
PubMed1990

The DNA polymerase from the archaebacterium Sulfolobus acidocaldarius: a thermophilic and thermoresistant enzyme which can perform automated polymerase chain reaction.

A DNA polymerase purified from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was used to perform automated DNA amplification at 70 degrees C as well as site directed mutagenesis by Polymerase Chain Reaction (P.C.R.). The yield of amplification performed at optimum MgCl2 concentration for the Taq or the S. acidocaldarius DNA polymerase, for the same DNA target, was equivalent. The ability of S. acidocaldarius DNA polymerase to perform P.C.R. under less stringent requirement of MgCl2 concentration gives this enzyme a non-negligible advantage over the Taq DNA polymerase.

Adenoviruses, Human↗
PubMed1990

Functional implications related to the gene structure of the elongation factor EF-Tu from Halobacterium marismortui.

The primary structure of the gene for the elongation factor EF-Tu from the halophilic archaebacterium Halobacterium marismortui (hEF-Tu) is described. It is the first gene of a halophilic elongation factor EF-Tu to be sequenced. When the sequence of hEF-Tu is compared to that of homologous proteins from other organisms, the highest identity (61%) is found with EF-Tu from Methanococcus vannielii, a non-halophilic archaebacterium. In the search for halophilic characteristics therefore the most appropriate comparison is with the M. vannielii sequence. The excess of acidic amino acid residues in the hEF-Tu sequence (already observed in the composition of other halophilic proteins) results to a large extent from changes of Lys, Asn or Gln to Asp or Glu. A structural analysis algorithm applied to the halophilic sequence places these acidic residues on the surface of the protein. The corresponding residues in the crystal structure of the first domain of EF-Tu from E. coli (the only EF-Tu structure available) are grouped in patches on the protein surface, in each of which several residues that may be far apart in the sequence come quite close to each other in the tertiary structure.

Amino Acid Sequence↗
PubMed1989

DNA polymerase from Sulfolobus acidocaldarius. Replication at high temperature of long stretches of single-stranded DNA.

The activity of a homogeneous DNA polymerase from the thermophilic archaebacterium, Sulfolobus acidocaldarius, on a singly primed, single-stranded recombinant phage M13 DNA has been examined. At the optimal temperature (70 to 75 degrees C) this template is efficiently replicated in ten minutes using a ratio of enzyme molecule to primed-template of 0.8. Analysis of DNA products during the course of polymerization shows that species of quite homogeneous size are observed and that the number of primers extended by the enzyme is constant, whatever the enzyme molecule to primed template ratio is in the range 1/50 to 2, indicating that the 100 x 10(3) Mr DNA polymerase from S. acidocaldarius is randomly recycled on the template molecules. At non-optimal temperature (60 degrees C and 80 degrees C) the distribution of products observed indicated the presence of arrest sequences; some have been shown to be reversible. One of these pausing signals detected at 80 degrees C has been further analysed, and has been found to be DNA sequence-dependent.

Archaea↗
PubMed1989

Expression of an enzymatically active murine retroviral reverse transcriptase in human cells.

The region of the pol gene of the Moloney murine leukemia virus (M-MuLV) encoding the reverse transcriptase and RNase H activities was inserted in an eukaryotic expression vector and transiently expressed in human cultured cells. This results in the expression of high levels of reverse transcriptase activity. This enzyme, partially purified, also carries a RNase H activity, has the biochemical requirements of the viral enzyme and is recognized and inhibited by antibodies directed against a M-MuLV reverse transcriptase expressed in Escherichia coli.

Animals↗
PubMed1989

Internal entry of ribosomes and ribosomal scanning involved in hepatitis B virus P gene expression.

The recent demonstration that the synthesis of duck hepatitis B virus (HBV) reverse transcriptase does not require translational frameshifting and the finding that poliovirus mRNA translation occurs in a cap-independent manner by internal binding of ribosomes in the 5' noncoding region led us to design experiments to test the hypothesis of internal entry of ribosomes on C gene mRNA for HBV P gene expression. We show that in human cells, translation can be initiated at the first AUG of the HBV P gene by entry of ribosomes in a region located upstream of the P gene. Moreover, the leaky scanning of ribosomes observed on the first AUG of the HBV P gene could be responsible for the synthesis of the two forms of reverse transcriptase described for HBV particles.

Amino Acid Sequence↗
PubMed1988

Novobiocin induces accumulation of a single strand of plasmid pGRB-1 in the archaebacterium Halobacterium GRB.

Treatment of Halobacterium GRB cells with the DNA topoisomerase II inhibitor novobiocin induces the accumulation of a circular single-stranded DNA form of the plasmid pGRB-1. This form corresponds to the transcribed strand of pGRB-1. A tiny amount of this form is detectable in untreated cells. The induction of single-stranded pGRB-1 molecules by novobiocin is abolished when cells are pretreated with aphidicolin or anisomycin, which inhibit halobacterial DNA replication and protein synthesis, respectively. These results suggest that the single-stranded form of pGRB-1 is generated in the course of plasmid replication.

Bacterial Proteins↗
PubMed1988

Novobiocin induces positive supercoiling of small plasmids from halophilic archaebacteria in vivo.

The halophilic archaebacterium Halobacterium strain GRB harbours a multicopy plasmid of 1.7 kb which is negatively supercoiled. After addition of novobiocin to culture medium all 1.7 kb plasmid molecules become positively supercoiled. Positive supercoiling occurs at the same dose of novobiocin inhibiting the eubacterial DNA gyrase in vitro. Novobiocin also induces positive supercoiling of pHV2, a 6.3 kb plasmid from Halobacterium volcanii. These results indicate the existence of a mechanism producing positive superturns in halobacteria. The 1.7 kb plasmid from Halobacterium GRB could be used to produce high amounts of pure positively supercoiled DNA for biophysical and biochemical studies.

DNA, Superhelical↗
PubMed1987

Antitumor drugs inhibit the growth of halophilic archaebacteria.

Permeability mutants of Escherichia coli have been used to prescreen antitumor drugs. However, most compounds active against eucaryotic proteins have no effect on isofunctional proteins of eubacteria. In contrast, we show that growth of halophilic archaebacteria, procaryotes as distantly related to eubacteria as to eucaryotes, is inhibited by several drugs known to interact with tubulin, actomyosin and DNA topoisomerase II of eucaryotes. Actually, different types of evidence indicate the presence of analogous proteins in halophilic archaebacteria: (a) a yeast actin probe hybridizes with DNA restriction digests of Halobacterium halobium; (b) antibodies against tubulin and actin from chicken react in a crude extract of H. halobium with polypeptides having Mr of 55,000 and 80,000, respectively; (c) the epipodophyllotoxin VP16, a eucaryotic DNA topoisomerase II inhibitor, induces DNA strand breaks with DNA-protein covalent linkage in H. halobium as in eucaryotes. Besides the evolutionary implications, these data indicate that halophilic archaebacteria can be used to prescreen antitumor drugs active on eucaryotic proteins.

Actins↗
PubMed1987

Effects of the antitumor drug VP16 (etoposide) on the archaebacterial Halobacterium GRB 1.7 kb plasmid in vivo.

The topoprofile of 1.7 kb plasmids from the archaebacterium Halobacterium GRB was analysed from cells growing with or without VP16 (etoposide). This drug interferes with the breakage-reunion reaction of eukaryotic DNA topoisomerase II by inhibiting the ligase activity of this enzyme. Addition of VP16 to the culture medium of Halobacterium GRB cells results in the introduction of single- and double-strand DNA breaks in part of the plasmid population, with proteins covalently associated at their 5' ends. While some of the remaining covalently closed circular DNA molecules are relaxed, VP16 treatment also gives rise to the production of positively supercoiled 1.7 kb plasmids. In contrast to adriamycin, VP16 does not intercalate into the 1.7 kb plasmid DNA in vivo. These results suggest that the VP16 target in halobacteria is a DNA topoisomerase II. Three major cleavage sites were detected on the 1.7 kb plasmid after VP16 treatment in vivo.

DNA, Bacterial↗
PubMed1982

Identification of a single-stranded DNA binding protein from rat liver with high mobility group protein 1.

The rat liver single-stranded DNA binding protein, S25 and HD25, isolated by differential DNA cellulose affinity chromatography was compared to the high mobility group proteins, HMG1 and HMG2, isolated from rat liver chromatin by the technique of Goodwin et al. (Goodwin, G. H., Sanders, C., and Johns, E. W. (1973) Eur. J. Biochem. 38, 14-19). Analysis of their amino acid composition, electrophoretic mobility, and tryptic peptide map reveal the identity of the single-stranded DNA binding protein with HMG1 protein, implying that the rat liver HMG1 protein becomes able both to destabilize a double helix of DNA and to stimulate homologous DNA polymerases only when rat liver cells enter a phase of DNA synthesis, possibly after a specific modification.

Amino Acids↗
PubMed1981

Single-strand deoxyribonucleic acid binding protein from rat liver changes the helical structure of deoxyribonucleic acid.

Incubation of rat liver single-strand DNA binding protein S25 with covalently closed relaxed SV40 DNA in the presence of rat liver topoisomerase I induced a decrease in the linking number LK of DNA, so that it appeared negatively supertwisted after removal of the protein. delta LK was found to be a linear function of protein to DNA ratio and reached a plateau corresponding to about 15 superhelical turns. The same result was obtained when S25 was incubated with form I or form Ir before addition of topoisomerase I or when SV40 was replaced by PM2 DNA. The observed reduction in the linking number of DNA when it is closed in the presence of rat liver protein S25 can be explained either by supercoiling of DNA induced by S25 or by detorsion or unwinding of DNA.

Animals↗
PubMed1981

Rat liver DNA polymerase-beta: thermal inactivation of RNA- and DNA-dependent activities.

Purified DNA polymerase-beta from rat liver was exposed to thermal inactivation and the remaining activities were then measured either with a hybrid template such as poly(A).(dT)12-18 (R-activity) or with a DNA template such as poly(dA).(dT)12-18 (D-activity). Time course of inhibition of R- and D-activities were identical. Neither activity was protected when the thermal treatment was performed in the presence of the template or dNTPs.

Animals↗