Biomedical subjects

G Lucchini

Publications and source records attributed to G Lucchini.

At least 55 records · Page 3Linked to original sources

PubMed1992

Control of DNA synthesis genes in budding yeast: involvement of the transcriptional modulator MOT1 in the expression of the DNA polymerase alpha gene.

Periodic transcription during the cell cycle of the budding yeast DNA polymerase alpha gene (POL1) requires the cis-acting element 5' ACGCGT 3', which has been found in the 5' non-coding region of all the DNA synthesis genes analyzed so far. Search for trans-acting mutations affecting POL1 expression led to the isolation of the temperature-sensitive reg1033 mutant, that showed increased levels of both DNA polymerase alpha and delta gene transcripts. Cloning of the REG1033 gene demonstrated that it is essential for cell viability and required for proper expression of the POL1 gene. DNA sequence comparison established that the REG1033 gene is identical to MOT1, a gene encoding a presumptive DNA helicase which modulates transcription of several yeast genes.

Base Sequence↗

PubMed1992

Theophylline transfer across human placental cotyledon during in vitro dual perfusion.

In vitro placental perfusion is widely used to investigate the placental transfer of endogenous compounds and, to a lesser extent, that of drugs. The aim of this study was to assess the suitability and reliability of such in vitro systems for application on drug placental transfer studies. We investigated the time course of theophylline (TH) transfer, a drug frequently used in the perinatal period. Eight experiments were performed with maternal and fetal circuits maintained in an open system, perfusing placentas for 160 min with Earle's enriched bicarbonate buffer containing two test substances, antipyrine (AP), (80 mg/L) and creatinine (CR), (150 mg/L), and the tool drug TH (15 mg/L). All substances equilibrated in our system with times proportional to the chemical-physical characteristics of each compound, being the time required to reach the steady state 5 to 12 min for AP, 12 to 31 min for CR and 10 to 35 min for TH. AP and CR clearances were 2.94 +/- 0.33 and 0.83 +/- 0.26 mL/min, respectively. The transfer profile of TH was similar to that of AP and its clearance was 2.39 +/- 0.37 mL/min, with a clearance index of 0.80 +/- 0.11. Transfer percentages of TH are in agreement with in vivo values for both humans and animals, and with results obtained during in situ placental perfusion in the rabbit. Physiological conditions and biochemical properties of the tissue were well maintained throughout perfusion. Glucose consumption and lactate release were, respectively, 0.65 +/- 0.16 and 0.73 +/- 0.11 mumoles/min/g. Oxygen consumption was 5.29 +/- 1.32 mL/min/kg and oxygen transfer from the maternal to fetal circuit was 0.99 +/- 0.43 mL/min/kg. The findings support the reliability of this technique to study transplacental passage of drugs, and the relevance of such a model to obtain information concerning potential therapeutic or toxicologic effects of drugs during the last trimester of pregnancy.

Female↗

PubMed1991

Mutations in conserved yeast DNA primase domains impair DNA replication in vivo.

To assess the role of eukaryotic DNA primase in vivo, we have produced conditional and lethal point mutations by random in vitro mutagenesis of the PR11 and PR12 genes, which encode the small and large subunits of yeast DNA primase. We replaced the wild-type copies of PRI1 and PRI2 with two pri1 and two pri2 conditional alleles. When shifted to the restrictive temperature, these strains showed altered DNA synthesis and reduced ability to synthesize high molecular weight DNA products, thus providing in vivo evidence for the essential role of DNA primase in eukaryotic DNA replication. Furthermore, mapping of the mutations at the nucleotide level has shown that the two pri1 and two pri2 conditional alleles and one pri2 lethal allele have suffered single base-pair substitutions causing a change in amino acid residues conserved in the corresponding mouse polypeptide.

Alleles↗

PubMed1991

Plasma protein binding of theophylline during development in the rabbit.

The plasma protein binding profile of theophylline was investigated in the rabbit during the perinatal and developmental period. The roles of unbound plasma theophylline fraction (THu), albumin, nonesterified fatty acids (NEFA) and plasma bilirubin levels were analyzed. Plasma total protein and albumin levels doubled with age from the 1st day of life to maturity (from 3.1 to 5.3 and 1.6 to 3.7 g/l, respectively). THu, NEFA and bilirubin levels were inversely related to age, and decreased from 81 to 37%, 2,136 to 239 microEq/l and 2.4 to 0.20 mg/dl, respectively. A linear correlation was shown between THu and albumin, NEFA, and bilirubin plasma concentrations. Stepwise multiple linear regression analysis, including albumin, NEFA and bilirubin values as independent variables, identified NEFA as the variable explaining most of the variability in plasma protein binding of theophylline. Findings support the need for careful interpretation (with a view to therapeutic utilization) of estimates of plasma protein unbound fraction of a drug during development. This fact highlights the importance of considering not only protein but NEFA concentrations too.

Age Factors↗

PubMed1990

Nucleotide sequence and characterization of temperature-sensitive pol1 mutants of Saccharomyces cerevisiae.

We have analyzed the effects of temperature-sensitivity (ts)-conferring mutations in the Saccharomyces cerevisiae DNA polymerase I-encoding gene on cell growth, in vivo DNA synthesis, intrachromosomal gene conversion and pop-out recombination. Also, we have identified the molecular defect responsible for the ts phenotype. Two mutant alleles (cdc17-1, cdc17-2) were originally identified as cell-cycle mutations, while a third mutation (hpr3) was found during a genetic screening for mutants with a hyper-recombination phenotype. Both cdc17-2 and hpr3 cells complete one round of cell division and DNA replication after shift to nonpermissive temperature, before being arrested as dumbbell-shaped cells. Conversely, the cdc17-1 mutation immediately blocks growth and DNA synthesis at 37 degrees C. No substantial difference was observed in the frequency of intrachromosomal gene conversion and pop-out recombination events, when hpr3 and cdc17-1 were compared to the previously characterized pol1-1 mutant. These two frequencies were ten- to 30-fold above wild-type level at semipermissive temperature. In each mutant, a single bp substitution, causing the replacement of Gly residues by either Asp (cdc17-1, cdc17-2) or Glu (hpr3) in yeast DNA polymerase I is responsible for the ts phenotype.

Amino Acid Sequence↗

PubMed1990

Expression of the yeast DNA primase gene, PRI1, is regulated within the mitotic cell cycle and in meiosis.

Mitotic cultures synchronised either by a feed-starve protocol or by elutriation have been used to show that the Saccharomyces cerevisiae DNA primase I gene is periodically expressed in the cell cycle. The transcript increases many-fold in late G1 and reaches a peak at the same time as four other genes essential for DNA synthesis, CDC8, CDC9, CDC21 and POL1. The primase I transcript is also regulated in meiosis, reaching maximal levels during premeiotic DNA synthesis.

DNA Primase↗

PubMed1989

Affinity labeling of the active center and ribonucleoside triphosphate binding site of yeast DNA primase.

A highly selective affinity labeling procedure has been applied to map the active center of DNA primase from the yeast Saccharomyces cerevisiae. Enzyme molecules that have been modified by covalent attachment of benzaldehyde derivatives of adenine nucleotides are autocatalytically labeled by incubation with a radioactive ribonucleoside triphosphate. The affinity labeling of primase requires a template DNA, is not affected by DNase and RNase treatments, but is sensitive to proteinase K. Both the p58 and p48 subunits of yeast DNA primase appear to participate in the formation of the catalytic site of the enzyme, although UV-photocross-linking with [alpha-32P]ATP locates the ribonucleoside triphosphate binding site exclusively on the p48 polypeptide. The fixation of the radioactive product has been carried out also after the enzymatic reaction. Under this condition the RNA primers synthesized by the DNA polymerase-primase complex under uncoupled DNA synthesis conditions are linked to both DNA primase and DNA polymerase. When DNA synthesis is allowed to proceed first, the labeled RNA chains are fixed exclusively to the DNA polymerase polypeptide. These results, in accord with previous data, have been used to propose a model illustrating the interactions and the putative roles of the polypeptides of the DNA polymerase-primase complex.

Adenosine Triphosphate↗

PubMed1989

A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae.

DNA primase activity of the yeast DNA polymerase-primase complex is related to two polypeptides, p58 and p48. The reciprocal role of these protein species has not yet been clarified, although both participate in formation of the active center of the enzyme. The gene encoding the p58 subunit has been cloned by screening of a lambda gt11 yeast genomic DNA library, using specific anti-p58 antiserum. Antibodies that inhibited DNA primase activity could be purified by lysates of Escherichia coli cells infected with a recombinant bacteriophage containing the entire gene, which we designate PR12. The gene was found to be transcribed in a 1.7-kilobase mRNA whose level appeared to fluctuate during the mitotic cell cycle. Nucleotide sequence determination indicated that PR12 encodes a 528-amino-acid polypeptide with a calculated molecular weight of 62,262. The gene is unique in the haploid yeast genome, and its product is essential for cell viability, as has been shown for other components of the yeast DNA polymerase-primase complex.

Amino Acid Sequence↗

PubMed1988

The yeast DNA polymerase-primase complex: genes and proteins.

The yeast DNA polymerase-primase complex is composed of four polypeptides designated p180, p74, p58 and p48. All the genes coding for these polypeptides have now been cloned. By protein sequence comparison we found that yeast DNA polymerase I (alpha) shares three major regions of homology with several DNA polymerases. A fourth region, called region P, is conserved in yeast and human DNA polymerase alpha. The site of a temperature-sensitive mutation in the POL1 gene which causes decreased stability of the polymerase-primase complex has been sequenced and falls in this region. We hypothesize that region P is important for protein-protein interactions. Highly selective biochemical methods might be similarly important to distinguish functional domains in the polymerase-primase complex. An autocatalytic affinity labeling procedure has been applied to map the active center of yeast DNA primase. From this approach we conclude that both primase subunits (p48 and p58) participate in the formation of the catalytic site of the enzyme.

Amino Acid Sequence↗

PubMed1988

Genetic mapping of the Saccharomyces cerevisiae DNA polymerase I gene and characterization of a pol1 temperature-sensitive mutant altered in DNA primase-polymerase complex stability.

The cloned DNA polymerase I gene has been used to map the POL1 locus on the left arm of chromosome XIV, between MET4 and TOP2. Temperature-sensitive mutants in POL1 have been obtained by in vitro mutagenesis of the cloned gene and in vivo replacement of the wild-type allele with the mutated copy. Physiological and biochemical characterization of one temperature-sensitive mutant (pol1-1) shows that cells shifted to the non-permissive temperature can complete one round of cell division and DNA replication before they arrest. Analysis of DNA polymerase I in crude extracts and in partially purified preparations indicates that the pol1-1 mutation results in a conformational change and affects the stability of the DNA primase-polymerase complex.

Chromosome Mapping↗

PubMed1988

DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases.

A 5600-base pair segment spanning the coding region of the Saccharomyces cerevisiae DNA polymerase I gene was sequenced and found to contain an open reading frame of 1468 codons, corresponding to a polypeptide of Mr 166,794. A pol1 temperature-sensitive mutation, encoding a DNA-polymerase-primase complex with altered stability, has a single base-pair substitution that changes the glycine at position 493 to a positively charged arginine. Protein sequence comparison with other prokaryotic and eukaryotic DNA polymerases reveals three major regions of homology. This observation suggests that certain DNA polymerases might require the conservation of critical amino acid residues for activity.

Amino Acid Sequence↗

PubMed1987

The nucleotide sequence of the PRI1 gene related to DNA primase in Saccharomyces cerevisiae.

The PRI1 gene of Saccharomyces cerevisiae encodes for the p48 polypeptide of DNA primase. We have determined the nucleotide sequence of a 1,965 bp DNA fragment containing the PRI1 locus. The entire coding sequence of the gene lies within an open reading frame, and there are 409 amino acids in the single polypeptide protein if translation is assumed to start at the first ATG in this frame. The 5' and 3' end-points of PRI1 mRNA have been determined by S1 mapping and primer extension analysis. The primary structure and the codon usage of PRI1 suggest that this essential gene is poorly expressed in yeast cells.

Amino Acid Sequence↗

PubMed1987

The yeast DNA polymerase I transcript is regulated in both the mitotic cell cycle and in meiosis and is also induced after DNA damage.

Using mitotic cultures synchronised by a feed-starve protocol or by elutriation, we have shown that the yeast DNA polymerase I gene is periodically expressed with its transcript increasing at least 100-fold in late G1 with a peak around the G1/S phase boundary. This is precisely the same interval of the cell cycle in which three other yeast DNA synthesis genes, CDC8, CDC9 and CDC21, have been found to be periodically expressed (White et al 1987. Expl. Cell. Res., in press). The polymerase I transcript is also regulated in meiosis, showing an overall fluctuation in level of some 20-fold, with a peak at about mid-S phase. In addition, following irradiation with 50J/m2 ultraviolet light, there was a 20-fold increase in the transcript, starting after 30 minutes and reaching a peak two hours later. These results indicate that DNA polymerase I is subject to a complex control and imply that it has a role in both DNA synthesis and DNA repair.

Cell Cycle↗

PubMed1987

Yeast DNA polymerase--DNA primase complex; cloning of PRI 1, a single essential gene related to DNA primase activity.

The immunopurified yeast DNA polymerase--DNA primase complex is constituted by DNA polymerase I polypeptides and by three other protein species, called p74, p58 and p48, which we show to be immunologically unrelated. The gene encoding the p48 polypeptide has been identified by immunological screening of a lambda gt11 yeast genomic DNA library. Antiserum specific for p48 inhibits DNA primase, and immunoreactive, inhibitory antibodies are affinity-purified by the clone-encoded protein, thus relating the p48 polypeptide to DNA primase activity. The entire gene has been cloned, and the 1.45-kb p48 mRNA is overproduced in cells containing the gene in high copy number. Gene disruption and Southern hybridization experiments demonstrate that the p48 protein is encoded by a single gene and it performs an essential function.

Cloning, Molecular↗

PubMed1986

Mechanism of initiation of in vitro DNA synthesis by the immunopurified complex between yeast DNA polymerase I and DNA primase.

The immunopurified yeast DNA-polymerase-I--DNA-primase complex synthesizes oligo(rA) and oligo(rG) molecules that are used as primer for replication of poly(dT) and poly(dC). Neither initiation nor DNA synthesis is observed with poly(dA) and poly(dI). Nitrocellulose-filter binding shows that the enzyme complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Although the yeast complex initiates DNA synthesis on deoxypyrimidine homopolymers, it prefers to elongate pre-existing primer molecules rather than to initiate de novo DNA replication. The size of the oligo(rA) and oligo(rG) primer molecules has been determined by urea/polyacrylamide gel electrophoresis: longer oligoribonucleotides are synthesized when their utilization is prevented by omitting dNTP. An oligodeoxythymidylate template with a chain length as short as five residues can support oligo(rA) synthesis catalyzed by the yeast DNA-polymerase--DNA-primase complex and the size of the oligoribonucleotide products synthesized with oligodeoxythymidylate of differing chain length has also been determined. The mechanistic properties of the DNA-polymerase--DNA-primase complexes, purified from different eukaryotic organisms, appear to be very similar. The possible biological implication of the studies on the mechanism and specificity of initiation of DNA synthesis in a well-defined model template system has been discussed.

DNA↗

PubMed1985

Identification of the yeast DNA polymerase I gene with antibody probes.

Partially overlapping fragments of the gene encoding yeast DNA polymerase I have been cloned by immunological screening of a yeast genomic library constructed in the phage lambda expression vector lambda gt11. The three gene fragments we analyzed in detail encode part of a yeast protein that has been identified as yeast DNA polymerase I, because it shares with this enzyme a number of antigenic determinants. In fact, the yeast protein fragments expressed by the recombinant phages react with both polyclonal and monoclonal antibodies raised against different, highly purified preparations of DNA polymerase I. Moreover, they can be used to affinity purify antibodies specifically reacting with active DNA polymerase I polypeptides and they compete with the yeast enzyme for binding to antibodies that inhibit catalytic activity. The gene is located on chromosome XIV in the yeast genome, and it is transcribed as a 5.2 kb mRNA.

Antibodies, Monoclonal↗

PubMed1985

A synthetic HIS4 regulatory element confers general amino acid control on the cytochrome c gene (CYC1) of yeast.

Hybrid promoters constructed from upstream sequences of the yeast HIS4 promoter and the downstream element of the yeast CYC1 promoter place iso-1-cytochrome c (CYC1) expression under the general amino acid control, typical of HIS4. HIS4 fragments that confer regulation contain at least one copy of the sequence T-G-A-C-T-C that is repeated at HIS4 and other genes subject to the general control. A 14-base-pair synthetic oligonucleotide containing a single copy of the HIS4 repeat places CYC1 under the general control. Two copies of this oligonucleotide produce a derepressed level of expression nearly equivalent to that conferred by the largest HIS4 5' noncoding fragments we examined and direct regulated expression of a set of transcripts with 5' ends typical of the CYC1 promoter. Comparison of the expression levels conferred by the short synthetic repeat and larger HIS4 5' fragments reveals additional promoter elements required for maintaining efficient gene expression under repressing growth conditions.

Amino Acids↗

PubMed1984

Positive regulatory interactions of the HIS4 gene of Saccharomyces cerevisiae.

The role of cis- and trans-acting elements in the expression of HIS4 has been examined by using HIS4-lacZ fusions in which lacZ expression is dependent upon the HIS4 5' noncoding region. The cis-acting sequences involved in regulation were defined by studying the effects of the wild-type and various deletions and their revertants on regulation via the general control of amino acid biosynthesis. The role of trans-acting genes was analyzed by studying the regulation of the HIS4-lacZ fusions in strains carrying mutations in the GCN (AAS) or GCD (TRA) genes and in strains carrying the GCN genes on high-copy-number plasmids. These studies have led to the following conclusions. (i) HIS4 is positively regulated by the general control. (ii) At least one copy of the 5'TGACTC3' repeat at -136 is required in cis for this regulation. (iii) Both the GCN4 gene and at least one copy of the repeated sequence are required for expression at the repressed level. (iv) The open reading frames in the 5' noncoding region are not required in either cis or trans for the regulation of HIS4.

Chromosome Deletion↗