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Biomedical subjects

R M Lacatena

Publications and source records attributed to R M Lacatena.

10 recordsLinked to original sources
PubMed1991

Detection of homologous recombination between yeast artificial chromosomes with overlapping inserts.

We have developed a system which facilitates the detection of recombination between Yeast Artificial Chromosomes (YAC's) carrying homologous inserts. The system consists of a classical YAC vector, a new YAC vector and two appropriately labelled yeast strains of opposite mating type. The new YAC vector differs in markers from the canonical YAC vector. To test whether homologous recombination takes place, phage lambda DNA was cloned in the two vectors to provide a region of homology. The two constructs were then introduced into yeast strains of opposite mating type in which the endogenous genes for the selective markers present in the vectors are not expressed. Artificial chromosomes obtained by meiotic recombination are detected in the spores resulting from the mating.

Bacteriophage lambda↗
PubMed1985

Analysis of dominant copy number mutants of the plasmid pMB1.

We characterize two dominant copy number mutants of a derivative of plasmid pMB1. One of the two mutations maps in the -35 region of the primer promoter and results in increased promoter activity. The analysis of the secondary structure in the proximity of the mutant sequence suggests a possible mechanism which could be the basis of the promoter-up phenotype. By comparing the properties of the mutant and the wild type plasmid in an in vitro system, we confirm that the primer and not its coding sequence is the target of RNA I inhibition. The second mutation affects the sequence of the primer so that it is less sensitive to inhibition by RNA I. We propose that this mutation stabilizes a secondary structure necessary for primer formation.

Base Sequence↗
PubMed1984

Control of pMB1 replication: inhibition of primer formation by Rop requires RNA1.

The rop gene participates in the control of plasmid copy number by interfering with transcripts originating from the primer promoter. Here we show that this inhibition mechanism requires RNA1 in trans. Mutations in the RNA1 coding sequence that result in plasmids with altered incompatibility properties do not affect the ability of the molecule to participate in the Rop inhibition mechanism. Furthermore we show that the target of the Rop-RNA1 inhibitory mechanism is located, at least in part, after the 52nd nucleotide of the sequence encoding the primer transcript.

Base Sequence↗
PubMed1984

How does Rop work?

Explore the source record for details and available documents.

DNA Replication↗
PubMed1984

Control of initiation of pMB1 replication: purified Rop protein and RNA I affect primer formation in vitro.

We show that a protein of 63 amino acids is the product of the rop gene, a gene which negatively regulates the copy number of plasmids of the ColE1 family. Rop protein purified to homogeneity inhibits ColE1 plasmid replication in a bacterial extract. Furthermore, we show that Rop inhibition requires RNA I. In a purified in vitro system that can support primer transcription and processing, Rop affects primer formation in two ways: first, it elicits transcription termination oat nucleotide 220, and second, it increases the ability of RNA I to inhibit RNAase H processing of the primer. The analysis of these data and the comparison with the results obtained in vivo with transcription fusion experiments allow us to propose a tentative model of the molecular mechanism underlying Rop-RNA I inhibition.

DNA Replication↗
PubMed1983

Interaction between RNA1 and the primer precursor in the regulation of Co1E1 replication.

The analysis of a large number of independent mutants in the target of one of the inhibitors of pMB1 replication suggests that RNA1 regulates primer formation by base-pairing with the complementary sequence in the primer precursor. We conclude that the number of bases that are involved in the hydrogen bonding responsible for the specificity of the mechanism that controls plasmid replication and incompatibility properties is not much larger than seven. Five of these bases are located in the central loop and two in loop I of the RNA primer cloverleaf structure. Twenty-two single, double or triple mutants, with different nucleotide sequences in these seven bases, maintain an active mechanism of control, though with altered specificity. The efficiency of the inhibition mechanism correlates with the delta G value of the hydrogen bonds between the nucleotides of the two heptamers postulated to be involved in the interaction. The implications of these findings are discussed, and a molecular model of the interaction between RNA1 and the primer precursor is presented.

Bacteriophage lambda↗
PubMed1982

An HMBA-resistant and heme-synthesizing variant clone of Friend leukemia cells.

A clone of Friend leukemia (FL) cells has been isolated which, in the presence of hexamethylene bisacetamide (HMBA) as inducer, is capable of growing and producing heme for long periods of time. The differentiation program expressed by this clone is very similar to that observed in the 5-86 parent, that, however, is arrested in its growth. The expression of the differentiated state in this clone depends on the continuous presence of an inducer; if the inducer is removed from a culture containing a high percentage of B+ (benzidine-positive) cells, the cells rapidly (5-7 days) revert to an undifferentiated state and appear to lose their differentiated character consecutively rather than simultaneously. The clone described in this paper is unstable; when grown for more than two months in the presence of an inducer it shifts toward a population containing a low percentage of heme-synthesizing cells. This instability seems to be due to the accumulation of a faster growing and more undifferentiated cell population.

Acetamides↗
PubMed1981

DNA polymerase activities in Friend cells during the differentiation process.

DNA synthesis and DNA polymerase activities were followed in FL cell cultures (clone 5.86) at different stages of differentiation. A temporary block of growth and DNA synthesis was observed after the addition of the inducers (DMSO or HMBA). This delay in the growth and in the DNA synthesis initiation is not observed in cultures of DMSO-resistant variants after treatment with DMSO. In both uninduced and induced cultures, during growth, the DNA gamma-polymerase activity is constant and the alpha-polymerase activity is strictly dependent on the DNA synthesis rate. On the other hand, a different behaviour between induced and uninduced cultures is observed for the DNA polymerase beta: its activity is constant in uninduced cultures, whereas it changes in induced cultures at various stages of differentiation, dropping to lower values at early times and rising to values similar to those observed in uninduced cultures at later times. This behaviour is not observed in cultures of a DMSO-resistant variant clone: in this case the beta-polymerase activity is constant in both the absence and the presence of DMSO or HMBA.

Animals↗