Association of the apolipoprotein B gene polymorphisms with cholesterol levels and response to fluvastatin in Brazilian individuals with high risk for coronary heart disease.

The influence of genetic polymorphism of the apolipoprotein B on lipid metabolism and coronary heart disease (CHD) risk has been demonstrated in different populations, but few studies have shown the contribution of this risk factor in individuals from Brazil. The Ins/del, Xbal and EcoRI polymorphisms of apo B were evaluated in 93 controls and in 104 Caucasian individuals presenting with a high risk lipid profile (HR1) for CHD; 54 of these subjects (HR2) were treated with fluvastatin during 16 weeks. DNA polymorphisms of the apo B gene were analyzed by polymerase chain reaction-restriction fragment length polymorphism. The X(-)X(-) genotype for Xbal polymorphism was associated with higher serum concentrations of total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) (p<0.01) in women of the HR1 group. The Ins/del and EcoRI polymorphisms were not associated with variation of lipid profile. After treatment with fluvastatin, TC and LDL-C levels of HR2 individuals were reduced by 23% and 30%, respectively. Individuals with II genotype had significantly greater reduction (34%) of LDL-C than those with ID/DD genotypes (27%). These results indicate that the Xbal polymorphism is associated with variation of serum TC and LDL-C levels in Brazilian women with lipid profile of risk for CHD and the Ins/del polymorphism is associated with the therapeutic response to fluvastatin.

Role of DNA methylation at GATC sites in the dnaA promoter, dnaAp2.

DnaA protein is required for the initiation of DNA replication at the bacterial chromosomal origin, oriC, and at the origins of many plasmids. The concentration of DnaA protein is an important factor in determining when initiation occurs during the cell cycle. Methylation of GATC sites in the dnaAp2 promoter, two of which are in the -35 and -10 sequences, has been predicted to play an important role in regulating dnaA gene expression during the cell cycle because the promoter is sequestered from methylation immediately following replication. Mutations that eliminate these two GATC sites but do not substantially change the activity of the promoter were introduced into a reporter gene fusion and into the chromosome. The chromosomal mutants are able to initiate DNA replication synchronously at both moderately slow and fast growth rates, demonstrating that GATC methylation at these two sites is not directly involved in providing the necessary amount of DnaA for precise timing of initiation during the cell cycle. Either sequestration does not involve these GATC sites, or cell cycle control of DnaA expression is not required to supply the concentration necessary for correct timing of initiation.

Chromosomal insertions localized around oriC affect the cell cycle in Escherichia coli.

The present work reports the effects of localized insertions around the origin of Escherichia coli chromosome, oriC, on cell cycle parameters. These insertions cause an increase of the C period with an inverse correlation to the distance from oriC. In addition, Omega insertion near oriC causes an increase in the number of replication forks per chromosome, n, and Tn10 insertion causes a decrease in growth rate. We found that the same insertion positioned in another region of the chromosome, outside of oriC, has a negligible effect on the C period. Marker frequency analysis suggests a slower replication velocity along the whole chromosome. We propose that the insertions positioned at less than 2 kbp from oriC could create a structural alteration in the origin of replication that would result in a longer C period. Flow cytometry reveals that asynchrony is not associated with these alterations.

Determining the optimal thymidine concentration for growing Thy- Escherichia coli strains.

Changes of thymidine concentration in the growth medium affect the chromosome replication time of Thy- strains without at the same time causing a detectable difference in the growth rate (R. H. Pritchard and A. Zaritsky, Nature 226:126-131, 1970). Consequently, the optimal thymidine concentration cannot be determined by ascertaining which concentration produces the highest growth rate. Here we present a method for determining the optimal thymidine concentration of any Thy- Escherichia coli strain. Using this method, we found that the E. coli "wild-type" strain MG1655 has a partial Thy- phenotype.

A calcium-binding protein that may be required for the initiation of chromosome replication in Escherichia coli.

Starvation for isoleucine but not for other amino acids in an ilv- strain or the addition of valine in an ilv+ strain inhibits initiation of chromosome and minichromosome replication in stringent (Rel+) Escherichia coli, but it does not inhibit replication in relaxed (relA) mutants (Guzman et al, 1988). From these results, we concluded that, (1) oriC initiation of replication is inhibited by ppGpp, and (2) isoleucine is not needed for the protein synthesis required at initiation. These results led us to find an isoleucine-free protein whose de novo synthesis is the sole protein synthesis requirement for oriC initiation. We also present evidence that this protein may be a calcium-binding protein located at 73 min in the genetic map.

Direct procedure for the determination of the number of replication forks and the reinitiation fraction in bacteria.

A direct method for calculating the average number of replication forks per chromosome in an exponentially growing bacterial culture and the fraction of reinitiation after an inductive treatment of the initiation step is presented. This method has allowed the development of REPLICON, a computer program designed for the resolution of the algorithm and simulation of the bacterial chromosome replication. Using REPLICON the following parameters can be obtained: average number of replication forks per chromosome, time required for the complete replication cycle, average amount of DNA per nucleotide, gene frequency of any chromosomal locus and reinitiation fraction. The use of this analysis also permits the determination of the uni- or bidirectionality of replication.

Heat stress in the presence of low RNA polymerase activity increases chromosome copy number of Escherichia coli.

A temperature shift-up accompanied by a reduction in RNA polymerase activity in Escherichia coli causes an increased rate of initiation leading to a 1.7- to 2.2-fold increase in chromosome copy number. A temperature shift-up without a reduction in polymerase activity induces only a transient non-scheduled initiation of chromosome replication caused by heat shock with no detectable effect on chromosome copy number.