Our previous DNA sequence comparisons of 3' terminal portions from equivalent herpes simplex virus type 1 (HSV-1) and HSV-2 genes identified a conserved sequence (consensus YGTGTTYY; Y = pyrimidine) located approximately 30bp downstream from the AATAAA signal. We report here that this signal is located downstream from 67% of the mammalian mRNA 3' termini examined. Using constructions with the bacterial chloramphenicol acetyl transferase (CAT) gene linked to an HSV 'terminator' fragment, we show that deletions in the 'terminator' reduce CAT activities and the levels of CAT mRNA 3' termini. Specifically: (1) deletions of downstream sequences which extend up to the consensus YGTGTTYY signal reduce CAT levels to values 35% of those obtained with undeleted plasmids, (2) a deletion of a further 14bp, which removes the YGTGTTYY consensus but not the poly A site, reduces CAT activities to 1%-4%. The levels of CAT mRNA 3' termini reflect the reductions in CAT activities however, levels of mRNA 5' termini are unaffected by these deletions. The RNA produced in the absence of the YGTGTTYY signal is present in the cytoplasm although no CAT activity is detectable.

This study investigated in a healthy population (n=220) the association of the Taql B restriction fragment length polymorphism (RFLP) in the cholesteryl ester transfer protein (CETP) gene with plasma high-density lipoprotein (HDL) cholesterol concentration and subfraction distribution. A raised HDL cholesterol level was found in B2B2 homozygotes (B2 cutting site absent) and was associated specifically with a 45% increase in HDL 2 compared with B1B1 homozygotes (B1B1, 77±39 mg/100 mL, mean±SD; B2B2, 112±59 mg/100 mL; / > <0.01). Total plasma, very-low-density lipoprotein, and HDL triglyceride levels did not differ among the genotype groups, nor did plasma apolipoprotein AI levels {B1B1, 1.45+0.35 mg/mL, mean±SD; B2B2, 1.56±0.33 mg/ mL). Thus, the genetic variation appeared to be independent of metabolic factors that are known to regulate HDL levels. Plasma CETP exchange activity was unlikely to be the cause of L ow plasma levels of high-density lipoprotein (HDL) are associated with increased coronary artery disease risk.1 -2 In addition, it has been found that clinical benefit is associated with a rise in HDL concentration in intervention trials. The Helsinki Heart Study 3 showed that a mean increase of 11% in HDL cholesterol levels was associated with a 34% reduction in coronary heart disease even after correction for other risk factors, including low-density lipoprotein (LDL) cholesterol and plasma triglyceride levels.Plasma HDL is composed of two main subfractions, HDL 2 (1.063

A method to isolate fragments of DNA that promote gene expression in Bacillus subtilis is described. The system is based on production ofcatechol 2,3-dioxygenase [CatO2ase; catechol:oxygen 2,3-oxidoreductase (decyclizing), EC 1. 13 Bacillus subtilis is an attractive alternative to Escherichia coli as a host for expression of cloned genes. The Gram-positive organism is nonpathogenic, free ofendotoxins, and an important producer of extracellular enzymes on a large industrial scale. Critical to the development ofthe microorganism as a host-vector system for recombinant DNA technology is the efficient expression of heterospecific genes. To express plasmid-borne genes in B. subtilis, transcriptional or translational signals that differ from those of E. coli are required (1). Plasmid vectors suitable for cloning fragments of DNA that carry transcriptional promoter or termination signals for Gram-negative bacteria into E. coli have been characterized (2-6). Detection in these systems is based on expression ofgenes that encode P-galactosidase (4-6) or confer antibiotic resistance to host cells (2, 3). An approach similar to the latter has been successful in B. subtilis using chloramphenicol acetyltransferase genes originating from Bacillus pumilus (7) or the transposable genetic element Tn9 (8). In the E. coli /-galactosidase system, selection ofDNA fragments that promote expression of the lacZ gene is based on an easily visualized color change ofbacterial colonies grown on indicator plates containing a chromogenic substrate (4). An analogous system that functions in B. subtilis would greatly facilitate the effort to decipher problems of heterospecific gene expression in Gram-positive bacteria.In this report, we present a method whereby fragments of DNA that promote expression of a foreign gene in B. subtilis are detected by a change ofcolor of bacterial colonies. The system is based on the cloning and expression, in B. subtilis, ofthe xylE gene, which originated from the TOL plasmid pWWO (9) of Pseudomonas putida mt-2. The assay is rapid and inexpensive, does not require special indicator plates but offers the advantages ofa genetic indicator test (10), and can be used for the development of efficient plasmid gene expression vectors. MATERIAL AND METHODSBacterial Strains and Plasmids. The B. subtilis strains used are derivatives of Marburg strain 168. Strains BZ2 cysB3 recE4 and TGB1 trpC2 recE4 spo331 were constructed by transformation (11). MI112 argl5 leuB thr5 r-mM recE4 was from T. Tanaka. Bacillus licheniformis 9945A and Bacillus pumilus BP1 were obtained from the Bacillus Genetic Stock Center (Ohio State University, Columbus). E. coli strain BZ18 was from W. Arber; C600 rj m' was from J. W. Little; Pseudomonas putida mt-2 was donated by K. Timmis. The bifunctional E. coli/B. subtilis plasmid pHV33 (12) was obtained from R. Dedonder. Plasmid DNA was prepared by an alkaline extraction procedure (13) or a cleared lysate method (14) followed by cesium chloride/ ethidium bromide density gradient centrifugati...

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A common CRP gene polymorphism is associated with important differences in CRP concentrations, free from confounding. The null association of this variant with coronary events suggests possible residual confounding (or reverse causation) in the CRP-coronary event association in observational studies, though the confidence limits are still compatible with a modest causal effect. Additional studies of genotype (or haplotype) and coronary events would help clarify whether or not the link between CRP and coronary events in observational studies is causal.