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.

Summary Background : Azathioprine is a useful agent in the management of inflammatory bowel disease. Its use is limited by its side‐effect profile. Marrow toxicity occurs in approximately 3.2% of patients and is known to be associated with diminished thiopurine methyltransferase enzyme activity resulting from genetic polymorphisms. Aim : To evaluate the cost‐effectiveness of screening for thiopurine methyltransferase gene polymorphisms prior to initiation of azathioprine therapy. Methods : Analysis of the literature was undertaken to calculate the expected frequency of leucopenia and its relationship with thiopurine methyltransferase polymorphisms in a model of theoretical inflammatory bowel disease patients. Decision analysis was then applied to assess the cost of a pre‐treatment genotyping strategy, taking account of direct costs and cost per life‐year saved. Results : In 1000 inflammatory bowel disease patients treated with azathioprine, 32 will develop myelosuppression and one will die because of this. Of those who develop myelosuppression during azathioprine therapy, 32% are attributable to lower thiopurine methyltransferase activity. Pre‐treatment genotyping costs £347 per life‐year saved for a 30 year old and £817 per life‐year saved for a 60 year old. This compares favourably with other health care technologies. Conclusion : The use of pre‐treatment screening for thiopurine methyltransferase polymorphisms in inflammatory bowel disease patients commencing azathioprine therapy represents good value for money.

In a survey of a healthy population (n = 197), LDL cholesterol, plasma triglycerides and VLDL triglycerides were found to be substantially increased and plasma HDL cholesterol decreased in smokers. The lipid-associated atherogenic risk in smokers as assessed by the LDL/HDL ratio was significantly higher [2.89 (SD 1.18, n = 63)] than in non-smokers [2.38 (SD 0.98, n = 86) P < 0.01]. The lower HDL level found in smokers was explained by a lower HDL-2 subfraction as determined by analytical ultracentrifugation. HDL 2b, 2a and 3a, measured by gradient gel electrophoresis, were all lower in the smokers but this was only significant for HDL 2a. Smoking had no effect on Lp(a) levels. HDL cholesterol and HDL-2 were strongly negatively correlated whereas LDL cholesterol and LDL/HDL ratio were strongly positively correlated with the plasma triglyceride concentration. There was a small but significant reduction in plasma CETP activity [non-smokers 49% t/microliter (SD 17, n = 90), smokers 43% t/microliter (SD 17, n = 66) P < 0.05] but CETP activity was not correlated with any measure of HDL in this population. Smoking was found to be an important independent contributor to the variation in plasma triglyceride, HDL, HDL-2 and LDL/HDL ratio. After correcting for sex, age, BMI, alcohol consumption, oral contraceptive use and plasma triglycerides smoking was still found to be significantly associated with HDL and the LDL/HDL ratio. Upon adjustment for covariant factors the mean differences between smokers and non-smokers for HDL cholesterol, HDL-2 and LDL/HDL were 0.15 mM, 16 mg dl-1 and 0.39 respectively. There appeared to be important sex differences in the influence of smoking on plasma lipoproteins. In women the main impact of smoking was on triglyceride levels and they in turn affected LDL and HDL. In contrast, in men, smoking had little impact on triglycerides and affected HDL more directly. We conclude that smoking cigarettes has an important effect on plasma lipoprotein metabolism through multiple mechanisms.

The apoB arginine-to glutamine change at codon 3500 has become established as a cause of failure of binding of the LDL particle to its receptor and the consequent hypercholesterolemia of familial defective apoB 100. A search for further similar mutations was undertaken by systematic screening of a candidate region of the apoB gene from individuals with hypercholesterolemia. Polymerase chain reaction and denaturing gradient gel electrophoresis were used. We describe two families in which a different mutation in the codon 3500 causes an arginine-to-tryptophan substitution. Most adults in these families who have this mutation have hypercholesterolemia. LDL derived from all who have inherited the mutation is dysfunctional in that it allows only poor growth of an LDL cholesterol-dependent cell line. We conclude that this arginine 3500 is essential to the function of apoB and that its loss and replacement by glutamine or tryptophan is responsible for the hypercholesterolemia of familial defective apoB 100.