ABSTRACT2fTGH is a human cell line containing the selectable marker guanine phosphoribosyltransferase regulated by a interferon (IFN-a). Two IFN-a-unresponsive mutants were isolated previously at a low frequency (ca. 10-8) by selecting mutagenized 2fTGH cells in selective medium containing 6-thioguanine and IFN-a. By using five rounds of mutagenesis, mutants can be isolated at an appreciably higher frequency, >3 x 10-7. Five new mutants have been isolated, and all are recessive, as are the two mutants we described previously. The seven mutants are in four complementation groups (U1-U4). Since several different types of mutants unresponsive to IFN-a have been isolated with high frequency, related approaches may succeed with other cytokines or growth factors. Mutants in the two new complementation groups U3 and U4 are unresponsive to IFN-a and, surprisingly, also unresponsive to IFN-y. They are also partially defective in response to double-stranded RNA. These results indicate that the signaling pathways for the two types of IFN and doublestranded RNA share common components or that their function depends on common enzymes or transcription factors. IFN receptors are unaffected in mutants U3A and U4A. A major defect appears to be in the synthesis or activation of E, the transcription factor mediating the primary response to type I (a/a) [FNs. Band-shift complementation assays show that U3A contains the Ey subunit but does not contain an active Ea subunit after treatment with IFN-a.

Risk of coronary artery disease (CAD) is determined by a combination of genetic and environmental factors which influence plasma lipid homeostasis, haemostasis and inflammation. Peroxisome proliferator activated receptor alpha (PPARa) is a ligand-inducible transcription factor [1] which regulates the expression of genes involved in fatty acid oxidation, extracellular lipid metabolism, haemostasis [2] and inflammation [3]. Ligands for PPARa include long chain fatty acids, eicosanoids, peroxisome proliferators, non-steroidal anti-inflammatory drugs and the fibrate class of hypolipidaemic drugs [4±6]. PPARa is highly expressed in tissues with a high rate of fatty Diabetologia (2000) Methods. The human PPARa gene was isolated and screened for variation by single strand conformation polymorphism analysis. Genotypes were determined for 129 Type II diabetic subjects and 2508 healthy men. The association with plasma lipid concentrations was examined. The function of the V162 variant was examined in co-transfection assays.Results. We identified two polymorphisms, one in intron 3 and a missense mutation, leucine 162 to valine, in the DNA binding domain. In Type II diabetic patients, V162 allele carriers had higher total cholesterol, HDL cholesterol and apoAI whereas intron 3 rare allele carriers had higher apoAI concentrations. By contrast, no effect was observed in healthy rare allele carriers. In vitro, the V162 variant showed greater transactivation of a reporter gene construct. Conclusion/interpretation. Naturally occurring variation alters PPARa function, influencing plasma lipid concentrations in Type II diabetic patients but not healthy people. This demonstrates that PPARa is a link between diabetes and dyslipidaemia, and so could influence the risk of coronary artery disease, the greatest cause of morbidity and mortality in Type II diabetes. [Diabetologia (2000) 43: 673±680]

Peroxisome proliferator-activated receptor alpha (PPARalpha) regulates genes responsible for skeletal and heart muscle fatty acid oxidation. Previous studies have shown that the PPARalpha intron 7 G/C polymorphism was associated with left ventricular growth in response to exercise. We speculated that GG homozygotes should be more prevalent within a group of endurance-oriented athletes, have normal fatty acid metabolism, and increased percentages of slow-twitch fibers. We have tested this hypothesis in the study of a mixed cohort of 786 Russian athletes in 13 different sporting disciplines prospectively stratified by performance (endurance-oriented athletes, power-oriented athletes and athletes with mixed endurance/power activity). PPARalpha intron 7 genotype and allele frequencies were compared to 1,242 controls. We found an increasing linear trend of C allele with increasing anaerobic component of physical performance (P=0.029). GG genotype frequencies in endurance-oriented and power-oriented athletes were 80.3 and 50.6%, respectively, and were significantly (P<0.0001) different compared to controls (70.0%). To examine the association between PPARalpha gene variant and fiber type composition, muscle biopsies from m. vastus lateralis were obtained and analyzed in 40 young men. GG homozygotes (n=25) had significantly (P=0.003) higher percentages of slow-twitch fibers (55.5+/-2.0 vs 38.5+/-2.3%) than CC homozygotes (n=4). In conclusion, PPARalpha intron 7 G/C polymorphism was associated with physical performance in Russian athletes, and this may be explained, in part, by the association between PPARalpha genotype and muscle fiber type composition.

Background-Left ventricular hypertrophy (LVH) occurs as an adaptive response to a physiological (such as exercise) or pathological (valvular disease, hypertension, or obesity) increase in cardiac work. The molecular mechanisms regulating the LVH response are poorly understood. However, inherited defects in fatty acid oxidation are known to cause severe early-onset cardiac hypertrophy. Peroxisome proliferator-activated receptor ␣ (PPAR␣) regulates genes responsible for myocardial fatty acid oxidation and is downregulated during cardiac hypertrophy, concomitant with the switch from fatty acid to glucose utilization. Methods and Results-The role of PPAR␣ in left ventricular growth was investigated in 144 young male British Army recruits undergoing a 10-week physical training program and in 1148 men and women participating in the echocardiographic substudy of the Third Monitoring Trends and Determinants in Cardiovascular Disease (MONICA) Augsburg study. A G/C polymorphism in intron 7 of the PPAR␣ gene significantly influenced left ventricular (LV) growth in response to exercise (Pϭ0.009). LV mass increased by 6.7Ϯ1.5 g in G allele homozygotes but was significantly greater in heterozygotes for the C allele (11.8Ϯ1.9 g) and in CC homozygotes (19.4Ϯ4.2 g). Likewise, C allele homozygotes had significantly higher LV mass, which was greater still in hypertensive subjects, and a higher prevalence of LVH in the Third MONICA Augsburg study. Conclusions-We

Background-Peroxisome proliferator-activated receptor ␣ (PPAR␣) regulates the expression of genes involved in lipid metabolism and inflammation, making it a candidate gene for atherosclerosis and ischemic heart disease (IHD). Methods and Results-We investigated the association between the leucine 162 to valine (L162V) polymorphism and a G to C transversion in intron 7 of the PPAR␣ gene and progression of atherosclerosis in the Lopid Coronary Angiography Trial (LOCAT), a trial examining the effect of gemfibrozil treatment on progression of atherosclerosis after bypass surgery and on risk of IHD in the second Northwick Park Heart Study (NPHS2), a prospective study of healthy middle-aged men in the United Kingdom. There was no association with plasma lipid concentrations in either study. Both polymorphisms influenced progression of atherosclerosis and risk of IHD. V162 allele carriers had less progression of diffuse atherosclerosis than did L162 allele homozygotes with a similar trend for focal atherosclerosis. Intron 7 C allele carriers had greater progression of atherosclerosis than did G allele homozygotes. The V162 allele attenuated the proatherosclerotic effect of the intron 7 C allele. Homozygotes for the intron 7 C allele had increased risk of IHD, an effect modulated by the L162V polymorphism Conclusions-The PPAR␣ gene affects progression of atherosclerosis and risk of IHD. Absence of association with plasma lipid concentrations suggests that PPAR␣ affects atherosclerotic progression directly in the vessel wall. (Circulation.

DNA clones encoding the 3 mouse neurofilament (NF) genes have been isolated by cross-hybridization with a previously described NF-L cDNA probe from the rat. Screening of a 2gtl0 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the entire coding region of 541 amino acids and of an NF-M cDNA that covers 219 amino acids from the internal a-helical region and the carboxy-terminal domains of the protein. These cDNA clones were used as probes to screen mouse genomic libraries, and cosmid clones containing both NF-L and NF-M sequences were isolated as well as overlapping cosmids containing the NF-H gene. This strongly suggests that the 3 neurofilament genes are organised in a cluster and derived by gene duplication of a common ancestral gene. RNA blot analyses using specific DNA probes for each of the genes indicate that NF mRNAs are differentially expressed during brain development. The NF-L and NF-M mRNAs are detected early in the embryonal brain, with a progressive increase in their levels during development, while the NF-H mRNA is barely detectable at embryonal stages and accumulates later in the postnatal brain.

Previously we described human cell line 2fTGH, in which expression of guanine phosphoribosyltransferase is tightly controlied by the upstream region of interferon (IFN)-stimulated human gene 6-16. After mutagenesis of 2fTGH and selection with 6-thioguanine and IFN-a, we isolated 11,1, a recessive mutant that does not respond to IFN-at. We now describe U2, a second recessive mutant, selected similarly, that complements 11,1. U2 had no response to IFN-a or IFN-,, and its response to IFN--y was partially defective. Although many genes did respond to IFN-,y in U2, the 9-27 gene did not and the antiviral response of U2 cells to IFN--y was greatly reduced. Band shift assays showed that none of the transcription factors normally induced in 2fTGH cells by IFN-a (E and M) or IFN--y (G) were induced in U2. However, extracts of untreated U2 cells gave rise to a novel band that was increased by treatment with IFN--y but not IFN-ca. Band shift complementation assays revealed that untreated and IFN-y-treated U2 cells lack the functional E-y subunit of transcription factor E and that IFN-aL-treated U2 cells do contain the functional Ea subunit.Pathways through which extracellular proteins such as cytokines and growth factors induce expression of mammalian genes are under intensive study in many laboratories, but none has been defined fully. In the case of alpha interferon (IFN-a)

Expression of the E1A oncogene of adenovirus type 5 inhibits the response of interferon (IFN)-inducible constructs to Type I (alpha,beta) and II (gamma) IFNs in transient transfection assays. In human cell lines stably expressing E1A mRNA and protein acquisition of an antiviral state and the induction of a number of genes in response to alpha- and gamma-IFNs is inhibited. A short IFN-stimulable response element (ISRE) present in the 5' flanking region of a number of genes mediates induction by alpha- and gamma-IFNs. In cells expressing E1A there is a substantial reduction in the levels of the ISRE-binding factors E and M, inducible by alpha-IFN, and of factor G, inducible by gamma-IFN. In E1A-expressing cells the E alpha subunit of factor E is activated normally in response to alpha-IFN; the defect is in the production or activation of the E gamma subunit. The inhibitory activity of E1A is lost upon deletion of the CR1 domain. The induction of HLA class II genes by gamma-IFN, which involves a different DNA response element(s), and of beta-IFN mRNA in response to double-stranded RNA are also inhibited by E1A. An essential component(s) of a number of signalling pathways must, therefore, be subject, directly or indirectly, to inhibition by E1A.