A HindIII polymorphism of fibronectin gene is associated with nephrolithiasis.

OBJECTIVES: To evaluate the association between fibronectin gene (FN1) polymorphisms and calcium oxalate nephrolithiasis as a genetic risk factor. METHODS: Genomic DNA of 143 patients with calcium oxalate nephrolithiasis and 154 healthy controls were screened for polymorphisms (HaeIII b, MspI, and HindIII) of the FN1 gene, using polymerase chain reaction-restriction fragments length polymorphism method. Allele and genotype frequencies were compared between the groups. RESULTS: Although the observed differences between distribution of genotypes of AA, AB, and BB (for HaeIII b), as well as CC, CD, and DD (MspI) were not significant, FF genotype for HindIII showed significant difference when compared with both EF and EE + EF genotype (P = .00202 and P = .00203, respectively). CONCLUSIONS: The results of our study revealed that HindIII polymorphism of the FN1 gene is highly associated with calcium oxalate stone disease. This association makes FN a good candidate for further studies about the etiology of stone disease, and in the future it could be a candidate marker for evaluating the genetic risks in patients with nephrolithiasis.

Lipoprotein lipase HindIII polymorphism influences HDL-cholesterol levels in statin-treated patients with coronary artery disease.

BACKGROUND: HDL-cholesterol (HDL-C) is a recognized athero-protective factor and low levels of HDL-C occur frequently in patients with coronary artery disease. Regulation of HDL-C level most probably results from the interaction of genes involved in lipoprotein metabolism and also from non-genetic factors. We studied associations and interactions among HindIII polymorphisms of the lipoprotein lipase gene LPL and selected non-genetic factors with respect to HDL-C levels in patients with coronary artery disease. PATIENTS AND METHODS: 288 Slovak patients (35% women) with documented coronary artery disease, age (mean +/- SEM) 60 +/- 1 years and BMI 29 +/- 0.3 kg/m(2), were examined and genotyped for LPL HindIII (rs320) using a PCR/RFLP method. HDL-C levels were determined in a direct enzymatic assay. RESULTS: In the sample overall there were no significant differences across the LPL genotypes in adjusted HDL-C levels or in other lipids, although a trend toward higher HDL-C and lower triglycerides in H-H- homozygotes was observed. Multiple linear regression identified a significant interaction between LPL HindIII and statin treatment, which together with sex and diabetes explained 12.1% of HDL-C variance. Accordingly, in statin-treated patients we observed significant stepwise increments of the HDL-C level related to the increasing number of H- alleles (P = 0.04 for linear trend), whereas no such association was observed in patients without hypolipidemic treatment. H-H- homozygotes had a 16% (0.19 mmol/l) higher level of HDL-C than the H+H+ homozygotes (P = 0.06). CONCLUSION: HDL-C may be influenced by an interaction between statin treatment and LPL HindIII genotype. However, the effect of this interaction appears to be small when compared with the effect of non-genetic factors. This finding requires replication in a pharmacogenetic study.

[Polymorphic Hind III restriction site of the Y chromosome and essential hypertension in Chinese Han People].

OBJECTIVE: To examine whether there is an association essential hypertension pressure and a polymorphic Hind III biallelic marker in the non-recombining region of Y chromosome in Chinese Han people. METHODS: Peripheral blood samples were collected from 402 males with essential hypertension pressure and 455 age- and body height-matched healthy males as control group. Genomic DNA was extracted from the white blood cells. Segments of polymorphic Hind III restriction site of the Y chromosome were amplified from the genomic DNA by polymerase chain reaction (PCR). The PCR products were restricted with 10 U of Hind III overnight at 37 degrees C. The digested products were subjected to electrophoresis in 3% agarose gels, and stained with ethidium bromide. RESULTS: The Hind III (+) genotype was found in 58.5% of the men with essential hypertension (235/402), significantly lower than that in the healthy men (64.4%, 302/455, P = 0.02). The systolic blood pressure of the men with Hind III (+) genotype was 133.8 mm Hg +/- 25.2 mm Hg, significantly lower than that of the Hind III (-) genotype (138.0 mm Hg +/- 27.0 mm Hg, P < 0.05), and the diastolic blood pressure (DBP) of the men with Hind III (+) genotype was 83.5 mm Hg +/- 13.3 mm Hg, significantly lower than that of the men with Hind III (-) genotype (85.9 mm Hg +/- 14.4 mm Hg, P = 0.01), and the mean arterial pressure of the men with Hind III (+) genotype was 100.2 mm Hg +/- 16.5 mm Hg, significantly lower than that of the of the men with Hind III (+) genotype was (103.3 mm Hg +/- 17.6 mm Hg, P = 0.01). CONCLUSION: Polymorphic Hind III restriction site of the Y chromosome is associated with essential hypertension in Chinese Han people.

Association of the human Y chromosome with cholesterol levels in the general population.

OBJECTIVE: Males are at higher risk of cardiovascular diseases than females. The aim of the study was to test whether the potential of the Y chromosome to affect cardiovascular risk could be attributed to its influence on lipids. METHODS AND RESULTS: 1288 Polish men (1157 subjects from young healthy cohort and 131 individuals from middle-aged hypertensive population) were phenotyped for determinants of cardiovascular risk including BMI, blood pressures, lipids, and testosterone. Each subject was genotyped for the HindIII(+/-) polymorphism within the nonrecombining region of the Y chromosome. Men with the HindIII(-) variant exhibited significantly higher total cholesterol (TC) and low-density lipoprotein cholesterol (LDL) levels than subjects with the HindIII(+) genotype in both populations. The differences between the genotypes were 0.15 mmol/L (P=0.0107) and 0.45 mmol/L (P=0.0377) in TC and 0.15 mmol/L (P=0.0059) and 0.41 mmol/L (P=0.0432) in LDL among young apparently healthy men and middle-aged hypertensive men, respectively. The HindIII(+) was associated with a significant increase in blood pressure of the middle-aged men. Testosterone serum concentrations correlated positively with HDL-cholesterol levels, and this association was independent of the Y chromosome. CONCLUSIONS: The results indicate that a locus/loci on the Y chromosome may influence LDL levels, independent of testosterone levels.

Genetic organization and molecular analysis of the EcoVIII restriction-modification system of Escherichia coli E1585-68 and its comparison with isospecific homologs.

The EcoVIII restriction-modification (R-M) system is carried by the Escherichia coli E1585-68 natural plasmid pEC156 (4,312 bp). The two genes were cloned and characterized. The G+C content of the EcoVIII R-M system is 36.1%, which is significantly lower than the average G+C content of either plasmid pEC156 (43.6%) or E. coli genomic DNA (50.8%). The difference suggests that there is a possibility that the EcoVIII R-M system was recently acquired by the genome. The 921-bp EcoVIII endonuclease (R. EcoVIII) gene (ecoVIIIR) encodes a 307-amino-acid protein with an M(r) of 35,554. The convergently oriented EcoVIII methyltransferase (M. EcoVIII) gene (ecoVIIIM) consists of 912 bp that code for a 304-amino-acid protein with an M(r) of 33,930. The exact positions of the start codon AUG were determined by protein microsequencing. Both enzymes recognize the specific palindromic sequence 5'-AAGCTT-3'. Preparations of EcoVIII R-M enzymes purified to homogeneity were characterized. R. EcoVIII acts as a dimer and cleaves a specific sequence between two adenine residues, leaving 4-nucleotide 5' protruding ends. M. EcoVIII functions as a monomer and modifies the first adenine residue at the 5' end of the specific sequence to N(6)-methyladenine. These enzymes are thus functionally identical to the corresponding enzymes of the HindIII (Haemophilus influenzae Rd) and LlaCI (Lactococcus lactis subsp. cremoris W15) R-M systems. This finding is reflected by the levels of homology of M. EcoVIII with M. HindIII and M. LlaCI at the amino acid sequence level (50 and 62%, respectively) and by the presence of nine sequence motifs conserved among m(6) N-adenine beta-class methyltransferases. The deduced amino acid sequence of R. EcoVIII shows weak homology with its two isoschizomers, R. HindIII (26%) and R. LlaCI (17%). A catalytic sequence motif characteristic of restriction endonucleases was found in the primary structure of R. EcoVIII (D(108)X(12)DXK(123)), as well as in the primary structures of R. LlaCI and R. HindIII. Polyclonal antibodies raised against R. EcoVIII did not react with R. HindIII, while anti-M. EcoVIII antibodies cross-reacted with M. LlaCI but not with M. HindIII. R. EcoVIII requires Mg(II) ions for phosphodiester bond cleavage. We found that the same ions are strong inhibitors of the M. EcoVIII enzyme. The biological implications of this finding are discussed.

Lack of association of lipoprotein lipase gene polymorphisms with coronary artery disease in the Saudi Arab population.

CONTEXT: Previous studies reported an association of certain polymorphisms in the lipoprotein lipase (LPL) gene with the risk of coronary artery disease (CAD); however, these studies were small and inconsistent. In addition, none of these studies attempted to establish such an association in the Arab population. OBJECTIVE: To determine whether 2 LPL polymorphisms (LPL-HindIII and LPL-PvuII located on introns 8 and 6, respectively, of the LPL gene) can be considered as independent risk factors or as predictors for CAD in Arabs. DESIGN: We used polymerase chain reaction and restriction enzyme digestion to determine the distribution of the LPL-HindIII and LPL-PvuII polymorphisms among healthy blood donors of Arabic origin (BD group) and angiographically confirmed CAD patients (CAD group) with identical ethnic backgrounds. RESULTS: For the HindIII genotypes, within the BD group (n = 410), the +/+ genotype was found in 206 individuals (50.2%), 173 (42.2%) carried the +/- genotype, and 31 (7.6%) carried the -/- genotype. Within the CAD group (n = 352), the +/+ genotype was found in 189 individuals (53.7%), 138 (39.2%) carried the +/- genotype, and 25 (7.1%) carried the -/- genotype. P values of.38,.45, and.92 were obtained for the +/+, +/-, and -/- genotypes, respectively. For the PvuII genotypes, within the BD group (n = 511), the +/+ genotype was found in 182 individuals (35.6%), 248 (48.5%) carried the +/- genotype, and 81 (15.9%) carried the -/- genotype. Within the CAD group (n = 431), the +/+ genotype was found in 138 individuals (32%), 225 (52.2%) carried the +/- genotype, and 68 (15.8%) carried the -/- genotype. P values of.28,.29, and.98 were obtained for the +/+, +/-, and -/- genotypes, respectively. The distribution and the allele frequency of these 2 LPL variants were similar in CAD and BD study groups and followed the Hardy-Weinberg equilibrium. CONCLUSION: There was no difference in the distribution of both LPL polymorphisms between the healthy group and the CAD group. Therefore, these 2 LPL polymorphisms cannot be considered as independent risk factors or as predictors for CAD in this population.

Associations of LPL and APOC3 gene polymorphisms on plasma lipids in a Mediterranean population: interaction with tobacco smoking and the APOE locus.

We conducted a cross-sectional study in a Spanish population (n = 1,029) to investigate associations between the LPL and APOC3 gene loci (LPL-HindIII, LPL-S447X, and APOC3-SstI) and plasma lipid levels and their interaction with APOE polymorphisms and smoking. Carriers of the H(-) or the X447 allele had higher levels of HDL cholesterol (HDL-C), and lower levels of TG, after adjustment for age, body mass index, alcohol, smoking, exercise, and education (P < 0.01). The APOC3 polymorphism presented additive effects to the LPL variants on TG and HDL-C levels in men, and on TG in women. The most and the least favorable haplotype combinations were H(-)/X447/S1 and H(+)/S447/S2, respectively. These combinations accounted for 7% and 5% of the variation in HDL-C and TG in men, and 3% and 4% in women. There was a significant interaction between APOE and LPL variants and HDL-C levels in both genders (P < 0.05). The increases in HDL-C observed for the rare alleles were higher in epsilon4 than in epsilon3 subjects, and absent in epsilon2 individuals. This effect was modulated by smoking (interaction HindIII-APOE-smoking, P = 0.019), indicating that smoking abolished the increase in HDL-C levels observed in epsilon4/H(-) subjects. Understanding this gene-gene-environmental interaction may facilitate preventive interventions to reduce coronary artery disease risk.

Evaluation of ribotyping for characterization and identification of Enterococcus haemoperoxidus and Enterococcus moraviensis strains.

Seven Enterococcus moraviensis and 16 Enterococcus haemoperoxidus as well as nine reference cultures of other enterococcal species obtained from the Czech Collection of Microorganisms were characterized using ribotyping with EcoRI and HindIII in the present work. The ribopatterns obtained by both restriction enzymes clearly distinguished all E. moraviensis and E. haemoperoxidus strains from the other enterococci (E. faecalis, E. faecium, E. avium, E. raffinosus, E. pseudoavium, E. malodoratus) and they differentiated both species from each other as well. Although all strains were isolated from different sampling sites, many strains shared the same band patterns. E. moraviensis formed four ribogroups using EcoRI and two ribogroups using HindIII restriction enzyme. E. haemoperoxidus gave six different patterns with EcoRI and five using the HindIII restriction enzyme.

The transposon A(R)4-24P[white, rosy] in Drosophila melanogaster is subject to position-effect variegation at a non-centromeric insertion site.

The white gene within the transposon A(R)4-24P[white,rosy] inserted at cytological location 24D1-2 in the euchromatic portion of the Drosophila melanogaster genome exhibits a mosaic pattern of expression which is modified by temperature and Y-chromosome number, as in cases of classical position-effect variegation (PEV). The eye colour of the flies in this variegated stock remains mosaic in the presence of the PEV modifier Su(var)3-6, slightly less so with Su(var)3-9 and Su(var)2-5, and full suppression of variegation occurs in the presence of Su(var)3-7. We have induced further transposition of A(R)4-24 and isolated two mosaic stocks with this transgene at new cytological locations. In these stocks, the A(R)4-24 transposon was flanked by the same genomic DNA fragments as in the original location. Spontaneous loss of these fragments leads to reversion of the variegated eye colour to wild-type. We suggest that the flanking DNA fragments from 24D1-2 are capable of inducing position-effect variegation without any association with centromeric heterochromatin. In situ hybridisation and Southern analysis demonstrate that the 5' flanking genomic fragment contains repeated sequences which are abundantly present in heterochromatin.

Tissue-specific developmental expression of OAX, a Xenopus repetitive element.

Approximately 1% of the Xenopus laevis genome consists of highly repetitive DNA known alternatively as OAX (for Oocyte Activation in Xenopus), Satellite I, or Repetitive HindIII Monomer 2. Present as tandemly repeated units of approximately 750 base pairs, OAX encodes a family of small RNA species transcribed by RNA polymerase III. Although the subject of many of the classic studies on early embryonic gene regulation, reports on OAX expression remain contradictory and incomplete. Using whole-mount in situ hybridization and RNase protection assays, we have therefore examined in detail the expression pattern of OAX in Xenopus embryos of various stages. OAX is initially expressed during gastrula stages; by tailbud stages embryos display discrete zones of expression at the dorsal boundary of the cement gland, in the developing somites and differentiating skeletal muscle, as well as in the dorsal aspect of the neural tube. These data demonstrate that OAX is expressed in a dynamic pattern under tight spatial and temporal regulation.

Ribosomal DNA replication fork barrier and HOT1 recombination hot spot: shared sequences but independent activities.

In the ribosomal DNA of Saccharomyces cerevisiae, sequences in the nontranscribed spacer 3' of the 35S ribosomal RNA gene are important to the polar arrest of replication forks at a site called the replication fork barrier (RFB) and also to the cis-acting, mitotic hyperrecombination site called HOT1. We have found that the RFB and HOT1 activity share some but not all of their essential sequences. Many of the mutations that reduce HOT1 recombination also decrease or eliminate fork arrest at one of two closely spaced RFB sites, RFB1 and RFB2. A simple model for the juxtaposition of RFB and HOT1 sequences is that the breakage of strands in replication forks arrested at RFB stimulates recombination. Contrary to this model, we show here that HOT1-stimulated recombination does not require the arrest of forks at the RFB. Therefore, while HOT1 activity is independent of replication fork arrest, HOT1 and RFB require some common sequences, suggesting the existence of a common trans-acting factor(s).

Mutational analyses of restriction endonuclease-HindIII mutant E86K with higher activity and altered specificity.

We have performed mutational analyses of restriction endonuclease HindIII in order to identify the amino acid residues responsible for enzyme activity. Four of the seven HindIII mutants, which had His-tag sequences at the N-termini, were expressed in Escherichia coli, and purified to homogeneity. The His-tag sequence did not affect enzyme activity, whereas it hindered binding of the DNA probe in gel retardation assays. A mutant E86K in which Lys was substituted for Glu at residue 86 exhibited high endonuclease activity. Gel retardation assays showed high affinity of this mutant to the DNA probe. Surprisingly, in the presence of a transition metal, Mo(2+) or Mn(2+), the E86K mutant cleaved substrate DNA at a site other than HindIII. Substitution of Glu for Val at residue 106 (V106E), and Asn for Lys at residue 125 (K125N) resulted in a decrease in both endonucleolytic and DNA binding activities of the enzyme. Furthermore, substitution of Leu for Asp at residue 108 (D108L) abolished both HindIII endonuclease and DNA binding activities. CD spectra of the wild type and the two mutants, E86K and D108L, were similar to each other, suggesting that there was little change in conformation as a result of the mutations. These results account for the notion that Asp108 could be directly involved in HindIII catalytic function, and that the substitution at residue 86 may bring about new interactions between DNA and cations.

Frequency and allelic association of common variants in the lipoprotein lipase gene in different ethnic groups: the Wandsworth Heart and Stroke Study.

The lower serum triglyceride (Tg), higher high density cholesterol (HDL-C) levels and low coronary heart disease (CHD) mortality in black populations, contrast with that in whites. By comparison, South Asian populations display a higher mortality from CHD associated with increased Tg and low HDL-C levels. Lipoprotein lipase (LPL) plays a major role in Tg metabolism. To determine if variation in the LPL gene contributes to the differences in lipid levels, we studied the frequencies and allelic associations of five common variants in the lipoprotein lipase (LPL) gene (-93T/G, D9N, N291S, S447X, and the HinddIII RFLP in intron 8) with serum Tg and HDL-cholesterol concentrations in population samples of middle-aged men and women of whites, South Asians, and blacks of African origin co-resident in South London. Significantly higher frequencies of the H(-) (P < 0.00001), N9 (P < 0.001), and -93G (P < 10(-10)) alleles were seen in blacks compared to the other two groups. Allelic association between -93G and N9, and H(+) and X447 was strong in all three groups. However, no association was observed between serum Tg and HDL-cholesterol concentrations and these variants in the three ethnic groups. A single common polymorphism in the LPL gene is unlikely to account for the differences in fasting serum Tg in populations of different ethnic background. The importance of the differences in frequencies and the mechanism(s) whereby these may contribute towards a beneficial LPL genotype in black populations remain to be determined.

Promoter interference in a bacteriophage lambda control region: effects of a range of interpromoter distances.

The p(R) and p(RM) promoters of bacteriophage lambda direct transcription in divergent directions from start sites separated by 83 phosphodiester bonds. We had previously shown that the presence of an RNA polymerase at p(R) interfered with open complex formation at p(RM) and that this effect was alleviated by the deletion of 10 bp between the two promoters. Here we present a detailed characterization of the dependence of the interference on the interpromoter distance. It was found that the reduced interference between the two promoters is unique to the 10-bp deletion. The relief of interference was demonstrated to be due to the facilitation of a step subsequent to RNA polymerase binding to the p(RM) promoter. A model to explain these observations is proposed. A search of known Escherichia coli promoters identified three pairs of divergent promoters with similar separations to those investigated here.

Site-directed mutagenesis of restriction endonuclease HindIII.

Site-directed mutagenesis by inverse PCR was done on the HindIII gene. Target residues to be mutated were chosen according to (i) the fact that a mutant obtained by sodium nitrite treatment showed almost no HindIII activity, where Asp-123 was replaced with Asn, and (ii) the model proposed by Stahl et al. (Stahl, F., Wende, W., Jeltsch, A. and Pingoud, A. Biol. Chem. 379, 467-473 (1998)). Seven kinds of mutants were obtained by the PCR, and their enzymatic and biochemical properties were examined. Three mutants, P50S, D108L, and D123N, showed fairly low HindIII activity. On the other hand, the other four, P84Q, E86K, V106E, and K125N, retained the activity. In particular, E86K showed higher activity than the wild enzyme. This fact was confirmed when activities of the purified wild and E86K enzymes were assayed. These results coincided fairly well with data using E. coli strains that carry the respective mutant plasmids, on their resistance to phage T7 and on growth rate. We conclude that the PE motif at residues 50 and 51, and DXK motif at residues 108-110, are responsible for the enzymic reaction of HindIII.

Analysis of murine Snrpn and human SNRPN gene imprinting in transgenic mice.

The SNRPN gene is known to be expressed exclusively from the paternal allele and to map to the critical region for the neurobehavioral disorder, Prader-Willi syndrome (PWS). As a means to investigate the mechanism of imprinting for the SNRPN gene, we have sought to recapitulate the imprinted expression of the endogenous gene. Using an 85-kb murine Snrpn clone, containing 33 kb of 5' and 30 kb of 3' flanking DNA, we obtained two intact transgenic lines. One line, containing two copies of the Snrpn transgene, recapitulated the imprinted expression pattern of the endogenous locus, whereas the other transgenic line, containing a single copy, was expressed upon both maternal and paternal inheritance. This suggests that a 6.6-kb region of maternal-specific DNA methylation that we have identified may be sufficient to confer imprinted expression, but not in a copy-number independent manner. Finally, we produced five lines of transgenic mice using a 76-kb human SNRPN clone containing 45 kb and 7 kb of 5' and 3' flanking DNA, respectively. We found all the lines were expressed upon both maternal and paternal inheritance, regardless of copy number, suggesting that the imprinting machinery in mouse and human may have diverged.

Construction and characterization of a sheep BAC library of three genome equivalents.

A sheep BAC library of over three genome equivalents was constructed and arrayed in superpools and row, column, and plate pools. The library contains 90,000 clones distributed in 39 superpools. The average insert size was estimated at 123 kb. The library was screened by PCR with 77 primer pairs corresponding to ovine microsatellites distributed throughout the genome. The probability of finding a random sequence in the library could be estimated at 0.96.

Cytogenetic and molecular characterization of a highly repeated DNA sequence in the peach potato aphid Myzus persicae.

Electrophoresis following digestion of Myzus persicae genomic DNA with HindIII showed the presence of a prominent band of approximately 200 bp whereas a faint electrophoretic band corresponding to DNA fragments of about 3000 bp was observed after digestion with ApaI. In situ digestion with restriction enzymes, followed by in situ nick translation, showed that ApaI targets are localized at the nucleolus organizer-bearing X telomeric region, whereas HindIII restriction sites are clustered in intercalary C-positive areas on the same X chromosome. Fluorescent in situ hybridization (FISH) carried out by using digoxygenin-labeled HindIII repeats as probe fully confirmed overlapping between the hybridization sites of this probe and the AT-rich intercalary heterochromatic bands on the X chromosome. These findings, together with published data, allow us to conclude that the M. persicae genome possesses three classes of C-positive heterochromatin: (i) a GC-rich argentophilic band located on one telomere of the X chromosome that contains ApaI targets; (ii) AT-rich intercalary bands located on the X chromosome containing clustered HindIII fragments; (iii) AT-rich telomeric bands located on autosomes, consisting of HaeIII repeats. Molecular analysis has shown that the length of the HindIII repeat consensus sequence is 189 bp with an AT content of 67%. Southern blotting with HindIII monomers revealed a regular ladder of bands composed of multimers of basic length that are characteristic of satellite DNAs. The HindIII repeat displays other features typical of eukaryotic satellite arrays such as overlapping with heterochromatic bands and a high degree of sequence similarity among monomers (84%-94%). A similarity plot showed that sequences were particularly variable in the 50-100 bp region whereas they proved to be highly conservative in the first 50 bp, thus suggesting that this portion of the repeat might be functionally important.

Genotype-specific transcriptional regulation of PAI-1 gene by insulin, hypertriglyceridemic VLDL, and Lp(a) in transfected, cultured human endothelial cells.

Plasminogen activator inhibitor-1 (PAI-1) has been shown to be an independent risk factor for coronary artery disease. Variations in plasma PAI-1 levels have been attributed to variations in the PAI-1 gene, and associations between PAI-1 levels and PAI-1 genotypes suggest that PAI-1 expression may be regulated in a genotype-specific manner by insulin, hypertriglyceridemic (HTG) very low density lipoprotein (VLDL), or lipoprotein(a) [Lp(a)]. Polymerase chain reaction-amplified 1106-bp fragments of the promoter of the 1/1 and 2/2 PAI-1 genotypes were sequenced and showed 5 regions of small nucleotide differences in the 1/1 versus 2/2 PAI-1 promoters that consistently occurred with high frequency. These fragments were ligated into the luciferase reporter gene, and 1/1 and 2/2 PAI-1 genotype human umbilical vein endothelial cell (HUVEC) cultures were transiently transfected with their respective p1PAI110/luc and p2PAI110/luc constructs and vice versa. Insulin induced an approximately 12- to 16-fold increase in luciferase activity in both the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with the p1PAI110/luc construct. HTG-VLDL and Lp(a) induced luciferase activity by approximately 14- to 16- and approximately 8- to 11-fold, respectively, in both the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with the p2PAI110/luc construct. The positive control interleukin-1 showed an approximately 7- to 12-fold response in the 1/1 and 2/2 PAI-1 genotype HUVEC cultures transfected with either of the constructs. These cross-over results demonstrate that regulation of either the 1/1 or 2/2 PAI-1 genotype by its respective inducer is due to the promoter itself and not to some factor(s) expressed differently in the 1/1 or 2/2 PAI-1 genotype HUVEC cultures.