Biomedical subjects

S S Deeb

Publications and source records attributed to S S Deeb.

At least 37 records · Page 2Linked to original sources

PubMed1999

Polymorphisms at the Werner locus: I. Newly identified polymorphisms, ethnic variability of 1367Cys/Arg, and its stability in a population of Finnish centenarians.

The Werner syndrome gene (WRN) encodes a novel helicase of 1,432 amino acids. Homozygous mutations, all of which result in the truncation of the protein, lead to Werner syndrome. However, little is known about the role of WRN in "normal" aging. We have identified four missense polymorphisms and four conservative polymorphsims in WRN gene. A single study showed that a polymorphism at amino acid 1367 Cys(TTG)/ Arg(CTG) is associated with a variation in risk of myocardial infarction among a Japanese population. The 1367 Cys/Arg polymorphism was examined during aging in three different populations: Finnish, Mexican, and North American. The frequencies of 1367 Cys were higher than those of 1367 Arg in all the populations examined, though the frequencies varied among populations. The frequency of the 1367 Arg allele, thought to be protective against myocardial infarction in a Japanese population, was approximately three times higher in the North American and Finnish adult populations. When newborns and centenarians were compared within the Finnish population, no differences were observed in the proportions of 1367 Cys/Arg across age groups. Within the Finnish population, we confirmed a significant decrease of the APOE epsilon2 allele and an increase in the epsilon4 allele in newborn infants compared with centenarians. Thus, unlike the APOE polymorphism, there is no evidence of an association of this WRN polymorphism with longevity.

Adult↗

PubMed1999

Murine phospholipid hydroperoxide glutathione peroxidase: cDNA sequence, tissue expression, and mapping.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), also known as glutathione peroxidase 4 (GPX4), is a 19-kDa, monomeric enzyme that protects cells from lipid peroxide-mediated damage by catalyzing the reduction of lipid peroxides. PHGPx is synthesized in two forms, as a 194-amino acid peptide that predominates in gonadal tissue and localizes to mitochondria, and as a 170-amino acid protein that predominates in most somatic tissues and localizes to the cytoplasm. With the rapid amplification of cDNA ends (RACE) procedure, an 876-bp PHGPx cDNA was amplified from mouse testis, and a 767-bp PHGPx cDNA was amplified from mouse heart. The cDNA sequences were identical except that the testis cDNA contained an additional 109 bp at its 5' end. With a partial cDNA with complete homology to both the testis and myocardial PHGPx cDNAs, the murine tissue distribution of PHGPx mRNA expression was determined by Northern blotting. Highest level of PHGPx expression was found in the testis, followed by the kidney, heart and skeletal muscle, liver, brain, lung, and spleen. Northern blotting performed with a cDNA specific for the longer PHGPx transcript demonstrated that this longer PHGPx transcript was present only in the testis. A 1.4-kb PHGPx genomic fragment was amplified from murine kidney DNA and used to map the PHGPx gene by linkage analysis of restriction fragment length variants (RFLVs). The murine PHGPx gene (Gpx4) was mapped to a region of murine Chromosome (Chr) 10, located 43 cM from the centromere, that is syntenic with the human locus, which is located at the terminus of the short arm of human Chr 19. This information may be valuable in characterizing the role of PHGPx in modulating susceptibility to lipid peroxide-mediated injury in inbred murine strains and for targeted disruption of the gene.

Amino Acid Sequence↗

PubMed1999

Analysis of red/green color discrimination in subjects with a single X-linked photopigment gene.

Many subjects despite having only a single X-linked pigment gene (single-L/M-gene subjects) are able to make chromatic discriminations by Rayleigh matching, especially when large fields are used. We used a combination of psychophysics (Rayleigh match), electroretinograms (ERG), and molecular genetic techniques to rule out several possible explanations of this phenomenon. Use of rods for chromatic discrimination was unlikely since strong adapting fields were employed and the large-field match results were not consistent with rod participation. A putative mid- to long-wavelength photopigment that escapes detection by current molecular genetic analysis was ruled out by finding only a single L/M photopigment in flicker ERGs from 16 single-L/M-gene subjects. Large-field match results were not consistent with participation of S cones. Amino acid sequence polymorphisms in the S-pigment gene that might have shifted the S cone spectrum towards longer wavelengths were not found on sequencing. The mechanism of chromatic discrimination in the presence of a single photopigment therefore remains unknown. Further possible explanations such as variations in cone pigment density and retinal inhomogeneities are discussed.

Adaptation, Ocular↗

PubMed1999

Position of a 'green-red' hybrid gene in the visual pigment array determines colour-vision phenotype.

The X-linked red- and green-pigment genes are arranged in a head-to-tail tandem array. The colour-vision defect of deuteranomaly (in 5% of males of European descent) is associated with a 5'-green-red-3' visual-pigment hybrid gene, which may also exist in males with normal colour vision. To explain why males with a normal red, a normal green and a green-red hybrid gene may have either normal or deutan colour vision, we hypothesized that only the first two genes are expressed and deuteranomaly results only if the green-red hybrid gene occupies the second position and is expressed preferentially over normal green-pigment genes occupying more distal positions. We used long-range PCR amplification and studied 10 deutan males (8 deuteranomalous and 2 deuteranopic) with 3 visual pigment genes (red, green and green-red hybrid) to investigate whether position of the hybrid gene in the array determined gene expression. The green-red hybrid gene was always at the second position (and the first position was always occupied by the red gene). Conversely, in two men with red, green and green-red hybrid genes and normal colour vision, the hybrid gene occupied the third position. When pigment gene mRNA expression was assessed in post-mortem retinae of three men with the red, green and green-red genotype, the green-red hybrid gene was expressed only when located in the second position. We conclude that the green-red hybrid gene will only cause deutan defects when it occupies the second position of the pigment gene array.

Color Perception↗

PubMed1999

A hepatic lipase gene promoter polymorphism attenuates the increase in hepatic lipase activity with increasing intra-abdominal fat in women.

High hepatic lipase (HL) activity is associated with an atherogenic lipoprotein profile of small, dense LDL particles and lower HDL(2)-C. Intra-abdominal fat (IAF) is positively associated with HL activity. A hepatic lipase gene (LIPC) promoter variant (G-->A(-250)) is associated with lower HL activity, higher HDL(2)-C, and less dense LDL particles. To determine whether the LIPC promoter polymorphism acts independently of IAF to regulate HL, 57 healthy, premenopausal women were studied. The LIPC promoter A allele was associated with significantly lower HL activity (GA/AA=104+/-34 versus GG=145+/-57 nmoles x mL(-1) x min(-1), P=0.009). IAF was positively correlated with HL activity (r=0.431, P<0.001). Multivariate analysis revealed a strong relationship between both the LIPC promoter genotype (P=0. 001) and IAF (P<0.001) with HL activity. The relationship between IAF and HL activity for carriers and noncarriers of the A allele was curvilinear with the carriers having a lower apparent maximum level of plasma HL activity compared with noncarriers (138 versus 218 nmoles x mL(-1) x min(-1), P<0.001). In addition, the LIPC A allele was associated with a significantly higher HDL(2)-C (GA/AA=16+/-7 versus GG=11+/-5 mg/dL, P=0.003). We conclude that the LIPC promoter A allele attenuates the increase in HL activity due to IAF in premenopausal women.

Abdomen↗

PubMed1999

Mutations in the lipoprotein lipase gene associated with ischemic heart disease in men. The Copenhagen city heart study.

The aim of this study was to test the hypothesis that the Asp9Asn substitution and the T(-93)-->G mutation in the promoter of the lipoprotein lipase gene affect plasma lipid levels and thereby the risk of ischemic heart disease (IHD). We genotyped 9033 men and women from a general population sample and 940 patients with IHD. The frequency of both the G allele and the Asn9 allele in the general population sample was approximately 0.015 for both men and women. These 2 mutations appeared together in 95% of carriers. The average triglyceride-raising effect associated with double heterozygosity for the T(-93)-->G mutation and the Asp9Asn substitution was 0.28 mmol/L (P=0.004) and 0.16 mmol/L (P=0.10) in men and women, respectively. On logistic regression analysis allowing for age, the risk of IHD for double heterozygous men and women was increased 90% (95% confidence interval [CI], 20% to 200%) and 30% (95% CI, -40% to 170%), respectively, compared with noncarriers. When, in addition, other conventional cardiovascular risk factors were allowed for, the risk of IHD for double heterozygous men and women was increased 70% (95% CI, 0% to 190%) and 20% (95% CI, -50% to 180%), respectively. Of the overall risk of IHD in men in the general population, the fraction attributable to double heterozygosity was 3%, similar to the 5% attributable to diabetes mellitus. These results demonstrate that the Asp9Asn substitution is in linkage disequilibrium with the T(-93)-->G mutation and that the double-heterozygous carrier status is associated with elevated plasma triglycerides and an increased risk of IHD in men.

Adult↗

PubMed1999

Two polymorphisms in the peroxisome proliferator-activated receptor-gamma gene are associated with severe overweight among obese women.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor that regulates adipocyte differentiation. Variations in the PPARgamma gene may affect the function of the PPARgamma and, therefore, body adipocity. We investigated the frequencies of the Pro12Ala polymorphism in exon B and the silent CAC478CAT polymorphism in exon 6 of the PPARgamma gene and their effects on body weight, body composition, and energy expenditure in obese Finns. One hundred and seventy obese subjects [29 men and 141 women; body mass index (BMI), 35.7 +/- 3.8 kg/m2; age, 43 +/- 8 yr; mean +/- SD) participated in the study. The frequencies of the Ala12 allele in exon B and CAT478 allele in exon 6 were not significantly different between the obese and population-based control subjects (0.14 vs. 0.13 and 0.19 vs. 0.21, respectively). The polymorphisms were associated with increased BMI [Pro12Pro, 34.5 +/- 3.8; Pro12Ala, 34.8 +/- 3.1; Ala12Ala, 39.2 +/- 4.6 kg/m2 (P = 0.011); CAC478CAC, 34.5 +/- 3.8; CAC478CAT, 34.5 +/- 3.3; CAT478CAT, 37.7 +/- 4.1 kg/m2 (P = 0.046)]. In addition, the women with both Ala12Ala and CAT478CAT genotypes (n = 5) were significantly more obese compared with the women having both Pro12Pro and CAC478CAC genotypes (n = 85; BMI, 40.6 +/- 3.3 vs. 34.4 +/- 3.9 kg/m2; P = 0.001), and they had increased fat mass (46.8 +/- 9.1 vs. 36.8 +/- 7.5 kg; P = 0.005). In conclusion, the Pro12Ala and CAC478CAT polymorphisms in the PPARgamma gene are associated with severe overweight and increased fat mass among obese women.

Adipose Tissue↗

PubMed1999

Analysis of mouse intron 7 DNA sequence of the APP gene: comparison with the human homologue.

Mutations in the beta-amyloid precursor protein gene (APP) cause Alzheimer disease (AD) in certain families. The mature protein (APP) exists in several different isoforms resulting from alternative splicing of the primary transcript. Several lines of evidence indicate that particular isoform(s) of APP may contribute to the etiology of AD. One of the isoforms, APP695, lacks the Kunitz protease inhibitor (KPI) domain encoded by exon 7. APP695 is expressed predominantly in neurons, whereas the KPI domain containing isoforms, APP751 and APP770, are expressed ubiquitously. The ratio of APP751/APP695 mRNA tends to increase in the brain of AD patients. Furthermore, this ratio in mouse brain is much lower than that in human brain, and mice are resistant to the spontaneous development of beta-amyloidosis. In addition, transgenic mice that develop pathological changes similar to those of AD expressed more KPI-domain containing APP mRNA than transgenic mice without the changes. Previous studies imply that the controlling elements exist in the flanking sequences of the alternatively-spliced exons. Therefore, we have determined the DNA sequences of intron 7 and made a comparison between mouse and human DNA sequences of intron 7. Mouse intron 7 shares about 50% sequence identity with the human homologue, with higher sequence identity (approximately 85%) mainly in the 5' end (approximately 250 bp) of the intron. A palindromic sequence was found in both human and mouse intron 7 and showed subtle differences in their structure between the two species. Whether this sequence plays any roles in regulating alternative splicing of exon 7 remains to be determined. Human intron 7 contains a Alu element, which possesses potential retinoic acid and thyroid hormone responsive elements that might be involved in the regulation of alternative splicing. Mouse intron 7 sequence also contains a few repeat sequences which are specific to the genome of mice and rats. Homologies shared between human and mouse intron 7 sequences may contribute to the common characteristics of neuron-specific splicing of APP in both species. The unique features of the intron may account for differences between human and mouse brain in fine tuning of alternative splicing of the APP transcript, which may lead to their different susceptibilities to beta-amyloidosis.

Alternative Splicing↗

PubMed1998

Functional consequences of naturally occurring variants of human hexokinase II.

Hexokinase II (HKII) catalyses a key step in glucose metabolism and can be regarded as a candidate gene for insulin resistance and type 2 (non-insulin-dependent) diabetes mellitus. We observed previously four amino acid substitutions among Finnish type 2 diabetic patients: Gln142His, Ala314Val; 0.9%, Arg353Cys; 2.7% and Arg775Gln; 2.7%. The Arg775Gln mutation was also observed in normal control subjects (2.1%) and the Gln142His substitution was found in both Type II diabetic and normal subjects with similar frequencies (approximately 20%). Since Gln at position 142, Ala at 314 and Arg at 775 are present in human and rat hexokinases and could be important for structure and function of the enzyme, we generated all four substitutions by site-directed mutagenesis and expressed them in E.coli. None of these substitutions had any effect on HKII catalytic activity, K(m) or Vmax for glucose values in vitro. Thus unless these substitutions have an impact on enzyme activity or regulation in vivo, it is unlikely that these substitutions contribute to the aetiology of Type II diabetes.

Animals↗

PubMed1998

The human FE65 gene: genomic structure and an intronic biallelic polymorphism associated with sporadic dementia of the Alzheimer type.

The FE65 protein binds to the intracellular domain of the beta-amyloid precursor protein (betaPP) and may modulate the internalization of betaPP. This gene is highly expressed in regions of the brain specifically affected in dementia of the Alzheimer type (DAT). As a prelude to further investigations of the role of FE65 in the metabolism of betaPP and in the pathogenesis of DAT, we have determined the entire genomic structure and sequence of human FE65 and have discovered several polymorphisms in this gene. Human FE65 contains 14 exons ranging in size from 6 to 735 bp. All splice sites conform to consensus sequences except for the donor site of intron 10. The 5' end of FE65 mRNA was identified by rapid amplification of the cDNA 5' end and is 31 bp longer than the previously published cDNA sequence. The 5'-flanking region of this gene is TATA-less and is very GC-rich with at least five putative Sp1 binding sites. In comparison to the genomic rat FE65 sequence, the human FE65 5'-untranslated region is 134 bp longer and has an extra exon (exon 1, 86 bp). To identify mutations/polymorphisms of the coding regions of this gene, we performed blinded analysis of 457 Caucasian case-control samples from a large epidemiological study of sporadic DAT. Screening was conducted by single-strand conformation polymorphism. Four minor variants were found within the coding region, with frequencies between 0.002 and 0.015; two of the four result in amino acid substitutions. The more informative biallelic polymorphism (a trinucleotide deletion and a single base substitution) was found within intron 13 (84 bp), which interrupts two exons encoding the betaPP binding site. The frequency of the minor allele in this intron was 0.097 in DAT cases and 0.161 in controls (chi2=7.78, P=0.0054). Having at least one copy of the minor allele was associated with a decreased risk for DAT (chi2=9.20, P<0.005, odds ratio=0.49, 95% CI 0.31-0.77). Multivariate analysis showed that this association was independent of the APOE genotype. These results suggest that either FE65 itself or a closely linked gene influences the pathogenesis of sporadic DAT. The interaction of FE65 with betaPP and the association of a FE65 polymorphism with DAT lend credence to the hypothesis that the metabolism of betaPP is central to the pathogenesis of common sporadic forms of DAT.

Aged↗

PubMed1998

Severity of color vision defects: electroretinographic (ERG), molecular and behavioral studies.

Earlier research on phenotype/genotype relationships in color vision has shown imperfect predictability of color matching from the photopigment spectral sensitivities inferred from molecular genetic analysis. We previously observed that not all of the genes of the X-chromosome linked photopigment gene locus are expressed in the retina. Since sequence analysis of DNA does not necessarily reveal which of the genes are expressed into photopigments, we used ERG spectral sensitivities and adaptation measurements to assess expressed photopigment complement. Many deuteranomalous subjects had L, M, and L-M hybrid genes. The ERG results showed that M pigment is not present in measurable quantities in deutan subjects. Using these results to determine gene expression improved the correlations between inferred pigment separation and color matching. Furthermore, we found a subject who had normal L and M genes and normal proximal promoter sequences, yet he had a single photopigment (M) by ERG and tested as a protanope. These results demonstrate the utility of ERG measurements in studies of molecular genetics of color vision deficiencies, and further support the conclusion that not all genes are expressed in color deficient subjects. In particular, deuteranomaly requires a presently unknown mechanism of selective expression which excludes normal M genes and allows expression of L-M hybrid genes in one cone type, and the normal L in another.

Adaptation, Ocular↗

PubMed1998

A Pro12Ala substitution in PPARgamma2 associated with decreased receptor activity, lower body mass index and improved insulin sensitivity.

The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a transcription factor that has a pivotal role in adipocyte differentiation and expression of adipocyte-specific genes. The PPARgamma1 and gamma2 isoforms result from alternative splicing and have ligand-dependent and -independent activation domains. PPARgamma2 has an additional 28 amino acids at its amino terminus that renders its ligand-independent activation domain 5-10-fold more effective than that of PPARgamma1. Insulin stimulates the ligand-independent activation of PPARgamma1 and gamma2 (ref. 5), however, obesity and nutritional factors only influence the expression of PPARgamma2 in human adipocytes. Here, we report that a relatively common Pro12Ala substitution in PPARgamma2 is associated with lower body mass index (BMI; P=0.027; 0.015) and improved insulin sensitivity among middle-aged and elderly Finns. A significant odds ratio (4.35, P=0.028) for the association of the Pro/Pro genotype with type 2 diabetes was observed among Japanese Americans. The PPARgamma2 Ala allele showed decreased binding affinity to the cognate promoter element and reduced ability to transactivate responsive promoters. These findings suggest that the PPARgamma2 Pro12Ala variant may contribute to the observed variability in BMI and insulin sensitivity in the general population.

Adult↗

PubMed1998

Common variants in the promoter of the hepatic lipase gene are associated with lower levels of hepatic lipase activity, buoyant LDL, and higher HDL2 cholesterol.

Increased hepatic lipase (HL) activity is associated with small, dense, low density lipoprotein (LDL) and low high density lipoprotein2 (HDL2) cholesterol (-C) levels. A polymorphism in the promoter region of the HL gene (LIPC) is associated with HDL-C levels. To test whether this association is mediated by differences in HL activity between different LIPC promoter genotypes, the LIPC promoter polymorphism at position -250 (G-->A), HL activity, LDL buoyancy, and HDL-C levels were studied in white normolipidemic men and men with coronary artery disease (CAD). The less common A allele (frequency=0.21 and 0.25 in normal and CAD subjects, respectively) was associated with lower HL activity (P<0.005 by ANOVA) and buoyant LDL particles (P</=0.01) in both groups. Normal and CAD subjects heterozygous for the A allele had lower HL activity (by 24% and 29%, respectively) and significantly more buoyant LDL particles. Homozygosity for this allele (AA) was associated with an even lower HL activity in normal (-26%) and CAD (-46%) subjects. The A allele was associated with higher HDL2-C in CAD patients (P=0.007); heterozygotes and homozygotes for the A allele had a 92% and a 140% higher HDL2-C level (P<0.01) than did GG individuals. In a small number of normolipidemic subjects, the same trend in HDL2-C was seen. In a univariate analysis, the LIPC genotype accounted for 20% to 32% of the variance in HL levels among normal subjects and CAD patients, respectively. After adjustment for HL, the association between LIPC genotype and LDL buoyancy was no longer significant, suggesting that the effect of LIPC genotype on LDL buoyancy is mediated by its effects on HL activity. The LIPC A allele was more frequent in Japanese-Americans and African-Americans than in whites. In summary, these results suggest that variants in the LIPC promoter may significantly contribute to the variance in levels of HL activity and consequently, to the prevalence of the atherogenic small, dense, LDL particles and low HDL2-C levels.

Adult↗

PubMed1998

Evidence against linkage of familial combined hyperlipidemia to the apolipoprotein AI-CIII-AIV gene complex.

Familial combined hyperlipidemia (FCHL) was originally described as a disorder characterized by elevated levels of either plasma cholesterol or triglyceride (TG) or both in members ofthe same family. More recent studies have indicated that apolipoprotein B levels (apoB) are also elevated in these individuals. Although a dominant mode of inheritance was originally proposed, recent studies have questioned this simple mode of inheritance, and the genetic basis of the disorder has eluded investigators. A study that reported evidence that FCHL is linked to the apolipoprotein AI-CIII-AIV region on chromosome 11 is therefore of interest. We have attempted to replicate this finding in three large, well-characterized FCHL kindreds by using a highly polymorphic marker in the apoCIII gene. Using the same definitions and parameters as were used in the initial report, we obtained strong evidence against linkage of FCHL to the apolipoprotein AI-CIII-AIV region on chromosome 11 (combined lod score of -7.87 at 0% recombination). Two other models, one based on total cholesterol (TC) levels alone and one based on the joint distribution of TC and apoB levels, also gave evidence against linkage of FCHL to this region (lod scores at 0% recombination of -8.95 and -2.58, respectively). An additional regression-based linkage analysis also gave no support for the existence of a locus in this region that influences these lipid levels in these pedigrees. Explanations for the differences in results between these studies include genetic heterogeneity, differences in clinical phenotype used to select the pedigrees, and ascertainment bias.

Apolipoprotein A-I↗

PubMed1998

Glucokinase gene islet promoter region variant (G-->A) at nucleotide -30 is not associated with reduced insulin secretion in Finns.

OBJECTIVE: To investigate the effect of the islet promoter region variant (G-->A) at nucleotide -30 of the glucokinase (GCK) gene on insulin levels in subjects with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and NIDDM. RESEARCH DESIGN AND METHODS: The study population included 294 subjects with NGT, 83 subjects with IGT, and 36 subjects with NIDDM. Oral glucose tolerance tests (OGTTs) were performed in all subjects, and intravenous glucose tolerance tests (IVGTTs) were performed in subjects with NGT. The islet promoter region of the GCK gene was amplified with polymerase chain reaction and screened for the variant (-30) using single-strand conformation polymorphism analysis. RESULTS: The islet promoter variant (-30) of the GCK gene was found in 17% of subjects with NGT, 23% of subjects with IGT, and 14% of patients with NIDDM (NS between the groups). Fasting, 1-h, and 2-h insulin levels, measured by OGTT, did not differ between subjects with and without this variant in any of the three groups. Furthermore, first-phase insulin secretion, determined by an IVGTT in subjects with NGT, did not associate with presence of the islet promoter region variant (-30) of the GCK gene. CONCLUSIONS: These results indicate that the variant (-30) of the islet promoter region of the GCK gene does not have a significant effect on insulin secretion in Finnish subjects with NGT, IGT, or NIDDM.

Administration, Oral↗

PubMed1998

Functional analysis of the promoters of the human red and green visual pigment genes.

PURPOSE: To delineate cis-acting DNA elements involved in the expression of the human red and green visual pigment genes and to correlate these with transcription factor binding sites. METHODS: Assays of promoter activity were accomplished by transient transfection into WERI cells. Nested deletion and block mutagenesis were undertaken to delineate critical elements. Transcription factor binding sites were determined by DNase I footprinting and electrophoretic mobility shift (EMSA) analyses. RESULTS: The human retinoblastoma cell line WERI, but not Y-79, was found to express the red and green pigment genes. Transfection assays in WERI cells revealed that the proximal region of the red pigment gene promoter had positive (-130 to -113 and -96 to -23) and negative (-190 to -130 and - 113 to -96) regulatory elements. The green pigment gene promoter was found to be 2 to 4 times more active than was that of the red pigment. This difference in activity was attributable mainly to a T to C substitution at position -3. DNase I protection and EMSA studies demonstrated the binding of several ubiquitous and WERI-enriched proteins to DNA sequences between - 130 and the TATA box. The locus control region (LCR) did not have any enhancer activity in transient transfection. CONCLUSIONS: The WERI cell line is a good model system for the analysis of gene expression of the human cone visual pigment genes. The expression of these genes in a cell-specific fashion seems to be controlled mainly by positive-acting elements in the region between - 130 and the TATA box. The higher activity of the green pigment gene promoter could have evolved to compensate for its longer distance from the activating LCR than that of the red pigment gene promoter (approximately 34 versus 3.5 kb). Although the LCR does not enhance transcription in transient transfection, it binds factors that also recognize the proximal promoter region. These interactions may be important for the establishment of a transcriptionally active domain in a chromatin context.

Base Sequence↗

PubMed1998

Transgenic mice expressing a functional human photopigment.

PURPOSE: Changes in retinal photopigments represent a fundamental step in the evolution of visual systems, in that addition of new pigment types or alterations in the spectral absorption properties of existing pigments modify visual capacities and thus open new visual worlds. To provide a tool that would allow direct examination of the changes caused by the presence of novel photopigments, this study was designed to determine whether a gene encoding a human cone photopigment introduced into the mouse genome would be expressed in a cone-specific manner and would support phototransduction. METHODS: Mice transgenic for the human long wavelength-sensitive (L) photopigment were generated by microinjection of fertilized mouse eggs. RNA expression in different tissues was monitored by reverse transcription-polymerase chain reaction analysis. Photopigment protein was localized in retinal cross sections and wholemounts by antibody staining. Light transduction of the cone photopigments was assessed by flicker photometric electroretinography (ERG). RESULTS: The human transgene was expressed specifically in the mouse cones in quantities comparable to those of the mouse middle wavelength-sensitive (M) pigment gene. Immunocytochemical analysis showed that the human L pigment was abundantly synthesized in most mouse cones, was translocated to the outer segments, and caused no detectable cone degeneration. Electroretinographic spectral sensitivity analysis showed that the human L pigment was efficient in eliciting an electrical response. The degree of expression of the transgene in the two founders correlated well with the spectral responsivity of the ERG. CONCLUSIONS: The human L photopigment transduces light efficiently in mouse cones, implying that all protein domains necessary for efficient interaction with intracellular transport and signal transduction machineries in mouse cones have been conserved through evolution. The expression of the human L photopigment gene in both classes of cone of the mouse retina indicates that the transgene did not have the regulatory elements necessary for restricting its expression to mouse M cones or that such elements are not recognized in mouse UV-sensitive cones.

Animals↗

PubMed1998

Sp1 and Sp3 transactivate the human lipoprotein lipase gene promoter through binding to a CT element: synergy with the sterol regulatory element binding protein and reduced transactivation of a naturally occurring promoter variant.

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein and energy metabolism and, therefore, regulation of its expression could have an important bearing on these processes. We have identified an evolutionarily conserved 5'-CCTCCCCCC-3' motif (from -91 to -83, CT element) in the human LPL gene promoter, deletion or mutation of which caused approximately 70-80% decrease in promoter activity. We found that Sp1 and Sp3 in THP-1 nuclear protein extracts bind specifically to this element. Co-transfection with Sp1 and Sp3 expression plasmids transactivated the LPL promoter via the CT element in Drosophila SL2 cells devoid of Sp proteins. Sp3 moderately repressed Sp1-mediated LPL promoter activation when both were co-expressed in SL2 cells. Furthermore, co-expression of an active sterol regulatory element binding protein (SREBP-1), with Sp1, but not with Sp3, synergistically activated the LPL promoter in SL2 cells. We previously reported a naturally occurring T-->G substitution at position -93 of the human LPL promoter which reduces promoter activity by 40-50% in transient transfection assays. In this study, we showed that this substitution results in reduced binding affinity to Sp1/Sp3 and in diminished transactivation by Sp1/Sp3 alone and by the synergistic action of Spl and SREBP-1 In conclusion, recruitment of Sp1/Sp3 by the CT element may play an important role in expression of the human lipoprotein lipase gene. Synergistic transcriptional activation by Sp1 and SREBP-1 may provide a mechanism for cross-talk between cholesterol and triglyceride metabolic pathways.

Base Sequence↗