Genomic organization of the 3' region of the human thyroglobulin gene.

The genomic organization of the 3' end of the human Thyroglobulin (Tg) gene has not previously been characterized. We isolated and characterized seventeen lambda phage clones from a human genomic library that included nucleotides 6263 to 8410 of the Tg mRNA, encompassing the last thirteen 3' exons of the Tg gene. The region contained exons ranging in size from 94 to 222 nucleotides, split by introns of 1 to 64 kb. We estimate a total of 48 exons in the Tg gene. All the intron-exon boundaries were sequenced. We found that the splicing sequences diverged considerably from the 3' and 5' consensus. However, the GT-AG rule was perfectly respected in all the exons. A total of 5788 intronic bases and most of the sequences contained in the 13 exons were analyzed (1846 bases). One sequence variation, TT to CC at positions 8377-8378, was found in the 3' untranslated segment. The three tyrosine residues involved in thyroid hormones synthesis (amino acids 2554, 2568, and 2747) at the carbosyl termini of Tg, are encoded by exons 44, 45, and 48. The knowledge of the precise organization of the Tg gene should help to direct studies of Tg gene mutations in families in which a defect in the synthesis of Tg occurs.     

A polymorphism in the 3' untranslated region of the gene for tumor necrosis factor receptor 2 modulates reporter gene expression

The gene encoding the human TNF alpha receptor (TNFR) 2 contains polymorphisms in the 3' untranslated region (UTR). Previous studies have shown that some variant alleles in this region are associated with obesity and insulin resistance. However, the effect of these polymorphisms on the expression of TNFR2 has not been studied to date. To examine the role played by different haplotypes in the control of TNFR2 expression (haplotypes A1-A5, referring to nucleotides 1663 G/A, 1668 T/G, and 1690 T/C), we introduced these sequences into the 3'-UTR of a heterologous reporter gene and expressed the corresponding constructs in a human T-cell line. We demonstrate that a 485-nt fragment of the TNFR2 3'-UTR that contains a U-rich region decreases reporter expression and that haplotypes A1-A4 exert a stronger effect than A5. Furthermore, time-course assays of mRNA stability using actinomycin D revealed that haplotypes A1-A4 destabilize the mRNA. The proximal TNFR2 3'-UTR, independently of haplotype differences, responded to T-cell activation by increasing mRNA decay. Electromobility shift analysis demonstrated that protein(s) found in T-cell extracts bind to the 485-nt fragment. We suggest that an increased rate of TNFR2 mRNA decay protects cells from unrestrained TNF alpha effects and that this protection is weakened in A5 subjects. These findings may explain the association of this haplotype with obesity and increased leptin levels.     

Genomic organization of the 5' region of the human thyroglobulin gene

OBJECTIVE: The purpose of the present work is to establish the intron-exon organization from exon 12 to exon 23 of the human thyroglobulin gene and to construct a physical map of the 5' terminal half of the gene. DESIGN: Screening of a genomic library and subsequent restriction map, hybridization and sequencing methods have been employed to characterize the recombinant positive phages. METHODS: A human genomic DNA library was screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. Intron/exon junction sequences were determined by the Taq polymerase-based chain terminator method. Finally, the thyroglobulin gene was mapped using the Gene Bridge 4 radiation hybrid clone panel. RESULTS: We isolated and characterized four lambda phage clones that include nucleotides 3002 to 4816 of the thyroglobulin mRNA, encompassing exons 12 to 23 of the gene. The exon sizes range between 78 and 219 nucleotides. We found that the GT-AG splicing sequences rule was perfectly respected in all the introns. A total of 7302 intronic bases was analyzed. Hormogenic tyrosine 5 and 1291 are encoded by exons 2 and 18. Also, seven alternative spliced variants are associated with the 5' region. Thyroglobulin gene maps to 5,5 centiRays from the AFMA053XF1 marker, in chromosome 8. CONCLUSIONS: The present study shows that the first 4857 bases of thyroglobulin mRNA are divided into 23 exons and the four phages isolated include 32.6 kb genomic DNA, covering 1815 nucleotides of exonic sequence distributed in 12 exons, from exon 12 to 23.     

No evidence for association between the polymorphism in the 3' untranslated region of interleukin-12B and human susceptibility to tuberculosis

Interleukin (IL)-12 plays a pivotal role in cell-mediated immunity to Mycobacterium tuberculosis infection. We tested the association between a biallelic single-nucleotide polymorphism in the 3' untranslated region (UTR) of IL-12B and human susceptibility to tuberculosis (TB), in a population-based case-control study of adult patients with TB from 2 ethnicities, African American and white, and in a family-based transmission/disequilibrium study of 60 informative families with at least 1 pediatric patient with TB and 1 heterozygous parent. Our results suggest that IL-12B 3' UTR has no effect or has a negligible effect on human susceptibility to TB.     

A novel single nucleotide polymorphism in the 3' untranslated region of human glutathione peroxidase 4 influences lipoxygenase metabolism

Selenium (Se) is an essential micronutrient for human health. The biological roles of the essential micronutrient Se are attributed to its presence in a range of 20-30 selenoproteins including the cytosolic and phospholipid hydroperoxide glutathione peroxidases (GPX1 and GPX4). It has been suggested that GPX4 may play a role in regulation of leukotriene biosynthesis and thus inflammation. In eukaryotes Se is incorporated into selenoproteins as the amino acid selenocysteine in a process requiring a stem-loop within the 3' untranslated region (3'UTR) of the mRNA. In this study the region of the GPX4 gene corresponding to the 3'UTR was scanned for mutations in a group of 66 volunteers. The data show a T/C variant at position 718. The distribution of this SNP in our population was 34% CC, 25% TT and 41% TC; i.e., it is in Hardy-Weinberg equilibrium. Individuals of different genotypes exhibited significant differences in the levels of lymphocyte 5-lipoxygenase total products, with C718 showing increased levels of those products compared to T718 and T/C718 (36% and 44% increases, respectively). The data suggest that the SNP718 that we have identified has functional effects and support the hypothesis that GPX4 plays a regulatory role in leukotriene biosynthesis.     

The 3' untranslated region of the lipoprotein lipase gene: haplotype structure and association with post-heparin plasma lipase activity

CONTEXT: Haplotypes comprising six single nucleotide polymorphisms (SNPs) (intron 7 to intron 9) of the lipoprotein lipase (LPL) gene appear to influence risk for atherosclerosis and insulin resistance in Mexican-Americans. OBJECTIVE: Based on rodent studies, we hypothesized that these haplotypes are in linkage disequilibrium with functional variants in the 3' untranslated region of LPL, which is encoded by exon 10, and that these variants influence phenotype by altering LPL expression. DESIGN: We sequenced exon 10 in subjects with divergent insulin sensitivity and divergent haplotypes. We also sequenced the other common LPL haplotypes. Variants identified by sequencing were genotyped in a large, family-based population along with the six SNPs spanning intron 7 to intron 9. We tested the potential functional significance of variation in exon 10 by evaluating association of haplotypes with post-heparin plasma LPL activity. SETTING: The study took place within the general community, with the Mexican-American Coronary Artery Disease Project cohort. PARTICIPANTS: Participants included 847 subjects from 163 families. MAIN OUTCOME MEASURES: We determined LPL haplogenotype and post-heparin plasma LPL activity. RESULTS: Exon 10 sequencing identified 15 variants. Thirteen of these variants were genotyped in large-scale along with the six SNPs spanning intron 7 to intron 9. LPL haplotypes and their relative frequencies in Mexican-Americans were determined. The fourth most common haplotype based on 19 SNPs (haplotype 19-4) was associated with increased LPL activity as well as multiple phenotypes related to the metabolic syndrome. CONCLUSIONS: These results support the possibility that variation in the 3' untranslated region of LPL affects LPL expression and activity, consequently influencing risk of atherosclerosis and insulin resistance, and provides important tools for further dissection of LPL regulation.     

Glucocorticoid receptor gene variant in the 3' untranslated region is associated with multiple measures of blood pressure

CONTEXT: The glucocorticoid receptor (GR) is a key hormone in the hypothalamus-pituitary-adrenal axis that regulates many pathways including blood pressure homeostasis. Thus, GR gene variation may influence interindividual differences in blood pressure in human populations. OBJECTIVE: We resequenced individual GR alleles for comprehensive discovery of GR variants and their chromosomal phase in three major American ethnic groups. We examined the influence of GR variants on blood pressure in large numbers of families using family-based association methods. DESIGN AND PARTICIPANTS: For association studies, we genotyped GR variants in family members from the Genetic Epidemiology Network of Arteriopathy (GENOA) study that were measured for multiple blood pressure traits. The GENOA families consisted of African-Americans, Mexican-Americans, and European-Americans. MAIN MEASUREMENTS: The blood pressure measurements for association studies included systolic blood pressure, diastolic blood pressure, mean arterial pressure, and pulse pressure. RESULTS: Single-nucleotide polymorphisms (SNPs) identified by resequencing were tested for associations with blood pressure measures in GENOA families. Analysis of individual SNPs identified significant associations of rs6198 A/G in exon 9beta with multiple blood pressure measures in European-Americans. Analysis of GR haplotypes found significant associations of a haplotype that is distinguished by rs6198 A/G. CONCLUSIONS: Significant associations of blood pressure with rs6198 A/G likely reflect allelic effects on GR signaling. This SNP disrupts a 3' untranslated region sequence element in exon 9beta that destabilizes mRNA, resulting in increased production of the inactive GRbeta isoform. Excess heterodimerization with the active GRalpha isoform may reduce GR signaling with subsequent physiological effects on blood pressure regulation.     

Structural organization of the human androgen receptor gene

The complete coding region of the human androgen receptor gene has been isolated from a genomic library. The information for the androgen receptor was found to be divided over eight exons and the total length of the gene exceeded 90 kb. The sequence encoding the N-terminal region is present in one large exon. The two putative DNA-binding fingers are encoded separately by two small exons. The information for the hormone-binding domain is split over five exons. Positions of introns are identical to those reported for the chicken progesterone receptor and the human oestrogen receptor genes. Southern blot analysis of genomic DNA with various specific probes reveal that the human androgen receptor is encoded by a single-copy gene.     

Structural analysis of the polymorphic 3' region of the human chorionic gonadotropin-alpha gene in normal placenta and choriocarcinoma cells

We previously examined the segregation patterns of two polymorphic restriction enzyme sites for EcoRI and HindIII in the 3'-flanking region of human CG-alpha gene in placenta and choriocarcinoma. We observed that a unique combination of restriction sites predominates in choriocarcinoma while this pattern was rare in placenta, suggesting a functionally important site in this region of genome. Genomic libraries were constructed from DNA derived from placenta and from the choriocarcinoma cell line JAr to examine the DNA sequence responsible for these polymorphic sites. Several clones were isolated; restriction fragments containing the polymorphic sites were subcloned into pBR322, and the stretch of DNA encompassing the polymorphic sites were sequenced. No apparent DNA rearrangements were observed; sequence analysis showed that both of the polymorphic sites were caused by single base pair mutation in the recognition sequence of each enzyme. Three new polymorphic sites were identified in the 3' region of the hCG-alpha gene. These data indicate that major DNA rearrangements in the 3' region of the gene are not associated with choriocarcinoma. However, analysis of the polymorphic sites revealed that there is a linkage disequilibrium among these sites which may extend to a putative oncogene which is responsible for developing choriocarcinoma.     

Long-term BRCA1 down-regulation by small hairpin RNAs targeting the 3' untranslated region

Mutations in the BRCA1 gene are responsible for the majority of hereditary breast/ovarian cancers. The functional significance of many mutations/splicing variants identified during the screening of high-risk individuals is difficult to predict due to the lack of in vitro functional tests correlating sequence variants with a risk of cancer development. RNA interference is a promising tool in analyzing functional properties of BRCA1 mutations. Here we designed and functionally analyzed shRNAs directed to 3'-UTR of BRCA1 mRNA that may be used to knock-down expression of endogenous BRCA1. Using retroviral infection, we achieved long-term down-regulation of BRCA1 in a cell-type specific manner. We propose that 3'-UTR-directed shRNAs, coupled with up-regulation of exogenous mutated BRCA1 variants, may constitute a versatile system for the functional analysis of BRCA1 gene alterations.     

Nucleotide sequences of the 3'-terminal untranslated region of messenger RNA for human beta globin chain.

In normal messenger RNA for the human beta-globin chain, nucleotide sequences have been identified which can be matched to the amino-acid sequence of the abnormally long segment of the beta-chain of hemoglobin Cranston. The finding of these sequences strengthens the hypothesis that the betaCranston chain arose by a frameshift mutation allowing the "readthrough" of the normal termination codon and translation of usually untranslated portions of the messenger RNA for the beta-globin chain. The oligonucleotides which match the amino-acid sequence of hemoglobin Cranston provide a sequence of 36 nucleotides which follows the normal beta-chain termination codon UAA.     

Mutations of the 3' untranslated region of the SDF1 gene in apes and monkeys: potential impact on sensitivity to AIDS induced by lentiviruses.

The comparison of the stromal cell-derived factor-1 (SDF1) gene 3' untranslated region (3'UTR) of four great ape and four monkey species with their human counterparts shows that the human SDF1-3'A mutation is present in primate species that are the most susceptible to lentivirus-induced AIDS and is absent in species that are particularly resistant to lentivirus-induced AIDS. The results enlighten the possible relationship between SDF1-3'UTR polymorphism and sensitivity to AIDS.     

Structure of the glucocorticoid receptor (NR3C1) gene 5' untranslated region: identification, and tissue distribution of multiple new human exon 1

The 5' untranslated region (UTR) of the glucocorticoid receptor (GR) plays a key role in determining tissue-specific expression and protein isoforms. Analysis of the 5' UTR of the human GR (hGR) has revealed 11 splice variants of the hGR exon 1, based on seven exon 1s, four of which (1-D to 1-F and 1-H) were previously unknown. All of the exon 1 variants have unique splice donor sites and share a common exon 2 splice acceptor site. Due to an upstream in-frame TGA stop codon the predicted translation from all splice variants is identical. The four new exon 1s show remarkable similarity with their rat homologues. Exon 1-D starts and finishes 17 and 36 bp upstream of the corresponding ends of the rat exon 1(4). Exon 1-E is only 6 bp longer than its homologue exon 1(5). Exon 1-F contains two short inserts of 11 and 6 bp when compared with the rat 1(7). 1-H is 18 bp longer than the corresponding rat 1(11). In addition to these new exons, we found that the human exon 1-C occurs as three distinct splice variants, covering the region homologous to the rat exons 1(9) and 1(10). All of the alternative hGR exons 1s presented here were found to be transcribed in human tissue. The human hippocampus expresses mRNA of all the exon 1 variants, while the expression of the other exon 1s seems to be tissue specific. While exon 1-D is only in the hippocampus, exons 1-E and 1-F are also detected in the immune system, and exon 1-H additionally in the liver, lung and smooth muscle. The 5' region of the hGR is more complex than previously thought, and we suggest that each of these untranslated first exons have a distinct proximal promoter region, providing additional depth to the mechanisms available for tissue-specific expression of the hGR isoforms.     

The A19G polymorphism in the 5' untranslated region of the human obese gene does not affect leptin levels in severely obese patients

Recently, the presence of different polymorphisms in the regulatory region of the ob gene has been associated with variations in leptin levels. However, the results of these studies are still contradictory. The aim of the present investigation was to evaluate the presence of the A19G polymorphism in an Italian population of obese patients and to verify its association with leptin levels and anthropometric, metabolic, and clinical parameters. Two hundred five obese patients [body mass index (BMI) > 36 kg/m2; 135 women and 70 men; mean age, 46.9+/-14.23 yr] were screened for presence of the polymorphism; 61 normal-weight controls (mean BMI, 21.05 kg/m2; 53 women, 8 men) were also screened to compare polymorphism frequency. For obese patients, BMI, waist-to-hip ratio, resting energy expenditure, body composition, fasting leptin, total cholesterol, high-density lipoproteins, triglycerides, and caloric intake were determined. Genotype frequencies in obese and control subjects were compared using the contingency table chi-square test; in obese subjects an ANOVA was performed to evaluate association between the polymorphism and several clinical parameters. No significant differences in genotype distribution between control and obese subjects were found. No significant correlations were found between this polymorphism and serum leptin levels and the other parameters considered. These findings confirm the results obtained in both a Finnish and a French population; taken together, these observations might rule out a significant role for the A19->G polymorphism in the regulation of leptin levels and other clinical, anthropometric, and metabolic parameters.