Genomic organization of the human thyroglobulin gene: The complete intron-exon structure

Objective: In order to complete the knowledge of the genomic organization of the human thyroglobulin gene, the present work was designed to establish the intron-exon organization from exon 24 to exon 35 and to construct a more complete physical map of the gene. Design: Screening of two genomic libraries, and subsequent restriction mapping, hybridization and sequencing were used to characterize the recombinant phages. Methods: Two human genomic DNA libraries were screened by in situ hybridization. Southern blotting experiments were performed to characterize the phage inserts. The Long PCR method was used to amplify the genomic DNA region containing exon 24. Intron-exon junction sequences were determined by using the Taq polymerase-based chain termination method. Results: We isolated and characterized five λ phage clones that include nucleotides 4933 to 6262 of the thyroglobulin mRNA, encompassing exons 25-35 of the gene. The remaining exon 24 (nucleotides 4817-4932) was sequenced from the amplified fragment. In total, 8010 intronic bases were analyzed. Conclusions: The present study shows that the five phages isolated and the amplified fragment include 59.4 kb genomic DNA, covering 1446 nucleotides of exonic sequence distributed over 12 exons, from exon 24 to exon 35. Using previous studies and our current data, 220 kb of the human thyroglobulin gene was analyzed, a physical map was constructed, and all exon-intron junctions were sequenced and correlated with the different domains of the protein. In summary, the thyroglobulin gene contains 48 exons ranging in size from 63 nucleotides to 1101 nucleotides.Fil: Chaparro Mendivelso, Jeffer Angel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; ArgentinaFil: Rivolta, Carina Marcela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Moya, Christian M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; ArgentinaFil: Vassart, Gilbert. Université Libre de Bruxelles; BélgicaFil: Targovnik, Hector Manuel. Université Libre de Bruxelles; Bélgica. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentin

Identification and characterization of a novel large insertion/deletion polymorphism of 1464 base pair in the human thyroglobulin gene

We identified a novel large insertion/deletion (Indel) polymorphism of 1464 bp localized in intron 18 of the human thyroglobulin gene. Data from sequence showed a high A+T content (62%), two 17-bp long motif repeats, and three different types of 10-bp long palindromic sequences. The comparison between these 1464 bp and sequences deposited in National Center for Biotechnology Information (NCBI)/GenBank database exhibit a nonsignificant degree of homology with any previously described sequences. The long polymerase chain reaction (PCR) method was used to amplify the genomic DNA region containing intron 17/exon 18/intron 18/exon 19/intron 19 by primers situated in the introns 17 and 19. The amplification generates two fragments of 3.5 and 5.0 kb that correspond to the exclusion or inclusion of a 1464-bp segment, respectively. Both variants are thus widely represented in the human population; giving allele frequencies of 0.56 (insertion) and 0.44 (deletion). Finally, the polymorphism was confirmed by sequence analysis of the 5.0- and 3.5-kb amplified fragments.Fil: Moya, Christian M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; ArgentinaFil: Varela, Viviana. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Rivolta, Carina Marcela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; ArgentinaFil: Mendive, Fernando M. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; ArgentinaFil: Targovnik, Hector Manuel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Genética y Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentin

Genetic Characterization of Thyroglobulin and Leptin Genes in Pasundan Cattle at West Java

The Thyroglobulin (TG) and Leptin (LEP) genes are two candidate genes that widely used for molecular selection to improve carcass traits in beef cattle. This research was carried out to identify the genetic characterization of TG and LEP genes from 47 heads of Pasundan cows at West Java using PCR-RFLP method. Research shows that TG gene of Pasundan cattle is monomorphic with C allele as the dominant allele (1.00). However, LEP gene of Pasundan cattle is polymorphic with C allele as the dominant allele (0.98) and T as the rare allele (0.02). The polymorphic informative content (PIC) and numberof effective allele (ne) values in the LEP gene in the animal studied were 0.04 and 1.04 respectively. It was concluded that TG/BstYI and LEP/Sau3AI gene in the present study can not be used as molecular selection in Pasundan cattle. These results are important as the basic information for preparing the molecular selection program in the future

BTN1A1 , FABP3 and TG genes polymorphism in East Anatolian red cattle breed and South Anatolian red cattle breed

The aim of the study was to determine butyrophilin, thyroglobulin and fatty acid binding protein genes in East Anatolian Red cattle breed and South Anatolian Red cattle breed. In the study, unrelated 50 South Anatolian red and 50 East Anatolian red cattle were used. Genomic DNA was isolated from whole blood using standart salt-out protocol. The polymerase chain reaction technique was used for gene amplification. Allele and genotype distribution and Hardy-Weinberg equilibrium was calculated by using PopGene32 software program. For BTN1A1 gene, A allele frequency was higher in East Anatolian red (EAR) and South Anatolian red (SAR) cattles. In TG gene, T allele frequency was higher in SAR breed but this frequency was lower in EAR cattle breed. For FABP3 gene, G allele frequency was lower in SAR breed but it was higher in EAR breed. The presented results should be confirmed in future investigations, taking into consideration all possible genotype at different loci and using other restriction enzymes for recognizing the variants.Keywords: BTN1A1, TG, FABP3, cattle, polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP)African Journal of Biotechnology Vol. 12(20), pp. 2802-280

Molecular detection of circulating thyroid specific transcripts (TSHR/Tg-mRNAs) in thyroid cancer patients: Their diagnostic significance

Thyroid cancer is the most prevalent endocrine malignancy. The preoperative diagnosis of differentiated thyroid cancer (DTC) that relies solely on fine-needle aspiration (FNAC) biopsy, sometimes possesses conflicting results. New molecular markers for thyroid cancer have been investigated with most of them based on the detection in thyroid nodules or tumor tissue specimens. Recently, it was possible to detect thyroid cancer cells in the circulation by measuring the mRNA of thyroid specific genes. Among these, thyroglobulin and more recently thyroid stimulating hormone receptor mRNAs, TSHR/Tg-mRNAs in peripheral blood might serve as cancer-specific markers. These have become promising new circulating markers for thyroid cancer. The purpose of this study is to assess TSHR/Tg-mRNAs as diagnostic molecular markers for thyroid cancer and if they can be used preoperatively in synergy with FNAC. This study was performed on 60 subjects; 20 healthy volunteers and 40 patients; including 16 patients with benign thyroid diseases, 24 patients with thyroid cancer; 18 patients with newly diagnosed (DTC) and 6 patients with recurrent thyroid cancer. Diagnosis of cancer was based on FNAC and histopathology of surgical specimens. All subjects had TSHR/Tg-mRNAs in peripheral blood measured by reverse transcriptase (RT)-PCR. Based on cytology/pathology; 18 patients had newly diagnosed DTC and 11 had benign thyroid disease. Preoperative FNAC was performed on 29 of 40 patients; FNAC was diagnostic in 11/18 of malignant lesions (61.1%), in 8/11 of benign lesions (72.7%), while 10/29 (34.5%) were indeterminate. TSHR/Tg-mRNAs correctly diagnosed DTC in 20/24 and 19/24 (sensitivity 83.3% and 79.1%) and benign disease in 14/16 and 13/16 (specificity 87.5% and 81.3%), respectively. With indeterminate FNA, TSHR/Tg-mRNAs correctly diagnosed DTC (follicular type) in 5/7 and benign disease in 2/3 (combined sensitivity 71.4%; specificity 66.7%). There was high concordance between RT-PCR results for TSHR-mRNA and Tg-mRNA. Of the controls 19/20 (95%) and 16/20 (80%) were negative for both TSHR- and Tg-mRNAs. With the use of a carefully selected primer pair and qualitative RT-PCR; our results indicate that TSHR/Tg-mRNAs in peripheral blood are both equally sensitive and specific markers for detection of thyroid cancer cells. Combining TSHR/Tg-mRNAs and FNAC and ultrasound enhances the preoperative detection of cancer in patients with thyroid nodules, reducing unnecessary surgeries and correctly classified most follicular cancers and could have spared surgery in patients with benign disease.Keywords: Differentiated thyroid Cancer; TSHR/Tg-mRNAs; Fine-needle aspiration cytology; Thyroid nodules; Indeterminate lesions; Molecular marke

Immunogenetics of Hashimoto's thyroiditis

Hashimoto's thyroiditis (HT) is an organ-specific T-cell mediated disease. It is a complex disease, with a strong genetic component. To date, significant progress has been made towards the identification and functional characterization of HT susceptibility genes. In this review, we will summarize the recent advances in our understanding of the genetic input to the pathogenesis of HT

A Large-Scale Zebrafish Gene Knockout Resource for the Genome-Wide Study of Gene Function

With the completion of the zebrafish genome sequencing project, it becomes possible to analyze the function of zebrafish genes in a systematic way. The first step in such an analysis is to inactivate each protein-coding gene by targeted or random mutation. Here we describe a streamlined pipeline using proviral insertions coupled with high-throughput sequencing and mapping technologies to widely mutagenize genes in the zebrafish genome. We also report the first 6144 mutagenized and archived F1’s predicted to carry up to 3776 mutations in annotated genes. Using in vitro fertilization, we have rescued and characterized ~0.5% of the predicted mutations, showing mutation efficacy and a variety of phenotypes relevant to both developmental processes and human genetic diseases. Mutagenized fish lines are being made freely available to the public through the Zebrafish International Resource Center. These fish lines establish an important milestone for zebrafish genetics research and should greatly facilitate systematic functional studies of the vertebrate genome

Influence of chondroitin 6-sulfate oligosaccharide unit addition on the immunopathogenicity of human thyroglobulin in cba/j(h-2k) mice. Multiple effects on antigen processing and t cell responses

The site of type D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg) was localized. Furthermore, hTg and its fractions endowed with chondroitin 6-sulfate oligosaccaride units (hTg-CS) and devoid of it (hTg-CS-), were compared, with respect to their ability to induce experimental autoimmune thyroiditis (EAT) in CBA/J(H-2k) mice, by subcutaneous administration, in the presence of complete adjuvant. HTg was chromatographically separated into hTg-CS and hTg-CS- molecules, on the base of their uronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6 percent. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondrotin-6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxy-terminal region, starting at residue 2513. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as being LTAGXGLRE (residues 2725-2733, X being Ser2729 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5’-triiodothyronine (T3) (171%) and 3,5,3’,5’-tetraiodothyronine (T4) (134%), than hTg-CS-. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T4 and, particularly, T3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Gly2713-Lys2714 from proteolysis, during the limited digestion of hTg-CS with trypsin. Although immunization with all forms of hTg was accompanied by thyroid cell damage, as judged from the increase of T4 in blood, a higher degree of mononuclear infiltration of the thyroid was associated with unfractionated hTg, in comparison both with hTg-CS and with hTg-CS-. Thus, it appears that both hTg subfractions contributed to the immunopathogenic potency of unfractionated hTg, as neither one reproduced fully the histological picture associated with the latter. Significant differences were observed also upon restimulation in vitro of splenic lymphocytes obtained from mice immunized in vivo with the different forms of hTg. Restimulation in vitro with hTg-CS of splenocytes from mice immunized with the same antigen was followed by low-level, dose-dependent proliferation and IFN-γ production, whereas cross-stimulation with hTg-CS- of the same cells was followed by proliferative and secretory responses of even lower degree. On the other hand, restimulation in vitro with hTg-CS- of splenocytes primed in vivo with the same antigen was followed by higher-level, dose-dependent increases of IFN-γ production, accompanied by proliferative responses of low degree and inversely related with the antigen dose, while cross-stimulation with hTg-CS of the same cells was followed by dose-dependent increases, both of proliferation and IFN-γ production, of the highest level observed in this study. Similar results were obtained when splenocytes, primed in vivo with hTg-CS-, were restimulated with purified glycopeptide hTg-CSgp, containing the chondroitin 6-sulfate unit, but not with its non-glycosylated, synthetic homologue. These data indicate that hTg-CS- was more effective than hTg-CS in priming autoreactive T lymphocytes, recognizing thyroiditogenic epitopes shared between murine and human Tg, whereas hTg-CS was a stronger inducer of proliferation of antigen-sensitized T cells. Moreover, different molecular signals, including structural determinant(s) associated with the chondroitin 6-sulfate chain, were required, in addition to epitope recognition, for the activation of T cell proliferation, together with IFN-γ production. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T4/T3 ratio in hTg, and may bear important implications in the processing and presentation of hTg as an autoantigen, and in the mechanisms of activation of Th-1-mediated and cytotoxic lymphocyte responses involved in EAT