Structure of the human hexokinase type I gene and nucleotide sequence of the 5′ flanking region

Abstract Abstract 1136 Introduction: Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation and variable bleeding tendency. Inherited genetic mutations in integrin alpha IIb and beta3 (ITGA2B, ITGB3) result in a heterogeneity of the thrombasthenia phenotypes. It is phenotypically expressed in homozygotes or compound heterozygotes, given that 50% of normal aIIbb3 is sufficient to guarantee unimpaired platelet function that result in asymptomatic carriers. Defects in ITGB3 result in failure of binding of B3 and alpha IIb. These defects had been reported in Arabs (Iraqi Jews). We are reporting some results of Saudi GT genotype project. Materials & Methods: In this study, we analyzed the entire coding region ITGB3 gene using polymerase chain reaction (PCR) and direct sequencing with primers specifically designed to amplify the coding region of exon 1–15 and exon /Intron boundaries in a cohort of 51 GT patients diagnosed and treated in our institute. Results: Out of 51 cases from 20 families had mutational screening of the ITGB3 gene with the aim to detect the causative pathogenic mutations to enable the pre-symptomatic diagnosis in at risk family members. In this study we detect 1 novel germline mutation c.2190delC (p.Ser703fs) in exon 13. The mutation is predicted to result in premature stop codon and protein truncation. The mutation was detected in 6 patients in homozygous stat (3 males and 3 females). Three tested samples from the patients family members detected the mutation in heterozygous state and all of them were asymptomatic with normal PFA and Intact expression of Platelet Glycoprotiens CD41(Gpllb), CD42a(GPIX), CD42b(GPlb), and CD61(Gpllla). All the GT patients with this mutation were type I GT with Prolonged PFA and complete absence of CD41(Gpllb) and CD61(Gpllla) glycoprotein. Conclusion: The result of this study represents the first Molecular analysis of ITGB3 gene in Saudi Arabia and displays the existence of novel pathogenic and possibly a founder effect in Saudi families. Disclosures: No relevant conflicts of interest to declare.

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Structure of the human aggrecan gene: exon-intron organization and association with the protein domains

Biochemical Journal ◽

10.1042/bj3090535 ◽

1995 ◽

Vol 309 (2) ◽

pp. 535-542 ◽

Cited By ~ 38

Author(s):

W B Valhmu ◽

G D Palmer ◽

P A Rivers ◽

S Ebara ◽

J F Cheng ◽

...

Keyword(s):

Yeast Artificial Chromosome ◽

Chondroitin Sulphate ◽

Direct Sequencing ◽

Regulatory Protein ◽

Protein Domains ◽

Artificial Chromosome ◽

Coding Region ◽

Rich Region ◽

Keratan Sulphate ◽

Protein Core

The complete exon-intron organization of the human aggrecan gene has been defined, and the exon organization has been compared with the individual domains of the protein core. A yeast artificial chromosome containing the aggrecan gene was selected from the Centre d'Etude du Polymorphisme Humaine yeast artificial chromosome library. A cosmid sulibrary was created from this, and direct sequencing of individual cosmids was used to provide the exon-intron organization. The human aggrecan gene was found to be composed of 19 exons ranging in size from 77 to 4224 bp. Exon 1 is non-coding, whereas exons 2-19 code for a protein core of 2454 amino acids with a calculated mass of 254379 Da. Intron 1 of the gene is at least 13 kb. Overall, the sizes of the 18 introns range from 0.5 to greater than 13 kb. Each intron begins with a GT and ends with an AG, thus obeying the GT/AG rule of splice-junction sequences. The entire coding region is contained in 39.4 kb of the gene. The organization of exons is strongly related to the specific domains of the protein core. The A loop of G1 and the interglobular domain are encoded by exons 3 and 7 respectively. The B and B' loops of G1 are encoded by exons 4-6, and those of G2 are encoded by exons 8-10. These sets of exons, coding for the B and B' loops, are identical in size and organization. This is supported by the intron classes associated with these exons. Exon 11 codes for the 5′ half of the keratan sulphate-rich region, and exon 12 codes for the 3′ half of the keratan sulphate-rich region as well as the entire chondroitin sulphate-rich region. G3 is encoded by exons 13-18, including the alternatively spliced epidermal growth factor-like and complement regulatory protein-like domains. The correspondence between the exon organization and the protein domains argues strongly for modular assembly of the aggrecan gene.

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Regulation of collagen I gene expression by ras

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4714-4723.1992 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4714-4723

Author(s):

J L Slack ◽

M I Parker ◽

V R Robinson ◽

P Bornstein

Keyword(s):

Type I Collagen ◽

The Body ◽

Transformed Cells ◽

Type I ◽

Mrna Levels ◽

Oncogenic Ras ◽

Genes Encoding ◽

Flanking Region ◽

Collagen Genes ◽

I Gene

Although transformation of rodent fibroblasts can lead to dramatic changes in expression of extracellular matrix genes, the molecular basis and physiological significance of these changes remain poorly understood. In this study, we have investigated the mechanism(s) by which ras affects expression of the genes encoding type I collagen. Levels of both alpha 1(I) and alpha 2(I) collagen mRNAs were markedly reduced in Rat 1 fibroblasts overexpressing either the N-rasLys-61 or the Ha-rasVal-12 oncogene. In fibroblasts conditionally transformed with N-rasLys-61, alpha 1(I) transcript levels began to decline within 8 h of ras induction and reached 1 to 5% of control levels after 96 h. In contrast, overexpression of normal ras p21 had no effect on alpha 1(I) or alpha 2(I) mRNA levels. Nuclear run-on experiments demonstrated that the transcription rates of both the alpha 1(I) and alpha 2(I) genes were significantly reduced in ras-transformed cells compared with those in parental cells. In addition, the alpha 1(I) transcript was less stable in transformed cells. Chimeric plasmids containing up to 3.6 kb of alpha 1(I) 5'-flanking DNA and up to 2.3 kb of the 3'-flanking region were expressed at equivalent levels in both normal and ras-transformed fibroblasts. However, a cosmid clone containing the entire mouse alpha 1(I) gene, including 3.7 kb of 5'- and 4 kb of 3'-flanking DNA, was expressed at reduced levels in fibroblasts overexpressing oncogenic ras. We conclude that oncogenic ras regulates the type I collagen genes at both transcriptional and posttranscriptional levels and that this effect, at least for the alpha 1(I) gene, may be mediated by sequences located either within the body of the gene itself or in the distal 3'-flanking region.

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Regulation of collagen I gene expression by ras.

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4714 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4714-4723 ◽

Cited By ~ 44

Author(s):

J L Slack ◽

M I Parker ◽

V R Robinson ◽

P Bornstein

Keyword(s):

Type I Collagen ◽

The Body ◽

Transformed Cells ◽

Type I ◽

Mrna Levels ◽

Oncogenic Ras ◽

Genes Encoding ◽

Flanking Region ◽

Collagen Genes ◽

I Gene

Although transformation of rodent fibroblasts can lead to dramatic changes in expression of extracellular matrix genes, the molecular basis and physiological significance of these changes remain poorly understood. In this study, we have investigated the mechanism(s) by which ras affects expression of the genes encoding type I collagen. Levels of both alpha 1(I) and alpha 2(I) collagen mRNAs were markedly reduced in Rat 1 fibroblasts overexpressing either the N-rasLys-61 or the Ha-rasVal-12 oncogene. In fibroblasts conditionally transformed with N-rasLys-61, alpha 1(I) transcript levels began to decline within 8 h of ras induction and reached 1 to 5% of control levels after 96 h. In contrast, overexpression of normal ras p21 had no effect on alpha 1(I) or alpha 2(I) mRNA levels. Nuclear run-on experiments demonstrated that the transcription rates of both the alpha 1(I) and alpha 2(I) genes were significantly reduced in ras-transformed cells compared with those in parental cells. In addition, the alpha 1(I) transcript was less stable in transformed cells. Chimeric plasmids containing up to 3.6 kb of alpha 1(I) 5'-flanking DNA and up to 2.3 kb of the 3'-flanking region were expressed at equivalent levels in both normal and ras-transformed fibroblasts. However, a cosmid clone containing the entire mouse alpha 1(I) gene, including 3.7 kb of 5'- and 4 kb of 3'-flanking DNA, was expressed at reduced levels in fibroblasts overexpressing oncogenic ras. We conclude that oncogenic ras regulates the type I collagen genes at both transcriptional and posttranscriptional levels and that this effect, at least for the alpha 1(I) gene, may be mediated by sequences located either within the body of the gene itself or in the distal 3'-flanking region.

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Organization of the human β-1,2-N-acetylglucosaminyltransferase I gene (MGAT1), which controls complex and hybrid N-glycan synthesis

Biochemical Journal ◽

10.1042/bj3210465 ◽

1997 ◽

Vol 321 (2) ◽

pp. 465-474 ◽

Cited By ~ 29

Author(s):

Betty YIP ◽

Shi-Hao CHEN ◽

Hans MULDER ◽

Jo W. M. HÖPPENER ◽

Harry SCHACHTER

Keyword(s):

Genomic Dna ◽

Transient Transfection ◽

Transcription Start ◽

Exon 2 ◽

Transcription Start Sites ◽

Exon 1 ◽

Dna Fragment ◽

Flanking Region ◽

Ribonuclease Protection ◽

I Gene

UDP-GlcNAc:α-3-d-mannoside α-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101; GlcNAc-T I) is a medial-Golgi enzyme which catalyses the first step in the conversion of oligomannose-type to N-acetyl-lactosamine- and hybrid-type N-glycans and is essential for normal embryogenesis in the mouse. Previous work indicated the presence of at least two exons in the human GlcNAc-T I gene MGAT1, exon 2 containing part of the 5ƀ untranslated region and the complete coding and 3ƀ untranslated regions, and exon 1 with the remainder of the 5ƀ untranslated region. We now report the cloning and sequencing of a human genomic DNA fragment containing exon 1, which is between 5.6 and 15 kb upstream of exon 2. Transient transfection, ribonuclease protection and reverse transcriptase-mediated PCR indicated the absence of transcription start sites in intron 1 between exons 1 and 2. Northern analysis, ribonuclease protection, primer extension analysis and rapid amplification of 5ƀ-cDNA ends showed that there are multiple transcription start sites for exon 1 compatible with the expression by several human cell lines and tissues of two transcripts, a broad band ranging in size from 2.7 to 3.0 kb and a sharper band at 3.1 kb. The 5ƀ flanking region of exon 1 has a GC content of 81% and has no canonical TATA or CCAAT boxes but contains potential binding sites for transcription factors Sp1, GC-binding factor and epidermal growth factor receptor-specific transcription factor. Chloramphenicol acetyltransferase (CAT) expression was observed on transient transfection into HeLa cells of a fusion construct containing the gene for CAT and a genomic DNA fragment from the 5ƀ flanking region of exon 1. It is concluded that MGAT1 is a typical housekeeping gene although there is, in addition, tissue-specific expression of the larger 3.1 kb transcript.

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Structure of the human hexokinase type I gene

Biochemical Society Transactions ◽

10.1042/bst025070s ◽

1997 ◽

Vol 25 (1) ◽

pp. 70S-70S ◽

Cited By ~ 1

Author(s):

ANNAMARIA RUZZO ◽

FRANCESCA ANDREONI ◽

MAURO MAGNANI

Keyword(s):

Type I ◽

I Gene ◽

Hexokinase Type I

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Polymorphism exon 1 variant at the locus of the scavenger receptor class B type I gene: influence on plasma LDL cholesterol in healthy subjects during the consumption of diets with different fat contents

American Journal of Clinical Nutrition ◽

10.1093/ajcn/77.4.809 ◽

2003 ◽

Vol 77 (4) ◽

pp. 809-813 ◽

Cited By ~ 42

Author(s):

Pablo Pérez-Martínez ◽

José M Ordovás ◽

José López-Miranda ◽

Purificación Gómez ◽

Carmen Marín ◽

...

Keyword(s):

Healthy Subjects ◽

Ldl Cholesterol ◽

Scavenger Receptor ◽

Type I ◽

Scavenger Receptor Class ◽

Class B ◽

Exon 1 ◽

I Gene

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A Deletion in the Gene for Transforming Growth Factor β Type I Receptor Abolishes Growth Regulation by Transforming Growth Factor β in a Cutaneous T-Cell Lymphoma

Blood ◽

10.1182/blood.v94.8.2854.420k07_2854_2861 ◽

1999 ◽

Vol 94 (8) ◽

pp. 2854-2861 ◽

Cited By ~ 91

Author(s):

William P. Schiemann ◽

Walther M. Pfeifer ◽

Edi Levi ◽

Marshall E. Kadin ◽

Harvey F. Lodish

Keyword(s):

Growth Factor ◽

Cell Line ◽

Transforming Growth Factor ◽

Cell Lymphoma ◽

Transforming Growth Factor Β ◽

Skin Lesions ◽

Cdna Clones ◽

Type I ◽

Exon 1 ◽

I Gene

Spontaneous regression of skin lesions is characteristic of lymphomatoid papulosis (LyP), a clonal cutaneous lymphoproliferative disorder. A minority of LyP patients progress to anaplastic large cell lymphoma (ALCL) in which skin lesions no longer regress and extracutaneous dissemination often occurs. In 1 such case, we developed a tumor cell line, JK cells, and show that these cells are resistant to the growth inhibitory effects of transforming growth factor β (TGF-β) due to the loss of cell surface expression of the TGF-β type I receptor (TβR-I). Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing of JK cell TβR-I cDNA clones identified a deletion that spanned the last 178 bp of exon 1, including the initiating methionine. Hybridization of a radiolabeled fragment internal to the deletion was detected in the genomes of TGF-β–responsive cells, but not in JK cells, indicating that they contain no wild-type TβR-I gene. PCR primers that flanked the deleted TβR-I region amplified a single band from JK cell genomic DNA that lacked the last 178 bp of exon 1 and all of the ≈ 5 kb of intron 1. This JK cell-specific genomic TβR-I PCR product was distinct from products amplified from TGF-β–responsive cells and was also readily detected in tumor biopsies obtained before the establishment of the JK cell line. Our results identify the first inactivating mutation in TβR-I gene in a human lymphoma that renders it insensitive to growth inhibition by TGF-β.

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Genomic organization, 5′-flanking region and chromosomal localization of the human glutathione transferase A4 gene

Biochemical Journal ◽

10.1042/bj3360437 ◽

1998 ◽

Vol 336 (2) ◽

pp. 437-442 ◽

Cited By ~ 22

Author(s):

Fabienne DESMOTS ◽

Claudine RAUCH ◽

Catherine HENRY ◽

André GUILLOUZO ◽

Fabrice MOREL

Keyword(s):

Transcription Initiation ◽

Glutathione Transferase ◽

Yeast Artificial Chromosome ◽

Stop Codon ◽

Artificial Chromosome ◽

Initiation Site ◽

Pcr Analysis ◽

Small Nuclear Rna ◽

Exon 2 ◽

Flanking Region

We have isolated and characterized a human glutathione transferase A4 (hGSTA4) subunit gene from a yeast artificial chromosome containing several other glutathione transferase alpha genes and pseudogenes. The homodimeric protein hGSTA4-4, is involved in the detoxification of 4-hydroxynonenal and other reactive electrophiles produced by oxidative metabolism, and may have a significant role in protecting intracellular components from oxidative damage. The hGSTA4 gene spans nearly 18 kb, contains seven exons, maps onto chromosome 6p12, and lies in close proximity to the 7SK small nuclear RNA gene in a head-to-tail orientation. The intron/exon borders conform to the standard rules, an open reading frame is present beginning at position 154 in exon 2, and the stop codon is at position 822 in exon 7. The transcription initiation site has been determined by primer extension analysis and is located 135 bp upstream of intron 1. Isolation and sequencing of the hGSTA4 gene 5´-flanking region revealed it to be devoid of TATA or CCAAT boxes but it does contain an initiator element overlapping the transcription start site, a GC box and putative binding sites for transcription factors AP1, STAT, GATA1 and NF-κB. Reverse transcription-PCR analysis revealed that hGSTA4 mRNA was present in all the tissues tested, although in low amounts, suggesting that this subunit may be ubiquitously expressed.

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