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

1997 ◽  
Vol 321 (2) ◽  
pp. 465-474 ◽  
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.

Genomic organization of the 5′ end of human β-ENaC and preliminary characterization of its promoter

2002 ◽  
Vol 282 (5) ◽  
pp. F898-F909 ◽  
Author(s):  
Christie P. Thomas ◽  
Randy W. Loftus ◽  
Kang Z. Liu ◽  
Omar A. Itani
Keyword(s):  
Genomic Organization ◽  
Human Kidney ◽  
Β Subunit ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Nuclear Extracts ◽  
Flanking Region ◽  
Rapid Amplification ◽  
Flanking Regions

The mRNA for the β-subunit of the epithelial Na+ channel (β-ENaC) is regulated developmentally and, in some tissues, in response to corticosteroids. To understand the mechanisms of transcriptional regulation of the human β-ENaC gene, we characterized the 5′ end of the gene and its 5′-flanking regions. Adaptor-ligated human kidney and lung cDNA were amplified by 5′ rapid amplification of cDNA ends, and transcription start sites of two 5′ variant transcripts were determined by nuclease protection or primer extension assays. Cosmid clones that contain the 5′ end of the gene were isolated, and analysis of these clones indicated that alternate first exons ∼1.5 kb apart and ∼ 45 kb upstream of a common second exon formed the basis of these transcripts. Genomic fragments that included the proximal 5′-flanking region of either transcript were able to direct expression of a reporter gene in lung epithelia and to bind Sp1 in nuclear extracts, confirming the presence of separate promoters that regulate β-ENaC expression.

Identification of multiple transcription start sites in the human insulin-like growth factor-I gene

1991 ◽  
Vol 78 (1-2) ◽  
pp. 115-125 ◽  
Author(s):  
E. Jansen ◽  
P.H. Steenbergh ◽  
D. LeRoith ◽  
C.T. Roberts ◽  
J.S. Sussenbach
Keyword(s):  
Growth Factor ◽  
Human Insulin ◽  
Insulin Like Growth Factor ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Factor I ◽  
Growth Factor I ◽  
I Gene

Alternate Promoters Direct Tissue-Specific Expression of Erythrocyte Ankyrin Transcripts with Novel NH2-Termini.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1710-1710
Author(s):  
Laurie A. Steiner ◽  
Jolinta Y. Lin ◽  
Ashley N. Owens ◽  
Jose I. Sangerman ◽  
David M. Bodine ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Genomic Dna ◽  
Hereditary Spherocytosis ◽  
Luciferase Expression ◽  
Erythroid Cells ◽  
Exon 2 ◽  
Tissue Specific ◽  
Exon 1 ◽  
Alternate Promoters

Abstract Mutations in erythrocyte ankyrin, ankyrin-1, are the most common cause of typical hereditary spherocytosis. Co-inheritance of cardiac, muscular, and neurologic diseases such as cardiomyopathy, psychomotor retardation, and spinocerebellar abnormalities with hereditary spherocytosis has been described. In the nb/nb mouse, an ankyrin-1 mutation manifests in erythroid cells with ankyrin deficiency and a spherocytosis phenotype, and in neural cells with an age-dependent psychomotor disorder due to loss of cerebellar Purkinje cells. These observations highlight the importance of understanding ankyrin-1 structure, function, and regulation in erythroid and nonerythroid cells. In erythroid cells, ankyrin expression is directed by a compact promoter controlled by a single GATA-1 site. Nonerythroid ankyrin-1 isoforms have been described with diversity arising from alternate splicing, alternate polyadenylation, and, in skeletal muscle, use of an alternate, tissue-specific promoter. Using 5′ RACE, we identified 2 additional alternate first exons of the ankyrin-1 cDNA. One encoded a first exon with an initiator methionine followed by 12 amino acids, designated exon 1A, that spliced in-frame to erythroid exon 2 sequences. The other, designated 1B, encoded a novel initiator methionine followed by 40 highly charged amino acids that also spliced in-frame to the erythroid exon 2. Both exons, found in human and mouse, link directly to the downstream exons encoding the ankyrin repeat and spectrin binding domains of ankyrin-1. Exon 1B mapped to a location 98.5 kb 5′ of erythroid exon 1 (1E) and exon 1A mapped 30.1 kb 3′ of exon 1E. Northern blot and quantitative RT-PCR analyses demonstrated that 1B was expressed in heart, skeletal muscle, and brain. Similar to what we previously reported for the promoter of the erythroid-specific exon 1, 1E, DNase I hypersensitive site (HS) mapping identified a pair of HS in genomic DNA, one immediately 5′ of exon 1B and one that mapped 6.6 kb downstream of this site (chr8:41,873,229 and 41,866,593, UCSC assembly, Mar 2006). Luciferase-reporter gene expression studies with plasmids containing either a 700bp or a 278bp fragment of the 1B flanking sequence directed high-level luciferase expression in RD cells (human rhabdomyosarcoma) but no luciferase expression in K562 (erythroid) or HeLa (fibroblastoid) cells. Consistent with its tissue-restricted pattern of expression, a polyclonal antipeptide antibody raised against novel sequence in exon 1B reacted with peptides of 220, 205, 45, and 40 kDa on immunoblots prepared from muscle and brain but not erythrocytes. In contrast to erythroid-specific exon 1E and tissue-restricted exon 1B, mRNAs containing exon 1A were detected in all 18 tissues examined. Like the 1E and 1B promoters, mapping identified an HS in the 5′ flanking genomic DNA/promoter region of exon 1A and another 6 kb downstream (chr8:41,873,229 and 41,866,593). An exon 1A anti-peptide antibody reacted with peptides of 205, 195, and 190 kDa on immunoblots prepared from numerous tissues, including erythrocytes ghosts. Regulation of ankyrin-1 expression by alternate promoters directing novel NH2-termini provides the basis for a complex pattern of tissue-specific ankyrin-1 isoform diversity. Characterization of the downstream alternate exon composition of the tissue-specific exon 1B and ubiquitous exon 1A-containing transcripts will allow a systematic evaluation of whether a specific spherocytosis-linked ankyrin-1 mutation could lead to a nonerythroid phenotype.

scholarly journals Cloning and characterization of the promoter for the liver isoform of the rat carnitine palmitoyltransferase I (L-CPT I) gene

1998 ◽  
Vol 330 (1) ◽  
pp. 217-224 ◽  
Author(s):  
A. Edwards PARK ◽  
L. Michelle STEFFEN ◽  
Shulan SONG ◽  
M. Vicki PARK ◽  
A. George COOK
Keyword(s):  
Genomic Library ◽  
Transcriptional Start Site ◽  
Carnitine Palmitoyltransferase ◽  
Proximal Promoter ◽  
Exon 2 ◽  
Exon 1 ◽  
Carnitine Palmitoyltransferase I ◽  
Specific Regulation ◽  
Cpt I ◽  
I Gene

Carnitine palmitoyltransferase I (CPT I) catalyses the transfer of long chain fatty acids to carnitine for translocation across the mitochondrial inner membrane. The cDNAs of two isoforms of CPT I, termed the hepatic and muscle isoforms, have been cloned. Expression of the hepatic CPT I gene (L-CPT I) is subject to developmental, hormonal and tissue specific regulation. We have cloned the promoter of the L-CPT I gene from a rat genomic library. In the L-CPT I gene, there are two exons 5ʹ to the exon containing the ATG that initiates translation. Exon 1 and the 5ʹ end of exon 2 contain sequences that were not previously described in the rat L-CPT I cDNA. There is an alternatively spliced form of the L-CPT I mRNA in which exon 2 is skipped. The proximal promoter of the L-CPT I gene is extremely GC rich and does not contain a TATA box. There are several putative Sp1 binding sites near the transcriptional start site. A 190 base pair fragment of the promoter can efficiently drive transcription of luciferase and CAT (chloramphenicol acetyltransferase) reporter genes transiently transfected into HepG2 cells. Sequences in both the first intron and the promoter contribute to basal expression. Our results provide the foundation for further studies into the regulation of L-CPTI gene expression.

scholarly journals Molecular characterization of erythrocyte glycophorin C variants

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 644-648
Author(s):  
S Chang ◽  
ME Reid ◽  
J Conboy ◽  
YW Kan ◽  
N Mohandas
Keyword(s):  
Molecular Characterization ◽  
Mechanical Stability ◽  
Exon 2 ◽  
Mrna Sequencing ◽  
Erythrocyte Shape ◽  
Altered Glycosylation ◽  
Exon 1 ◽  
Dna Fragment ◽  
Glycophorin C

Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. Immunochemical and serologic studies have identified a number of glycophorin C variants that include the Yus, Gerbich, and Webb phenotypes. We report here the molecular characterization of these variants. Amplification of glycophorin C mRNA from the Yus phenotype, using two oligonucleotide primers that span the coding domain, generated a 338-bp fragment compared with a 395-bp fragment generated by amplification of normal glycophorin C mRNA. Sequencing of the mutant 338-bp fragment identified a 57-bp deletion that corresponds to exon 2 of the glycophorin C gene. Similar analysis showed deletion of 84-bp exon 3 in the Gerbich phenotype. In contrast to the generation of shorter than normal DNA fragments from mRNA amplification in the Yus and Gerbich phenotypes, amplification of mRNA from the Webb phenotype generated a normal-sized fragment. Sequencing of this DNA fragment showed an A----G substitution at nucleotide 23 of the coding sequence, resulting in the substitution of asparagine by serine. This modification accounts for the altered glycosylation of glycophorin C seen in this phenotype. These results have enabled us to characterize glycophorin C variants in three different phenotypes that involve deletions of exons 2 and 3 of the glycophorin C gene, as well as a point mutation in exon 1 that results in altered glycosylation of this protein.

scholarly journals The promoter and first, untranslated exon of the human glucocorticoid receptor gene are GC rich but lack consensus glucocorticoid receptor element sites.

1990 ◽  
Vol 10 (10) ◽  
pp. 5580-5585 ◽  
Author(s):  
J Zong ◽  
J Ashraf ◽  
E B Thompson
Keyword(s):  
Glucocorticoid Receptor ◽  
Receptor Gene ◽  
Flanking Sequence ◽  
Exon 2 ◽  
Transcription Start Sites ◽  
Untranslated Exon ◽  
Glucocorticoid Response ◽  
Glucocorticoid Receptor Gene ◽  
Exon 1 ◽  
Glucocorticoid Receptor Mrna

Glucocorticoid receptor mRNA is regulated by glucocorticoids. We found no consensus glucocorticoid response element, TATA box, or CAAT box but many GC boxes in approximately 3 kilobases of the 5'-flanking sequence of the human glucocorticoid receptor gene. We identified several transcription start sites, an untranslated exon 1, and the coding content of exon 2.

scholarly journals Molecular characterization of erythrocyte glycophorin C variants

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 644-648 ◽  
Author(s):  
S Chang ◽  
ME Reid ◽  
J Conboy ◽  
YW Kan ◽  
N Mohandas
Keyword(s):  
Molecular Characterization ◽  
Mechanical Stability ◽  
Exon 2 ◽  
Mrna Sequencing ◽  
Erythrocyte Shape ◽  
Altered Glycosylation ◽  
Exon 1 ◽  
Dna Fragment ◽  
Glycophorin C

Abstract Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. Immunochemical and serologic studies have identified a number of glycophorin C variants that include the Yus, Gerbich, and Webb phenotypes. We report here the molecular characterization of these variants. Amplification of glycophorin C mRNA from the Yus phenotype, using two oligonucleotide primers that span the coding domain, generated a 338-bp fragment compared with a 395-bp fragment generated by amplification of normal glycophorin C mRNA. Sequencing of the mutant 338-bp fragment identified a 57-bp deletion that corresponds to exon 2 of the glycophorin C gene. Similar analysis showed deletion of 84-bp exon 3 in the Gerbich phenotype. In contrast to the generation of shorter than normal DNA fragments from mRNA amplification in the Yus and Gerbich phenotypes, amplification of mRNA from the Webb phenotype generated a normal-sized fragment. Sequencing of this DNA fragment showed an A----G substitution at nucleotide 23 of the coding sequence, resulting in the substitution of asparagine by serine. This modification accounts for the altered glycosylation of glycophorin C seen in this phenotype. These results have enabled us to characterize glycophorin C variants in three different phenotypes that involve deletions of exons 2 and 3 of the glycophorin C gene, as well as a point mutation in exon 1 that results in altered glycosylation of this protein.

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

1998 ◽  
Vol 331 (2) ◽  
pp. 607-613 ◽  
Author(s):  
Annamaria RUZZO ◽  
Francesca ANDREONI ◽  
Mauro MAGNANI
Keyword(s):  
Yeast Artificial Chromosome ◽  
Direct Sequencing ◽  
Artificial Chromosome ◽  
Type I ◽  
Coding Region ◽  
Exon 1 ◽  
Flanking Region ◽  
The Individual ◽  
I Gene ◽  
Hexokinase Type I

This study reports the precise intron/exon boundaries and intron/exon composition of the human hexokinase type I gene. A yeast artificial chromosome containing the hexokinase type I gene was isolated from the yeast artificial chromosome library of the Centre d'Étude du Polymorphisme Humaine. A cosmid sublibrary was created and direct sequencing of the individual cosmids was used to provide the exon/intron organization. The human hexokinase type I gene was found to be composed of 18 exons ranging in size from 63 to 305 bp. Intron 1 is at least 15 kb in length, whereas intron 2 spans at least 10 kb. Overall, the length of the 17 introns ranges from 104 to greater than 15 kb. The entire coding region is contained in at least 75 kb of the gene. The structure of the gene reveals a remarkable conservation of the size of the exons compared with glucokinase and hexokinase type II. Isolation of the 5´ flanking region of the gene revealed a 75–90% identity with the rat sequence. Direct evidence of an alternative red-blood-cell-specific exon 1 located upstream of the 5´ flanking region of the gene is also provided.

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