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 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 Molecular analysis of glycophorin C deficiency in human erythrocytes

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2799-2803
Author(s):  
R Winardi ◽  
M Reid ◽  
J Conboy ◽  
N Mohandas
Keyword(s):  
Mechanical Stability ◽  
Mutant Gene ◽  
Erythrocyte Shape ◽  
Normal Individuals ◽  
Pcr Products ◽  
Protein Isoform ◽  
Polymerase Chain ◽  
Exon 4 ◽  
Glycophorin C

Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. We report here the characterization of the glycophorins C and D deficiency in erythrocytes of the Leach phenotype. Glycophorin C gene is encoded by 4 exons. Amplification of reticulocyte cDNA from Leach phenotype and normal individuals generated a 140-bp fragment when using primers spanning exons 1 and 2. However, no polymerase chain reaction (PCR) products were detected in the Leach phenotype using primers flanking either exons 1 and 3 or exons 1 and 4, suggesting that the 3' end of the mRNA was missing or altered. Exon 4 also appeared to be missing from Leach genomic DNA, based on both Southern hybridization and PCR. These results indicate that an absence of glycophorin C and glycophorin D in erythrocytes from these Leach phenotype individuals is a consequence of a deletion or marked alteration of exon 3 and exon 4 of their glycophorin C gene. Surprisingly, the mutant gene encodes an mRNA stable enough to be detected in circulating reticulocytes. Although this mRNA could encode an N-terminal fragment of glycophorin C, these protein isoform(s) would not be expressed in the membrane because they lack the transmembrane and cytoplasmic domains.

scholarly journals Molecular analysis of glycophorin C deficiency in human erythrocytes

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2799-2803 ◽  
Author(s):  
R Winardi ◽  
M Reid ◽  
J Conboy ◽  
N Mohandas
Keyword(s):  
Mechanical Stability ◽  
Mutant Gene ◽  
Erythrocyte Shape ◽  
Normal Individuals ◽  
Pcr Products ◽  
Protein Isoform ◽  
Polymerase Chain ◽  
Exon 4 ◽  
Glycophorin C

Abstract Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. We report here the characterization of the glycophorins C and D deficiency in erythrocytes of the Leach phenotype. Glycophorin C gene is encoded by 4 exons. Amplification of reticulocyte cDNA from Leach phenotype and normal individuals generated a 140-bp fragment when using primers spanning exons 1 and 2. However, no polymerase chain reaction (PCR) products were detected in the Leach phenotype using primers flanking either exons 1 and 3 or exons 1 and 4, suggesting that the 3' end of the mRNA was missing or altered. Exon 4 also appeared to be missing from Leach genomic DNA, based on both Southern hybridization and PCR. These results indicate that an absence of glycophorin C and glycophorin D in erythrocytes from these Leach phenotype individuals is a consequence of a deletion or marked alteration of exon 3 and exon 4 of their glycophorin C gene. Surprisingly, the mutant gene encodes an mRNA stable enough to be detected in circulating reticulocytes. Although this mRNA could encode an N-terminal fragment of glycophorin C, these protein isoform(s) would not be expressed in the membrane because they lack the transmembrane and cytoplasmic domains.

Molecular characterization of 9p21 deletions shows a minimal common deleted region removing CDKN2A exon 1 and CDKN2B exon 2 in diffuse large b-cell lymphomas

2011 ◽  
Vol 50 (9) ◽  
pp. 715-725 ◽  
Author(s):  
Suzan Guney ◽  
Philippe Bertrand ◽  
Fabrice Jardin ◽  
Philippe Ruminy ◽  
Jean Pierre Kerckaert ◽  
...  
Keyword(s):  
B Cell ◽  
Molecular Characterization ◽  
B Cell Lymphomas ◽  
Exon 2 ◽  
Exon 1 ◽  
Large B Cell

The mouse carbonic anhydrase I gene contains two tissue-specific promoters

1989 ◽  
Vol 9 (8) ◽  
pp. 3308-3313
Author(s):  
P Fraser ◽  
P Cummings ◽  
P Curtis
Keyword(s):  
Carbonic Anhydrase ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Exon 2 ◽  
Tissue Specific ◽  
Isolation And Characterization ◽  
Carbonic Anhydrase I ◽  
Erythroleukemia Cells ◽  
Exon 1

We report the isolation and characterization of the mouse carbonic anhydrase I (CAI) gene. Direct RNA sequence analysis of the 5' nontranslated regions of CAI mRNA from mouse colon and mouse erythroleukemia cells demonstrated tissue specificity in the lengths and sequences of CAI transcripts. Analysis of several mouse CAI genomic clones showed that the transcripts arose from a single CAI gene with two tissue-specific promoters and eight exons. CAI transcripts in the colon were found to initiate just upstream of the erythroid exon 2 of the CAI gene region sequence. Erythroid transcripts originated from a novel promoter upstream of exon 1, which was located more than 10 but less than 250 kilobases upstream of exon 2. Erythroid exon 1 contained only a nontranslated sequence, which was spliced to exon 2 via a cryptic splice acceptor site located in the region that encoded the colon mRNA 5' nontranslated sequence. The remaining exon-intron junctions were conserved in comparison with those of the CAII and CAIII genes.

Characterization of NRG1 gene fusion events in solid tumors.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 3113-3113
Author(s):  
Sushma Jonna ◽  
Rebecca Feldman ◽  
Sai-Hong Ignatius Ou ◽  
Misako Nagasaka ◽  
Jeffrey Swensen ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Fusion Partner ◽  
Exon 2 ◽  
Downstream Signaling ◽  
Chimeric Transcripts ◽  
Exon 1 ◽  
Tumor Types ◽  
Fusion Products ◽  
Ig Domain

3113 Background: NRG1 fusions are actionable genomic alterations detected across tumor types. The NRG1 gene encode for neuregulin, which serves as a ligand for ERBB3 and ERBB4 receptors and activates downstream signaling through the MAPK and PI3K pathways. Here, we update the detection of NRG1 gene fusions across tumor types and further describe fusion characteristics. Methods: Samples submitted for clinical molecular profiling that included RNA-sequencing (Archer Dx or Caris MI transcriptome) were retrospectively analyzed for NRG1 fusion events. All NRG1 fusions with ≥ 3 junction reads were identified for manual review and for characterization of fusion class, intact functional domains, domain prediction, breakpoints, frame retention and co-occurring alterations by NGS. Results: A total of 82 NRG1 fusion events (0.2% of 44,570) were identified. Among the fusions identified, the distribution across tumor types was as follows: non-small cell lung cancer (NSCLC, 54%), breast cancer (11%), ovarian cancer (7%), pancreatic cancer (7%), cholangiocarcinoma (6%), colorectal cancer (5%), and other (10%). Forty-two unique fusion partners were identified, the most common being CD74 (23%), ATP1B1 (9%), SLC3A2 (7%), RBPMS (6%) and SDC4 (4%). Almost half (47%) of all fusion events are expected to include the transmembrane domain contributed by the NRG1 fusion partner. Lung and pancreatobilliary cancers had the highest rates of transmembrane domain retention from their fusion partners (63.6% and 54.5%, respectively). In all other tumor groups, most fusion partners lacked transmembrane domains. In 15% of cases, the chimeric transcripts are predicted to lead to increased expression of NRG1. The most commonly reported breakpoints in NRG1 occur in exon 6 and exon 2. While fusions with the NRG1 breakpoint at exon 2 retain the immunoglobulin (Ig) domain and all downstream portions (including EGF-like domain), those at exon 6 do not contain the Ig portion and result in shorter chimeric proteins. The breakpoints in all CD74:NRG1 fusions, the most common fusions in NSCLC, occur at exon 5 or 6 and cause truncation of domains upstream of the EGF-like domain. In ATP1B1:NRG1 fusions, the most common fusions in pancreatobilliary cancers, the breakpoints are at exon 1 or 2 and retain the Ig domain. Conclusions: NRG1 fusion products are diverse across tumor types, but the significance of these variations is not clear. The biological and clinical implications of retaining certain domains of NRG1 (such as the Ig domain) and of fusion partners warrants further investigation.

scholarly journals Molecular Characterization of the PceA Reductive Dehalogenase of Desulfitobacterium sp. Strain Y51

2002 ◽  
Vol 184 (13) ◽  
pp. 3419-3425 ◽  
Author(s):  
Akiko Suyama ◽  
Masaki Yamashita ◽  
Sadazo Yoshino ◽  
Kensuke Furukawa
Keyword(s):  
Escherichia Coli ◽  
Molecular Characterization ◽  
Specific Activity ◽  
Chlorinated Ethenes ◽  
Reductive Dehalogenation ◽  
Reductive Dehalogenase ◽  
Dna Fragment

ABSTRACT The tetrachloroethene (PCE) reductive dehalogenase (encoded by the pceA gene and designated PceA dehalogenase) of Desulfitobacterium sp. strain Y51 was purified and characterized. The expression of the enzyme was highly induced in the presence of PCE and trichloroethene (TCE). The purified enzyme catalyzed the reductive dehalogenation of PCE via TCE to cis-1,2-dichloroethene at a specific activity of 113.6 nmol · min−1 · mg of protein−1. The apparent Km values for PCE and TCE were 105.7 and 535.3 μM, respectively. Chlorinated ethenes other than PCE and TCE were not dehalogenated. However, the enzyme exhibited dehalogenation activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane. The pceA gene of Desulfitobacterium sp. strain Y51 was identified in a 2.8-kb DNA fragment and used to express the protein in Escherichia coli for the preparation of antibodies. Immunoblot analyses located PceA in the periplasm of the cell.

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.

Molecular characterization of Ovar-DRB1 exon2 gene in Garole sheep resilient to gastrointestinal nematodes

2015 ◽  
Author(s):  
C. Paswan ◽  
L. L.L. Prince ◽  
R. Kumar ◽  
C. P. Swarnkar ◽  
D. Singh ◽  
...  
Keyword(s):  
Disease Resistance ◽  
Molecular Characterization ◽  
Molecular Mechanisms ◽  
Gastrointestinal Nematodes ◽  
Exon 2 ◽  
Pcr Rflp

Ovine MHC DRB1 exon 2 (Ovar-DRB1.2) gene is arguably one of the critical genes, responsible for disease resistance against parasite in animals. This study was carried out for indepth analysis of polymorphism in MHC DRB1.2 gene and to explore the underlying molecular mechanisms for the development of disease resistance in Garole. PCR-RFLP study revealed that the gene is polymorphic in nature. The frequency of allele ‘A’ for endonuclease SacI and Allele ‘B’ for endonuclease Hin1I were significantly higher in Garole population.

scholarly journals Molecular characterization of novel germline deletions affecting SDHD and SDHC in pheochromocytoma and paraganglioma patients

2009 ◽  
Vol 16 (3) ◽  
pp. 929-937 ◽  
Author(s):  
Jean-Pierre Bayley ◽  
Marjan M Weiss ◽  
Anneliese Grimbergen ◽  
Bernadette T J van Brussel ◽  
Frederik J Hes ◽  
...  
Keyword(s):  
Nonsense Mutations ◽  
Gene Deletions ◽  
Exon 2 ◽  
Gene Insertion ◽  
Large Deletions ◽  
Exon 1 ◽  
Long Range Pcr ◽  
Intron 2 ◽  
Dependent Probe

A major cause of paraganglioma and pheochromocytoma is germline mutation of the tumor suppressor genes SDHB, SDHC, and SDHD, encoding subunits of succinate dehydrogenase (SDH). While many SDH missense/nonsense mutations have been identified, few large deletions have been described. We performed multiplex ligation-dependent probe amplification deletion analysis in 126 point mutation-negative patients, and here we describe four novel deletions of SDHD and SDHC. Long-range PCR was used for the fine mapping of deletions. One patient had a 10 kb AluSg–AluSx-mediated deletion including SDHD exons 1 and 2, the entire TIMM8B gene, and deletion of exons of C11orf57. A second patient had a deletion of SDHD exons 1 and 2 and exon 1 of the TIMM8B gene. A third patient showed a deletion of exon 2 of SDHD, together with a 235 bp MIRb–Tensin gene insertion. In a fourth patient, a deletion of exons 5 and 6 of the SDHC gene was found, only the second SDHC deletion currently known. The deletions of the TIMM8B and C11orf57 genes are the first to be described, but do not appear to result in an additional phenotype in these patients. Four of the eight breakpoints occurred in Alu sequences and all three SDHD deletions showed an intron 2 breakpoint. This study underlines the fact that clinically relevant deletions may encompass neighboring genes, with the potential to modify phenotype. Gene deletions of SDHD and SDHC represent a substantial proportion of all mutations, and must be considered in paraganglioma patients shown to be negative for mutations by sequencing.

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