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E D Wieben

Publications and source records attributed to E D Wieben.

At least 19 recordsLinked to original sources
PubMed1995

Abnormal processing of the human cholecystokinin receptor gene in association with gallstones and obesity.

BACKGROUND & AIMS: Cholesterol gallstone disease and obesity are often associated and share the potential, yet unreported, common etiology of cholecystokinin (CCK) dysfunction. While cloning the human CCK-A receptor complementary DNA (cDNA), we found predominance of a 262-base pair coding region deletion in a cDNA library prepared from a patient with this phenotype. The aim of this study was to determine the abundance, functional significance, and mechanism for generating this gene product. METHODS: Relative abundance of CCK receptor gene products was determined using polymerase chain reaction and hybridization analysis. Constructs were expressed in COS cells and studied for radioligand binding and intracellular calcium responses. A human genomic clone for this receptor was sequenced, and the critical regions were compared with those of the patient. RESULTS: Ninety-three percent of the patient's CCK receptor transcripts contained the 262-base pair deletion, whereas only 1.5% +/- 0.9% of control patients had the deletion. This encoded a receptor that did not bind or signal. The deletion corresponded with the third exon; however, this sequence and flanking introns were normal in the patient. CONCLUSIONS: Abnormality of processing an apparently normal CCK receptor gene yields the predominant product with an absent third exon and encoding a nonfunctional receptor, probably reflecting a defective trans-acting splicing factor. An atypical lariat region in the third intron may explain the presence of small amounts of this product in control patients.

Adult↗
PubMed1995

Human dehydroepiandrosterone sulfotransferase gene: molecular cloning and structural characterization.

Dehydroepiandrosterone sulfotransferase (DHEA ST) catalyzes the sulfate conjugation of DHEA and other steroids. From 20 to 25% of subjects are included in a subgroup with high levels of hepatic DHEA ST activity, raising the possibility that this enzyme activity might be controlled by a genetic polymorphism. To understand the molecular mechanisms involved in regulating levels of DHEA ST activity in human tissue, we cloned the human DHEA ST gene, STD. STD spans at least 17 kb and is composed of 6 exons and 5 introns. The locations of the splice junctions for several of the introns are identical to those present in the rat phenol or aryl ST gene, the only other cytosolic ST gene for which the entire exon/intron structure has been reported, as well as those present in two partially characterized genes for the rat senescence marker protein, genes that are also thought to encode ST enzymes. The 5'-flanking region of the human STD gene does not contain canonical TATA or CCAAT elements, but this region is capable of promoting transcription of a reporter gene in Hep G2 cells. Molecular cloning and structural characterization of the human STD gene will make it possible to study genetic mechanisms involved in the regulation of DHEA ST activity in human tissue.

Amino Acid Sequence↗
PubMed1995

Thiopurine methyltransferase pharmacogenetics. Cloning of human liver cDNA and a processed pseudogene on human chromosome 18q21.1.

Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. This genetic polymorphism is an important factor responsible for individual variation in thiopurine drug response. A cDNA for TPMT has been cloned from T84 human colon carcinoma cells. Northern blot analysis of multiple human tissues was performed with the T84 human colon carcinoma TPMT cDNA open reading frame (ORF) as a probe as one step toward understanding the molecular basis for the TPMT genetic polymorphism. Three mRNA species (approximately 1.4, 2.0, and 3.6 kb in length) were present in all tissues studied, including liver. However, none of these mRNAs matched the length of the 2.7 kb T84 TPMT cDNA. Therefore, it was important to clone a TPMT cDNA from a human drug-metabolizing organ such as the liver to determine whether its sequence matched that of the cDNA cloned from the T84 cell line. A human liver cDNA library was screened with the T84 TPMT cDNA ORF as a probe, and a 1.8 kb cDNA was isolated with a coding region sequence identical to that of the T84 TPMT cDNA. The TPMT cDNA ORF was then used to screen a human lymphocytes genomic DNA library in an attempt to clone the TPMT gene(s) in humans. Three intronless clones were isolated with identical ORF sequences that were 96% identical to that of the TPMT cDNA, but which contained multiple nucleotide substitutions and one deletion. The 3'- and 5'-flanking regions of one of the genomic DNA clones were sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Overexpression of the erythrocyte plasma membrane Ca2+ pump in COS-1 cells.

A full-length cDNA corresponding to the hPMCA4 plasma membrane Ca2+ pump was assembled and expressed in COS-1 cells. The original sequence of hPMCA4 gave a very low expression. The mutation of the initiation translation site of this sequence to the consensus A/G-X-X-AUG-G increased the production of the protein. The Ca2+ pump activity in transfected cells was 1.5-3.5-fold higher than in controls. The Ca(2+)-dependence and the calmodulin stimulation of hPMCA4 expressed in COS-1 cells were comparable with those of the erythrocyte Ca2+ pump. Immunohistochemistry experiments showed that most of the expressed protein remained in intracellular membranes. Possible explanations for this targeting of the pump are discussed.

Animals↗
PubMed1992

Molecular diversity of neuronal-type calcium channels identified in small cell lung carcinoma.

Using the polymerase chain reaction (PCR), we identified RNA transcripts for two distinct classes of neuronal-type voltage-gated Ca2+ channels (VGCC) in a prototypic small cell lung carcinoma (SCLC) cell line, SCC-9. Oligonucleotide primers were designed to encode amino acid sequences common to alpha 1-subunits of known neuronal VGCC classes. Sequencing of complementary DNA (cDNA) clones derived from two independent PCR products revealed that one corresponded to a brain class A VGCC fragment predicted to encode a P-type VGCC (insensitive to dihydropyridines and omega-conotoxin) characteristic of cerebellar Purkinje cells but not previously identified in humans. The second PCR product was identical (except for one conservative nucleotide difference) to a fragment of the class D VGCC of neurons and neuroendocrine cells, which encodes an L-type VGCC (sensitive to dihydropyridines). By Northern blot analyses, both cDNAs hybridized to messenger RNAs (mRNAs) obtained from SCC-9; class D hybridized additionally to human cerebral cortical mRNA, but neither hybridized to mRNA from the skeletal muscle cell line TE671. Although no cDNA corresponding to class B VGCC (N-type) was identified, SCLCs are known to express VGCC that are sensitive to omega-conotoxin and coprecipitate with 125I-labeled-omega-conotoxin when complexed with serum IgG from patients with the Lambert-Eaton myasthenic syndrome. The multiple classes of neuronal-type VGCC expressed in SCLC could conceivably have both unique and related antigenic determinants that may give rise to antineuronal autoimmune responses. This would account for a spectrum of paraneoplastic neurologic disorders including the Lambert-Eaton syndrome and subacute cerebellar degeneration.

Base Sequence↗
PubMed1992

Conservation of coding and transcriptional control sequences within the snRNP E protein gene.

The snRNP E protein is one of several proteins associated with the U family of small nuclear RNAs that are involved in RNA processing. Isolation and characterization of the snRNP E protein cDNA sequences from mouse and chicken revealed a 100% conservation of the predicted amino acid sequence when compared to that of the human homologue. Further characterization of a genomic clone for the mouse snRNP E protein gene revealed that the 5' untranslated region and the immediate 5' upstream region have also been highly conserved: 72 and 70%, respectively. Conserved 5' regions include multiple copies of the CTTCCG hexamer sequence which are involved in regulating transcription of the human snRNP E protein gene. Mobility shift assays using corresponding DNA fragments from both human and mouse reveal that both fragments can compete for binding of at least one common transcription factor. These studies demonstrate that along with the amino acid sequence conservation between human and mouse, the snRNP E protein gene has also maintained a high DNA sequence conservation within its basal promoter structure.

Animals↗
PubMed1992

Human liver dehydroepiandrosterone sulfotransferase: molecular cloning and expression of cDNA.

Sulfation is an important pathway in the metabolism of many hormones and drugs. Human liver contains at least three well characterized sulfotransferase (ST) enzymes, i.e., dehydroepiandrosterone (DHEA) ST and two forms of phenol sulfotransferase (PST). Our goal was to purify, to obtain partial amino acid sequence for, and to clone and express cDNA for human liver DHEA ST. Polymerase chain reaction primers were designed on the basis of homology among rat liver hydroxysteroid ST, rat liver PST, and bovine estrogen ST. These primers amplified a unique sequence from human liver cDNA, and this polymerase chain reaction product was used to screen a human liver cDNA library. Two clones were isolated that contained identical open reading frames, of 855 nucleotides, that encoded a protein of 285 amino acids. The deduced amino acid sequence of the encoded protein included two separate 27- and 23-amino acid sequences that were identical to those obtained by microsequencing of proteolytic fragments from purified human liver DHEA ST. Translation, in a rabbit reticulocyte lysate system, of mRNA transcribed in vitro from the two cDNA clones resulted in a 35-kDa translation product that comigrated with purified human liver DHEA ST during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This translation product also catalyzed the sulfation of DHEA but not the sulfation of model substrates for the two forms of PST found in human liver. The two cDNA clones were also used to create expression constructs with the eukaryotic expression vector P91023(B), and these constructs were used to transfect COS-1 cells. The transfected cells expressed a high level of DHEA ST activity, and this enzyme activity displayed a pattern of inhibition by the ST inhibitor 2,6-dichloro-4-nitrophenol identical to that of human liver DHEA ST. Cloning of cDNA for this important human sulfate-conjugating enzyme will enhance understanding of the relationship between DHEA ST and other human liver STs, as well as ST enzymes in other species.

Amino Acid Sequence↗
PubMed1991

Transcriptional regulation of the small nuclear ribonucleoprotein E protein gene. Identification of cis-acting sequences with homology to genes encoding ribosomal proteins.

The 5'-upstream region of the small nuclear ribonucleoprotein E protein (snRNP E) gene has multiple sequence similarities to elements found to be integral in the transcriptional regulation of some mouse ribosomal protein genes (G + C block, CTTCCG motifs) as well as U1 snRNA genes (U1 "B," U1 "D," and SPH enhancers). Furthermore, the immediate upstream region of the snRNP E protein gene lacks typical TATA and CAAT sequence motifs but contains one copy of a GC box characteristic of a housekeeping promoter. By transfection of constructs containing various amounts of the 5'-upstream region of the snRNP E protein gene linked to the bacterial gene for chloramphenicol acetyl transferase (CAT), we determined that the 5' boundary of sequence needed for transcription was within the first 153 nucleotides upstream of the transcription start site. Further deletions to within 51 base pairs reduced CAT expression by 2.5-fold. Mutational analysis within this 51-base pair region revealed that direct repeats of the hexamer CTTCCG were essential for CAT expression. Gel shift assays and DNase I footprinting experiments confirmed this conclusion by demonstrating specific binding of proteins to the CTTCCG motifs. This suggests that the direct repeats of the CTTCCG hexamer sequence play a key role in transcriptional regulation of the snRNP E protein gene.

Animals↗
PubMed1991

A U6 snRNA gene with an internal promoter is juxtaposed to an snRNP protein sequence within an intron of a human G protein gene.

A complex locus on human chromosome 1 brings together sequences homologous to a G protein and two components of the RNA processing machinery of eukaryotic cells. Specifically, the seventh intron of the human Gi3 alpha gene contains a fusion of a partial snRNP E protein pseudogene to a variant U6 snRNA gene. The novel U6 sequence contains nine point mutations and a one nucleotide deletion relative to the major U6 genes from humans. Unlike all other vertebrate U6 genes characterized to date, the variant U6 gene is efficiently transcribed by RNA polymerase III even in the absence of all natural flanking sequences. The union of elements from the signal transduction pathway and the RNA processing machinery suggests the possibility of functional interplay.

Amino Acid Sequence↗
PubMed1991

The snRNP E protein multigene family contains five pseudogenes with common mutations.

Sequence data from three previously-uncharacterized members of the snRNP E protein multigene family suggest that each is a non-transcribed processed pseudogene, even though one clone has the potential to code for a full-length protein with greater than 90% similarity to previously-characterized E protein cDNAs. Each of the newly-analyzed family members is without introns, contains a tract of polyadenylic acid residues, and is flanked by short direct repeats. In addition, the three sequences all contain point mutations that distinguish them from the E protein coding sequence. Seven point mutations are common to the three sequences described here and to two previously-described E protein pseudogenes. Although all of these mutations are transitions, only 5 of 7 could have been generated by deamination of methylated cytosines in inactive genes. Thus, the common mutations in the pseudogenes suggest an origin other than the expressed gene that we have described. Allelic variants for two of the pseudogenes were detected and repetitive elements are located near four of the five E protein pseudogenes that have been characterized.

Amino Acid Sequence↗
PubMed1990

Assignment of the gene for the small nuclear ribonucleoprotein E (SNRPE) to human chromosome 1q25-q43.

Small nuclear ribonucleoproteins (snRNPs), which are composed of various U RNAs and several proteins, are components of the mRNA splicing apparatus. The snRNP protein E is encoded by a multigene family which consists of a single expressed gene and several processed pseudogenes. We have used somatic cell hybridization, in situ hybridization, and linkage analysis to both physically and genetically map the expressed E protein gene to human chromosome 1q25-43, with the most probable location being band 1q32. In addition to the snRNP E protein gene, two other snRNP components--the U1 RNA true multigene family and a group of class I U1 pseudogenes--are located on human chromosome 1.

Animals↗
PubMed1989

Database pollution.

Explore the source record for details and available documents.

Bacteriophage lambda↗
PubMed1989

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. I. Evidence for androgen-regulated proteolytic processing.

The guinea pig seminal vesicle epithelium synthesizes and secretes four major secretory proteins (SVP-1-4). Previous work has established that these four proteins are cleaved from two primary translation products in a complex series of protein processing reactions. The present studies suggest that these protein processing reactions are regulated by androgens. In vitro labeling of seminal vesicle proteins revealed significant differences in the patterns of secretory protein intermediates produced by tissue from intact and castrated animals. Seminal vesicle tissue explants from castrated animals secreted a subset of the processing intermediates secreted by tissue from intact animals. The changes in the patterns of secretory protein intermediates became more pronounced with increasing time after castration, and were fully reversible by treatment of castrated animals with testosterone, suggesting that androgens were affecting the processing or secretion of secretory protein precursors. Amino-terminal protein sequencing of secretory protein processing intermediates that accumulate in the seminal vesicle lumen after castration suggests that the guinea pig seminal vesicle contains an androgen-regulated proteolytic processing activity.

Animals↗
PubMed1989

Androgens affect the processing of secretory protein precursors in the guinea pig seminal vesicle. II. Identification of conserved sites for protein processing.

The guinea pig seminal vesicle epithelium is an androgen-dependent tissue that synthesizes and secretes four major secretory proteins (SVP-1, SVP-2, SVP-3, and SVP-4). Sequencing of near full-length cDNA clones corresponding to the two most abundant mRNAs produced by the seminal vesicle reveals that all four secretory proteins are cleaved from two secretory protein precursors. Amino acid sequences from purified SVP-2 match the central region of the predicted amino acid sequences from the smaller cDNA clone, GP2 (581 nucleotides). Similar analysis demonstrates that the predicted amino acid sequence from the longer cDNA clone, GP1 (1368 nucleotides), codes for the related proteins SVP-3 and SVP-4 as well as SVP-1. The 43.2 kilodalton polyprotein precursor coded by GP1 contains two different sets of 24 amino acid tandemly repeated sequences. The two secretory protein precursors have extensive regions of peptide sequence homology, particularly in regions where protein processing must occur to produce the mature secretory proteins. Analysis of the predicted secondary structure of the two precursor polypeptides revealed a strong correlation between structural features and sites of protein processing.

Amino Acid Sequence↗
PubMed1988

The complete primary structure of the human snRNP E protein.

The snRNP E protein is one of four "core" proteins associated with the snRNAs of the U family (U1,U2,U4,U5, and U6). Screening of a human teratoma cDNA library with a partial cDNA for a human autoimmune antigen resulted in the isolation of a cDNA clone containing the entire coding region of this snRNP core protein. Comparison of the 5' end of this cDNA with the sequences of two processed pseudogenes and primer extension data suggest that the cDNA is nearly full length. The longest open reading frame in this clone codes for a basic 92 amino acid protein which is in perfect agreement with amino acid sequence data obtained from purified E protein. The predicted sequence of this protein reveals no extensive similarity to other snRNP proteins, but contains regions of similarity to a eukaryotic ribosomal protein.

Amino Acid Sequence↗
PubMed1988

The small nuclear ribonucleoprotein E protein gene contains four introns and has upstream similarities to genes for ribosomal proteins.

The human small nuclear ribonucleoprotein E protein is an 11,000-dalton basic protein which is an integral component of several small nuclear ribonucleoprotein complexes involved in RNA processing reactions. Sequence analysis of the E protein multigene family reveals that at least one gene for this component of the RNA splicing machinery is interrupted by four introns. The exons of this gene are identical to two cDNA clones isolated from independent tissue sources and span approximately 9 kilobase pairs. Primer extension data indicated the presence of two major transcription start sites. The upstream region of the small nuclear ribonucleoprotein E protein gene does not contain TATA or CCAAT sequences within 175 nucleotides of the transcription start sites. However, the proximal upstream region does contain several similarities to the promoter regions of both snRNA genes and vertebrate ribosomal protein genes.

Amino Acid Sequence↗