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At least 685 records · Page 38Linked to original sources
PubMed1998

Functional GHRH receptor carboxyl terminal isoforms in normal and dwarf (dw) rats.

GHRH plays a critical role in pituitary somatotroph development and function, actions which are mediated by a G-protein coupled receptor (GHRHr) that has been recently cloned. PCR amplification of rat pituitary mRNA using primers that span the GHRHr coding region resulted in two distinct products. When sequenced, the two isoforms were identical through bp 1278 of the GHRH coding region. However, the novel variant, which we have termed GHRHrbeta, contains a 131 bp deletion (1279-1408) and resumes at bp 1409 in the 3'UTR of the previously identified transcript (GHRHralpha). The identical isoforms were present in pituitaries from dwarf (dw) rats. The predicted amino acid sequence for the alternate receptor isoform differs from the published amino acid sequence at the extreme carboxyl terminus, with the last 5 amino acids of the published sequence replaced and an additional 17 amino acids added to the sequence. When translated in vitro or expressed as an epitope-tagged construct in non-GHRHr containing cell lines, the GHRHrbeta mRNA produces a 42 kDa protein product, appropriately larger than the 40 kDa product of GHRHralpha mRNA. Furthermore, GHRHrbeta retains the ability to promote cAMP generation in response to GHRH when expressed in non-GHRHr containing cell lines. These results indicate the presence of a splice variant of rat GHRHr mRNA present in normal and dw rat pituitary that codes for a functional receptor protein with an alternate carboxyl terminal domain. These findings raise the possibilities of target cell regulation of GHRH response, modulation of response through receptor isoform interactions and the involvement of multiple intracellular signaling pathways.

Alternative Splicing↗
PubMed1999

DGGE method for the mutational analysis of the coding and proximal promoter regions of the Alzheimer's disease presenilin-1 gene: two novel mutations.

Many different mutations that cause Alzheimer's disease (AD) have been found in the presenilin-1 gene (PSEN1) and are associated with the most aggressive forms of the disease. With the aim of screening for PSEN1 genetic variations, we developed a method based on denaturing gradient gel electrophoresis (DGGE) that allows the mutational analysis of all the coding exons and the proximal promoter of PSEN1 using only four DGGE gels. The analysis by this methodology of a sample of 58 early-onset AD (EOAD) patients nonselected for family history resulted in finding four genetic variants within the PSEN1 coding region, two of which are novel mutations (M233L and A409T), whereas the other two have been reported previously (L282R and E318G). We also found a novel mutation within the PSEN1 proximal promoter (-280 C-->G) that, interestingly, provokes significant changes in the transcriptional activity of the gene in cell lines of neuronal and astrocytic, but not hepatic origin. These data strongly suggest that the region around -280 of PSEN1 promoter contains a regulatory element that controls its transcription specifically in neural cells.

Adult↗
PubMed2006

Prohibitin mutations are uncommon in prostate cancer families linked to chromosome 17q.

BACKGROUND: Linkage studies have provided evidence for a prostate cancer susceptibility locus on chromosome 17q. The mitochondrial protein prohibitin (PHB) is a plausible candidate gene based on its chromosomal location (17q21) and known function. METHODS: All coding regions and intron/exon junctions of the PHB gene were sequenced in 32 men from families participating in the University of Michigan Prostate Cancer Genetics Project that demonstrated evidence of linkage to 17q markers. RESULTS: Although a number of nucleotide variants were identified, no coding region substitutions were identified in any of the 32 men with prostate cancer from 32 unrelated multiplex prostate cancer families. CONCLUSIONS: PHB mutations do not appear to account for the linkage signal on 17q21-22 detected in PCGP families. Fine mapping of this region is in progress to refine the candidate region and highlight additional candidate prostate cancer susceptibility genes for sequence analysis.

Adult↗
PubMed2000

Cloning of the full-length coding sequence of rat liver-specific organic anion transporter-1 (rlst-1) and a splice variant and partial characterization of the rat lst-1 gene.

The full-length coding sequence of rat liver-specific organic anion transporter-1 (lst-1) and its splice variant have been cloned. The full-length rat lst-1 (designated rlst-1a) encodes a protein containing 687 amino acids and has 12-putative transmembrane domains, multiple potential N-glycosylation and phosphorylation sites. Therefore, rat lst-1a has 35 additional amino acid residues compared to the previously reported rat lst-1. A splice variant (designated rlst-1c) reported in this communication encodes a protein containing 654 amino acids and has 10-putative transmembrane domains. PCR analysis suggests that rlst-1a is the most abundant form in liver. Phylogenetic analysis reveals that rat lst-1a is an ortholog of human LST-1 (hLST-1) and mouse lst-1 (mlst-1). The rlst-1 gene is composed of 15 exons and 14 introns. Analysis of exon-intron boundary reveals that the splice variant rlst-1c lacks the entire exon 7, while the previously reported rat lst-1 (designated herein as rlst-1b) lacks approximately half of exon 10, and the splicing has occurred through alternative usage of a splice donor site within exon 10.

Alternative Splicing↗
PubMed1994

Transcription of a variant human U6 small nuclear RNA gene is controlled by a novel, internal RNA polymerase III promoter.

Promoter elements in the 5' flanking regions of vertebrate U6 RNA genes have been shown to be both necessary and sufficient for transcription by RNA polymerase III. We have recently isolated and characterized a variant human U6 gene (87U6) that can be transcribed by RNA polymerase III in vitro in the absence of any natural 5' or 3' flanking sequences. This gene contains 10 nucleotide differences from the previously characterized human U6 gene (wtU6) within the coding region but has no homology to wtU6 in the upstream promoter region. By constructing chimeras between these two genes, we have shown that mutation of as few as two nucleotides in the coding region of the human U6 RNA gene is sufficient to create an internal promoter that is functional in vitro. A T-to-C transition at position 57 and a single T deletion at position 52 produce an internal U6 promoter that is nearly as active in vitro as the external U6 polymerase III promoter utilized by wtU6. Neither of these residues is absolutely conserved during evolution, and both of these nucleotide changes occur within the previously noted A box homology. Deletion and linker scanning mutations within the coding region of this variant U6 gene suggest that, in addition to the central region including bp 52 and 57, sequences at the extreme 5' end of the gene are critical for efficient transcription. In contrast, flanking sequences have a minor effect on transcriptional efficiency. This arrangement is unique among internal RNA polymerase III promoters and may indicate unique regulation of this gene.

Base Sequence↗
PubMed1985

Replacement variant histone genes contain intervening sequences.

The nucleotide sequences of two chicken histone genes encoding replacement variant H3.3 polypeptides are described. Unlike the replication variant genes of chickens (and almost all other organisms), these genes contain intervening sequences; introns are present in both genes in the 5' noncoding and coding sequences. Furthermore, the replacement variant histone mRNAs are post-transcriptionally polyadenylated. The locations, but not the sizes, of the two introns within the coding segments of the two genes have been exactly conserved, whereas the intron positions in their respective 5' flanking regions differ. Although both H3.3 genes predict the identical histone polypeptide sequence, they are as different from one another as each of them is from a more common replication variant H3.2 gene in silent base substitutions within the coding sequences. Thus, the H3.3 polypeptide sequence has been precisely maintained over a great evolutionary period, suggesting that this class of histones performs a strongly selected biological function. Although replacement variant histones can account for more than 50% of the total H3 protein in the nuclei of specific chicken tissues, the steady-state level of H3.3 mRNA is nearly the same (and is quite low) in all tissues and ages of animals examined. These properties suggest novel mechanisms for the control of the basal histone biosynthesis which takes place outside of the S phase of the cell cycle.

Age Factors↗
PubMed2006

Functional genetic diversity in the high-affinity carnitine transporter OCTN2 (SLC22A5).

Systemic carnitine deficiency (SCD) is a rare autosomal recessive disease resulting from defects in the OCTN2 (SLC22A5) gene, which encodes the high-affinity plasma membrane carnitine transporter. Although OCTN2 is fairly well studied in its relationship with SCD, little is known about the carrier frequency of disease-causing alleles of OCTN2, or of more common functional polymorphisms in this gene. To address these issues, we screened for genetic variants in the OCTN2 coding region by direct sequencing of the exons and flanking intronic region of OCTN2 in a large sample (n = 276) of ethnically diverse subjects. In addition, we established lymphoblastoid cell lines from subjects homozygous for either allele of the previously identified promoter region variant, -207G>C. We found eight amino acid sequence variants of OCTN2, of which three (Phe17Leu, Leu144Phe, and Pro549Ser) were polymorphic in at least one ethnic group. When assayed for functional activity by expression in human embryonic kidney 293 cells, using as probes both the endogenous substrate (l-carnitine) and the organic cation tetraethylammonium, three variants showed functional differences from the reference OCTN2 (Phe17Leu, Tyr449Asp, Val481Phe; p < 0.05). Further studies of the Phe17Leu polymorphism showed a reduced V(max) for l-carnitine transport to approximately 50% of the reference OCTN2. Confocal microscopy studies using an OCTN2-GFP fusion protein showed that Phe17Leu had distinct subcellular localization from the reference OCTN2, with diffuse cytoplasmic retention of Phe17Leu, in contrast to reference OCTN2, which localized specifically to the plasma membrane. Lymphoblasts from subjects homozygous for the -207G allele showed increased l-carnitine transport compared with the -207C/C homozygotes (p < 0.05). This study suggests that although loss-of-function mutations in OCTN2 are likely to be rare, common variants of OCTN2 found in healthy populations may contribute to variation in the disposition of carnitine and some clinically used drugs.

Amino Acid Sequence↗
PubMed1996

Retarded and aberrant splicings caused by single exon mutation in a phosphoglycerate kinase variant.

The molecular abnormality of a phosphoglycerate kinase variant which was associated with severe tissue enzyme deficiency and episodes of muscle contractions and myoglobinuria was examined. Analysis of the patient's DNA showed the existence of a nucleotide transversion A/T - C/G in exon 7. No other nucleotide change was detected in the coding region of the variant gene. The mutation should produce a single amino acid substitution Glu - Ala at protein position 251 counting from the NH2-terminal acetyl serine residue. The protein abnormality caused by the amino acid substitution cannot explain the enzyme deficiency. Northern blot hybridization indicated that the PGK mRNA content of the patient's lymphoblastoid cells was only about 10% of that of normal. Nucleotide sequence analysis revealed the existence of two PGK mRNA components in the patient's cells. The major component corresponds to the normal PGK mRNA except for A - C change at nucleotide position 755 counting from adenine of the chain initiation codon. The minor component contains 5' region (52 bases) of intron 7 between exon 7 and exon 8. An inframe chain termination codon exists in the minor mRNA component, and the COOH-terminal half is expected to be deleted in the translation product. These results indicate that the low PGK activity in the patient's tissues is mainly due to retarded and aberrant pre-mRNA splicings caused by the change of the consensus 5' splice sequence AGgt to a nonconsensus sequence CGgt at the junction between exon 7 and intron 7 of the variant gene.

Adolescent↗
PubMed2005

Human cortical responses during one-bit delayed-response tasks: an fMRI study.

Neuroimaging study of cognition across aging requires simple tasks ensuring: (i) high rate of correct performances in neurophysiological settings; and (ii) significant modulation of cortical activity. As a preliminary step, the present functional magnetic resonance imaging (fMRI) study tested the hypothesis that very simple delayed-response tasks fit these requirements in normal young adults. The short-term memory (STM) variant included a sequence of cue stimulus (two vertical bars), delay period (blank screen for only 5s), go stimulus, and motor response compatible with the taller vertical bar. Noteworthy, the retention (only one bit) could be based on visuo-spatial, phonological, and somatomotor coding. In the control variant (no STM, NSTM), the cue stimulus was present during the delay period. Results showed high rate of correct performances in both tasks (about 95%). Compared to the NSTM task (delay period), the STM task enhanced cortical responses in bilateral dorsolateral prefrontal (Brodmann area 8-9 (BA 8-9)), lateral premotor (BA 6L), medial premotor (BA 6M), inferior parietal (BA 40), and superior parietal (BA 7) areas. In the STM task, cortical responses were stronger in right than left BA 8-9 and BA 6L. These results indicate that, in normal young adults, a simple STM variant of delayed-response tasks (one bit to be retained) is correctly performed and enhances bilateral fronto-parietal responses. Therefore, it may be used for future cognitive neuroimaging studies on aging.

Adult↗
PubMed1987

Cloacal exstrophy and cloacal exstrophy variants: a proposed system of classification.

A coding system that documents the abnormalities within the cloacal exstrophy complex is presented. Analysis allows the classification of these abnormalities into classical and variant series in a logical manner. The application of the coding system to selected reported material is described and it demonstrates the similarities between apparently divergent cases.

Abnormalities, Multiple↗
PubMed2003

Variation in the FABP2 promoter affects gene expression: implications for prior association studies.

AIMS/HYPOTHESIS: An Ala54Thr polymorphism in the FABP2 gene has previously been associated with insulin resistance and lipid oxidation rates in Pima Indians. Ala54Thr functionally alters the protein's ability to bind and transport dietary fatty acids. In the current report, we sought additional functional variation in FABP2 by sequencing putative regulatory regions. METHODS: More than 1.2 Kb of the putative promoter of FABP2 was sequenced in 20 Pima subjects. Variations were genotyped in 84 additional Pima Indian subjects to assess haplotype combinations. Functional activities of variant and nonvariant promoters were compared in Caco-2 cells transfected with luciferase reporter constructs. RESULTS: Seven variations were identified in the FABP2 promoter in Pima Indians. Genotypes of these variants were in complete concordance with each other, and were in complete concordance with Ala54Thr. Therefore, only two promoter alleles were observed in Pima Indians, an Ala54-associated promoter and a Thr54-associated promoter. In contrast, genotyping of these variants in Caucasian DNA showed multiple genotypic combinations. In vitro reporter assays indicated that the Thr54-associated promoter in Pima Indians resulted in a threefold reduction in promoter activity as compared to Ala54-associated promoter. CONCLUSION/INTERPRETATION: Two functional variations exist in FABP2--the coding Ala54Thr and the variant promoter. In the Pima Indian population, but not the Caucasian population, these two functional variants are always carried on the same allele. Therefore, some of the in vivo phenotypic associations previously attributed to the Ala54Thr substitution, which alters binding characteristics of the protein, could instead be due to promoter variation, which alters expression levels.

Alanine↗
PubMed2001

Prostanoid receptors: subtypes and signaling.

Cyclooxygenases metabolize arachidonate to five primary prostanoids: PGE(2), PGF(2 alpha), PGI(2), TxA(2), and PGD(2). These autacrine lipid mediators interact with specific members of a family of distinct G-protein-coupled prostanoid receptors, designated EP, FP, IP, TP, and DP, respectively. Each of these receptors has been cloned, expressed, and characterized. This family of eight prostanoid receptor complementary DNAs encodes seven transmembrane proteins which are typical of G-protein-coupled receptors and these receptors are distinguished by their ligand-binding profiles and the signal transduction pathways activated on ligand binding. Ligand-binding selectivity of these receptors is determined by both the transmembrane sequences and amino acid residues in the putative extracellular-loop regions. The selectivity of interaction between the receptors and G proteins appears to be mediated at least in part by the C-terminal tail region. Each of the EP(1), EP(3), FP, and TP receptors has alternative splice variants described that alter the coding sequence in the C-terminal intracellular tail region. The C-terminal variants modulate signal transduction, phosphorylation, and desensitization of these receptors, as well as altering agonist-independent constitutive activity.

Amino Acid Sequence↗
PubMed2003

Novel splice variants of the receptor for advanced glycation end-products expressed in human vascular endothelial cells and pericytes, and their putative roles in diabetes-induced vascular injury.

The binding of advanced glycation end-products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications. In the present study, we show that the cellular constituents of small vessels, endothelial cells (EC) and pericytes express novel splice variants of RAGE mRNA coding for the isoforms that lack the N-terminal V-type immunoglobulin-like domain (N-truncated) or the C-terminal transmembrane domain (C-truncated), as well as the known full-length mRNA. The ratio of the expression of the three variants was different between EC and pericytes; the content of the C-truncated form was highest in EC, whereas the full-length form was the most abundant in pericytes. Transfection experiments with COS-7 cells demonstrated that those variant mRNAs were translated into proteins as deduced; C-truncated RAGE was efficiently secreted into the culture media, and N-truncated RAGE was located mainly on the plasma membrane. The three isoforms were also detected in primary cultured human EC and pericytes. Further, full-length and C-truncated forms of RAGE bound to an AGE-conjugated column, whereas N-truncated RAGE did not. The AGE induction of extracellular-signal-related kinase phosphorylation and vascular endothelial growth factor in EC and of the growth and cord-like structure formation of EC was abolished completely by C-truncated RAGE, indicating that this endogenous secretory receptor (endogenous secretory RAGE) is cytoprotective against AGE. The results may contribute to our understanding of the molecular basis for the diversity of cellular responses to AGE and for individual variations in the susceptibility to diabetic vascular complications.

Alternative Splicing↗
PubMed1999

Characterization of a brain-specific nuclear LIM domain protein (FHL1B) which is an alternatively spliced variant of FHL1.

We have amplified and sequenced a novel, alternatively spliced variant of a human gene coding for the four-and-a-half LIM domain protein 1 (FHL1). This gene is located at chromosome Xq27 and the spliced variant is named FHL1B. The ORF of FHL1B cDNA codes for a LIM-only protein that possesses a zinc finger and three tandem repeats of LIM domains at the N-terminus with an active bipartite nuclear localization signal (NLS) motif and a possible RBP-J binding region at the C-terminus. FHL1B and FHL1 have the same N-terminal three-and-a-half LIM domains but different C-terminal protein sequences, due to the presence of an additional alternative exon 4b in FHL1B causing a frame-shift in the 3'coding region. RT-PCR results revealed that the expression of FHL1 is not restricted in skeletal muscle and heart, but is widely distributed in other tissues, including brain, placenta, lung, liver, kidney and pancreas, albeit as a low abundance transcript. In contrast, FHL1B is specifically expressed in brain. The C-terminal alternative region in FHL1B is sufficient to localize FHL1B in the nucleus of mammalian cell. FHL1B is probably related to neural differentiation and certain fragile X syndrome.

Alternative Splicing↗
PubMed1998

Multiple transcripts coding for the menkes gene: evidence for alternative splicing of Menkes mRNA.

We isolated cDNA fragments from four human cell lines that had sequences for the Menkes Cu-transporting ATPase (ATP7A). Primers designed to generate a 4.8 kb cDNA with the complete open reading frame generated a 1.9 kb cDNA in addition to the expected 4.8 kb product. Sequence analysis revealed that the 1.9 kb cDNA encoded one of the six Cu-binding sites and two of the eight transmembrane domains of ATP7A. Stop and start codons were also present. More striking, however, was an unusual union between exons 2 and 16 that retained an in-frame reference to exon 23. The 1.9 kb cDNA thus appeared to be a truncated Menkes mRNA that coded for an ATP7A variant that lacked exons 3-15. A 530 bp probe specific for exon 23 that avoided sequences in the exon 3-15 region hybridized to a 5.5 kb band on Northern blot analysis. Western blotting provided immunochemical evidence for the presence of both a 170 kDa and a 57 kDa protein with ATP7A sequences in detergent extracts of Caco-2 and induced BeWo cells. Extracts from non-induced BeWo cells, which lack the capacity to express the Menkes gene (MNK), showed neither protein. In a cell-free reticulocyte lysate, a plasmid containing the 1.9 kb cDNA insert directed the synthesis of a 59 kDa protein with antigenic properties of ATP7A. These studies provide evidence that non-Menkes cells have the capacity to synthesize more than one MNK mRNA. The one characterized in this report codes for a 57-59 kDa protein that lacks the core structure of the ATP7A protein. The smaller variant could be an alternative spliced form of MNK mRNA.

Adenosine Triphosphatases↗
PubMed2003

A novel genetic variant in the apolipoprotein A5 gene is associated with hypertriglyceridemia.

The apolipoprotein A5 gene (APOA5 ) has been shown to play an important role in determining plasma triglyceride concentrations in humans. We describe here a novel variant, c.553G>T, in the apolipoprotein A5 gene that is associated with hypertriglyceridemia. In contrast to some other polymorphisms, which occur in non-coding regions of the gene, this variant occurs within the coding region and causes the change of amino acid sequence (a substitution of a cysteine for a glycine residue). The minor allele frequencies were 0.042 and 0.27 (P<0.001) for control and hypertriglyceridemic patients, respectively. The serum triglyceride level was significantly different among the genotypic groups (G/G 92.5+/-37.8 mg/dl, G/T 106.6+/-34.8 mg/dl, T/T 183.0 mg/dl, P=0.014) in control subjects. Multiple logistic regression revealed individuals carrying the minor allele had age, gender and BMI (body mass index)-adjusted odds ratio of 11.73 (95% confidence interval of 6.617-20.793; P<0.0001) for developing hypertriglyceridemia in comparison to individuals without that allele. These findings suggest the possible use of c.553G>T polymorphisms in APOA5 as prognostic indicators for hypertriglyceridemia susceptibility in Chinese.

Age Factors↗
PubMed2004

Allelic association analysis of phospholipase A2 genes with schizophrenia.

Several studies suggest increased activity of phospholipase A2 in schizophrenic patients. In the present study, variants of four genes coding for phospholipase A2 enzyme groups (sPLA2, cPLA2, iPLA2 and PAFAH) were analysed in a case-control sample using 240 schizophrenic patients and 312 healthy controls. No difference was observed on the allelic and genotypic distribution of cPLA2 and sPLA2 gene polymorphisms among the groups. The PAFAH variant was very rare in our population and therefore not informative. A significant allelic (chi2=5.86, P=0.0085, odds ratio=1.38, 95% confidence interval, 1.08-1.77) and genotypic (chi2=5.4, P=0.02) association with the iPLA2 gene polymorphism was found. In conclusion, our data suggested that iPLA2 may play a role as a susceptibility gene for schizophrenia in our sample; however, confirmatory studies in independent samples are needed.

Alleles↗
PubMed2002

Human alcoholism studies of genes identified through mouse quantitative trait locus analysis.

Coding region DNA sequence variants have been recently identified in several QTL candidate genes in a mouse model of differential sensitivity to alcohol [inbred long-sleep (ILS) and inbred short-sleep (ISS)]. This work has been extended into a human population characterized for their initial level of response to alcohol (LR). The coding region of one of the most promising of these candidate genes, zinc finger 133 (Znf133), has been sequenced completely in 50 individuals who participated in alcohol challenges at approximately age 20 and have been followed subsequently for the last 15 years. PCR products were obtained for the protein coding region of ZNF133 using human genomic DNA and directly sequenced using automated sequencers. Novel single nucleotide polymorphisms (SNPs) were detected by analyzing the sequence data using a suite of bioinformatics programs including Consed, Phred, Phrap and Polyphred. Five human SNPs were detected, two that correspond to amino acid changes in the protein, two that are silent DNA changes and one located in an intron. In this small sample, no significant association between any of the SNPs and alcohol diagnosis was detected. A follow-up of these SNPs in a larger sample should allow a more definitive conclusion to be reached. Significantly, the data presented here demonstrate the feasibility of directly testing genes in human alcoholic populations that had been identified first by comparative DNA sequencing of candidate genes located within mouse alcohol-related QTLs, even without detailed knowledge of the gene's function.

Adult↗