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At least 1,135 records · Page 63Linked to original sources
PubMed1994

Ancient, highly polymorphic human major histocompatibility complex DQA1 intron sequences.

A 438 basepair intron 1 sequence adjacent to exon 2 in the human major histocompatibility complex DQA1 gene defined 16 allelic variants in 69 individuals from wide ethnic backgrounds. In contrast, the most variable coding region spanned by the 247 basepair exon 2 defined 11 allelic variants. Our phylogenetic human intron 1 tree derived by the Bootstrap algorithm reflects the same relative allelic relationships as the reported DQA1 exon 2 tree [Gyllensten and Erlich, Hum Immunol 36:1-10, 1989]. Thus 3' DQA1 intron 1 and exon 2 have cosegregated since divergence of the human races. Comparison of human alleles to a Rhesus monkey DQA1 first intron sequence found only 10 nucleotide substitutions unique to Rhesus, with the other 428 positions (98%) found in at least one human allele. This high degree of homology reflects the evolutionary stability of intron sequences since these two species diverged over 20 million years ago. Because more intron 1 alleles exist than exon 2 alleles, these polymorphic introns can be used to improve tissue typing for transplantation, paternity testing, and forensics and to derive more complete phylogenetic trees. These results suggest that introns represent a previously underutilized polymorphic resource.

Algorithms↗
PubMed2006

Sequence analysis of cDNA encoding rabbit follicle-stimulating hormone beta-subunit precursor protein.

To understand the molecular basis of the rabbit's efficient superovulation, we determined the cDNA sequence of the follicle-stimulating hormone (FSH) beta-subunit precursor protein using a combination of 5'- and 3'-rapid amplification of cDNA ends (RACE) with pituitary cDNA libraries of the Japanese White rabbit and compared it with those of other mammals. RACE experiments detected at least three transcripts for the FSHbeta precursor protein in the libraries. The transcripts had lengths of 457, 1,621, and 1,767 bp, from the 5'-end to the poly(A) site. The shortest and mid-length transcripts had the putative polyadenylation signal sequence AATAAA at nucleotides 436 and 1,601, respectively, whereas the longest form had an ATTAAA sequence at nucleotide 1,745 of the cDNA sequence. These transcripts are likely to be polyadenylation variants of one large transcript because they share the same coding sequence for the precursor protein (130 amino acid residues in length). However, only a few shortest variants seem to be formed because the shortest variants were not detected by Northern blot analysis. Phylogenetic analysis of the deduced amino acid sequence indicates that the rabbit is phylogenetically closer to humans than to the other mammals, suggesting that an FSH preparation from human sources would be superior as a follicle stimulant for the induction of superovulation.

Amino Acid Sequence↗
PubMed2000

Alternative splicing and gene structure of the transforming growth factor beta-activated kinase 1.

We have identified a fourth splice variant of the TGF beta-activated kinase (TAK1), called TAK1-d, and identified an error in the previously published TAK1-c sequence. Our data shows that the c and d variants encode proteins whose carboxyl ends differ markedly from those of variants a and b. Analysis of the human TAK1 gene sequence, located at 6q16.1-q16.3, shows that the coding sequence is organised in 17 exons. The four splice variants result from alternative splicing of exons 12 and 16, the reading frame of exon 17 being determined by the presence or absence of exon 16. Study of the relative levels of expression of the four splice variants showed significant variations between tissues. Our evidence suggests that the alternative splicing of the TAK1 mRNA may have important functional implications.

Alternative Splicing↗
PubMed2000

Identification of novel, functional genetic variants in the human matrix metalloproteinase-2 gene: role of Sp1 in allele-specific transcriptional regulation.

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix and nonmatrix proteins, particularly basement membrane constituents. Thus, any naturally occurring genetic variants that directly affect gene expression and/or protein function would be expected to impact on progression of pathological processes involving tissue remodeling. We scanned a 2-kilobase pair promoter region and all 13 exons of the human MMP-2 gene, from a panel of 32 individuals, and we identified the position, nature, and relative allele frequencies of 15 variant loci as follows: 6 in the promoter, 1 in the 5'-untranslated region, 6 in the coding region, 1 in intronic sequence, and 1 in the 3'-untranslated region. The majority of coding region polymorphisms resulted in synonymous substitutions, whereas three promoter variants (at -1306, -790, and +220) mapped onto cis-acting elements. We functionally characterized all promoter variants by transient transfection experiments with 293, RAW264.7, and A10 cells. The common C --> T transition at -1306 (allele frequency 0.26), which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with the T allele. Electrophoretic mobility shift assays confirmed that these differences in allelic expression were attributable to abolition of Sp1 binding. These data suggest that this common functional genetic variant influences MMP-2 gene transcription in an allele-specific manner and is therefore an important candidate to test for association in a wide spectrum of pathologies for which a role for MMP-2 is implicated, including atherogenesis and tumor invasion and metastasis.

3' Untranslated Regions↗
PubMed1994

Circularly permuted dihydrofolate reductase of E. coli has functional activity and a destabilized tertiary structure.

Three circularly permuted variants of Escherichia coli dihydrofolate reductase genes were constructed. Linkers coding tri- and pentapeptides were used to connect the natural 5'- and 3'-terminal ends. Only one variant of circularly permuted protein with tripeptide linker and the cleavage of the peptide bond between 107 and 108 amino acid residues was produced in a good yield. The expressed protein was insoluble in the cells, but at pH 8.0 and higher the isolated protein was soluble. Enzymatic assay and physical studies have shown that permuted dihydrofolate reductase has a destabilized tertiary structure. Only the addition of the natural substrates or inhibitors lead to the protein with the native-like structure and functional activity.

Amino Acid Sequence↗
PubMed2001

Uniquely conserved non-translated regions are involved in generation of the two major transcripts of protein phosphatase 2Cbeta.

Partial cDNAs of different isoforms of protein phosphatase 2Cbeta (PP2Cbeta or PPM1B) have been characterized in mammals. We disclose here the full cDNAs of two major PP2Cbeta isoforms from human, rat and mouse. These cDNAs (2.6 and 3.3 kb) are able to encode 53 kDa (PP2Cbetal) and 43 kDa (PP2Cbetas) polypeptides, respectively. The isoforms are co-expressed ubiquitously with the highest level in skeletal muscle, as assessed by Northern-blot analysis. Western and in situ analyses using monoclonal antibodies against PP2Cbeta confirmed the existence of two isoforms in the cytoplasm. Comparative sequence analysis revealed that both cDNAs consist of six exons with an alternate usage of the 3' exons that underlies the differences between them. The genomic structure of PP2Cbeta is similar to that of other PP2C paralogs and includes a non-coding first exon followed by a large intron and a large second exon that encoded most of the catalytic domain. Both variants of the ending exon include large non-coding regions. All non-translated regions (NTRs) are highly conserved between the orthologous genes, indicating their regulatory function. The 5'-NTR is long (379 bp), includes upstream start codons and is predicted to contain stable secondary structures. Such features inhibit translation initiation by the scanning mechanism. Introduction of this NTR element into a bi-luciferase expression-cassette enabled expression of the second cistron, suggesting that it might serve as an internal ribosome entry site, or it contains a cryptic promoter. Overexpression of PP2Cbeta under CMV-promoter in 293 cells led to cell-growth arrest or cell death.

Alternative Splicing↗
PubMed2002

Structure of the human carboxypeptidase M gene. Identification of a proximal GC-rich promoter and a unique distal promoter that consists of repetitive elements.

The human carboxypeptidase M (CPM) gene was found to encompass about 112.6 kb of genomic sequence, containing 11 exons of which eight (exons 2-9) are common to all transcripts and contain the entire coding region. We have cloned several alternative variants of CPM transcripts that result from differential promoter usage and alternative splicing. Although CPM belongs to the same metallocarboxypeptidase subfamily as CPE, their intron/exon structures differ significantly. Multiple transcription start sites were found in the CPM gene that cluster in two regions separated by about 30 kb and are flanked by two unique functional promoters. One ('proximal') is immediately upstream of the coding region and contains GC-rich sequences and a typical TATA box whereas the other ('distal') consists almost entirely of repetitive elements. Luciferase reporter assays with constructs of the promoter regions showed they were both quite active in several cell lines. However, the proximal promoter was much stronger than the distal one in two of the human cell lines tested (HepG2 and HEK293) whereas both promoters were highly and equally active in the human monocytic cell line THP-1, which has high constitutive expression of CPM.

3' Untranslated Regions↗
PubMed1988

The avian retrovirus env gene family: molecular analysis of host range and antigenic variants.

The nucleotide sequence of the env gp85-coding domain from two avian sarcoma and leukosis retrovirus isolates was determined to identify host range and antigenic determinants. The predicted amino acid sequence of gp85 from a subgroup D virus isolate of the Schmidt-Ruppin strain of Rous sarcoma virus was compared with the previously reported sequences of subgroup A, B, C, and E avian sarcoma and leukosis retroviruses. Subgroup D viruses are closely related to the subgroup B viruses but have an extended host range that includes the ability to penetrate certain mammalian cells. There are 27 amino acid differences shared between the subgroup D sequence and three subgroup B sequences. At 16 of these sites, the subgroup D sequence is identical to the sequence of one or more of the other subgroup viruses (A, C, and E). The remaining 11 sites are specific to subgroup D and show some clustering in the two large variable regions that are thought to be major determinants of host range. Biological analysis of recombinant viruses containing a dominant selectable marker confirmed the role of the gp85-coding domain in determining the host range of the subgroup D virus in the infection of mammalian cells. We also compared the sequence of the gp85-coding domain from two subgroup A viruses, Rous-associated virus type 1 and a subgroup A virus of the Schmidt-Ruppin strain of Rous sarcoma virus. The comparison revealed 24 nonconservative amino acid changes, of which 6 result in changes in potential glycosylation sites. The positions of 10 amino acid differences are coincident with the positions of 10 differences found between two subgroup B virus env gene sequences. These 10 sites identify seven domains in the sequence which may constitute determinants of type-specific antigenicity. Using a molecular recombinant, we demonstrated that type-specific neutralization of two subgroup A viruses was associated with the gp85-coding domain of the virus.

Alpharetrovirus↗
PubMed1992

Virulence phenotypes of Shigella flexneri 2a avirulent mutant 24570 can be complemented by the plasmid-coded positive regulator virF gene.

Avirulent mutation of an opaque colony variant of Shigella flexneri 2a designated 24570 has been believed to be linked with the glpK locus of the chromosome. However, avirulent phenotypes of the 24570 strain could be complemented by the invasion plasmid-coded virF gene, a positive regulator for invasion genes. The 24570 strain had a DNA structural alteration upstream of the virF gene.

Antigens, Bacterial↗
PubMed1998

Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV.

Human Monocyte Chemotactic Protein (MCP)-2 has originally been isolated from stimulated osteosarcoma cells as a chemokine coproduced with MCP-1 and MCP-3. Here, a 5'-end extended MCP-2 cDNA was cloned from a human testis cDNA library. It encoded a 76 residue MCP-2 protein, but differed from the reported bone marrow-derived MCP-2 cDNA sequence in codon 46, which coded for a Lys instead of a Gln. This MCP-2Lys46 variant, caused by a single nucleotide polymorphism (SNP), was biologically compared with MCP-2Gln46. The coding regions were subcloned into the bacterial expression vector pHEN1, and after transformation of Escherichia coli, the two MCP-2 protein variants were recovered from the periplasm. The recombinant proteins were purified to homogeneity by heparin-Sepharose affinity chromatography and reversed-phase HPLC. Edman degradation revealed a Gln residue at the NH2 terminus instead of a pGlu. To evaluate the influence of the cyclization, this Gln was chemically converted into pGlu in both MCP-2 variants. The conversion was confirmed by electrospray mass spectrometry. rMCP-2Gln46 and rMCP-2Lys46 and the NH2-terminal cyclic counterparts were tested on monocytic cells in calcium mobilization and chemotaxis assays. No significant difference in biological activity was observed between the rMCP-2Gln46 and rMCP-2Lys46 isoforms. However, for both MCP-2 variants the NH2-terminal pyroglutamate was shown to be essential for chemotaxis, but not for calcium mobilization. NH2-terminal truncation of rMCP-2Lys46 by the serine protease CD26/dipeptidyl peptidase IV (CD26/DPP IV) resulted in the cleavage of the NH2-terminal Gln-Pro dipeptide, whereas synthetic MCP-2 with an amino-terminal pGlu remained unaffected. CD26/DPP IV-clipped rMCP-2Lys46(3-76) was almost completely inactive in both chemotaxis and signaling assays. These observations indicate that the NH2-terminal pGlu in MCP-2 is necessary for chemotactic activity but also that it protects the protein against degradation by CD26/DPP IV.

Alleles↗
PubMed1979

Genetic mapping of the coding regions for three heat-shock proteins in Drosophila melanogaster.

We describe variants of three heat-shock proteins of Drosophila melanogaster and their use to map the chromosome regions that contain the coding sequences for these proteins. All three map to a region on chromosome 3L that includes only one heat-shock puff, designated as 67B. The results imply that the genes coding for at least three heat-shock proteins are included within the 67B region.

Animals↗
PubMed1999

Analysis of coding sequences for tissue inhibitor of metalloproteinases 1 (TIMP1) and 2 (TIMP2) in patients with aneurysms.

Aneurysms are characterized by dilation, i.e. expansion and thinning of all the arterial wall layers, which is accompanied by remodeling of the connective tissue. Genes involved in the regulation of tissue remodeling are therefore candidate genes. We analyzed TIMP1 and TIMP2 coding sequences in 12 individuals with abdominal aortic aneurysms (AAA), one individual with AAA and intracranial aneurysms (IA), four individuals with IA and two clinically unaffected individuals. We identified two nucleotide variants in both the TIMP1 and the TIMP2 coding sequences. All differences occurred in the third base positions of codons and were neutral polymorphisms. A significant difference was observed in the frequency of TIMP2 nt 573 polymorphism between 168 alleles from AAA patients and 102 control alleles.

Alleles↗
PubMed2003

Functional characterization of three human cytochrome p450 2E1 variants with amino acid substitutions.

1. Cytochrome p450 (p450) 2E1 is a hepatic enzyme of importance for the metabolism of xenobiotics such as drugs and environmental toxicants. Genetic polymorphisms of CYP2E1 in 5'-flanking and coding regions have been found previously in Caucasian and Chinese populations. 2. In order to investigate the effects of amino acid substitutions on the function of CYP2E1, the enzymes of all known CYP2E1 variants in the coding region (CYP2E1.2, CYP2E1.3 and CYP2E1.4) with Arg76His, Val389Ile and Val179Ile substitutions, respectively, as well as the wild-type CYP2E1 (CYP2E1.1) were expressed in COS-1 cells, and their chlorzoxazone 6-hydroxylation and 4-nitrophenol 2-hydroxylation activities were determined. 3. The protein level of CYP2E1.2 was reduced to 29% compared with that of CYP2E1.1. The profiles of the level of activity relative to CYP2E1.1 for chlorzoxazone 6-hydroxylation (300 microM substrate) and 4-nitrophenol 2-hydroxylation (150 microM substrate) were very similar. 4. Although the K(m) values were not significantly different among wild-type and variant CYP2E1s in any oxidation metabolism, the V(max) and V(max)/K(m) of CYP2E1.2 on the basis of the CYP2E1 protein level were 2.7-3.0-fold higher than those of CYP2E1.1. In contrast, the levels of CYP2E1 protein and catalytic activity of CYP2E1.3 and CYP2E1.4 were not affected by the corresponding amino acid substitutions. 5. The findings suggest that Arg76 is closely associated with the function of CYP2E1, and that the genetic polymorphism of CYP2E1 is one cause of interindividual differences in the toxicity of xenobiotics.

Amino Acid Substitution↗
PubMed1998

Salivary acidic proline-rich proteins in rheumatoid arthritis.

In an ongoing attempt to investigate qualitative salivary parameters in diseases affecting salivary glands, patients with rheumatoid arthritis (RA) were examined. Patients were selected from the Oslo RA register for the present study if they fulfilled the following criteria: age 52-74 years, disease duration 10-20 years, and disability score as assessed by the Modified Health Assessment Questionnaire < or = 2.5. From these 105 patients, two subgroups of patients were selected, one group with pronounced sicca symptoms from eyes and mouth, and one group without such symptoms. Sicca symptoms were assessed using a postal questionnaire with the questions on dry mouth and dry eyes of the European classification criteria for Sjögren's syndrome. Patients were excluded from further examinations if they used medication that could cause dryness in eyes or mouth. Thus, nine patients remained in the sicca group (having four or more sicca symptoms), and ten matched RA patients were selected for the nonsicca group. A healthy sex- and age-matched control group (n = 10) was also examined. In a preliminary report we have shown that differences in flow rates between sicca and nonsicca RA patients were limited to lower values of unstimulated whole saliva. To further evaluate salivary changes in RA, a disease frequently associated with secondary Sjögren's syndrome, we have studied qualitative salivary parameters in these patients,' including secretory rates of proline-rich proteins (PRPs), statherins, and histatins. In the present report, phenotypes of PRPs, the ratio of PRPs derived from the two loci (PRH1 and PRH2), and PRP concentration and output in parotid and submandibular saliva derived from the two loci are presented. Parotid (PS) and submandibular saliva (SS) were collected from all individuals using 2% citric acid as a saliva stimulus. PRPs in PS and SS were identified using a SMART microchromatographic system with a Mono Q column and a Tris-HCl/NaCl gradient (method adapted from ref. 5). For PRPs, the primary polypeptide products are coded for on two loci (PRH1 and PRH2), which have three and two commonly occurring gene variants, respectively. On PRH1, the proteins PIF-s, Db-s, and Pa are coded for, whereas PRP-1 and PRP-2 are coded for on the PRH2 locus. As each protein variant has a postranscriptional cleavage product, individuals will exhibit four, six, or eight PRPs in their saliva, depending on whether they are homozygous at both, one, or neither of the two loci. Accordingly, 18 possible phenotypes may exist, but as few as three phenotypes were found in 79% of the 127 healthy individuals examined by Hay et al. The SMART system allows the determination of the different acidic PRPs present in saliva. Concentrations of the various phenotypes were calculated by peak integration versus pure PRP standards. Total PRP concentration derived from each locus was calculated as the sum of the concentrations of PRP variants from that locus.

Aged↗
PubMed2002

HOX11L1: a promoter study to evaluate possible expression defects in intestinal motility disorders.

Intestinal Neuronal Dysplasia (IND) is a congenital disorder characterized by intestinal motility defects associated with hyperplasia of enteric ganglia. A phenotype resembling human IND has been observed in mice knocked-out for a member of the Hox11 homeobox gene family, Hox11l1, suggesting that the human homologue of this gene could be responsible for congenital disorders of intestinal innervation. However, previous mutation analysis of the coding sequence of the HOX11L1 gene in patients affected with IND detected neither mutations nor other nucleotide variants. In the present work, a detailed study of the non coding promoter region of this gene was undertaken in patients affected with IND, with Hirschsprung associated IND and with neurogenic chronic intestinal pseudo-obstruction. No alterations potentially impairing expression of HOX11L1, such as nucleotide variants, small deletions or cytogenetic alterations, could be identified thus further excluding the direct involvement of this gene in the pathogenesis of human intestinal motility disorders.

Autonomic Nervous System Diseases↗
PubMed1991

Molecular defect of a phosphoglycerate kinase variant (PGK-Matsue) associated with hemolytic anemia: Leu----Pro substitution caused by T/A----C/G transition in exon 3.

We have identified the mutation in a phosphoglycerate kinase variant (PGK-Matsue) associated with severe enzyme deficiency, congenital nonspherocytic hemolytic anemia, and mental disorders. The mRNA coding for PGK was reverse transcribed and amplified by the polymerase chain reaction. Nucleotide sequencing of the variant cDNA showed a point mutation, a T/A----C/G transition in exon 3 of the variant gene. No other mutation was found in all coding regions of PGK-Matsue. The nucleotide change created an additional NciI cleavage site in the variant gene; thus, the NciI fragment types detected by Southern blot hybridization differ in the variant DNA and normal DNA. The mutation should cause Leu----Pro substitution at the 88th position from the NH2-terminal Ser of PGK. Because the Leu----Pro substitution is expected to induce serious perturbation and instability in the protein structure, the severe enzyme deficiency is mainly caused by more rapid in vivo denaturation and degradation of the variant enzyme.

Adenine↗
PubMed1999

[Molecular analysis of the human "growth hormone secretagogue"-receptor].

BACKGROUND: Growth hormone secretagogues (GHS) are highly potent synthetic peptides which release growth hormone (GH) by activation of a growth hormone-releasing hormone-independent signal cascade. A specific growth hormone secretagogue receptor (GHS-R) has been isolated, its endogenous ligand is still unknown. It might represent another major endocrine pathway controlling GH secretion. To gain insight into the specific function of the human GHS-R we studied the gene structure. Two variants, type 1a and 1b, have been described, but their specific functions are unknown. METHODS AND RESULTS: A specific probe for the GHS-R was cloned following reverse transcription and PCR amplification of pituitary mRNA. A genomic human placenta library was screened for the GHS-R gene. Positive clones were identified and further characterized by Southern blotting and sequencing. A genomic clone of 18 kb in size was determined to include the coding sequence of both GHS-R variants. Here we show that GHS-R type 1a and type 1b are encoded by a single gene. Sequencing of the immediate 5'-flanking region suggests a number of transcription factor binding sites, but their functional significance remains to be investigated. CONCLUSION: A genomic clone encoding for the two known variants of the human GHS-R was isolated. Further studies will determine physiological relevance and regulation of GHS-R. This will facilitate studies of GHS as diagnostic and therapeutic agents in GH disorders.

GTP-Binding Proteins↗
PubMed1996

Naturally occurring splicing variants of the hMSH2 gene containing nonsense codons identify possible mRNA instability motifs within the gene coding region.

We have identified certain unusually spliced cDNA species following PCR amplification of peripheral blood lymphocyte (PBL) mRNA from the hMSH2 gene. A naturally occurring transcript containing a nonsense codon due to the skipping of 5 exons was amplified from PBLs of several healthy individuals. A feature of this and another unusual splicing product was the presence of sequence motifs which bore significant similarity to mRNA instability determinants in the region immediately downstream of the stop codon. In particular, the rare tetranucleotide GAUG, previously identified in yeast as being of critical importance to the rapid degradation of nonsense-containing mRNAs was situated 23 base pairs downstream of the stop codon. Furthermore the region downstream of the stop codon was A:U rich and contained 2 copies of the AUUUA motif. As other forms of alternative splicing would not result in the same juxtaposition of stop codons and instability motifs, we suggest that the stop codons may have been deliberately introduced by the splicing process for their proximity to these destabilising motifs, and that splicing may play a role in channeling mRNAs into degradative pathways. These results are consistent with the hypothesis that nuclear factors may scan pre-mRNAs prior to splicing.

Adenosine Triphosphatases↗