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A Metspalu

Publications and source records attributed to A Metspalu.

At least 19 recordsLinked to original sources
PubMed2000

Human N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (human meprin): genomic structure of the alpha and beta subunits.

N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), a zinc-metalloendopeptidase of the astacin family, consists of two similar subunits. As well as in small-intestinal epithelial cells, the enzyme is found in lamina propria leucocytes, human cancer cells and colorectal cancer tissue, making it a potential candidate for a role in tumour formation and cancer progression. To elucidate the mechanisms that control PPH gene expression and to gain more insights into the evolutionary relationship of the two subunits, we analysed the complete exon-intron organization and searched for putative regulatory elements in 3 kb of the upstream region of both genes. The human gene for the alpha subunit is approx. 35 kb in size and contains 14 exons. The gene for the beta subunit is organized in 15 exons and spans approx. 27 kb. A comparison of both genes indicates strong structural similarities. The exons are almost identical in size, except exon 13 in PPHalpha, which codes for an additional I domain not present in PPHbeta. The locations of the respective exon-intron junctions and the intron phases are almost identical; five of them contain conserved split codons. The main variation is in the intron lengths. It can be concluded that PPHalpha and PPHbeta are derived from a common ancestor. Sequence analysis of the 5' flanking DNA with a computer search for promoter elements and different promoter constructs transfected into Caco-2 cells revealed a number of potential regulatory motifs and suggests that each of the two genes is regulated independently.

Amino Acid Sequence↗
PubMed2000

Mutation 985A>G in the MCAD gene shows low incidence in Estonian population.

Medium-chain acyl-CoA dehydrogenase (MCAD) is an enzyme responsible for large part of mitochondrial beta-oxidation of fatty acids and therefore stays on key position of cellular energy supply. In case of its deficiency, starvation, rapid growth periods or infections may cause fatal lack of energy, especially in the first years of life. MCAD deficiency is inherited in an autosomal recessive manner and it has been shown to be rather common in some European countries (Great Britain 1 in 6,000, Switzerland 1 in 10,000). In Caucasoid populations one mutation, the 985A>G transition, causing the amino acid substitution K329E, accounts for about 90% of all mutant MCAD alleles. Here we present data about screening the Estonian population for this mutation. We analyzed the DNA from 1,098 persons from all regions of Estonia (all newborns born in one month) and found 5 heterozygotes for 985A>G, that makes the carrier frequency 1 in 220 and the frequency of possibly affected homozygotes 1 out of 193,000. No mutant alleles were found among the samples of the children, who had unclear diagnosis for death during the years 1994 and 1995.

Acyl-CoA Dehydrogenase↗
PubMed2000

High carrier frequency of the 35delG deafness mutation in European populations. Genetic Analysis Consortium of GJB2 35delG.

Congenital deafness accounts for about 1 in 1000 infants and approximately 80% of cases are inherited as an autosomal recessive trait. Recently, it has been demonstrated that connexin 26 (GJB2) gene is a major gene for congenital sensorineural deafness. A single mutation (named 35delG) was found in most recessive families and sporadic cases of congenital deafness, among Caucasoids, with relative frequencies ranging from 28% to 63%. We present here the analysis of the 35delG mutation in 3270 random controls from 17 European countries. We have detected a carrier frequency for 35delG of 1 in 35 in southern Europe and 1 in 79 in central and northern Europe. In addition, 35delG was detected in five out of 376 Jewish subjects of different origin, but was absent in other non-European populations. The study suggests either a single origin for 35delG somewhere in Europe or in the Middle East, and the possible presence of a carrier advantage together with a founder effect. The 35delG carrier frequency of 1 in 51 in the overall European population clearly indicates that this genetic alteration is a major mutation for autosomal recessive deafness in Caucasoids. This finding should facilitate diagnosis of congenital deafness and allow early treatment of the affected subjects.

Connexin 26↗
PubMed2000

Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology.

The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 5' end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common beta-thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identified. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.

DNA↗
PubMed2000

Unravelling genetic data by arrayed primer extension.

We have developed a method for arrayed primer extension (APEX) on an oligonucleotide microchip together with the 4-color fluoresence imaging equipment and supporting software, that allows analysis of the DNA sequence and changes in it. Mutation analysis of BRCA1 gene and single nucleotide polymorphism (SNP) chip for genotyping were used as a model system. Chip surface chemistry, template preparation and APEX reaction conditions were optimised and the assay is ready to be implemented in variety of DNA analysis from SNP testing to DNA resequencing.

Alleles↗
PubMed1999

U82, a novel snoRNA identified from the fifth intron of human and mouse nucleolin gene.

A novel snoRNA, designated as U82, was found from the sequence analysis of the 5th intron of human and mouse nucleolin gene. The snoRNA U82 has characteristic boxes C, D and D' and 11 nucleotides (nt) antisense complementarity to the 18S rRNA. Presumably U82 functions as a guide in the methylation of residue A1678 in human 18S rRNA. Northern blot analysis with various oligodeoxynucleotide probes showed that human and mouse U82 is expressed as RNA variants with length of 70 (+/- 1) and 67 (+/- 1) nt in HeLa and mouse C127 cells. Most probably, the 70 nt variant of U82 is encoded by nucleolin gene 5th intron. The 67 nt variant of U82 could be a transcript of another gene, the genomic locus of which remains unknown.

Animals↗
PubMed1999

Genomic organization of the human complex I 13-kDa subunit gene NDUFA5.

We have characterized the human gene NDUFA5 encoding a 13-kDa subunit of the mitochondrial respiratory chain complex I (NADH: ubiquinone oxidoreductase). The gene contains 5 exons and 4 introns, and spans 14 kb of genomic DNA. In the untranscribed region we observed potential transcription factor binding sites. We determined a single nucleotide variant (C/T) at -318, and its frequency in the German population. The functional gene was localised by FISH to 7q31 and by radiation hybrid panel near marker D7S648 in YAC 883_a_2.

Animals↗
PubMed1998

Nuclear import and nucleolar accumulation of the human ribosomal protein S7 depends on both a minimal nuclear localization sequence and an adjacent basic region.

In the course of the eukaryotic ribosomal biogenesis, both the nuclear import and export are involved. We have studied the nuclear and nucleolar localization of the human ribosomal protein S7. We examined the subcellular distribution of the S7:beta-galactosidase fusion protein in SAOS-2 cells. We have identified two evolutionarily conserved domains, both of which are necessary for S7 nuclear and nucleolar targeting: amino acids 98 to 109 and 115 to 118. Out of the S7 protein context, a fragment 98...118, containing these domains, is sufficient for nuclear transport and nucleolar accumulation. Interestingly, a tetrapeptide 115KRPR118, which can act as an independent nuclear localization signal (NLS), is not sufficient for exclusively nuclear accumulation of the S7 protein if the adjacent region 98...109 is deleted. In addition, site-directed mutagenesis revealed that critical residues for nuclear targeting in this tetrapeptide and in the full-length S7 protein are different. While mutation of a Pro117 significantly impaired nuclear import of S7, similar substitution within the tetrapeptide-NLS had no effect on nuclear targeting. This suggests that to function perfectly, proper secondary structure of the S7 nuclear targeting domain is required.

Amino Acid Sequence↗
PubMed1998

A novel snoRNA (U73) is encoded within the introns of the human and mouse ribosomal protein S3a genes.

The mouse ribosomal protein S3a-encoding gene (mRPS3a) was cloned and sequenced in this study. mRPS3a shares identical exon/intron structure with its human counterpart. Both genes are split to six exons and exhibit remarkable conservation of the promoter region (68.8% identity in the 250 bp upstream of cap site) and coding region (the proteins differ in two amino acids). mRPS3a displays many features common to other r-protein genes, including the CpG-island at 5'-end of the gene, cap site within an oligopyrimidine tract and no consensus TATA or CAAT boxes. However, mRPS3a represents a rare subclass of r-protein genes that possess a long coding sequence in the first exon. Comparison of human and mouse S3a genes revealed sequence fragments with striking similarity within introns 3 and 4. Here we demonstrate that these sequences encode for a novel small nucleolar RNA (snoRNA) designated U73. U73 contains C, D and D' boxes and a 12-nucleotide antisense complementarity to the 28S ribosomal RNA. These features place U73 into the family of intron-encoded antisense snoRNAs that guide site-specific 2'-O-ribose methylation of pre-rRNA. We propose that U73 is involved in methylation of the G1739 residue of the human 28S rRNA. In addition, we present the mapping of human ribosomal protein S3a gene (hRPS3a) and internally nested U73 gene to the human chromosome 4q31.2-3.

Amino Acid Sequence↗
PubMed1998

Isolation and characterization of the mouse ribosomal protein S7 gene.

We have isolated and characterized the single intron-containing gene encoding the mouse ribosomal protein S7. The mouse 129SV S7 gene has seven exons and six introns spanning about 5000 nucleotides. The exon-intron structure of the gene is similar to other vertebrate homologues. Southern blot analysis showed that in addition to the isolated single-copy intron-containing gene, there are 10-12 members in the mouse S7 gene family, which are all most probably processed pseudogenes. The promoter region of rpS7 contains several evolutionarily conserved putative regulatory elements. The main transcription start site was mapped to a T residue within a polypyrimidine tract, 79 nucleotides upstream from AUG codon.

Amino Acid Sequence↗
PubMed1998

Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Neonatal screening for the cystic fibrosis main mutation delta F508 in Estonia.

In this pilot study the frequency of delta F508 mutation carriers, their geographic distribution, and the prevalence of cystic fibrosis (CF) in Estonia were investigated. During the screening programme 7396 newborns were tested for delta F508 mutation and 88 were found to carry this deletion. The mean frequency of delta F508 mutation carriers in Estonia was thus estimated as 1 out of 84 live births. In eight separate districts of Estonia the heterozygote frequencies differed significantly (p = 0.0369), with the highest incidence (1:36) on the Baltic Sea islands and western coastal region and with the lowest in south eastern parts of Estonia.

Cystic Fibrosis↗
PubMed1998

Development of the phenylketonuria screening programme in Estonia.

OBJECTIVE: To develop the phenylketonuria (PKU) screening programme in Estonia. METHOD: All data about patients with PKU, born during 1980-92, were documented to establish its prevalence at birth in Estonia. Newborn screening for the diagnosis and treatment of PKU was started in Estonia in 1993 and the prevalence at birth established by screening. Phenylalanine was determined from filter paper blood by a modified fluorometric method based on enhancement of the fluorescence of a phenylalanine-ninhydrin reaction product by L-leucyl-L-alanine. RESULTS: During three years (1993-5) 36,074 newborns (85% of the total) were screened for PKU. PKU was diagnosed in six cases during the first four to six weeks of life. All investigated cases could be classified as classical PKU. No cases of mild forms of hyperphenylalaninaemia were diagnosed. The retrospective study showed an average incidence of PKU of 1 in 8090, the prospective study identified a comparable incidence of 1 in 6010 live births. CONCLUSION: The prevalence at birth of classic PKU in Estonia is higher than the average in Europe and similar to that of some eastern and middle European countries.

Estonia↗
PubMed1997

A human cDNA encoding the homologue of NADH: ubiquinone oxidoreductase subunit B13.

A cDNA encoding the human homologue of bovine NADH:ubiquinone oxidoreductase (complex I of mitochondrial respiratory chain) subunit B13 has been isolated. The clone contains an open reading frame of 348 bp, 23 bp of 5'-untranslated sequence (UTR) and a long 3'UTR of 1088 bp. The deduced amino-acid sequence is 87% identical to bovine B13. Human B13 mRNA expression was observed in all tissues examined with highest levels in heart, skeletal muscle and brain. Southern analysis of human genomic DNA revealed the presence of multigene family.

Amino Acid Sequence↗
PubMed1997

Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays.

We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.

DNA-Directed DNA Polymerase↗
PubMed1996

Structural characterization of the mouse ribosomal protein S6-encoding gene.

The gene encoding mouse ribosomal protein (r-protein) S6 is 2.7 kb in length, and is composed of five exons. The intron positions of the mouse S6 (Rps6) coincide exactly to those of the homologous human S6 (RPS6), but the last intron present in the human is absent in the mouse gene. The latter displays higher G + C content than the RPS6, both in the overall sequenced region and at the 3rd codon position. The promoter area is highly conserved between mouse and human, and contains several putative cis-acting elements. Comparison of the intronic sequences of both genes revealed surprisingly a high degree of identity (63%) within 350 bp of the first intron. Besides the single-copy Rsp6 there are up to 15 S6 family members, most likely processed pseudogenes. Characterization of the Rps6 provides a basis to study the functions of the mammalian S6 by gene targeting.

Amino Acid Sequence↗