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A Metspalu

Publications and source records attributed to A Metspalu.

At least 19 recordsLinked to original sources
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↗
PubMed1996

Mutation detection by solid phase primer extension.

A mutation analysis method based upon a wild-type DNA sequence is presented. Oligonucleotides were utilized for primer extension by T7 DNA polymerase to discriminate between wild-type and mutant sequences in two solid phase approaches. 1. Oligonucleotides were annealed to an immobilized template, extended with fluorescent dideoxynucleotides (ddNTPs), and analyzed on an automated fluorescent DNA sequencer. The oligonucleotide length identified the known mutation site, and the fluorescence emission of the ddNTP identified the mutation. 2. Template DNA was annealed to an oligonucleotide array, extended with alpha-32P dNTPs, and analyzed with a Phosphor Imager. The grid position of the oligonucleotide identified the mutation site and the extended base identified the mutation.

Base Sequence↗
PubMed1996

Phenylalanine hydroxylase gene mutation R408W is present on 84% of Estonian phenylketonuria chromosomes.

Phenylalanine hydroxylase (PAH) is the enzyme which converts phenylalanine into tyrosine. In case of its deficiency, hyperphenylalaninemia is observed, which leads to phenylketonuria (PKU), a disease causing mental retardation, unless treated with a low-phenylalanine diet since early childhood. In Estonia, PKU is among the most common inherited metabolic diseases. The data from retrospective study and newborn screening show an approximate incidence of 1 in 6,000 newborns. Molecular analysis of 34 Estonian patients has revealed high genotypic homogeneity in this group, as 84% of the mutant alleles carry the R408W mutation. The high rate of this mutation in the Estonian population rises the speculation of Finno-Ugric contribution to the East European pool of mutant PAH alleles. Five more mutations-IVS12nt1, R261Q, R252W, R158Q, S349P-have been detected. The mutation detection rate was 92% among the studied patients.

Estonia↗
PubMed1995

The human ribosomal protein S7-encoding gene: isolation, structure and localization in 2p25.

We have identified a gene encoding the human ribosomal protein (r-protein) S7. The S7 gene contains seven exons and six introns spanning about 6 kb. Organization of the gene is similar to that of Xenopus laevis S8, the only homologous intron-containing gene isolated so far. An mRNA transcribed from this gene has an open reading frame (ORF) of 582 nucleotides (nt), which encodes a protein of 194 amino acids (22.1 kDa). The transcription start point (tsp) was mapped by a primer extension assay to a C residue within a pyrimidine-rich tract. Human S7 (hS7) is identical to rat S7 (rS7) and exhibits significant similarity with the X. laevis, insect and plant homologs. We have used fluorescence in situ hybridization (FISH) to localize S7 to chromosome 2p25.

Amino Acid Sequence↗
PubMed1995

An RNA stem-loop structure involved in the packaging of bovine leukemia virus genomic RNA in vivo.

An RNA secondary structure of the bovine leukemia virus (BLV) 5'-terminal RNA sequence was constructed by computer-assisted RNA secondary structure analysis. Mutations were created in the noncoding region (NCR) of BLV, which contains a conserved consensus sequence, to disrupt predicted secondary structure of this region. After transfection of these constructs into FLK-BLV cells and analysis of viral particles a reduction in mutant RNA content was observed relative to that of unmutated vector RNA. The packaging efficiency of the mutant with a substitution in the consensus sequence was reduced threefold and that of the mutant with a deleted 5' NCR was reduced fivefold. We conclude that predicted RNA secondary structure and/or nucleotide sequence of the BLV noncoding region is essential for BLV RNA packaging in vivo.

Animals↗
PubMed1992

The human ribosomal protein S6 gene: isolation, primary structure and location in chromosome 9.

Using PCR cloning we isolated the first intron of the human ribosomal protein S6 gene (hRPS6). By screening the human HeLa cell cDNA library in lambda ZAPII vector (Stratagene, La Jolla, CA), we identified and sequenced a partially spliced pre mRNA copy of hRPS6. The complete hRPS6 gene was isolated from a lambda DASH library with an intron-specific probe. The gene and flanking regions were sequenced, and the mRNA 5' end was mapped by primer extension experiments. The hRPS6 gene has 6 exons and 5 introns and is 3.6 kb long. Using intron-specific primers in PCR and a panel of human-hamster cell lines we localized the hRPS6 gene in human chromosome 9.

Amino Acid Sequence↗
PubMed1992

Human ribosomal protein S3a: cloning of the cDNA and primary structure of the protein.

The amino acid (aa) sequence of human ribosomal protein S3a (hRPS3a) was deduced partially from the nucleotide sequence of the corresponding cDNA and confirmed by direct aa sequencing from the N terminus of the purified hRPS3a protein. The cDNA clone was isolated from a cDNA expression library in the pEX vector using antibodies. The hRPS3a protein has 263 aa and its calculated M(r) is 29 813.

Amino Acid Sequence↗
PubMed1988

A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells.

A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.

Animals↗
PubMed1987

Structure and expression of the genes coding for human alpha 1-acid glycoprotein.

alpha 1-acid glycoprotein (alpha AGP) is a well-characterized human plasma protein. Its structural properties have been studied for many years but little is known about its function. Amino acid sequence analysis of purified human alpha AGP from plasma pooled from several individuals showed considerable heterogeneity. We have cloned the genomic DNA segment encoding alpha AGP and we show that it contains three adjacent alpha AGP coding regions, AGP-A, B and B', identical in exon--intron organization but with slightly different coding potential. These results account for the heterogeneity observed by protein sequencing. Southern blot analysis indicates that the cloned cluster contains all the alpha AGP coding sequences present in the human genome. The larger majority of alpha AGP mRNA in human liver is transcribed from AGP-A, whose promoter and cap site have been determined while the level of AGP-B and B' mRNA in human liver is very low. Using Hep3B hepatoma cells as a model system for the in vitro study of the acute phase reaction, we show that only AGP-A is strongly induced by treatment with culture medium of LPS stimulated monocytes.

Amino Acid Sequence↗
PubMed1981

Location of single-stranded and double-stranded regions in rat liver ribosomal 5S RNA and 5.8S RNA.

Rat liver 5S rRNA and 5.8S rRNA were end-labelled with 32P at 5'-end or 3'-end of the polynucleotide chain and partially digested with single-strand specific S1 nuclease and double-strand specific endonuclease from the cobra Naja naja oxiana venom. The parallel use of these two structure-specific enzymes in combination with rapid sequencing technique allowed the exact localization of single-stranded and double-stranded regions in 5S RNA and 5.8 S RNA. The most accessible regions to S1 nuclease in 5S RNA are regions 33-42, 74-78, 102-103 and in 5.8 S RNA 16-20, 26-29, 34-36, 74-80 and a region around 125-130. The cobra venom endonuclease cleaves the following areas in 5S RNA: 7-8, 17-20, 28-30, 49-51, 56-57, 60-64, 69-70, 81-82, 95-97, 106-112. In 5.8S RNA the venom endonuclease cleavage sites are 4-7, 10-13, 21-22, 33-35, 43-45, 51-55, 72-74, 85-87, 98-99, 105-106, 114-115, 132-135. According to these results the tRNA binding sequences proposed by Nishikawa and Takemura [(1974) FEBS Lett. 40, 106-109], in 5S RNA are located in partly single-stranded region, but in 5.8S RNA in double-stranded region.

Animals↗