Biomedical subjects

W Dackowski

Publications and source records attributed to W Dackowski.

8 recordsLinked to original sources

PubMed1993

Chromosomal localization of glutamate receptor genes: relationship to familial amyotrophic lateral sclerosis and other neurological disorders of mice and humans.

Receptors for the major excitatory neurotransmitter glutamate may play key roles in neurodegeneration. The mouse Glur-5 gene maps to chromosome 16 between App and Sod-1. The homologous human GLUR5 gene maps to the corresponding region of human chromosome 21, which contains the locus for familial amyotrophic lateral sclerosis. This location, and other features, render GLUR5 a possible candidate gene for familial amyotrophic lateral sclerosis. In addition, dosage imbalance of GLUR5 may have a role in the trisomy 21 (Down syndrome). Further characterization of the murine glutamate receptor family includes mapping of Glur-1 to the same region as neurological mutants spasmodic, shaker-2, tipsy, and vibrator on chromosome 11; Glur-2 near spastic on chromosome 3; Glur-6 near waltzer and Jackson circler on chromosome 10; and Glur-7 near clasper on chromosome 4.

Amyotrophic Lateral Sclerosis↗

PubMed1992

Molecular analysis of human chromosome 16 cosmid clones containing NotI sites.

To test the feasibility of using cloned NotI sites as markers for physical mapping, we have screened for cosmid clones spanning the NotI sites on human Chromosome (Chr) 16. Fluorescence in situ hybridization analysis of these clones confirms the previously reported cluster of NotI sites on 16p13.3. Methylation status of the cloned NotI sites on genomic DNA was established by hybridization of the cosmids to Southern blots containing EcoRI and EcoRI/NotI digest of genomic DNA. These results indicated that four of six clones included in our study can be used as linking clones for physical mapping. Two clones have NotI sites which are not cleavable in the cell lines tested. In one clone, the NotI site exists as an isolated rare-cutting restriction enzyme site, whereas in the other clone the NotI site appears to be island-related.

Blotting, Southern↗

PubMed1992

Multicolor fluorescence in situ hybridization for the simultaneous detection of probe sets for chromosomes 13, 18, 21, X and Y in uncultured amniotic fluid cells.

The most frequent aneuploidies in newborns involve the autosomes 13, 18 and 21 as well as both sex chromosomes. Fluorescence in situ hybridization readily allows the detection of numerical chromosomal aberrations throughout all stages of the cell cycle. Using a multicolor fluorescence in situ hybridization approach based on combinatorial probe labeling and digital imaging microscopy we demonstrate the simultaneous visualization of probe sets specific for chromosomes 13, 18, 21, X and Y. This approach enables one to evaluate aberrations of multiple chromosomes in a single hybridization experiment using metaphase chromosomes and interphase nuclei from a variety of cell types, including lymphocytes and amniocytes.

Amniotic Fluid↗

PubMed1990

Expression of completely gamma-carboxylated and beta-hydroxylated recombinant human vitamin-K-dependent protein S with full biological activity.

Human anticoagulant vitamin-K-dependent protein S was expressed in mouse C127 cells using a bovine papilloma virus vector system. A full-length cDNA construct was introduced into the vector in the 5' untranslated region of the mouse metallothionein-I gene. Transfected cells expressed approximately 10 micrograms/ml of the recombinant protein which was purified by ion-exchange chromatography followed by affinity chromatography using Ca2(+)-dependent monoclonal antibodies against the region of protein S containing 4-carboxyglutamic acid. Recombinant protein S was structurally and functionally similar to protein S purified from plasma. On SDS/polyacrylamide-gel electrophoresis recombinant protein S had a slightly higher molecular mass than plasma protein S. After treatment with endoglycosidase F, the proteins comigrated suggesting the observed molecular mass difference to be due to alterations in the N-linked carbohydrate side chains. Recombinant and plasma protein S demonstrated identical amino-terminal sequences, similar amino acid composition and number of 4-carboxyglutamyl and 3-hydroxyaspartyl/asparaginyl residues. Recombinant protein S had the same affinity for Ca2+ as protein S from plasma and the two proteins had the same activated protein C cofactor activity in a functional assay. In addition, both forms of protein S formed complexes with C4b-binding protein with the same apparent Kd. Protein S is the most extensively post-translationally modified vitamin-K-dependent protein, and all the modifications were carried out in the recombinant DNA system yielding a recombinant protein S with full biological activity.

Amino Acid Sequence↗

PubMed1989

Identification of a putative isoform of the Na,K-ATPase beta subunit. Primary structure and tissue-specific expression.

We have isolated cDNA clones from rat brain and human liver encoding a putative isoform of the Na,K-ATPase beta subunit. The rat brain cDNA contains an open reading frame of 870 nucleotides coding for a protein of 290 amino acids with a calculated molecular weight of 33,412. The corresponding amino acid sequence shows 98% identity with its human liver counterpart. The proteins encoded by the rat and human cDNAs exhibit a high degree of primary sequence and secondary structure similarity with the rat Na,K-ATPase beta subunit. We have therefore termed the polypeptides these cDNAs encode a beta 2 subunit with the previously characterized rat cDNA encoding a beta 1 subunit. Analysis of rat tissue RNA reveals that the beta 2 subunit gene encodes a 3.4-kilobase mRNA which is expressed in a tissue specific fashion distinct from that of rat beta 1 subunit mRNA. Cell lines derived from the rat central nervous system shown to lack beta 1 subunit mRNA sequences were found to express beta 2 subunit mRNA. These results suggest that different members of the Na,K-ATPase beta subunit family may have specialized functions.

Amino Acid Sequence↗

PubMed1986

Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation.

Protein S is a cofactor of activated protein C; together they function as a regulator of blood coagulation. A human liver cDNA library constructed in bacteriophage lambda gt11 was screened with DNA fragments from a full-length bovine cDNA clone encoding protein S. Several cDNA clones were isolated and sequenced. The combined cDNA sequences encoded the mature protein and 15 residues of the leader sequence when compared to bovine protein S. Human protein S is a single-chain protein consisting of 635 amino acids with 82% homology to bovine protein S. After an NH2-terminal gamma-carboxyglutamic acid-containing region, there is a short region with thrombin-sensitive bond(s), followed by a region with four repeat sequences that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the COOH-terminal portion of human protein S does not show any resemblance to serine proteases.

Amino Acid Sequence↗

PubMed1981

Somatically generated mouse myeloma variants synthesizing IgA half-molecules.

Whereas mouse myelomas that secrete IgA half-molecules have been shown to arise in vivo, their origin has not been definitely established. We show that somatic variants secreting phenotypically similar molecules can arise directly from the normal IgA-secreting myelomas S107 and W3082. In addition to being improperly assembled, the variant proteins have distinct carboxy-terminal deletions and an aberrant heavy-light chain disulfide bond. For at least one of the variants, variable region serology and affinity for hapten are both unaffected by these changes. Southern and Northern blot analyses indicate normal size DNA restriction fragments and mRNA, suggesting premature termination as the mechanism of deletion. These results are discussed in relation to possible mutational hot spots and long-range interdomain interactions.

Animals↗

PubMed1981

Two alpha heavy chain disease proteins with different genomic deletions demonstrate that nonexpressed alpha heavy chain genes contain methylated bases.

Two independently arising alpha heavy chain mutants have been found to synthesize heavy chains with CH1 deletions of approximately equal extent. Both were isolated from heavy chain-producing variants of the mouse myeloma W3129 and demonstrate that it is possible to arrive at the heavy chain disease phenotype by the pathway H + L leads to H leads to delta H. Analysis of genomic DNA by digestion with restriction endonucleases followed by molecular hybridization showed that one mutant (delta 37) had a deletion of approximately 0.2 kilobase and the second mutant (delta 15) had a deletion of approximately 0.5 kilobase. Mouse myeloma cells contain several alpha chain alleles but only one is expressed; the presence of the deletion in delta 37 and delta 15 made it possible to identify the restriction fragments from the expressed allele. Analysis of the fragments produced after cleavage with an isoschizomeric pair of restriction enzymes, Msp I and Hpa II, indicated that, in the W3129 cell line and its variants, the unexpressed alpha alleles contain methylated bases. The influence of methylation on gene expression remains to be elucidated.

Alleles↗