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Biomedical subjects

B D Halligan

Publications and source records attributed to B D Halligan.

15 recordsLinked to original sources
PubMed2001

Variations in site and levels of expression of chondrocyte nucleotide pyrophosphohydrolase with aging.

The aim of this study was to identify changes in cartilage intermediate layer protein/nucleotide pyrophosphohydrolase (CILP/NTPPH) expression in articular cartilage during aging. Adult (3-4 years old) and young (7-10 days old) porcine articular hyaline cartilage and fibrocartilage were studied by Northern blot analysis, in situ hybridization, and immunohistochemistry using a complementary DNA (cDNA) probe encoding porcine CILP/NTPPH and antibody to a synthetic peptide corresponding to a CILP/NTPPH sequence. Northern blot analysis of chondrocytes showed lower expression of CILP/NTPPH messenger RNA (mRNA) in young cartilage than in adult cartilage. In adult cartilage, extracellular matrix from the surface to the middeep zone was immunoreactive for CILP/NTPPH, especially in the pericellular matrix surrounding the middeep zone chondrocytes. In young cartilage, chondrocytes were moderately immunoreactive for CILP/NTPPH throughout all zones except the calcified zone. The matrix of young cartilage was negative except in the superficial zone. In young cartilage, CILP/NTPPH mRNA expression was undetectable. In adult cartilage, chondrocytes showed strong mRNA expression for CILP/NTPPH throughout middeep zones. Protein and mRNA signals were not detectable below the tidemark. CILP/NTPPH secretion into matrix around chondrocytes increases with aging. In this extracellular site it may generate inorganic pyrophosphate and contribute to age-related calcium pyrophosphate dihydrate crystal deposition disease.

Aging↗
PubMed1999

V(D)J recombinational signal sequence DNA binding activities expressed by fetal bovine thymus.

V(D)J recombination, or immunoglobulin gene rearrangement is a developmentally regulated, cell type specific, site directed recombination event that brings either immunoglobulin or T-cell receptor gene segments together to form mature, expressible Ig or TCT genes. This DNA recombination is directed by the recombinational signal sequences or RSS elements present adjacent to Ig and TCR gene segments. The RSS element is composed of a conserved nonamer element and a conserved heptamer element separated by a conserved length spacer region. In this report, we examine the expression of DNA binding proteins that interact with the RSS element in the bovine fetal thymus using EMSA assays. Our data indicates that the nonamer portion of the RSS element is the primary site of recognition for RSS binding proteins expressed in the bovine fetal thymus. We also show that these proteins are expressed from early stages of bovine fetal development through to full term development.

Animals↗
PubMed1999

Characterization of a 3'-5' exonuclease associated with VDJP.

VDJP (V(D)J RSS Dependent DNA Joining Protein) was cloned based on binding to the nonamer portion of the V(D)J recombinational signal sequence (RSS), and genetic analysis revealed that VDJP is encoded by the same gene as the large subunit of Replication Factor C (RF-C). Recombinant VDJP has a site directed DNA joining activity and is capable of forming a covalent bond between DNA fragments containing an RSS element near their ends and exhibits 3' to 5' exonuclease activity. In this report, we examine the biochemical properties of the VDJP exonuclease activity such as directionality of nuclease action (3' to 5' or 5' to 3'), single-strand substrate preference, cleavage products, dependence on cofactors and metal cations, and optimal reaction conditions. From this analysis, we conclude that VDJP has an intrinsic 3'-5' exonuclease activity that produces mononucleotide products.

DNA↗
PubMed1997

Molecular cloning and expression of a porcine chondrocyte nucleotide pyrophosphohydrolase.

The porcine 127-kDa nucleotide pyrophosphohydrolase (NTPPHase) had been previously purified from the conditioned culture media of porcine articular cartilage. Protein sequencing of an internal 61-kDa proteolytic fragment of NTPPHase (61-kDa NTPPHase) determined the 26 N-terminal amino acids. This sequence was used to amplify a DNA fragment, which was used as a probe to clone the gene encoding the 61-kDa NTPPHase from a porcine chondrocyte cDNA library. DNA sequence analysis showed the cDNA insert to be 2509 bp, corresponding to a predicted open reading frame (ORF) encoding 599 amino acids. The 26 N-terminal amino acids of the 61-kDa NTPPHase were located within the ORF immediately downstream of a putative protease recognition region, RRKRR. This is consistent with this cDNA insert representing an internal proteolytic fragment of the full length 127-kDa NTPPHase. BLAST and FASTA analysis confirmed that the deduced amino acid sequence of 61-kDa NTPPHase was unique and did not possess a high degree of homology to sequence in the non-redundant protein and nucleotide databases. Proteins that possess limited homology (< 17%) with the 61-kDa NTTPPHase include several prokaryotic and eukaryotic ATP pyrophosphate-lyases (adenylate cyclase). Northern blot analysis of porcine chondrocyte RNA showed that the DNA encoding the 61-kDa NTPPHase hybridized to a single 4.0-kb RNA transcript. This DNA probe also hybridized to a single species of human chondrocyte RNA. Expression of a 61-kDa protein was detected by coupled in-vitro transcription/translation. Western blot analysis of this in-vitro transcription/translation reaction detected a 61-kDa protein, using an antibody raised against the peptide sequence that was originally used to clone the 61-kDa NTPPHase. These data indicate the successful in-vitro cloning and expression of the porcine chondrocyte 61-kDa NTPPHase. Future studies that utilize the gene encoding the 61-kDa NTPPHase may allow the characterization of the role of NTPPHase in calcium pyrophosphate dihydrate (CPPD) crystal deposition disease.

Amino Acid Sequence↗
PubMed1997

Site directed DNA joining.

We have previously identified a cDNA encoding part of the amino terminal portion of the large subunit of replication factor c (RF-C, AP-1), which we have named VDJP. Analysis of VDJP demonstrated that it has amino acid homology to bacterial DNA ligases and specific binding to the nonamer portion of the V(D)J recombination signal sequence motif. In this report, we demonstrate that VDJP is capable of forming a covalent bond between DNA fragments in a sequence dependent fashion. The VDJP mediated DNA ligation reaction is neither dependent on the presence of compatible DNA ends nor on sequence homology between the DNA fragments that are joined. Furthermore, we show that the covalent junction between the DNA fragments is resistant to proteases and phenol, and therefore not protein linked.

DNA Ligases↗
PubMed1995

Cloning of the murine cDNA encoding VDJP, a protein homologous to the large subunit of replication factor C and bacterial DNA ligases.

A putative full-length 1.7-kb cDNA, encoding a murine protein that specifically binds to the nonamer portion of the V(D)J recombinational signal sequence (RSS) element, has been cloned. By its sequence analysis, this cDNA is identical to a portion of the 4.5-kb murine replication factor C large-subunit-encoding cDNA. By Northern blot analysis, the 1.7-kb mRNA species is observed in murine immature B cells but not in non-lymphoid cells and tissues, while the 4.5-kb replication factor C-encoding cDNA is expressed in all cell types. The deduced VDJP amino-acid sequence includes a region of homology with bacterial DNA ligases at the C terminus of each of the proteins. VDJP has been synthesized as a fusion protein in bacteria, and the purified protein has been previously shown to mediate the joining of DNA fragments in a V(D)J RSS-dependent fashion (Guilliams et al., Biochem. Biophys. Res. Commun. 202 (1994) 1134-1141).

Amino Acid Sequence↗
PubMed1994

Distance and end configuration effects on VDJP-mediated DNA joining.

We have previously reported the cloning of a protein, VDJP, that is capable of binding the nonamer element of the V(D)J Recombinational Signal Sequence (RSS) as well as joining linear DNA fragments containing RSS elements in vitro. We show here that the linearized DNA molecules must contain a 5' extension or blunt end in order to be joined by VDJP. DNAs with 3' extensions are not efficiently joined by VDJP. Furthermore, the joining activity of DNAs with 5' extensions is significantly increased as the distance between the end and the RSS decreases. It is not yet clear what role VDJP plays in vivo, because our assay may not mimic exactly the in vivo DNA intermediates.

Animals↗
PubMed1993

Nonamer binding protein induces a bend in the immunoglobulin gene recombinational signal sequence.

DNA bending has been shown to play a critical role in conservative site-specific DNA recombination reactions such as lambda integration. V(D)J recombination, the only mammalian site directed recombination system, is directed by recombinational signal sequences composed of heptamer, nonamer and spacer elements. The nonamer element, GGTTTTTGT, is similar to the consensus sequence for bent DNA. Using the circular permutation electrophoretic mobility assay, we show that the nonamer sequence has a detectable intrinsic bend. The nonamer sequence has been shown to be the binding site for nonamer binding protein (NBP). Binding of NBP to the nonamer site increases the apparent angle of the bend from 32 degrees to 66 degrees. The identification of a protein induced DNA bend near the site of V(D)J recombination may have implications for our understanding of the mechanism of V(D)J recombination.

Animals↗
PubMed1987

Identification of a DNA binding protein that recognizes the nonamer recombinational signal sequence of immunoglobulin genes.

Extracts of nuclei from B- and T-lymphoid cells contain a protein that binds specifically to the conserved nonamer DNA sequence within the recombinational signals of immunoglobulin genes. Complexes with DNA fragments from four kappa light-chain joining (J) segments have the same electrophoretic mobility. Nonamer-containing DNA fragments from heavy-chain and light-chain genes compete for binding. Within the 5'-flanking DNA of the J kappa 4 gene segment, the binding site has been localized to a 27-base-pair interval spanning the nonamer region. The binding activity is recovered as a single peak after ion-exchange chromatography. The site of binding of the protein and its presence in nuclei of lymphoid cells suggest that it may function in the assembly of immunoglobulin genes.

Animals↗
PubMed1985

Purification and characterization of a type II DNA topoisomerase from bovine calf thymus.

We report here the large scale purification of DNA topoisomerase II from calf thymus glands, using the unknotting of naturally knotted P4 phage DNA as an assay for enzymatic activity. Topoisomerase II was purified more than 1300-fold as compared to the whole cell homogenate, with 22% yield. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 125 and 140 kDa. Tryptic maps of the two bands indicated that they derive from the same protein. Using these fragments, specific polyclonal antisera to topoisomerase II were raised in rabbits. Immunoblotting of whole cell lysates from various species indicated that topoisomerase II is well conserved among mammals and has a native subunit molecular mass of 180 kDa. Analytical sedimentation and gel filtration were used to determine a sedimentation coefficient of 9.8 S and a Stokes radius of 68 A. The calculated solution molecular mass of 277 kDa implies a dimer structure in solution. The purified topoisomerase II unknots P4 DNA in an ATP-dependent manner and is highly stimulated in its relaxation activity by ATP. A DNA-stimulated ATPase activity, as has been found with other type II topoisomerases, is associated with the purified enzyme. Approximate kinetic parameters for the ATPase reaction were determined to be: a Vmax of 0.06 nmol of ATP/(micrograms of protein) (min) and Km of 0.2 mM in the absence of DNA, and a Vmax of 0.2 nmol of ATP/(micrograms of protein) (min) and Km of 0.4 mM ATP in the presence of supercoiled plasmid DNA.

Adenosine Triphosphate↗
PubMed1984

Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II.

Many intercalative antitumor drugs have been shown to cleave DNA indirectly through their specific effect on the stabilization of a cleavable complex formed between mammalian DNA topoisomerase II and DNA (Nelson, E.M., Tewey, K.M., and Liu, L.F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1361-1365). Antitumor epipodophyllotoxins (VP-16 and VM-26) which do not intercalate DNA can similarly induce protein-linked DNA breaks in cultured mammalian cells. In vitro studies using purified mammalian DNA topoisomerase II show that epipodophyllotoxins interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II by stabilizing a cleavable complex. Treatment of this stabilized cleavable complex with protein denaturants results in DNA strand breaks and the covalent linking of a topoisomerase subunit to the 5'-end of the broken DNA. Furthermore, epipodophyllotoxins also inhibit the strand-passing activity of mammalian DNA topoisomerase II, presumably as a result of drug-enzyme interaction. The agreement between the in vivo and in vitro studies suggests that mammalian DNA topoisomerase II is a drug target in vivo. The similarity between the effect of epipodophyllotoxins on mammalian DNA topoisomerase II and the effect of nalidixic acid on Escherichia coli DNA gyrase suggests that the cytotoxic action of epipodophyllotoxins may be analogous to the bactericidal action of nalidixic acid.

Animals↗
PubMed1984

Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II.

Adriamycin (doxorubicin), a potent antitumor drug in clinical use, interacts with nucleic acids and cell membranes, but the molecular basis for its antitumor activity is unknown. Similar to a number of intercalative antitumor drugs and nonintercalative epipodophyllotoxins (VP-16 and VM-26), adriamycin has been shown to induce single- and double-strand breaks in DNA. These strand breaks are unusual because a covalently bound protein appears to be associated with each broken phosphodiester bond. In studies in vitro, mammalian DNA topoisomerase II mediates DNA damage by adriamycin and other related antitumor drugs.

Adenosine Triphosphate↗
PubMed1982

Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA.

Eukaryotic DNA topoisomerase I introduces transient single-stranded breaks on double-stranded DNA and spontaneously breaks down single-stranded DNA. The cleavage sites on both single and double-stranded SV40 DNA have been determined by DNA sequencing. Consistent with other reports, the eukaryotic enzymes, in contrast to prokaryotic type I topoisomerases, links to the 3'-end of the cleaved DNA and generates a free 5'-hydroxyl end on the other half of the broken DNA strand. Both human and calf enzymes cleave SV40 DNA at the identical and specific sites. From 827 nucleotides sequenced, 68 cleavage sites were mapped. The majority of the cleavage sites were present on both double and single-stranded DNA at exactly the same nucleotide positions, suggesting that the DNA sequence is essential for enzyme recognition. By analyzing all the cleavage sequences, certain nucleotides are found to be less favored at the cleavage sites. There is a high probability to exclude G from positions -4, -2, -1 and +1, T from position -3, and A from position -1. These five positions (-4 to +1 oriented in the 5' to 3' direction) around the cleavage sites must interact intimately with topo I and thus are essential for enzyme recognition. One topo I cleavage site which shows atypical cleavage sequence maps in the middle of a palindromic sequence near the origin of SV40 DNA replication. It occurs only on single-stranded SV40 DNA, suggesting that the DNA hairpin can alter the cleavage specificity. The strongest cleavage site maps near the origin of SV40 DNA replication at nucleotide 31-32 and has a pentanucleotide sequence of 5'-TGACT-3'.

Animals↗
PubMed1982

Intra- and intermolecular strand transfer by HeLa DNA topoisomerase I.

The major type I DNA topoisomerase (topo I) has been purified from HeLa cell nuclei to a homogeneous, monomeric protein (Mr = 100,000). Similar to the nicking-closing enzyme (Mr = 67,000) from rat liver (Been, M. D., and Champoux, J. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2883-2887), HeLa topo I has the following properties: (a) HeLa topo I breaks down single-stranded DNA to smaller fragments, each with an enzyme-linked 3'-phosphoryl end and a free 5'-OH end. This cleavage is not dependent upon protein denaturant or protease treatment. (b) HeLa topo I produces single-stranded DNA circles from linear single-stranded DNA. Such DNA circles are believed to be produced by the intramolecular cyclization of topo I-linked, single-stranded DNA fragments. (c) HeLa topo I-linked, single-stranded fragments (donors) can join covalently to double-stranded DNA possessing a 5'-OH group (acceptors). The donor is transferred to the 5'-OH end of the acceptor, independent of the position of the end (internal nick or end of linear DNA) or the configuration of the end (flush, 5'-protruding, or 5'-recessed end) of the acceptor. (d) HeLa topo I cleavage of single-stranded DNA is site-specific, but no special sequence at the ends of the acceptor molecule is apparently required for a successful heterologous strand transfer. These results suggest that HeLa topo I may be involved in DNA sequence rearrangements in addition to its possible role as a swivelase for transcription and replication.

DNA Topoisomerases, Type I↗