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Biomedical subjects

T J Kelly

Publications and source records attributed to T J Kelly.

At least 55 records · Page 3Linked to original sources
PubMed1994

The DNA-activated protein kinase is required for the phosphorylation of replication protein A during simian virus 40 DNA replication.

The 32-kDa subunit of replication protein A (RPA) is phosphorylated during the S phase of the cell cycle in vivo and during simian virus 40 DNA replication in vitro. To explore the functional significance of this modification, we purified a HeLa cell protein kinase that phosphorylates RPA in the presence of single-stranded DNA. By several criteria we identified the purified enzyme as a form of the DNA-activated protein kinase (DNA-PK), a previously described high molecular weight protein kinase that is capable of phosphorylating a number of nuclear DNA binding proteins. Phosphorylation of RPA by DNA-PK is stimulated by natural single-stranded DNAs but not by homopolymers lacking secondary structure. Studies with the simian virus 40 model system indicate that DNA-PK is required for DNA-replication-dependent RPA phosphorylation. Depletion of the kinase activity, however, has no effect on the extent of DNA replication in vitro. Our data support a model in which phosphorylation of RPA by DNA-PK is activated by formation of replication intermediates containing single- and double-stranded regions. This event may be involved in a signaling mechanism that coordinates DNA replication with the cell cycle.

DNA Repair↗
PubMed1994

High-resolution genomic mapping of the three human replication protein A genes (RPA1, RPA2, and RPA3).

Human replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, homologous recombination, and excision repair. We have previously reported the cloning and bacterial overexpression of the three RPA genes and have mapped them to chromosome 1 (RPA2), chromosome 7 (RPA3), and chromosome 17 (RPA1). We have now obtained yeast strains with artificial chromosomes carrying the three human RPA genes and report the more detailed genomic mapping of RPA. RPA1 was mapped to chromosome 17p13.3 using a combination of PCR amplification of somatic cell hybrids and radiation hybrids containing chromosome 17 fragments. RPA2 was mapped to chromosome 1p35 by PCR amplification of somatic cell hybrids of chromosome 1 and by fluorescence in situ hybridization. RPA3 was mapped to chromosome 7p22 by Southern analysis and PCR amplification of somatic cell hybrids of chromosome 7 as well as fluorescence in situ hybridization. Since RPA is an essential component of major metabolic events affecting DNA, the physical mapping of the genes for it may help elucidate the biochemical basis of genetic disorders involving DNA metabolism.

Blotting, Southern↗
PubMed1994

Replication and transcription. Silence of the ORCs.

The origin recognition complex, a multi-protein complex known to bind to replication origins, has now been implicated in transcriptional silencing, providing another link between DNA replication and transcription.

Base Sequence↗
PubMed1994

Factors influencing bone lead concentration in a suburban community assessed by noninvasive K x-ray fluorescence.

OBJECTIVE: To determine the influence of demographic, exposure and medical factors on the bone lead concentration of subjects with background (nonindustrial) environmental lead exposure. DESIGN: Survey. SETTING: Suburban residential community. PARTICIPANTS: A total of 101 subjects (49 males, 52 females; aged 11 to 78 years) were recruited from 49 of 123 households geographically located in a suburban residential neighborhood unexposed to any major source of industrial lead emissions. MAIN OUTCOME MEASUREMENTS: Cortical bone lead concentrations in the midshaft of the tibia were noninvasively measured by in vivo K x-ray fluorescence. Blood lead concentrations were measured by anodic stripping voltammetry. An administered questionnaire assessed potential sources of lead exposure and medical conditions affecting bone metabolism. RESULTS: After the exclusion of one outlier, log-transformed bone lead concentration was highly correlated with age (r = .71; P < or = .0001). Bone lead concentration showed no significant change up to age 20 years, increased with the same slope in men and women between ages 20 and 55 years, and then increased at a faster rate in men older than 55 years. In addition to the variables age and sex, the best fitting multiple regression model for bone lead concentration (R2 = .66; P < or = .0001) revealed a positive correlation with total pack-years of cigarette smoking and a negative correlation with a history of having nursed an infant for longer than 2 weeks. Blood lead concentrations of the subjects were low (geometric mean, 0.24 mumol/L [4.9 micrograms/dL]) and after log transformation were weakly correlated with log-transformed bone lead concentration (r = .23; P = .02). CONCLUSIONS: The age- and sex-related increases in bone lead concentration found by K x-ray fluorescence concur with published postmortem studies of bone lead concentration and are consistent with the kinetics of bone turnover and secular trends in lead exposure. These data help to establish a reference range for assessing the lead burden of other populations with environmental or occupational lead exposure.

Adolescent↗
PubMed1994

Complete base sequence for the mitochondrial large subunit ribosomal RNA of the gypsy moth Lymantria dispar (L.).

A 1355 bp sequence (accession number L32141) isolated from a gypsy moth (Lymantria dispar) cDNA library showed 68-74% sequence identity to mitochondrial large subunit ribosomal RNA (mt IrRNA) sequences of Locusta migratoria, Apis mellifera, Aedes albopictus, Anopheles gambiae and two Drosophila species. A comparison of the primary sequences of the mt IrRNAs from the above insects in four orders and from Esherichia coli demonstrated regions of conservation which presumably correspond to regions of functional and/or structural homology. A secondary structure for the gypsy moth mt IrRNA sequence was derived based on the proposed secondary structures of Drosophila yakuba and Aedes albopictus mt IrRNAs (Gutell & Fox, Nucleic Acid Res 16 (Suppl.), r175-r269, 1988). This sequence was found to hybridize to about 10-15% of the clones in several (eleven) gypsy moth cDNA libraries.

Animals↗
PubMed1994

Formate in serum and urine after controlled methanol exposure at the threshold limit value.

Methanol will be present as a new air pollutant when methanol-powered vehicles are introduced in the United States. Little is known about the effect of low-dose methanol exposure. It is controversial whether or not formate, the main metabolite responsible for methanol's acute toxicity, is a sensitive biological marker of toxicity or exposure. We studied the effect of a 4-hr exposure at rest to 200 ppm of methanol vapors on endogenous serum formate and on urinary formic acid excretion. A randomized, double-blind study of human exposure to a constant concentration of methanol was performed in a whole-body exposure chamber. Twenty-six healthy volunteers, each serving as his or her own control, participated in sham and methanol exposures. Urine (at 0, 4, 8 hr) and serum specimens (15 time points over 8 hr) collected before, during, and after the exposure were measured for formate. We found no significant differences in serum formate concentration between exposure and control conditions either at any time point or for area under the curve. Mean concentrations at the end of the exposure were: exposed 14.28 +/- 8.90 mg/l and control 12.68 +/- 6.43 mg/l. A slight, but nonsignificant (p = 0.08), increase in urine formate excretion rate was found at 4 hr (exposed 2.17 +/- 1.69 mg/4 hr and control 1.67 +/- 1.02 mg/4 hr). Age, sex, folic acid level, and smoking were not significant covariates. At 200 ppm, methanol exposure does not contribute substantially to endogenous formate quantities. Serum and urine formate determinations are not sensitive biological markers of methanol exposure at the threshold limit value.

Absorption↗
PubMed1993

The fission yeast cdc18+ gene product couples S phase to START and mitosis.

Commitment to the cell cycle in fission yeast requires the function of the cdc10+ transcriptional activator at START. The product of the cdc18+ gene is a major downstream target of cdc10+, and transcription of cdc18+ is activated by cdc10+ during passage through START. The cdc18+ function is required for entry into S phase. In addition, the product of the cdc18+ gene is part of the checkpoint control that prevents mitosis from occurring until S phase is completed. Thus, cdc18+ plays a key role in coupling S phase to START and mitosis.

Amino Acid Sequence↗
PubMed1993

Cloning, overexpression, and genomic mapping of the 14-kDa subunit of human replication protein A.

Replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, recombination, and repair. We have previously reported the cloning and bacterial expression of the 70- and 32-kDa subunits of human RPA (hRPA). We have now cloned the 14-kDa subunit (hRPA3) from a HeLa cell cDNA library. The hRPA3 cDNA is a 692-base pair sequence that contains an open reading frame encoding a protein of 121 amino acids with a calculated molecular mass of 13.6 kDa. The deduced amino acid sequence shows only limited similarity to the small subunit of yeast RPA and is unrelated to any other protein in the current data banks. A recombinant protein containing a short histidine tag at the NH2 terminus has been purified in good yield from Escherichia coli by metal-chelate affinity chromatography. Antibodies prepared against recombinant hRPA3 recognize the native protein and inhibit SV40 DNA replication in vitro. We have localized the genes for the 70-, 32-, and 14-kDa subunits to chromosomes 17, 1, and 7, respectively, using polymerase chain reaction amplification of genomic DNA from rodent-human hybrid cell lines. Since RPA appears to be involved in several fundamental cellular processes, the physical mapping of the RPA genes may be useful in identifying possible human genetic defects associated with RPA deficiency or dysfunction.

Amino Acid Sequence↗
PubMed1993

Insect neuropeptides: discovery and application in insect management.

New approaches to the development of insect control agents have been revealed through the molecular description of neuropeptides, their biogenesis, action, and degradation. Prerequisite to the exploitation of a neuropeptide as a lead to control agent development is a thorough understanding of the biochemistry of the neuropeptide and appreciation of its physiological impact. Reliable bioassays must be coupled with advanced biochemical and molecular genetic technologies to overcome limitations imposed by the typically low endogenous levels of individual neuropeptides. Purification, amino acid sequencing, and gene cloning provide the molecular tools necessary for studies on neuropeptide synthesis, processing, secretion, receptor binding, and inactivation. Each of these areas consists of a number of amino acid sequence-, and enzyme-dependent steps which may be considered as targets for the development of highly specific control agents. These agents will include antagonist and superagonists, peptidomimetics, recombinant peptides delivered through the baculovirus technology, receptor blockers, and enzyme inhibitors.

Amino Acid Sequence↗
PubMed1993

The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.

DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV40 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and expressed it as a fusion protein in bacteria. The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusion protein promotes formation of a complex containing T antigen and the catalytic subunit (subunit A) of DNA polymerase alpha, suggesting that it serves to tether the two proteins. These physical interactions are functionally significant, since the ability of T antigen to stimulate the activity of the catalytic subunit of DNA polymerase alpha is highly dependent upon the B subunit. We suggest that the interactions mediated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating the subsequent priming and synthesis of DNA chains on the lagging strand template. The protein may play similar roles in cellular DNA replication.

Amino Acid Sequence↗
PubMed1993

Measurement of hemoglobin-acetaldehyde adduct in alcoholic patients.

A sensitive and specific test for chronic alcohol abuse is useful in the diagnosis and management of alcoholic patients. Herein, we report the measurement of hemoglobin-acetaldehyde adducts (Hb-AA) in alcoholic patients by a sandwich ELISA using different antibodies. Keyhole limpet hemocyanin (KLH), a peptide consists of eight amino acid residues (8-pep, V1 to K8) at the N-terminus of beta-chain of human sickle-cell Hb and a segment of HbA beta-chain consists of 11 amino acids rich in lysine or K (11-pep, G56-K66) were incubated with acetaldehyde and NaCNBH3 to form protein-AAs. 8-Pep-AA and 11-pep-AA were individually conjugated to unmodified KLH as the carrier. Anti-protein-AA IgGs were raised in rabbits using these three protein-AA immunogens. When anti-KLH-AA IgG was used in ELISA, optical densities for alcoholic patients and controls were 0.311 +/- 0.124 and 0.147 +/- 0.042 (means +/- SD, n = 40/group, p < 0.001), respectively. Using mean value +/- 2 SD of controls as the cut-off, sensitivities to detect alcoholic patients were 78, 75, and 43%, respectively, when anti-KLH-AA, anti-11-pep-AA, and anti-8-pep-AA were used. Correlation among optical densities obtained from the first two IgGs was excellent (R2 = 0.905). We conclude that: (1) Hb-AA has the potential of being a good marker for alcohol abuse, and (2) the site of Hb that is modified by acetaldehyde in vivo is primarily located in a surface-accessible domain near the center of the beta-chain of HbA where several lysine residues are clustered.

Acetaldehyde↗
PubMed1992

The fate of parental nucleosomes during SV40 DNA replication.

The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.

Cell-Free System↗
PubMed1992

Regulation of DNA replication in vitro by the transcriptional activation domain of GAL4-VP16.

Studies of DNA viruses have provided evidence that eukaryotic transcriptional activator proteins can enhance the efficiency of DNA replication as well as transcription. The mechanism of this effect was studied in vitro using the chimeric transcription factor GAL4-VP16 and a DNA template containing GAL4 binding sites adjacent to the simian virus 40 origin of DNA replication. The binding of GAL4-VP16 prevented the repression of DNA replication which otherwise occurred when the template was assembled into chromatin. Relief of repression by GAL4-VP16 required both its DNA-binding and transcriptional activation domains but did not require RNA synthesis. The results are consistent with a general model in which transcriptional activators stimulate eukaryotic DNA replication by modifying the outcome of the competition between initiation factors and histones for occupancy of the origin.

DNA Replication↗
PubMed1992

Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen.

The purified human single-stranded DNA binding protein, replication protein A (RP-A), forms specific complexes with purified SV40 large T antigen and with purified DNA polymerase alpha-primase, as shown by ELISA and a modified immunoblotting technique. RP-A associated efficiently with the isolated primase, as well as with intact polymerase alpha-primase. The 70 kDa subunit of RP-A was sufficient for association with polymerase alpha-primase. Purified SV40 large T antigen bound to intact RP-A and to polymerase-primase, but not to any of the separated subunits of RP-A or to the isolated primase. These results suggest that the specific protein-protein interactions between RP-A, polymerase-primase and T antigen may play a role in the initiating of SV40 DNA replication.

Amino Acid Sequence↗
PubMed1992

Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A.

The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.

Antigens, Polyomavirus Transforming↗
PubMed1991

Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication.

Replication protein A (RP-A) is a three-subunit single-stranded DNA-binding protein that has been isolated from human cells. RP-A is essential for SV40 DNA replication and may also be important in genetic recombination. The sequence of a cDNA encoding the 70-kDa subunit of human RP-A is reported. The 616-amino acid predicted open reading frame of the human protein is 31% identical with the 621-amino acid open reading frame of the 70-kDa subunit of RP-A from the yeast Saccharomyces cerevisiae. Both proteins share a highly conserved putative metal binding domain of the 4-cysteine type. The human cDNA directs production in Escherichia coli of a 70-kDa protein that reacts with a monoclonal antibody directed against the 70-kDa subunit of human RP-A. The recombinant 70-kDa subunit, purified from bacteria, exhibits single-stranded DNA binding activity comparable to that of the complete RP-A complex. The 70-kDa subunit is able to substitute for the complete human RP-A complex in stimulating the activity of DNA polymerase alpha-primase on a poly(dA).oligo(dT) template. However, the 70-kDa subunit alone cannot substitute for the complete RP-A complex in SV40 DNA replication in vitro, suggesting an important functional role for the other subunits.

Amino Acid Sequence↗