Biomedical subjects

T W Myers

Publications and source records attributed to T W Myers.

7 recordsLinked to original sources

PubMed1992

A 5' to 3' exonuclease functionally interacts with calf DNA polymerase epsilon.

Analysis of fractions containing purified DNA polymerase epsilon from calf thymus has revealed the presence of a 5' to 3' exonuclease activity that is specific for a single strand of duplex DNA. This activity is capable of degrading a 3'-labeled oligonucleotide hybridized to M13mp18 DNA. When a second oligonucleotide primer is annealed 3 bases upstream, degradation of the downstream primer is strictly dependent on DNA synthesis from the upstream primer. Replacement of the downstream primer by an oligoribonucleotide of identical sequence results in a similar pattern of exonucleolytic activity. The activity has been highly purified and found to cosediment in glycerol gradients with a peptide of 56 kDa as judged by SDS/PAGE analysis. Effects of calf DNA polymerase alpha and delta on exonuclease activity are also observed but with differences in the pattern of products.

Animals↗

PubMed1992

A novel DNA helicase from calf thymus.

We report the purification and characterization of a novel DNA helicase from calf thymus tissue. This enzyme partially copurifies with DNA polymerase epsilon* through many of the chromatographic procedures used to isolate it. The enzyme contains an intrinsic DNA-dependent ATPase activity. It can displace short oligonucleotides annealed to long single stranded substrates, in an ATP-dependent reaction. Use of this assay indicates that the DNA helicase translocates in a 3' to 5' direction with respect to the substrate strand to which it is bound. Maximal efficiency of displacement is accomplished by hydrolysis of (d)ATP as cofactor, however, (d)CTP can also be utilized resulting in a 5-fold decrease in the level of displacement. Displacement activity is enhanced by the presence of saturating amounts of Escherichia coli single stranded DNA-binding protein, not affected by the presence of phage T4 gene 32 protein, and inhibited by human replication factor A. The DNA helicase has a molecular mass of approximately 104 kDa as measured by denaturing gel electrophoresis, and an S value of 5.4 obtained from glycerol gradient sedimentation. Direct [alpha-32P]ATP cross-linking labels a protein of molecular mass approximately 105 kDa, providing further evidence that this polypeptide contains the helicase active site. In view of the differences in the properties of this helicase from four others recently identified in calf and designated A through D, we propose the name helicase E.

Adenosine Triphosphate↗

PubMed1992

Structural relationships between two forms of DNA polymerase epsilon from calf thymus.

We previously reported purification of two forms of DNA polymerase epsilon from calf thymus (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). We have now used the "polymerase trap" photolabeling method to identify the polypeptides containing the polymerase active site in each enzyme preparation. The molecular mass of these polypeptides are 210 and 145 kDa for the polymerases now designated epsilon and epsilon*, respectively. Renaturation of polymerase activity from denaturing gel electrophoresis corroborates the polymerase trap results. Photolabeling of polymerase fractions suggests that the smaller subunit is derived by proteolysis of the larger subunit during purification. Native sedimentation coefficient measurements of polymerase-containing column fractions further suggest a precursor/product relationship between the two polymerases. Response of polymerization activity to a battery of inhibitors normally used to distinguish mammalian nuclear DNA polymerases was found to be essentially identical for polymerases epsilon, epsilon*, and the epsilon* generated in fractions initially containing epsilon. These latter results demonstrate that the loss of the protease-sensitive domain of the active site subunit does not affect catalytic function as measured in a standard DNA polymerase assay. The sole apparent functional difference observed here between the epsilon and epsilon* forms is evidence that only the full-length epsilon form can be directly photocrosslinked to dATP, independent of DNA synthesis. Photolabeling of the post-microsomal supernatant fraction from thymus glands obtained from fetal calves reveals the presence of both the epsilon and epsilon* polypeptide.

Animals↗

PubMed1991

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1991

Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase.

A recombinant DNA polymerase derived from the thermophilic eubacterium Thermus thermophilus (Tth pol) was found to possess very efficient reverse transcriptase (RT) activity in the presence of MnCl2. Many of the problems typically associated with the high degree of secondary structure present in RNA are minimized by using a thermostable DNA polymerase for reverse transcription, and predominantly full-length products can be obtained. The cDNA can also be amplified in the polymerase chain reaction (PCR) with the same enzyme. The Tth pol was observed to be greater than 100-fold more efficient in a coupled RT/PCR than the analogous DNA polymerase from Thermus aquaticus (Taq pol). The sensitivity of the reactions performed by Tth pol allowed for the detection of ethidium bromide stained products starting with as little as 100 copies of synthetic cRNA. Similar results were also obtained with RNA from a Philadelphia-chromosome positive cell line. Detection of IL-1 alpha mRNA was possible starting with 80 pg of total cellular RNA. The ability of Tth pol to perform both reverse transcription and DNA amplification will undoubtedly prove useful in the detection, quantitation, and cloning of cellular and viral RNA.

Base Sequence↗

PubMed1991

Polymerization and RNase H activities of the reverse transcriptases from avian myeloblastosis, human immunodeficiency, and Moloney murine leukemia viruses are functionally uncoupled.

The functional interaction between the RNA-dependent DNA polymerase and the RNase H activities of reverse transcriptases (RTs) were examined using a 272 nucleotide long plasmid-derived RNA transcript primed in a specific location. Properties of the avian myeloblastosis virus (AMV) RT, the human immunodeficiency virus RT and the Moloney murine leukemia virus RT were examined. All three enzymes formed stable complexes with the primer-template with half-lives ranging from about 16 to 41 s. Each enzyme synthesized full-length primer extension products and cleaved the RNA template at least once during DNA synthesis. Polymerization was then assayed in the presence of challenger RNA that effectively sequestered RTs after one round of processive DNA synthesis. This assay allowed measurement of the number of endonucleolytic cleavages catalyzed by the RT during one encounter with the primer-template. Results indicated that each of the three RTs cut the transcript before dissociating from the primer-template, whether or not deoxynucleoside triphosphates were present to allow synthesis. During synthesis, the extent of RNA degradation differed among the RTs, with AMV-RT generating mostly large segments of RNA-DNA hybrid, and virtually no small RNA cleavage products. Human immunodeficiency virus and Moloney murine leukemia virus-RT generated more small degradation products than AMV-RT, but still left much of the potentially degradable hybrid undigested. Results demonstrate that the RNase H function is much less active than the polymerization function during processive DNA synthesis and that the activities are not strictly coupled.

Avian Myeloblastosis Virus↗

PubMed1978

Nutritional programming for the elderly.

It is impossible to talk about nutrition and the elderly without reviewing related factors such as program funding levels, income and purchasing power, education, medical implications, and resulting social costs.Although we have witnessed a tremendous increase in federal appropriations for Title VII programs, many senior citizens have not and will not benefit from nutrition programs. As the ratio of elderly citizens to total population increases, we may expect: (1) an increase in program budgets with a concomitant decrease in the proportion of senior citizens served; and (2) a regressed government attitude toward income-related support programs, causing decreased purchasing power, ineffective nutrition programs, and little relative improvement in the health of our elderly population.

Aged↗