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Biomedical subjects

L A Loeb

Publications and source records attributed to L A Loeb.

At least 181 records · Page 10Linked to original sources
PubMed1985

Nucleotide sequence preservation of human mitochondrial DNA.

Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mp11; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both between- and within-individual differences were identified. Between-individual differences were identified in approximately 1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation of mammalian mitochondrial DNA sequences in single organisms.

Adult↗
PubMed1985

Fidelity of DNA polymerase-beta in neurons from young and very aged mice.

Neurons do not divide during adult life and thus they provide a unique system to study the effects of age-accumulated damage to DNA in the absence of DNA replication. We have analyzed DNA polymerase activity in neurons isolated from young adult and very aged mice. The predominant catalytic activity is DNA polymerase-beta and it is present in similar amounts in neurons from young and old mice. This polymerase is highly error-prone in copying phi X174 DNA, the error frequency being about 1/7,000 and not significantly different when obtained from young and old animals. This high infidelity is considered with respect to DNA repair and the protein synthesis error catastrophe theory of aging.

Aging↗
PubMed1985

Nucleotide sequence preservation of human leukemic mitochondrial DNA.

Nucleotide sequence variation in mitochondrial DNA isolated from human leukemic cells has been analyzed by recombinant DNA techniques. Three hundred eighty-seven independent recombinant DNA clones, each containing one of three defined segments of mitochondrial DNA isolated from the neoplastic cells of four leukemic patients, were analyzed. Partial nucleotide sequence determination of the 387 clones yielded a total of 81.7 kilobases of nucleotide sequence information. The only evidence of within-individual nucleotide sequence divergence consisted of three clones containing deletions of one or two nucleotides in one mitochondrial DNA region. These clones were three of 113 independent clones isolated from a patient with acute lymphocytic leukemia. The low level of nucleotide sequence divergence in the mitochondrial DNA population of neoplastic cells from individual leukemic patients suggests that a mechanism or mechanisms exist that limit the development of nucleotide sequence divergence in mammalian mitochondrial DNA. The results further suggest that this mechanism does not appear to be abrogated by neoplastic transformation in leukemic patients.

Adolescent↗
PubMed1984

Transfection of the DNA polymerase-alpha gene.

DNA polymerase-alpha is the major replicative DNA polymerase in animal cells. The gene coding for a mutant DNA polymerase-alpha was transferred from one cell to another by transfection of DNA from mutant cells. The DNA was isolated from a mutant hamster cell line resistant to aphidicolin, a specific inhibitor of DNA polymerase-alpha, and transferred into an aphidicolin-sensitive cell line. The resulting transfectants exhibited increased survival in the presence of aphidicolin and contained an aphidicolin-resistant DNA polymerase-alpha.

Animals↗
PubMed1984

Site specific mutagenesis: insertion of single noncomplementary nucleotides at specified sites by error-directed DNA polymerization.

We have utilized infidelity of DNA synthesis as a basis for site-directed mutagenesis. Both an endonuclease restriction fragment and a synthetic oligonucleotide were used as primers. DNA polymerase from bacteriophage T4 was used to elongate primer termini to a position immediately adjacent to two different preselected positions on phiX174 DNA templates. Then, the error-prone DNA polymerase from avian myeloblastosis virus was used to insert single non-complementary nucleotides at the designated positions at high efficiency. DNA sequence analysis confirmed that the mutant phage produced as a result of each site-specific mutagenesis reaction contained the nucleotide that was complementary to the one provided during the DNA copying reaction. The general applicability of this methodology to cloned DNAs will be discussed.

Base Sequence↗
PubMed1984

On the fidelity of DNA replication. Lack of primer position effect on the fidelity of mammalian DNA polymerases.

Mechanisms for the fidelity of DNA replication in eucaryotes are not adequately understood. Certain hypotheses can be tested by examining whether the first nucleotide inserted is incorporated with a significantly higher error rate than subsequent nucleotides. Using synthetic oligodeoxynucleotides, we have measured the effect of primer position on single-base misinsertion frequencies at an amber site in phi X174 DNA. Our results show a lack of position effect, indicating that processivity and the most direct "energy relay" proofreading mechanisms are not important determinants in eucaryotic replication fidelity.

Bacteriophage phi X 174↗
PubMed1984

On the fidelity of DNA replication. The accuracy of T4 DNA polymerases in copying phi X174 DNA in vitro.

The fidelity with which wild type T4 DNA polymerase copies phi X174 amber 3 plus strand DNA at position 587 in vitro has been measured. Synthesis is initiated by hybridizing to the template a HaeIII restriction fragment whose 3'-OH terminus is 83 nucleotides from the amber 3 site. Based on gel electrophoresis of product DNA molecules and genetic marker rescue data, T4 DNA polymerase copies significantly beyond the mutant site. Transfection analysis shows that the A X T leads to G X C mutation at position 587 occurs 10- to 100-fold less frequently with T4 DNA polymerase than with E. coli DNA polymerase I. The aberrant incorporation of cytosine opposite adenine at position 587 by the T4 polymerase alone is occurring at a frequency not greater than about 10(-7) which, for this particular locus, may be similar to the fidelity exhibited by the T4 accessory proteins plus the polymerase comprising the replication complex. A comparison of the accuracy of mutator L56 and antimutator L141 T4 DNA polymerases relative to wild type shows at most a 2- to 4-fold decrease and increase, respectively, in fidelity. When compared to 10- to 1000-fold effects on mutation frequencies that these same mutant alleles have in vivo, these results suggest that the wide range in expression of mutator and antimutator phenotypes in vivo may be dependent on an abnormal interaction of the aberrant DNA polymerases with other protein components of the replication complex.

Bacteriophage phi X 174↗
PubMed1984

Delayed and reduced cell replication and diminishing levels of DNA polymerase-alpha in regenerating liver of aging mice.

Kinetics of cell replication was compared in regenerating livers of Mus musculus at ages ranging between 6 and 32 months. Incorporation of [3H]-thymidine into DNA and autoradiographic analysis showed that the maximal extent of DNA replication following partial hepatectomy became delayed with age. Furthermore, the total fraction of parenchymal and nonparenchymal cells in S phase at different intervals during regeneration diminished as mice aged. The specific activity of DNA polymerase-alpha, the putative replicative enzyme, declined progressively during aging. The specific activity of DNA polymerase-beta, the purported repair enzyme, declined to an appreciably lesser extent during the lifespan of the mouse. No evidence was found for the appearance of a specific inhibitor of polymerase-alpha in senescent mouse liver. Also, the bulk of the activities of both hepatic DNA polymerase-alpha and -beta remained localized in the cell nucleus throughout the lifetime of the animal and were mainly associated with chromatin.

Aging↗
PubMed1984

Assessment of the carcinogenic potential of a proposed food coloring additive, laccaic acid, using short-term assays.

Laccaic acid is a red colored natural dye produced by the insect Laccifer lacca or Coccus lacca. It is obtained in large amounts as a by-product of the shellac industry and has been considered for general use as a food coloring agent. Laccaic acid is found to have no mutagenic activity as assessed by two short-term assays: the Salmonella/microsome mutagenicity test, and the phi X fidelity assay. However, laccaic acid did inhibit metabolic cooperation in Chinese hamster V79 cells. These results suggest that laccaic acid should be tested in animals with particular emphasis on in vivo models for tumor promotion.

Animals↗
PubMed1984

Smoking and lung cancer: an overview.

This position paper summarizes the overwhelming evidence that tobacco smoking is the cause of 30 to 40% of deaths from cancer. The focus is on lung cancer because of the sheer magnitude of this disease in males and the likelihood of a similar epidemic in females. There are two categories of evidence that indicate smoking to be the major cause of human lung cancer. Without exception, epidemiological studies have demonstrated a consistent association between smoking and lung cancer in men and now suggest a similar association in women. Chemical analyses of cigarette smoke reveal a multitude of known mutagens and carcinogens. Moreover, these chemicals are absorbed, are metabolized, and cause demonstrable genetic changes in smokers. Two consequences of smoking are evaluated. The results of treatment of lung cancer are not encouraging; despite vigorous therapy, the 5-year survival rate remains less than 10%. The social and economic costs of lung cancer and the smoking habit impinge on the productiveness of our society.

Adult↗
PubMed1983

Rapid changes in deoxynucleoside triphosphate pools in mammalian cells treated with mutagens.

In this communication we describe the rapid increase in cellular deoxynucleoside triphosphate (dNTP) concentrations in Chinese Hamster cell line V79 after exposure to known mutagens. With this cell line an expansion of dATP and dTTP pools was detected; changes in dCTP were not large; changes in dGTP were either not significant or too low to quantitate. This situation may reflect the existence of imbalances in dNTP pools at the DNA replication fork. The expansion of dATP and dTTP pools occurred within 2 to 4 hours after exposure of cultured cells to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Ultraviolet light (UV), mitomycin C, and cytosine arabinoside also caused similar dNTP pool changes.

Animals↗
PubMed1983

Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro.

Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.

Animals↗
PubMed1983

Metal content of DNA polymerase I purified from overproducing and wild type Escherichia coli.

DNA polymerase I purified from both E. coli strain B, and from an overproducing E. coli stain lysogenized with a lambda pol A phage were analyzed for metal content. After gel filtration to remove loosely bound metals, DNA polymerase I from both strains contained less than or equal to 0.2 gm atoms Zn2+/mole enzyme and 0.09 to 0.7 Mg2+/mole enzyme. Substoichiometric amounts of Fe, Co, Ni (less than or equal to 0.2 gm atoms), and Mn (less than or equal to 0.1 gm atoms) were detected. Since the metal content does not correlate with enzymatic activity, we conclude that DNA polymerase I is not a metalloenzyme.

Chemical Phenomena↗
PubMed1983

On the fidelity of DNA replication. Specificity of nucleotide substitution by intercalating agents.

The effects of intercalating agents on the fidelity of DNA synthesis in vitro have been investigated. The accuracy of DNA synthesis with Escherichia coli DNA polymerase I with both the poly[d(A-T)] and poly[d(G-C)] templates is decreased in the presence of the intercalating agents proflavin, ethidium bromide, acridine orange, ICR-170, and ICR-191. Nearest neighbor analyses of the product of the reaction indicate that two different types of misincorporations occur in the presence of intercalating agents, frameshifts, and single-base substitutions. With alternating polynucleotide templates, frameshifts involving pyrimidines are the most frequent change in sequence observed. Overall, frameshift misincorporations occur with frequencies of one complementary pyrimidine for each intercalated site and one noncomplementary pyrimidine for each 150 sites. From analysis of nearest neighbor frequencies in the product, it is inferred that the intercalating agents interact specifically with pyrimidine (3' leads to 5') purine sequences. An analysis of ratios of correct nucleotide incorporations as a function of intercalator concentration indicates that frameshifts are predominantly additions; however, one cannot rule out infrequent deletions. Base substitutions in the presence of intercalators occur less frequently than frameshifts. From the results of reaction kinetics and nearest neighbor frequencies, it is concluded that the noncomplementary nucleotides are incorporated in phosphodiester linkage and are present as single-base substitutions. Taken together, the results of these studies suggest at least two different modes of action for intercalating agents on the accuracy of DNA synthesis: one leading to frameshift misincorporations and the other leading to single-base substitutions.

DNA Polymerase I↗
PubMed1983

On the fidelity of DNA replication. Mechanisms of misincorporation by intercalating agents.

Two distinct mechanisms of action for intercalating agents have been delineated: one leading to the production of frameshift misincorporations and the other leading to the production of single-base substitutions. Addition misincorporations are competitive with respect to DNA template (a measure of classical intercalation) but are not competitive with respect to deoxynucleotide substrates. Single-base substitutions are not competitive with template, polymerase, or deoxynucleotide as tested individually, but are proportional to the absolute drug concentration, indicating a ternary complex involving intercalator, polymerase, and template. Increased frequencies of single-base substitutions have not been considered as a general property of intercalators. Using a mutant phi X174 DNA, we demonstrate that intercalators also induce single-base substitutions with natural DNA templates. Reversion of am3 phi X174 DNA occurs only by single-base substitutions at position 587; this is increased 8-fold when the DNA is copied in vitro in the presence of intercalators.

Animals↗
PubMed1983

Mouse teratocarcinoma cells resistant to aphidicolin and arabinofuranosyl cytosine: isolation and initial characterization.

With the long-range goals of elucidating the biochemical genetics and the phenotypic expression, both in vitro and in vivo, of mutator strains of mammalian cells, we have isolated and partially characterized a series of drug-resistant mutants from a subline a feeder-dependent parental mouse teratocarcinoma line (PSA-1) known to be capable of chimerizing host blastocysts via the injection of inner cell mass. Two series of stable mutants were isolated--one (Aphr) selected on the basis of resistance to aphidicolin (Aph), a specific inhibitor of DNA polymerase-alpha, and the other (AraCr) selected by resistance to arabinofuranosyl cytosine, an analogue of cytosine. Irrespective of the method of selection, most of the 32 mutants isolated were resistant to both agents, although to different degrees, and with variations in growth characteristics, deoxynucleoside sensitivities, and sensitivities to mutagens (5-bromodeoxyuridine, N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet light). A protocol of multi-step selection provided stable mutants highly resistant to aphidicolin and only mildly resistant to AraC, even in the absence of prior mutagenesis, and is thus recommended as an approach to the isolation of candidate mutator strains of multipotent teratocarcinomas.

Animals↗
PubMed1983

Infidelity of DNA synthesis associated with bypass of apurinic sites.

The mutagenic potential of apurinic sites in vivo has been studied by transfection of depurinated phi X174 DNA containing amber mutations into SOS-induced Escherichia coli spheroplasts. Mutagenicity is abolished by treatment of the depurinated DNA with an apurinic endonuclease from Hela cells, establishing the apurinic site as the mutagenic lesion. The frequency of copying apurinic sites in vitro was analyzed by measuring the extent of DNA synthesis using E. coli DNA polymerase I and avian myeloblastosis DNA polymerase. The inhibition of DNA synthesis by apurinic sites was less with avian myeloblastosis DNA polymerase, suggesting that this error-prone enzyme copies apurinic sites with greater frequency. Consistent with this conclusion is the observation that, upon transfection into (normal) spheroplasts, the reversion frequency of depurinated phi X174 am3 DNA copied with avian myeloblastosis virus DNA polymerase is much greater than that of the same DNA copied with E. coli DNA polymerase I. Sequence analysis of the DNA of 33 revertant phage produced by depurination indicates a preference for incorporation of deoxyadenosine opposite putative apurinic sites. The combined results indicate that mutagenesis resulting from apurinic sites is associated with bypass of these noncoding lesions during DNA synthesis.

Apurinic Acid↗