Isolation and characterization of a 1-beta-D-arabinofuranosylcytosine-resistant Chinese hamster ovary cell mutant that is also X-ray sensitive and is noncomplementary with ataxia telangiectasia cells.

In order to study the mechanism of induction of mutations and chromosome aberrations by ionizing radiations, it is particularly useful to have available radiation-sensitive mutants. While several X-ray-sensitive rodent cell lines are available, they have been selected rather nonspecifically. It was determined that selection for resistance to the DNA replication/repair inhibitor, 1-beta-D-arabinofuranosylcytosine (ara-C), would permit production of a set of X-ray-sensitive mutant cell lines that would be defective in the resynthesis step of excision or recombination repair.

Cloning and expression in Escherichia coli of a human cDNA encoding the DNA repair protein N-methylpurine-DNA glycosylase.

A 871-base pair cDNA encoding the human N-methylpurine-DNA glycosylase (MPG) was cloned from a HeLa S3 cDNA expression library in a pUC vector by phenotypic screening of MPG-negative (tag- alkA-) Escherichia coli cells exposed to methylmethane sulfonate. The active MPG is expressed as a 31-kDa fusion protein. The human cDNA-encoded MPG releases 3-methyladenine, 7-methylguanine, and 3-methylguanine from DNA and thus has a substrate range similar to that of the indigenous enzyme and the E.

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations.

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli.

Mutational analysis of leucine 47 in human epidermal growth factor.

Seven site-specific mutants (including changes to other hydrophobic, charged, and heterocyclic amino acids) of leucine 47 of human epidermal growth factor (EGF) were generated by protein engineering and characterized for their activity in three assays: radioreceptor competition binding in membrane fractions, the stimulation of the EGF receptor's tyrosine kinase activity, and the stimulation of thymidine uptake in tissue culture cells. K1/2 (concentration required for half maximum response) values for each of the mutants are reported in the three assays. The results show that the native leucine residue is quite important for EGF activity.

Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

The third disulfide loop (amino acids 33 to 42) of human epidermal growth factor (hEGF) encompasses the region of highest amino acid conservation among all of the EGF-like family of molecules. The importance of some of these highly conserved residues for the maintenance of biological activity, especially the aromatic amino acid tyrosine at position 37, has until now been considered essential on the basis of previous studies with the EGF-like molecule transforming growth factor alpha. Variants at the Tyr-37 position of hEGF were constructed by site-directed mutagenesis.

Female-specific mutagenic response of mice to hycanthone.

Male and female gametogeneses differ markedly in all mammals. While male germ cells are continuously being produced from stem cells throughout the reproductive life span, the number of female germ cells is fixed during prenatal development and, soon after birth, all of the oocytes are arrested in a modified diplotene, or dictyate, stage. Following puberty, dictyate oocytes are hormonally triggered to mature either singly or in groups, resulting in ovulation and the completion of the first meiotic division.

Physicochemical studies of human O6-methylguanine-DNA methyltransferase.

O6-Methylguanine-DNA methyltransferase, present in most organisms, removes mutagenic and carcinogenic O6-alkylguanine from DNA by accepting the alkyl group in a stoichiometric reaction. The protein has been partially purified from human placenta. It reacts with second-order rate constants of 2.

Biochemical properties of site-directed mutants of human epidermal growth factor: importance of solvent-exposed hydrophobic residues of the amino-terminal domain in receptor binding.

Eight analogues of human epidermal growth factor (hEGF) having specific amino acid substitutions in the beta-sheet structure (residues 19-31) of the amino-terminal domain were generated by site-directed mutagenesis. Affinity of the epidermal growth factor (EGF) receptor for each of these mutant hEGF analogues was measured by both radioreceptor competition binding and receptor tyrosine kinase stimulation assays. The relative binding affinities obtained by these two methods were generally in agreement for each hEGF species.

Affinity labeling of spinach phosphoribulokinase subsequent to S-methylation at Cys16.

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification.

Uptake and metabolism of putrescine in confluent LLC-PK1 cells.

Putrescine is taken up by confluent pig kidney (LLC-PK1) cells at roughly equal rates over both Na(+)-dependent and Na(+)-independent pathways. The former is sensitive to 1 mM amiloride, but the latter is not. Uptake rates are similar at both the apical and basolateral surfaces.

The aggregation states of spinach phosphoribulokinase.

Phosphoribulokinase (PRK; EC 2.1.7.

Isolation and structural characterization of a cDNA clone encoding the human DNA repair protein for O6-alkylguanine.

O6-Methylguanine-DNA methyltransferase (MGMT; DNA-O6-methylguanine:protein-L-cysteine S-methyltransferase, EC 2.1.1.

Structure of the nucleosome core particle at 8 A resolution.

The x-ray crystallographic structure of the nucleosome core particle has been determined using 8 A resolution diffraction data. The particle has a mean diameter of 106 A and a maximum thickness of 65 A in the superhelical axis direction. The longest chord through the histone core measures 85 A and is in a non-axial direction.

Tomographic reconstruction from energy-filtered images of thick biological sections.

Energy filtration makes it possible to image an approximately 0.5 microm biological section at 80 kV in the electron microscope. Based on spectra taken at different tilt angles, we chose the most probable energy loss, deltaEp +/- 10eV for each tilt angle, as the imaging energy window.

A comparison of stem cell populations and hemoglobin switching in normal versus beta-thalassemic mice.

The hematopoietic stem cell concentrations in tissues of homozygous beta-thalassemic and non-thalassemic fetuses and neonates were compared by using the spleen colony-forming units (CFU-S) assay. The relative quantities of embryonic and adult hemoglobins were also determined for fetuses. Beta-thalassemic fetuses had a reduced incidence of CFU-S in the liver throughout gestation, but after birth the beta-thalassemic neonates maintained a greater number of CFU-S in the liver for an extended period.

Age-dependent modulation of tissue-specific repair activity for 3-methyladenine and O6-methylguanine in DNA in inbred mice.

3-Methyladenine-DNA N-glycosylase (MAG) and O6-methylguanine-DNA methyltransferase (MGMT) activities were assayed in liver, lungs, brain and ovaries of female mice of two inbred stocks, C3Hf and C57BL/E, as a function of age. In addition to differences in the enzyme levels between the two stocks for each organ, the suckling animals (9-day-old) have consistently lower levels of both MAG and MGMT than young adults (7- or 8-week-old). While the MGMT levels in adults did not decrease with age, the MAG levels in 15- to 17-month-old animals were, in general, significantly lower than those in young adults.

A domain that assumes a Z-conformation includes a specific deletion in some cloned variants of a complex satellite.

Sequence analyses show that deletions of 10 and 12 bp occur at homologous sites in a domain that is rich in alternating purines and pyrimidines (Pu/Py) in B42 and EXT, two cloned variants of a complex satellite DNA. A 3-bp deletion occurs 27 bp upstream from the site of the specific deletions in B42 and RU, a third cloned satellite variant that has not suffered the 10-bp deletion. Under torsional stress, the Pu/Py-rich domain adopts a Z-conformation as shown by (i) inhibition of cutting at a BssHII site that accounts for 2/5 of a 15-bp tract of pure Pu/Py in the domain; (ii) binding of polyclonal and monoclonal anti-Z-DNA antibodies to the domain; and (iii) antibody stabilization and subsequent relaxation of the Z-region.

DNA sequence patterns in precisely positioned nucleosomes.

Several investigators have recognized the importance of non-periodic DNA sequence information in determining the translational position of precisely positioned nucleosomes. The purpose of this study is to determine the extent of such information, in addition to the character of periodic information present. This is accomplished by examining the half-nucleosome DNA sequences of a considerable number of precisely positioned nucleosomes, and determining the probability of occurrence of each dinucleotide type as a function of position from the nucleosome center to the terminus (positions 0 to 72).

Cloning and sequencing of cDNA encoding the mature form of phosphoribulokinase from spinach.

Phosphoribulokinase (PRK) is a key enzyme in the Calvin cycle of autotrophic organisms. We have constructed a spinach leaf cDNA library in the phage expression vector, lambda gt11, and used a rabbit polyclonal antibody raised against spinach PRK to identify PRK clones. Analyses of the nucleotide sequences of two antibody-positive clones, 1.

Changes in hepatic differentiation following treatment of rat fetuses with 5-azacytidine.

Rat fetuses of 20 days gestational age were treated in utero with 5-azacytidine. Within 14 to 18 h after treatment several significant changes in the fetal livers were observed, including a dramatic maturation of hepatocyte morphology with little alteration in hematopoietic elements. Assessment of mRNA levels by hybridization to cloned cDNAs, together with other measures of gene expression, established that the change in hepatocyte morphology was associated with strong activation of expression of genes normally activated later in development, including those coding for the liver enzymes tyrosine aminotransferase and phosphoenolcarboxykinase and a gene of unknown specificity that is regulated in liver much like the aminotransferase.

Effect of polyvalent metal ions on the reactivity of human O6-methylguanine-DNA methyltransferase.

Polyvalent metal ions are highly effective in inhibiting human O6-methylguanine-DNA methyltransferase, the repair protein responsible for the removal of the promutagenic and presumably procarcinogenic adduct, O6-alkylguanine, in DNA. The sulfhydryl group-reacting metal ions (Cd2+, Zn2+, Hg2+, Pb2+) completely inhibited the reaction at concentrations of 100-500 microM while other metal ions (Al3+, Fe3+) required concentrations of 1 mM or greater for significant reduction of the reaction rate. Inhibition by the former group of metals could be reversed by dithiothreitol but not by EDTA, while the opposite was true for the second group.

Biphasic response of Na+-dependent amino acid transport to tumor promoting phorbol esters in cultured renal epithelial cells, LLC-PK1.

Confluent, nongrowing renal epithelial cells, LLC-PK1, have a low rate of Na+-dependent (A-system) amino acid transport. Following a brief period of amino acid and serum deprivation, but with glucose provided as an energy source, such cells respond to the tumor promoter TPA with a brief enhancement of A-system activity that returns to control levels within 10-20 min. The response is followed some 30 min later by a large and prolonged elevation of transport activity (delayed response).

Lectin-like components in the macronuclear replication bands of Euplotes eurystomus.

Upon incubation with fluoresceinylated neoglycoproteins, isolated macronuclei from the ciliated protozoan Euplotes eurystomus display different labelling patterns depending on the nature of the sugar bound to the neoglycoproteins. Specific sugar-binding components (i.e.

The induction of chromosome aberrations by restriction endonucleases that produce blunt-end or cohesive-end double-strand breaks.

Restriction endonucleases have been used to study the involvement of specific types of DNA damages in the production of chromosome aberrations. In this study restriction endonucleases were introduced into viable CHO cells using osmolytic shock of pinocytic vesicles. We compared two cohesive-end cutters, Msp I (CCGG-2-base overlap) and Sau3A I (GATC-4-base overlap) with two blunt-end cutters, Alu I (AGCT) and Rsa I (GTAC).