Joseph A. Mazrimas

Direct evidence for the role of incorporated BUdR in the induction of sister chromatid exchanges.

Abstract The level of bromodeoxyuridine (BUdR) substitution for thymidine (dThd) in nascent DNA of Chinese hamster cells increases rapidly with increasing BUdR concentration at low BUdR/base pair (BU/bp) ratios and then increases much more slowly at higher ratios. The relationship between percentage substitution and BU/bp indicates that BUdR competes with dThd for incorporation into DNA and that BUdR concentration has no effect on the effective dThd/base pair ratio in these cells. Equal amounts of BUdR are incorporated during both the first and second cell cycles (S phases) after provision of the dThd analog. Incorporation of BUdR induces sister chromatid exchanges (SCEs), and induction appears to occur during the cell cycle (S phase) in which incorporation occurs, with incorporated BUdR from previous cycles having little effect. BUdR-induced SCE frequencies are linearly proportional to the percentage substitution of BUdR for dThd. Extrapolation to zero substitution indicates that SCEs occur spontaneously at a frequency of 0.045 per chromosome per cycle, and that many so-called “spontaneous” SCEs are actually induced by BUdR.

Satellite DNA and cytogenetic evolution

The genusDipodomys (kangaroo rats) exhibits major interspecies variations in the proportions of highly reiterated satellite DNA sequences in the genome as well as in the chromosome number and the proportions of uni-armed and bi-armed chromosomes. For nearly all of the approximately 22 species of the genus and several subspecies, liver DNA was distributed in neutral CsCl buoyant density gradients into four fractions: principal DNA (1.698 g/ml), intermediate-density DNA (1.702 g/ml), MS satellite (1.707 g/ml) and HS (heavy) satellites (1.713 g/ml). The total nuclear DNA content of diploid liver cells measured in eleven species by quantitative cytophotometry, ranged from 6.9 to 10.9 pg. These data were correlated with known features of the karyotypes of individual species. The salient findings were: (1) that interspecies variations in diploid chromosome number cluster at 52–54, 60–64 and 70–72 (2) that high total nuclear DNA was associated with high chromosome number, and with relatively large amounts of satellite DNA (3) that a high ratio of HS satellites to intermediate-density DNA was generally correlated with a predominance of metacentric and submetacentric chromosomes (high fundamental number). The relationships of satellite DNA to karyotype structure reveal a new level of hierarchy in the genome that appears capable of exerting global control over environmental adaptation and the evolution of new species. This mechanism is consistent with recent hypotheses that changes in the macro-structure of the genome are more important than point mutations in facilitating the rapid phases of animal evolution.

Physical studies on synthetic DANs containing 5-methylcytosine

Abstract In synthetic DNAs in which cytosine or 5-methylcytosine is base-paired with guanine or hypoxanthine, the physical properties depend both on base composition and base sequence. The absorption spectra of poly(dI) · poly(m5dC) and poly(dI-m5dC) · poly(dI-m5dC) closely resemble the spectra of an equimolar mixture of nucleotides; the maxima near 250 nm corresponds to dIMP absorption and the shoulder at 278 nm corresponds to 5-methyldeoxyCMP absorption. In contrast, when dIMP is replaced by dGMP, the maxima remain near 250 nm, but there is no shoulder; the pyrimidine absorption appears to be blue-shifted by approx. 28 nm. The fluorescence of 5-methyldeoxyCMP is unperturbed when incorporated into randomcoil poly(m5dC) and is blue-shifted by approx. 8 nm when incorporated into poly(dI-m5dC) · poly(dI-m5dC). Incorporation of 5-methyldeoxyCMP into poly(dG-m5dC) · poly(dG-m5dC) reduces the fluorescence yield several-fold and blue-shifts the emission by approx. 50 nm. Methylation of pyrimidines raises the melting temperature of both poly(dR-dY) · poly(dR-dY) and Poly(dR) · poly(dY) and reverses the order of melting: methylation (and also bromination) gives the homopolymer pair a higher melting temperature than the alternating copolymer. Methylation also invariably reduces the buoyant density of the synthetic DNAs in neutral and alkaline CsCl, but the decrement is not constant. Methylation of poly(dI-dC) also recudes its buoyant density in neutral and alkaline Cs2SO4. Methylation of poly(dG-dC) increases its buoyant density in neutral Cs2SO4 and reduces it in alkaline Cs2SO4.

A corrected primary sequence for bull protamine

We have redetermined the primary sequence for bull protamine using HPLC peptide mapping and automated amino-acid sequencing techniques and report, on the basis of these findings, that the previously published amino-acid sequence for this protein is incorrect. The correct protamine sequence is 50 amino acids in length and differs from the original published sequence by the tripeptide -Cys-39-Arg-40-Arg-41-. Analyses of protamine tryptic peptides derived from nine diverse breeds of Bos tarus and Bos indicus indicate that this sequence is present in the protamine of each breed and that it does not represent a variant or mutation.

Location of satellite DNAs in the chromosomes of the kangaroo rat (Dipodomys ordii)

The location of satellite DNA sequences in metaphase chromosomes has been studied in the kangaroo rat by the in situ hybridization technique, staining techniques and phase contrast microscopy. The HS-β satellite DNA is located at the kinetochores of all but three chromosome pairs. The HD satellite is located predominantly in the short arms of the chromosomes containing HS-β and in the kinetochores of chromosome pairs that lack HS-β. The regions that contain the satellite DNA sequences can also be identified by the Giemsa staining technique, and can be visualized with phase contrast microscopy or following Feulgen staining of fixed chromosome preparations.

Formation of intraprotamine disulfides in vitro.

When mammalian protamine is dissolved in aqueous buffers at neutral or alkaline pH, both ends of the protein fold inward toward the center of the molecule and form disulfide crosslinks that stabilize several different structures. In the absence of reducing agents, these folded forms of protamine may be visualized and quantitated by gel electrophoresis. Using this technique, we have examined the formation of bull protamine disulfides in solution and describe a variety of factors that affect this process. At pH 8, disulfide-stabilized folded forms of protamine appear within minutes after solubilization of the fully reduced protein. Five different monomers are detected by electrophoresis. Each of these monomers is stabilized by at least one disulfide crosslink and migrates with a distinct mobility, ahead of the fully reduced and extended protein. Under certain conditions, dimers of these folded structures crosslinked by interprotamine disulfides are also formed. The appearance of these disulfide-crosslinked forms of protamine is effected by air oxidation, accelerated at alkaline pH, inhibited upon lowering the pH below pH 7 and eliminated by modifying the proteins cysteine residues. Similar intramolecular disulfides are also produced after the protamine molecule binds to DNA. These results suggest that only those cysteines located within the amino- and carboxyterminal ends of the protein appear to participate in forming intramolecular disulfides in vitro.

Tritiation of animals from tritiated water.

Mice were given tritiated water (HTO) to drink for 40-147 days at a specific activity of 94 ± 4.8 μCi/ml (mean ± SD). Tissue HTO was 77.8 ± 10.1 μCi/ml in liver, testis and intestinal mucosa, reflecting the input of H2 O in unlabeled food and metabolic H2 O. Bound T specific activity was 15.8 ± 6.0 μCi/g dry matter. Kangaroo rats (Dipodomys merriami) were given periodic HTO injections and fed a dry diet, with similar results. In both species the specific activity of nonaqueous tissue hydrogens was 25-40% that of hydrogen in tissue HTO. The total soft tissue radiation dose was estimated to be over 2000 rads. In the mouse nonexchangeable T in liver DNA had a specific activity 12% that of hydrogen in tissue HTO; and all four bases were labeled. An apparent tritium isotope effect was observed in the lungs. The specific activity of expired HTO vapor relative to tissue HTO was 0.44 in kangaroo rats and 0.64 in mice.

High-performance liquid chromatographic analysis of cytosine arabinoside and metabolites in biological samples

A rapid, non-radioactive method to quantitate therapeutically realistic levels of 1-beta-D-arabinofuranosylcytosine (Ara-C) and its metabolites would be useful both in the clinic, for monitoring drug levels, and in the laboratory for correlating drug levels with cellular and molecular perturbations. Liquid chromatographic analysis of arabinose-nucleoside analogs in biological samples is complicated by the presence of interfering nucleosides and nucleotides. We report the development of two analytic procedures to measure Ara-C and metabolite levels in biological samples. One method uses a quaternary ammonium type anion-exchange resin to achieve isocratic separation in less than one hour. The second method utilizes a boronate-derivatized polyacrylamide column which binds cis-diols to selectively retain cytosine and uridine, while arabinose compounds are eluted with recovery approaching 100%. The eluted compounds are then easily quantitated on a reversed-phase C18 column. The sensitivity of both procedures was sufficient to obtain pharmacokinetic data on Ara-C and uracil-arabinose levels in serum and urine and on Ara-C triphosphate levels in tumor cells.

S phase patterns of replication of different satellite DNAs in three species of Dipodomys (kangaroo rat)

The pattern of synthesis of four different DNA components, main band, MS, HS-α and HS-β satellites has been studied in each of three different species of kangaroo rat: Dipodomys ordii, Dipodomys panamintinus and Dipodomys merriami . All DNA components are synthesized to some extent at all S phase intervals, but they all show rapid changes in the rate of synthesis at defined points in the S phase. Within one species, different satellite DNAs are replicated at their maximal rate during different intervals of the S phase and are not always late replicating. The time of synthesis of HS-α satellite DNAs is independent of species type, but HS-β satellite DNA is synthesized at the maximal rate during a different interval of the S phase in each of the three species.

Reactivity and adduct formation of a polyaromatic hydrocarbon, 7-bromomethylbenz[a]anthracene, with chromatin histone proteins.

The alkylation of histones by the direct-acting carcinogen 7-bromomethylbenz[a]anthracene was demonstrated both in vivo and in vitro. The relative molar reactivity for mouse liver histones in vivo was H3 greater than H1 greater than H2b greater than H4 greater than H2a. The in vitro modification of histone H3 was examined in detail. Amino acid adducts stable to acid hydrolysis were separated after acetylation by reversed-phase high-performance liquid chromatography and characterized using ultraviolet absorbance spectra and synthetic amino acid adduct standards. Three major adducts were observed and tentatively identified as cysteinyl, lysyl and histidinyl adducts of histone H3.