Role of 3-methyladenine-DNA glycosylase in host-cell reactivation of methylated T7 bacteriophage

Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway. For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1. These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage. Quantitative analysis of the data, using the results of Verly et al. (Verly, W.G., Crine, P., Bannon, P. and Forget, A. (1974) Biochim. Biophys. Acta 349, 204–213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5–10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant. Thus, 3-methyladenine may be as toxic a lesion as an apurinic site.     

Oligosaccharides accumulated in the kidney of a goat with β-mannosidosis: Mass spectrometry of intact permethylated derivatives

Two oligosaccharides accumulate in the kidney of a goat with β-mannosidosis. These oligosaccharides were isolated and purified from kidney extracts by Bio-Gel P2 gel permeation column chromatography. Their structures were characterized as Manβ1 → 4GlcNAc and Manβ1 → 4G1cNAcβ1 → 4G1cNAc by mass spectrometry of the permethylated intact oligosaccharide alcohols and permethylated native oligosaccharides. Carbohydrate composition analysis, methylation linkage studies, and enzymatic hydrolysis were also performed. Stored in 1 g of kidney were 1.6 μmol of disaccharide and 7.6 μmol of trisaccharide, which was three times that found in the brain of this affected animal (M. Z. Jones and R. A. Laine, 1981, J. Biol. Chem., 256, 5181–5184). In both the brain and kidney of the affected goat, oligosaccharide accumulation was evidently represented by membrane-bound, electron-lucent vacuoles in numerous cell types. While lesions in the brain were associated with profound neurological deficits, functional impairment of the kidney was not apparent. Similar oligosaccharides excreted in urine may be derived from those stored in the kidney. The mass spectrometric methods utilized in this investigation will facilitate comparison of oligosaccharide composition in different tissues and biological samples in β-mannosidosis and other disorders of glycoprotein catabolism.     

Induction of congenital anomalies in offspring of female mice exposed to varying doses of X-rays

Female mice were exposed to varying absorbed doses (108–504 rad) of X-rays and mated at different intervals after irradiation (1–7, 8–14, 15–21 and 22–28 days). Uterine contents were examined at late pregnancy in order to detect early fetal deaths (dominant lethality) and malformations in the live fetuses.Two trends were apparent from data on abnormal fetuses. At each weekly interval, the incidence of abnormalities tended to rise with increase in dose, and, at any given dose, the incidence tended to increase with time after irradiation. Dwarfism and exencephaly were the two most common malformations found.The changes in incidence of dominant lethality and of abnormal fetuses with time and with dose follow each other closely, the highest incidence for both being reached in week 3 (59±4.7% for dominant lethals and 12.5±3.1% for abnormal fetuses, after 504 rad) indicating increased radiosensitivity of less mature oocytes. These results parallel those obtained from known genetic effects reported by other workers and suggest that testing for incidence of congenital malformations among offspring of treated animals may prove a useful means of assessing genetic hazards of radiation of chemicals.     

Turnaround of Axoplasmic Transport of Selected Particle-Specific Enzymes at an Injury in Control and Diisopropylphosphorofluoridate-Treated Rats

: Reversal of direction (turnaround) of axonal transport of particle specific enzyme activities was studied at a ligature placed on rat sciatic nerve. In the principal experiment, the ligature remained on the nerve in vivo several hours, allowing enzyme activities (acetylcholinesterase, acid phosphatase, and monoamine oxidase) to accumulate immediately proximal to the tie. The nerve was then tied a second time, proximal to the first tie, and incubated in vitro for several more hours. Accumulation of enzyme activities just distal to the second tie was measured. This second accumulation, of activities traveling in the retrograde direction, was shown to be the result of turnaround in several ways. (1) The increase in activity distal to the second tie was equal to the decrease in activity proximal to the first. (2) The increase in enzyme activities distal to the second tie was greatly reduced when the accumulation proximal to the first tie was trapped by placing a third tie between the first and second ties. (3) It was shown that the activity that accumulated distal to the second tie could not have been in retrograde motion at the time of the first tie. (4) Accumulation distal to the second tie was not a function of the length of nerve segment included between the two ties. In contrast to the consistent occurrence of turnaround of orthograde flow, turnaround of retrograde flow could not be demonstrated. Turnaround transport was blocked by incubation in the cold and in the presence of NaCN or vinblastine. The turnaround process operated on all three enzymes studied, suggesting that it operates on lysosomes and mitochondria, as well as on the endoplasmic reticulum-like material bearing acetylcholinesterase. Evidence for the participation of the transport process in the renewal of AChE in the distal portions of the axon was obtained in experiments using diisopropylphosphorofluoridate and cycloheximide.     

Origins of immunoglobulin heavy chain domains

Summary Using computer programs that analyze the evolutionary history and probability of relationship of protein sequences, we have investigated the gene duplication events that led to the present configuration of immunoglobulin C regions, with particular attention to the origins of the homology regions (domains) of the heavy chains. We conclude that all of the sequenced heavy chains share a common ancestor consisting of four domains and that the two shorter heavy chains, alpha and gamma, have independently lost most of the second domain. These conclusions allow us to align corresponding regions of these sequences for the purpose of deriving evolutionary trees. Three independent internal gene duplications are postulated to explain the observed pattern of relationships among the four domains: first a duplication of the ancestral single domain C region, followed by independent duplications of the resulting first and last domains. In these studies there was no evidence of crossing-over and recombination between ancestral chains of different classes; however, certain types of recombinations would not be detectable from the available sequence data.     

Study on the dehydrating effect of the red cell Na+/K+-pump in nystatin-treated cells with varying Na+ and water contents

Using the antibiotic Nystatin, we have developed a systematic method for the preparation of red blood cells with independently selected levels of intracellular Na⁺ concentrations and water content. Such cells provided an experimental model to study the effect of Na⁺/K⁺ pump stimulation on red cell water content. Even in initially dehydrated cells, stimulation of the Na⁺/K⁺ pump by elevated intracellular Na⁺ caused subsequent further loss of cell water. Cell water loss was reflected in decreased monovalent cation content per unit mass of hemoglobin and by a shift in the density distribution of the cell populations to higher densities on discontinuous Stractan gradients. We conclude that the 3 Na_out⁺ : 2 K_in⁺ stoichiometry of the Na⁺/K⁺ pump results in a net desalting effect with increased pump activity. Under the conditions of these experiments, the cell appears to have no effective mechanism to compensate for a net loss of ions and water.     

Histone acetylation and heterochromatin content of cultured Peromyscus cells

In order to explore the relationship between unacetylated arginine-rich histones and condensed chromatin structure, the extent of histone acetylation was examined in cultured cell lines derived from three species of deer mice. These species differ considerably in their genomic content of heterochromatin but contain essentially the same euchromatin content. Cells of Peromyscus eremicus, containing 34–36% more constitutive heterochromatin than Peromyscus boylii or Peromyscus crinitus cells were found to contain 28–35% more unacetylated histone H4, 22–29% more unacetylated histone H3, and 18–22% more unacetylated histone H2B. This relationship between unacetylated histones and heterochromatin content was further explored by inducing hyperacetylation of P. eremicus and P. boylii histones through treatment of cells with 15 mM sodium butyrate for 24 h. It was found that the percentages of unacetylated histones H3 and H4 remaining after butyrate treatment were proportional to the amount of constitutive heterochromatin in the genome. These data support the concept that a small core of histones in constitutive heterochromatin is inaccessible to acetylation. It was also found that the acetylated state of isolated histones was sensitive to the method of histone extraction. Thus concern must be given to preparative procedures when studying histone acetylation in order to minimize these acetate losses.     

A surprising new protein superfamily containing ovalbumin,antithrombin-III,and alpha1-proteinase inhibitor

A distant relationship between chicken ovalbumin and two human plasma protease inhibitors was revealed by computer analyses. We propose a new protein superfamily containing at least three families: ovalbumin (and probably gene X and gene Y proteins), antithrombin-III, and alpha₁-proteinase inhibitor. Although these families may have diverged from a common ancestor more than 500 million years ago, they may still share similarity in gene structure as well as in protein sequence.     

The effects of antimicrotubule agents on cell motility in fibroblast aggregates

Spherical aggregates of chick heart, sclera and skin fibroblasts were fused with tritiated thymidine-labelled aggregates of the identical cell type. After being placed in contact, the two aggregates cohered and broadened the area of contact to form a single aggregate with a planar interface between the labelled and unlabelled halves. The motility of cells in the aggregate was determined by measuring the movement of labelled cells across the boundary into the unlabelled half. Exposure to pharmacological doses of antimicrotubule agents resulted in a significant reduction in fibroblast motility in the three-dimensional aggregates.