Alternate pathways of DNA replication in Escherichia coli

We have described the pcbA1 mutation which enables E. coli cells to replicate DNA in the absence of a functional dnaE gene product if DNA polymerase I (the polA gene product) is present. The pcbA1 mutation phenotypically suppresses multiple dnaEts and dnaEam alleles. The pcbA1/PolI replication pathway differs from normal in sensitivity to certain DNA-damaging agents such as methylmethane sulfonate (MMS) and a lack of damage-directed mutagenesis. We report here cloning of the pcbA1 gene in a multicopy plasmid. The pcbA1 mutation is detected only in cis; therefore, cloning necessitated gene eviction. The pcbA1 gene lies closely- linked to gyrB. We have demonstrated the physical presence of DNA polymerase I in the replicating holoenzyme complex by immunoblotting using dnaEam strains. We conclude that E. coli has two alternate replisome structures: REP-A, in which DNA polymerase I is the functional synthetic subunit; and REP-E, in which the alpha-subunit, product of the dnaE gene, is functional. To investigate further the role of individual DNA polymerases in replication, we have isolated the polB gene on multicopy plasmids.     

Effects of colchicine on cardiac cell function indicate possible role for membrane surface tubulin

The effects of the tubulin-binding drug colchicine on cultured neonate cardiac cell function were investigated. Application of low doses of colchicine (but not lumicolchicine) caused an early reversible increase in beating rate with a concomitant decrease in amplitude. Treatment of the cells with trypsin at a dose that removes surface tubulin but does not inhibit spontaneous beating, diminished the colchicine effect. Surface radio-iodination of the live cultures followed by two-dimensional gel electrophoresis and radioautography revealed that two spots were heavily labeled. These spots co-migrated with purified brain tubulin. Fibroblasts derived from the cardiac cultures did not label over the tubulin spots. Trypsin treatment removed the presumptive tubulin from the radioautographs but only removed the most basic portion of the alpha-tubulin spot from the stained gel. These results are consistent with a surface membrane role for an iso-form of tubulin in neonate cardiac cells.     

Inability of Pseudomonas stutzeri denitrification mutants with the phenotype of Pseudomonas aeruginosa to grow in nitrous oxide.

Pseudomonas aeruginosa PAO1 reduced nitrous oxide to dinitrogen but did not grow anaerobically in nitrous oxide. Two transposon insertion Nos- mutants of Pseudomonas stutzeri exhibited the P. aeruginosa phenotype. Growth yield studies demonstrated that nitrous oxide produced in vivo was productively respired, but nitrous oxide supplied exogenously was not. The defect may be in electron transport or in nitrous oxide uptake.     

Mitogen-like monoclonal anti-actin antibodies

Monoclonal antibodies (IgM kappa) have been produced to actin isolated electrophoretically from L cell extracts. These monoclonal anti-actin antibodies bind to intact L cells and modulate DNA synthesis and cell proliferation, much like affinity-purified polyclonal rabbit antibody to the same Mr 42,000 actin. In addition, monoclonal antibodies specific for actin from Entamoeba histolytica also bound to and modulated the growth of L cells. A monoclonal antibody directed against a neuroblastoma surface antigen did not produce stimulation of L cells, and the binding activity of anti-actin monoclonal antibody to L cells was removed by absorption with actin covalently coupled to Sepharose. These observations demonstrate the specificity of interaction between the anti-actin monoclonal antibodies and the surface of intact L cells. We conclude that a surface actin-like molecule on the L cell, when bound by specific monoclonal antibody, initiates a stimulatory signal which results in enhanced cellular metabolism.     

Ontogenetic and individual variation in size, shape and speed in the Australian agamid lizard Amphibolurus nuchalis

The present study investigates relationships among size, shape and speed in the Australian agamid lizard Amphibolurus nuchalis . Maximal running speed, body mass, snout-vent length, tail length, fore- and hind limb spans and thigh muscle mass were measured in 68 field-fresh individuals spanning the entire ontogenetic size range (1.3 48 g). Relative lengths of both foreand hind limbs decrease with increasing body mass (= negative allometry), whereas relative tail length and thigh muscle mass increase with body mass (= positive allometry). Repeatable and significant differences in maximal running speed exist among individuals. Maximal running speed scales as (body mass)^0.161, and 59% of the variation in maximal speed was related to body mass. Based on the results of the present and previous studies, data on scaling of body proportions alone appear inadequate to infer scaling relationships of functional characters such as top speed.
Surprisingly, individual variation in maximal speed is not related to individual variation in shape (relative limb, tail and body lengths). These components of overall shape are not independent; individuals tended to have either relatively long or relatively short limbs, tails and bodies for their body mass. Even the significant difference in multivariate shape between adult males and females has no measurable consequences for maximal speed. Speeds of field-fresh animals did not vary on a seasonal basis, and eight weeks of captivity had no effect on maximal running speeds. Gravid females and long-term (obese) captive lizards were both approximately 12% slower than field-fresh lizards.     

Catalytic properties of the resolved flavoprotein and cytochrome B components of the NADPH dependent O2−• generating oxidase from human neutrophils

The resolved flavoprotein and cytochrome b₅₅₉ components of the NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b₅₅₉, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b₅₅₉, depleted of flavoprotein, demonstrated no measureable NADPH dependent $\text{O}{}_2{}^{\mathrm{?}}{}_{\mathrm{?}}$ generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b₅₅₉ was rapidly oxidized by oxygen, as was the cytochrome b₅₅₉ in the intact oxidase.     

Activation of tyrosinase in mouse melanoma cell cultures

Summary Tyrosinase activity increased in Cloudman S-91 mouse melanoma cell homogenates incubated at 37°C for a minimum of 8 h. Enzyme activity continued to increase for 48h at which time the maximal level of activation was observed. Activation did not occur at 4°C and did not occur in the cytosol fraction of the cell, suggesting that the response was localized to melanosomes. The activated enzyme was resistant to solubilization with the nonionic detergent, Triton X-100, and preparation of homogenates in this detergent did not inhibit the temperature-dependent activation of the melanosomal fraction of the cell. The activation process increased the V _Max of tyrosinase 10-fold and lowered the K _M by a factor of 2 as determined by the tyrosine hydroxylase assay. The increase in tyrosinase activity was detectable by three assay methods: tyrosine hydroxylation, melanin synthesis, and by tyrosine decarboxylation. The formation of melanin, however, was found to be 1/20 that of either tyrosine hydroxylation or decarboxylation, a finding which suggests that the melanin pathway may be blocked at 5,6-dihydroxyindole. The “self-activation” response could not be mimicked by incubating cell homogenates with cyclic AMP-dependent protein kinase. Activated tyrosinase could be inhibited by the addition of fresh cell extracts, a finding which suggests that tyrosinase inhibitors may be present in these cells. This investigation was supported by Public Health Service grants CA41425 and CA30393 awarded by the National Cancer Institute, Bethesda, MD and by a research grant from the Proctor and Gamble Company.     

Enhancer sequences responsible for DNase I hypersensitivity in polyomavirus chromatin.

DNase I preferentially cleaves polyomavirus minichromosomes at two sites in the enhancer, each of which comprises the sequence AAGCAPuPuAAG flanked by short inverted repeats. A tandem duplication of this sequence generates an additional hypersensitive locus. Mutations which alter either the AAGCAPuPuAAG or flanking repeats diminish hypersensitivity. This region must determine the chromatin conformation recognized by DNase I.