Replication and recombination in adenovirus-infected cells are temporally and functionally related.

We have studied the temporal and functional relationships between DNA replication and recombination in adenovirus-infected cells by using Southern blot hybridization to detect recombinant products among intracellular viral genomes. The data show that recombination can be detected soon after DNA replication has commenced and that the proportion of recombinant products increases thereafter. To determine the functional relationship between DNA replication and recombination, replication was blocked with the protein synthesis inhibitor anisomycin, the replication inhibitor cytosine arabinoside, and conditionally lethal mutations in either the virus-specified DNA-binding protein or the DNA polymerase. All treatments that directly or indirectly blocked DNA replication caused a delay in the appearance of recombinant products and a marked decline in their abundance relative to products of parental genotype. These data strongly suggest that DNA replication and recombination are interrelated, either because both processes share functions or because DNA structures produced by replication are suitable substrates for recombination. In addition, we have shown that some recombination function(s) is intrinsically thermolabile at 40.9 degrees C, even in wild-type crosses, since the appearance of recombinant products is delayed and their extent is reduced compared with that from crosses performed at 39.9 degrees C.     

Intracellular sorting of alcohol dehydrogenase isoenzymes in yeast: a cytosolic location reflects absence of an amino-terminal targeting sequence for the mitochondrion.

The yeast Saccharomyces cerevisiae contains three alcohol dehydrogenase isoenzymes (ADHI-ADHIII), two in the cytoplasm (ADHI and ADHII) and one in the mitochondrion (ADHIII). Sequence comparison of the corresponding nuclear genes showed that these three proteins are 80-90% identical except for a 27-amino acid extension at the amino terminus of ADHIII. Here we demonstrate that ADHIII is located inside the mitochondrial inner membrane. We also show, using gene fusions, that the amino terminus of ADHIII contains the information for targeting the protein to and transporting it into the mitochondrion. The mitochondrial isoenzyme ADHIII can be converted into a cytosolic protein by deleting its first 28 amino acids. Conversely, the cytoplasmic isoenzyme ADHII can be converted into a mitochondrial isoenzyme by replacing its first 21 amino acids with the first 48 amino acids of ADHIII. We conclude that ADHII is a cytosolic protein because it lacks an amino-terminal targeting sequence for the mitochondrion and that ADHIII is a mitochondrial protein because it contains a mitochondrial targeting sequence.     

Posttranscriptional control of human gamma interferon gene expression in transfected mouse fibroblasts.

Human gamma interferon genomic DNA was introduced into NIH 3T3 fibroblasts by calcium phosphate precipitation and was not expressed in these cells at the cytoplasmic mRNA or protein level. Treatment of the transfected cells with cycloheximide (1 microgram/ml) induced the accumulation of cytoplasmic gamma interferon mRNA and biologically active human gamma interferon. Analysis of the nuclear enriched RNA from untreated cells indicated that human gamma interferon mRNA was present, suggesting that cycloheximide may act by inhibiting a specific nuclease or may enhance the processing or transport of the RNA from the nucleus to the cytoplasm.     

Substrate recognition by the 2 micron circle site-specific recombinase: effect of mutations within the symmetry elements of the minimal substrate.

The minimal substrate for the 2 microns circle site-specific recombinase FLP consists of a nearly perfect 13-base-pair dyad symmetry with an 8-base-pair core. By using a series of chemically synthesized FLP substrates in in vitro FLP recombination and FLP-binding assays, we have identified four positions within each of the symmetry elements that are important contact points for the FLP protein. Furthermore, the binding and recombination data provide evidence for cooperativity between the two symmetry elements of a substrate and between the symmetry elements of two partner substrates during FLP recombination.     

The complete coding sequence of the human raf oncogene and the corresponding structure of the c-raf-1 gene.

The complete 648 amino acid sequence of the human raf oncogene was deduced from the 2977 nucleotide sequence of a fetal liver cDNA. The cDNA has been used to obtain clones which extend the human c-raf-1 locus by an additional 18.9 kb at the 5' end and contain all the remaining coding exons.     

Stability of tissue culture medium pH as a function of autoclaving,time, and cultured plant material

Autoclaving is a standard procedure for sterilizing nutrient media for plant tissue cultures. Most tissue cultures are grown at pH 5.2 to 5.8 with pH adjustments being made prior to autoclaving. This paper reports that there are significant differences between initial pH levels and pH levels following autoclaving, particularly in the pH range of 5.7 to 8.5. This effect is noted with and without agar. In addition, we report that with time the pH of the medium drifts into the acid range. When Cucumis callus was added to the medium, the pH was changed significantly within 48 hours. The amount and direction (increase or decrease of pH) was significantly correlated with the original pH. This suggests that researchers should be wary of the true pH situation in their medium. In addition, in publications authors should specify whether their medium pH value was determined before or after autoclaving.     

The effect of sub-lethal doses of dieldrin on resting and active metabolism in two species of shrimps

The effects of dieldrin on the active (VaO2) and resting (VrO2) oxygen consumption rates of the shrimps Macrobrachium faustinum (De Sassure) and Macrobrachium amazonicum (Heller) were studied during exposure for 4 days at 0.01 p.p.b. and 7 days at 0.0002 p.p.b., respectively. The VrO2 of M. faustinum increased significantly (P less than 0.01) by 48% but the VaO2 decreased by 13%. The VrO2 and VaO2 decreased by 43 and 70%, respectively, in M. amazonicum. Thus, in both species the aerobic metabolic scope for activity decreased. The increased resting metabolic rate of the indigenous M. faustinum is ascribed to energy consuming responses which allow compensation for the effects of the stressor. The stressor may be said to have moved this species into a metabolic "zone of compensation". The decreased resting metabolic rate of the pond-cultured M. amazonicum is ascribed to greater susceptibility, more extensive metabolic breakdown and failure of compensatory responses. This species might be said to have been forced by the stressor into a metabolic "zone of collapse".