Specificity of deletion events in pBR322

The reversion of mutations due to inserts of identical palindromic DNAs just 1-bp apart in the amp gene of plasmid pBR322 varied up to 3000-fold (U. DasGupta, K. Weston-Hafer, and D.E. Berg (1987) Genetics 115, 41-49). The experiments reported here show that the intrinsic frequencies of deletion from these sites are truly very different. Deletions were selected by the joint loss of sacB (sucrose sensitivity) and lacZ alpa genes cloned together at these sites, without requiring restoration of the ampr allele. We found that greater than 90% of deletions at each of these sites do restore the ampr allele. This result reinforces the view that the probability of forming a particular deletion depends strongly on the DNA sequence at its prospective endpoints.     

Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins

The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.     

Study of the amniotic fluid from smokers and non-smokers in the Ames test

Amniotic fluid from smokers and non-smokers was tested by the Salmonella/mammalian microsome test. Concentrated amniotic fluid from heavy smokers at term showed an increase in the number of revertants with increasing exposure to tar. However, some of the non-smokers had a higher number of revertants than the smokers. No significant differences were found between second-trimester samples from smokers and non-smokers, but the limited volumes available at this stage of pregnancy may be a source of error.     

Effect of chlorine dioxide on selected membrane functions of Escherichia coli

The mode of action of chlorine dioxide on Escherichia coli was assessed by studying outer membrane permeability to macromolecules and potassium, and observing effects on respiration. The results indicate that gross cellular damage involving significant leakage of intracellular macromolecules does not occur. There was a substantial efflux of potassium, however, and respiration was inhibited even at sublethal doses. It was concluded that the inhibition of respiration, which could be due to the damage to the cell envelope, was not the primary lethal event. Observations of the efflux of K⁺ strongly implicate the loss of permeability control as the primary lethal event at the physiological level, with nonspecific oxidative damage to the outer membrane leading to the destruction of the trans-membrane ionic gradient.     

Homologous and nonhomologous recombination in monkey cells.

Though recombinational events are important for the proper functioning of most cells, little is known about the frequency and mechanisms of recombination in mammalian cells. We have used simian virus 40 (SV40)-pBR322 hybrid plasmids constructed in vitro as substrates to detect and quantitate intramolecular homologous and nonhomologous recombination events in cultured monkey cells. Excision of wild-type or defective SV40 DNAs by recombination from these plasmids was scored by the viral plaque assay, in either the absence or the presence of DNA from a temperature-sensitive helper virus. Several independent products of homologous and nonhomologous recombination have been isolated and characterized at the DNA sequence level. We find that neither DNA replication of the recombination substrate nor SV40 large T antigen is essential for either homologous or nonhomologous recombination involving viral or pBR322 sequences.