Cholesterol requirement of mycoplasmas

Cholesterol requirement for growth of mycoplasmas was tested in a serum-free medium supplemented with albumin, l-arginine, palmitic acid, and various concentrations of cholesterol dissolved in Tween 80. In cases in which Tween 80 was shown to inhibit growth, the test medium was supplemented with cholesterol dissolved in ethanol. Of the 31 species examined, all but Mycoplasma laidlawii, M. granularum, and Mycoplasma species strain S-743 exhibited a growth response to cholesterol. No requirement for cholesterol could be shown with the stable L-phase variants of Streptobacillus moniliformis and Proteus species. The results provide experimental support for the view that the large majority of the established Mycoplasma species require cholesterol for growth.     

Formaldehyde fixation of cells does not greatly reduce the ability to amplify cellular DNA

Formaldehyde fixation of cells is routinely used to study DNA-protein interactions in vivo. In these studies, DNA is often analyzed using a polymerase chain reaction technique. Although it is known that formaldehyde can damage DNA, no studies have been performed so far to compare the efficiency of DNA amplification between normal and fixed cells. Here we show that formaldehyde fixation results in a 15% to 20% reduction in the ability to amplify cellular DNA. The loss of amplifiability is independent of the length of the amplification region and the degree to which DNA is compacted on packaging into chromatin.     

DNA repair in human promyelocytic cell line, HL-60.

The human promyelocytic cell line, HL-60, shows large changes in endogenous poly(ADP-ribose) and in nuclear ADP-ribosyl transferase activity (ADPRT) during its induced myelocytic differentiation. DNA strand-breaks are an essential activator for this enzyme; and transient DNA strand breaks occur during the myelocytic differentiation of HL-60 cells. We have tested the hypothesis that these post-mitotic, terminally differentiating cells are less efficient in DNA repair, and specifically in DNA strand rejoining, than their proliferating precursor cells. We have found that this hypothesis is not tenable. We observe that there is no detectable reduction in the efficiency of DNA excision repair after exposure to either dimethyl sulphate or gamma-irradiation in HL-60 cells induced to differentiate by dimethyl sulphoxide. Moreover, the efficient excision repair of either dimethyl sulphate or gamma-irradiation induced lesions, both in the differentiated and undifferentiated HL-60 cells, is blocked by the inhibition of ADPRT activity.     

DNA fragments which specifically bind to isolated nuclear matrix in vitro interact with matrix-associated DNA topoisomerase II

A matrix-associated region (MAR)-containing fragment has been selected from the library of cloned chicken nuclear matrix-associated DNA fragments. Factors, which determine the specific binding of DNA fragments have been studied. Using topoisomerase II-specific inhibitor VM 26 we established that nuclear matrix-associated topoisomerase II interacted with the MAR-containing DNA fragment producing specific cleavage sites on DNA of the fragment.     

Thrombin and IgE antigen induce formation of inositol phosphates by mouse E-mast cells

Stimulation of murine chondroitin sulfate E containing mast cells (E-MC) in vitro either by thrombin or immunologically resulted in a rapid formation of inositol phosphates (IPs). Increase in all of the three IPs (IP1, IP2 and IP3) could be detected 20 s after stimulation. The depletion of Ca2+ from the medium resulted in more than 80% reduction in beta-hexosaminidase release from either thrombin or IgE antigen stimulated cells. However, both thrombin and IgE antigen increased the formation of IP3 under these conditions independent of the presence of extracellular Ca2+.     

Biological role of DNA methylation: sequence-specific single-strand breaks associated with hypomethylation of GATC sites in Escherichia coli DNA.

The effect of methylation of GATC sites in Escherichia coli DNA on the formation of single-strand breaks was studied with dam+, dam mutant, and Dam-overproducer strains. Single-strand breaks have been observed in dam mutant cells predominantly at TpT and, to a lesser extent, at CpC. In dam mutant cells harboring pTP166 (a plasmid containing the dam gene), no such nicks were observed.