Extent of CpG methylation is not proportional to the in vivo spontaneous mutation frequency at transgenic loci in Big Blue rodents.

The lacI transgene used in the Big Blue (BB) mouse and rat mutation assays typically displays spontaneous mutation frequencies in the 5x10(-5) range. Recently, the bone marrow and bladder of the Big Blue rat were reported to have, by an order of magnitude, the lowest spontaneous mutation frequencies ever observed for lacI in a transgenic animal, approaching the value for endogenous targets such as hprt ( approximately 10(-6)). Since spontaneous mutations in transgenes have been attributed in part to deamination of 5-methylcytosine in CpG sequences, we have investigated the methylation status of the lacI transgene in bone marrow of BB rats and compared it to that present in other tissues including liver, spleen, and breast. The first 400 bases of the lacI gene were investigated using bisulfite genomic sequencing since this region contains the majority of both spontaneous and induced mutations. Surprisingly, all the CpG cytosines in the lacI sequence were fully methylated in all the tissues examined from both 2- and 14-week-old rats. Thus, there is no correlation between 5-methylcytosine content at CpG sites in lacI and the frequency of spontaneous mutation at this marker. We also investigated the methylation status of another widely used transgenic mutation target, the cII gene. The CpG sites in cII in BB rats were fully methylated while those in BB mice were partially methylated (each site approximately 50% methylated). Since spontaneous mutation frequency at cII is comparable in rat and mouse, the methylation status of CpG sequences in this gene also does not correlate with spontaneous frequency. We conclude that other mechanisms besides spontaneous deamination of 5-methylcytosine at CpG sites are driving spontaneous mutation at BB transgenic loci.

Analysis of sequence alterations in a defined DNA region: comparison of temperature-modulated heteroduplex analysis and denaturing gradient gel electrophoresis.

The ability to detect DNA sequence heterogeneity quickly and reliably is becoming increasingly important as more genes involved in disease processes are discovered. We have assessed the ability of a high pressure liquid chromatography technique (HPLC) termed temperature-modulated heteroduplex analysis (TMHA) to detect a collection of 20 point mutations distributed throughout a 279 base pair fragment spanning the exon 8 region of the human HPRT gene. All mutant/wild type heteroduplexes formed from mutations in the lowest temperature melting domain of the fragment were easily resolved from the corresponding mutant and wt homoduplexes, while those generated from mutants in the next higher melting domain barely resolved from their parental homoduplexes. For comparison, identical heteroduplex samples were subjected to denaturing gradient gel electrophoresis (DGGE). Heteroduplexes in the lowest temperature melting domain were easily resolved, while no resolution was achieved with those in the next higher melting domain. These results suggest that TMHA and DGGE are measuring similar melting characteristics in heteroduplex molecules. TMHA appears to be a robust approach for detecting and/or purifying a wide variety of mutations in a defined region of DNA, provided that the melting characteristics of the fragment under study are carefully considered.

A comparative study of in vivo mutation assays: analysis of hprt, lacI, cII/cI and as mutational targets for N-nitroso-N-methylurea and benzo[a]pyrene in Big Blue mice.

We have compared the response of the native hprt gene and the lacI, cII, and cI transgenes in Big Blue B6C3F1 mice following treatment with either N-nitroso-N-methylurea (MNU) or benzo[a]pyrene (BaP). Three weeks after mutagen treatment splenic T cells were isolated from the animals, and samples were either cultured to measure mutation at the native hprt locus or used to extract genomic DNA for transgene mutation analysis. Phage rescued from extracted DNA were plated in the presence of 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) to score lacI mutations, or plated on a hflAB lawn to score cII and cI mutants. With MNU hprt mutant frequency increased in a dose-related, sublinear manner up to 78-fold above background at the highest dose tested (20 mg/kg). In comparison, the lacI transgene yielded only a 3.1-fold increase at this dose, and the cII and cI transgenes did not show any increase. With 150 mg/kg BaP a 5.8- and 8.7-fold increase in mutant frequency was observed at hprt and lacI, respectively, while only a 1.3-fold increase was observed at cII. DNA sequencing revealed an increase in GC-->TA transversions among the cII mutants, suggesting that the increase was related to BaP exposure. No significant increase in cI mutant frequency was observed. Therefore, the order of mutation assay sensitivity was hprt>lacI>cII/cI with MNU, and hprt approximately lacI> cII/cI with BaP. While the hflAB selection system offers significant advantages with respect to cost and effort when compared to the lacI assay, additional evaluation of its sensitivity is warranted.

Pretreatment with 17 beta-estradiol attenuates basal- and PTH-stimulated membrane adenylyl cyclase activity in human osteoblast-like SAOS-2 cells.

Parathyroid hormone (PTH)-stimulated production of cAMP by intact human osteoblast-like SaOS-2 cells is diminished by pretreatment with 17 beta-estradiol (E2). The goal of the present study was to determine whether E2, affected adenylyl cyclase activity in cell membranes. Cells were deprived of steroid hormones for 24 h and then incubated with E2 (1 nM) or vehicle for 12 h. Cell membranes were prepared and incubated with [alpha-32P]-ATP in the absence or presence of agonist, and the amount of [32P]-cAMP produced was measured to quantify adenylyl cyclase activity. There was less cAMP produced by membranes from E2-treated cells in response to PTH, GTP gamma S, forskolin, and Mn2+. E2 had no effect on the amounts of Gs or Gi. We propose that pretreatment of SaOS-2 cells with E2 reduced membrane cyclase activity, at least in part, by actions on the PTH-sensitive adenylyl cyclase and/or the G protein-adenylyl cyclase complex.

Palytoxin modulates cytosolic pH in human osteoblast-like Saos-2 cells via an interaction with Na(+)-K(+)-ATPase.

Palytoxin (PTx) at nanomolar concentrations enhances the permeability of mammalian cell membranes to both Na+ and Ca2+. In basal human osteoblast-like Saos-2 cells, PTx (8 nM) caused a persistent decrease in cytosolic pH (pHi) of about 0.2 units, which required the presence of extracellular Ca2+ (Cae2+) and Na+ (Nae+). We acidified Saos-2 cells by incubation with nigericin to examine the action of PTx in cells with an activated Na+/H+ antiporter. Under these conditions, PTx increased the pHi without requiring Cae2+ or Nae+, and the alkalinization was unaffected by hexamethylene amiloride. We conclude that the PTx-induced rise in pHi did not involve the Na+/H+ antiporter. PTx increased the rate of 86Rb+ efflux. We propose that PTx induced alkalinization in nigericin-acidified cells by collapsing the K+ gradient. Exposure to ouabain had no effect on pHi, but it prevented the actions of PTx on PHi in both basal and nigericin-acidified cells. Ouabain-resistant mutant cells were less sensitive to PTx in extruding 86Rb+ than their ouabain-sensitive parents. We conclude that PTx interacts with the Na(+)-K(+)-adenosinetriphosphatase to regulate pHi in both basal and nigericin-acidified Saos-2 cells.

Actions of palytoxin on Na+ and Ca2+ homeostasis in human osteoblast-like Saos-2 cells.

Palytoxin (PTx) is a potent membrane-active agent produced by marine coelenterates that acts to stimulate bone resorption in organ culture at nanomolar concentrations. We report here the actions of PTx on Na+ and Ca2+ homeostasis in human osteoblast-like Saos-2 cells. PTx induced a rise in the cytosolic free Na+ concentration ([Na]i) by causing entry of extracellular Na+ (Na(e)+). PTx also caused a concentration-dependent biphasic rise in the cytosolic free Ca2+ concentration ([Ca2+]i) by enhancing entry of extracellular Ca2+ (Ca(e)2+). Entry of Na+ was dependent on the presence of Ca(e)2+ and was prevented by the Na+/Ca2+ exchange antagonist 3,4-dichlorobenzamil (DCB). Entry of Ca2+ was dependent on the presence of Na(e)+ but was not prevented by DCB. The actions of PTx on [Na+]i and [Ca2+]i were completely inhibited by pretreatment of the cells with ouabain. Ouabain alone had no acute effect on [Na+]i or [Ca2+]i in Saos-2 cells. We propose that interaction of PTx with the Na+ pump created a channel that allowed influx of Na(e)+ and Ca(e)2+. The rise in [Ca2+]i then stimulated the activity of the plasma membrane Na+/Ca2+ exchanger, which further enhanced Na(e)+ entry.

Clathrin light chains LCA and LCB are similar, polymorphic, and share repeated heptad motifs.

The clathrin light chains fall into two major classes, LCA and LCB. In an intact clathrin triskelion, one light chain, of either class, is bound to the proximal segment of a heavy chain leg. Analysis of rat brain and liver complementary DNA clones for LCA and LCB shows that the two light chain classes are closely related. There appear to be several members of each class having deletions of varying length aligned at the same position. A set of ten heptad elements, characteristic of alpha-helical coiled coils, is a striking feature of the central part of each derived amino acid sequence. These observations suggest a model in which the alpha-helical segment mediates binding to clathrin heavy chains and the amino- and carboxyl-terminal segments mediate interactions with other proteins. They also suggest an explanation for the observed tissue-dependent size variation for members of each class.