Methods for study of mutations and mutagenesis in human lymphocytes

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.     

SCENEDESMUS WALL ORNAMENTATION. I. SCENEDESMUS PARISIENSIS CULTURES1

Four strains of Scenedesmus parisiensis Chodat were studied in xenic and axenic culture in 3 media as well as in cultures incubated in sterile vessels in nature. Organized coenobia are usually produced but these may have merely short spines, spines and serrate edges, or lack wall ornamentation. Because the serrate edge is either not formed or cannot be readily detected in most cases, it is not a satisfactory morphological feature for delimiting this species. In laboratory studies it is noted that S. parisiensis might be confused with S. denticulatus rather than S. brasilien-sis. Inasmuch as both xenic and axenic cultures of the -f strains produced similar results, S. parisiensis can be readily characterized.     

Mutations in human lymphocytes studied by an HLA selection system

Human lymphocytes mutated at the HLA-A2 or HLA-A3 alleles were enumerated and studied by primary selection using antibody and complement, followed by limiting dilution cloning and secondary selection using immunofluorescence or antibody and complement. The geometric mean frequency of in vivo mutant lymphocytes was 3.08 X 10(-5) for the HLA-A2 allele and 4.68 X 10(-6) for the HLA-A3 allele. Mutagenesis by X-radiation or mitomycin produced a dose-related increase in mutant frequency. HLA-B phenotyping and Southern Analysis of the HLA-A gene suggested that mutation was frequently due to gene deletion, which was often substantial.     

Delayed DNA methylation is an integral feature of DNA replication in mammalian cells

In the majority of sites of methylation in the DNA of mammalian cells, the symmetry of methylation is restored within a few minutes of the passage of a replication fork. However, it has been shown that daughter strand methylation in immortalised cell lines is delayed in a substantial minority of sites for up to several hours after replication. We report here the results of two new approaches to the determination of the functional significance of delayed DNA methylation in mammalian cells. Firstly, we demonstrate that normal, nontransformed cells (human peripheral lymphocytes in short-term primary culture) have comparable proportions of delayed DNA methylation to many immortalised cell lines, showing that delayed DNA methylation is not just a secondary consequence of abnormally high methionine requirements commonly observed in transformed cells and that delayed DNA methylation would be unlikely not to occur in vivo. Secondly, we have used 5-aza-2'-deoxycytidine (5azadCyd) to derive subclones of cells from the Chinese hamster ovary cell line which have stably hypomethylated DNA. In three of these subclones which had lost on average one fourth of the methylation sites from their genomes, the proportion of daughter strand methylation which was delayed after replication was reduced by less than 10%. If delayed DNA methylation were site-specific, this implies that of the order of twice the number of "immediate" methylation sites than delayed methylation sites had been lost from the genomes of these hypomethylated subclones. Thus, delayed DNA methylation is an integral part of the process whereby replicating mammalian cells maintain the pattern of methylation in their genomes. These observations are discussed in relation to the significance of delayed DNA methylation for the accurate maintenance of methylation patterns in the genome and the consequent implications for the possible role of methylated deoxycytidines in mammalian gene control.     

A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells.

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.     

CAPITATE APPENDAGES ON SCENEDESMUS CULTURE 16 WALLS1

A capitate appendage was detected on the cell wall of Scenedesmus strain 16 with the electron microscope, using the negative staining technique. The mushroom-like structures, from 450 to 650 mμ long, possessed an elongate stipe and a circular cap. These are attached to ridges or the cell wall of both spiny and spineless colonies.     

Diffusion effects in calcium-regulated strain fields

The Goodwin-Trainor equations for cellular morphogenesis, based on calcium ion regulation of the visco-elastic properties of the cellular cortex, are generalized to the situation where the concentration of calcium ions (free plus bound) is allowed to change locally. A stability analysis is presented which shows that the generalized equations are also stable against perturbations at low and high wave lengths and may, for certain parameter values, develop instabilities at intermediate wave lengths.