Occurrence of copper resistant mutants in the toxic cyanobacteria Microcystis aeruginosa: characterisation and future implications in the use of copper sulphate as algaecide.

Werner syndrome (WRN; MIM #277700) is an uncommon autosomal recessive disease in which features of premature aging are associated with genetic instability and an elevated risk of cancer. The Werner syndrome clinical phenotype mimics premature aging: following puberty, patients rapidly develop premature graying and loss of hair, scleroderma-like skin changes, osteoporosis, atherosclerosis, bilateral cataract formation, diabetes mellitus, and hypogonadism (Epstein et al. 1966; Goto 2000). Spontaneous genetic instability has been identified in multiple cell lineages in WRN patients, and may be an important determinant of the increased risk of cancer (for review, see Goto et al. 1996; Moser et al. 2000a; van Brabant et al. 2000). Most Werner patients die prematurely, of either cancer or cardiovascular disease, with an average age at death of 47 yr (Epstein et al. 1966; Goto 2000).

The chromosome 8p WRN gene encodes a 162 kD member of the human RecQ helicase family that is unique in possessing an N-terminal 3′ → 5′ exonuclease domain in addition to the central, conserved 3′ → 5′ RecQ helicase consensus domain (for review, see Shen and Loeb 2000b). A majority of WRN patients have mutations in the WRN gene that truncate the WRN protein with loss of a C-terminal nuclear localization signal. Cells containing these mutant alleles produce little or no detectable protein (Goto et al. 1999; Moser et al. 1999, 2000b). There is growing evidence that WRN, like most other helicases, functions as part of a multiprotein complex. Other proteins that may associate or act with WRN include replication protein A (RPA), DNA polymerase δ, PCNA, DNA topoisomerase I, p53 and Ku (for review, see Shen and Loeb 2000b). Mutations in genes encoding these or other WRN-interacting proteins may be responsible for the ∼10% of patients who fulfill clinical and diagnostic criteria for WRN, but lack WRN mutations (Goto et al. 1999; J. Oshima, pers. comm.). One example is a well-documented 29-year-old Japanese female who had clinical and cellular phenotypes that were indistinguishable from WRN patients with truncating WRN mutations, but lacked inactivating WRN mutations (Matsumura et al. 1985; Fukuchi et al. 1989; Prince et al. 1999; see below).

RecQ helicases play a role in recombination or genome stability assurance in many organisms (for review, see Chakraverty and Hickson 1999; Shen and Loeb 2000b; van Brabant et al. 2000). Thus we reasoned that WRN might play a comparable role in human somatic cells, and we explored this possibility by using two different chromosomally integrated mitotic recombination reporter plasmids to measure spontaneous mitotic recombination rates, infer potential recombination pathways and determine the outcome of mitotic recombination events in WRN and control fibroblast cell lines.

http://depts.washington.edu/monnatws/pdf/2001_Prince.pdf

Loss of Werner syndrome protein function promotes aberrant mitotic recombination

Genome instability is a recurring feature of tumorigenesis. Mutation in MLL2, encoding a histone methyltransferase, is a driver in numerous different cancer types, but the mechanism is unclear. Here, we present evidence that MLL2 mutation results in genome instability. Mouse cells in which MLL2 gene deletion can be induced display elevated levels of sister chromatid exchange, gross chromosomal aberrations, 53BP1 foci, and micronuclei. Human MLL2 knockout cells are characterized by genome instability as well. Interestingly, MLL2 interacts with RNA polymerase II (RNAPII) and RECQL5, and, although MLL2 mutated cells have normal overall H3K4me levels in genes, nucleosomes in the immediate vicinity of RNAPII are hypomethylated. Importantly, MLL2 mutated cells display signs of substantial transcription stress, and the most affected genes overlap with early replicating fragile sites, show elevated levels of γH2AX, and suffer frequent mutation. The requirement for MLL2 in the maintenance of genome stability in genes helps explain its widespread role in cancer and points to transcription stress as a strong driver in tumorigenesis.

/pdf/mutation-of-cancer-driver-mll2-results-in-transcription-55bev33zki.pdf

Mutation of cancer driver MLL2 results in transcription stress and genome instability

The increasing rates of global extinction due to human activities necessitate studies of the ability of organisms to adapt to the new environmental conditions resulting from human disturbances. We investigated the evolutionary adaptation of a microalga to sudden environmental change resulting from exposure to novel toxic chemical residues. A laboratory strain of Dictyosphaerium chlorelloides (Naum.) Kom. and Perm. (Chlorophyceae) was exposed to increasing concentrations of the modern contaminant 2,4,6-trinitrotoluene (TNT). When algal cultures were exposed to 30 mg.L - 1 TNT, massive lysis of microalgal cells was observed. The key to understanding the evolution of microalgae in such a contaminated environment is to characterize the TNT-resistant variants that appear after the massive lysis of the TNT-sensitive cells. A fluctuation analysis demonstrated unequivocally that TNT did not facilitate the appearance of TNT-resistant cells; rather it was found that TNT-resistant cells appeared spontaneously by rare mutations under nonselective conditions, before exposure to TNT. The estimated mutation rate was 1.4 X 10 - 5 mutants per cell division. Isolated resistant mutants exhibited a diminished fitness in the absence of TNT. Moreover, the gross photosynthetic rate of TNT-resistant mutants was significantly lower than that of wild-type cells. Competition experiments between resistant mutants and wild-type cells showed that in small populations, the resistant mutants were driven to extinction. The balance between mutation rate and the rate of selective elimination determines the occurrence of about 36 TNT-resistant mutants per million cells in each generation. These scarce resistant mutants are the guarantee of potential for adaptation.

Evolution of microalgae in highly stressing environments: an experimental model analyzing the rapid adaptation of dictyosphaerium chlorelloides (chlorophyceae) from sensitivity to resistance against 2,4,6‐trinitrotoluene by rare preselective mutations

A murine B cell lymphoma (38C13) was subjected to immunoselection with mAbs directed against the idiotypic determinants of its cell surface Ig. Variants emerged with altered Ig receptors containing identical heavy chains but different light chains. The functional light chain genes in these variants were composed of V kappa segments drawn from the V kappa Ox-1 family, which had replaced the V kappa gene expressed by the parental tumor by rearranging to downstream J kappa segments. Rearrangement at the kappa locus continued to occur spontaneously, giving rise to secondary and tertiary variants at a rate of 1.9 x 10(-4) per cell per generation. Variants were isolated that had ceased production of surface Ig but went on to rearrange again and to become surface Ig+. The Ig- state may be an intermediate step providing a stimulus for continued rearrangement. This process provides an additional mechanism for generating diversity within B cell clones and expands the use of the available repertoire of Ig genes.

/pdf/functional-immunoglobulin-light-chain-genes-are-replaced-by-2j8idkp6ke.pdf

Functional immunoglobulin light chain genes are replaced by ongoing rearrangements of germline V kappa genes to downstream J kappa segment in a murine B cell line.

The factors that affect reliable estimations of mutation rates (g) in cultured mammalian somatic cell populations by fluctuation analysis are studied experimentally and statistically. We analyze the differential effect of the final cell population size in each culture (N t) and the number of parallel cultures (C) on the variation in the rate estimates (tl) inferred from the P0 method. The analysis can be made after the derivation of the variance of/i, which is a measure of variation of tl for a given combination of N t and C in a number of repeat experiments. The variance of ti is inversely proportional to C and to the square of N t. N t determines the probability of occurrence of mutations in a cell culture. By influencing the size of P0, Nt also determines whether a rate estimate is obtainable from the experiment. Since Po is estimated from the fraction of cultures containing no mutation in a set of C cultures, C becomes a determining factor for the accuracy of/2. The rate estimated from/5 o is biased, but the bias is in general 2 orders of magnitude smaller than /i. By the selection of an appropriate combination of N t and C for the experiment, this bias can be reduced even further. Based on the notion of comparing two proportions, we propose a test statistic and have applied it to experimental results for a test of equality of mutation rates in different cell lines. This development places the comparison of mutation rates on a statistical basis. Since the publication of Luria and Delbriick's paper [19] on the distribution of the number of mutant colonies in a series of parallel cultures, fluctuation analysis has been widely applied as a method for the determination of mutation rates (#) in cell populations. The analysis has since been further developed methodologically by Lea and Coulson [15], Newcombe [22], and Armitage [2,3] and modified theoreti

Yung-Tai Hung

Papers

Estimation of mutation rates in cultured mammalian cells