Intraclonal polymorphism in the bacterium Streptomyces ambofaciens ATCC23877: evidence for a high degree of heterogeneity of the wild type clones

In Streptomyces ambofaciens a genetic instability generates a high degree of polymorphism consisting of four main phenotypes: pigmented colonies (Pig(+) qualified as WT phenotype), pigment-defective colonies, pigmented colonies with pigment-defective sector and pigmented colonies with pigment-defective papillae. Molecular analysis of Pig(col)(-) and Pig(sec)(-) (pigment-defective mutant derived from a colony and a sector, respectively) produced by genetic instability and isolated in five Pig(+) subclones progenies revealed a new aspect of polymorphism in S. ambofaciens ATCC23877. Frequencies of Pig(col)(-) and Pig(sec)(-) mutants deleted at the chromosome ends varied from one WT progeny to another. Two main types of deleted mutants were observed: deleted for one or both chromosomal extremities. The relative proportion of these two categories differed according to the WT progeny. These results argue for heterogeneity of the WT clones, i.e., Pig(+) colonies, originated from S. ambofaciens ATCC23877.     

Clustering of mutations blocking synthesis of xanthan gum by Xanthomonas campestris.

Mutations that block the synthesis of xanthan gum by Xanthomonas campestris B1459S-4L-II were isolated as nonmucoid colonies after treatment with ethyl methanesulfonate. Complete libraries of DNA fragments from wild-type X. campestris were cloned into Escherichia coli by using a broad-host-range cosmid vector and then transferred into each mutant strain by conjugal mating. Cloned fragments that restored xanthan gum synthesis (Xgs+; mucoidy) were compared according to restriction pattern, DNA sequence homology, and complementation of a subset of Xgs- mutations. Groups of clones that contained overlapping homologous DNA were found to complement specific Xgs- mutations. The results suggest clustering of the genetic loci involved in xanthan synthesis. The clustering occurred within three unlinked regions. Two forms of complementation were observed. In most instances, independently isolated cosmid clones that complemented a single mutation were found to be partially homologous. Less frequent was the second form of complementation, in which two cosmid clones that lacked any homologous sequences restored the mucoid phenotype to a single mutant. Finally, xanthan production was measured for wild-type X. campestris carrying multiple plasmid copies of the cloned xanthan genes.     

Unstable lysogeny and pseudolysogeny in Vibrio harveyi siphovirus-like phage 1

Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.     

An unusual complementation in non-excitable mutants in Paramecium

A non-excitable behavioural mutant, d4-662, was previously characterized as the fourth pawn locus mutant pwD in Paramecium tetraurelia. We now provide data demonstrating that d4-662 is in fact controlled by a pwB allele that has the unusual feature of complementing other pwB alleles in heterozygous F1 progeny. Neither the cytoplasm nor the nucleoplasm of d4-662 cured the mutational defects of pwB and in the reverse combination of d4-662 and pwB, the result was the same. On the other hand, pwA, another non-excitable mutant, was cured upon cross-injection with d4-662 and mutants carrying trichocyst non-discharge marker genes were also cured. This evidence suggests that d4-662 is a new mutant belonging to pwB, and would be better designated as pwB662. Extensive crossbreeding analyses, however, showed an unusual genetic relationship between d4-662 and pwB (pwB95 or pwB96). When d4-662 was crossed with pwB mutants, many progeny expressing wild-type phenotype or mixed clones of wild-type and pawn cells were obtained in the F1. Less than 12.5% expressed the pawn phenotype. The appearance of wild-type progeny in this F1 strongly suggests that an inter-allelic interaction between pwB662 and other pwB alleles may occur during development of the macronucleus.     

Genetic hypervariability of telomere-related sequences is associated with meiosis in Plasmodium falciparum.

Sequences related to those near chromosome telomeres in the human malaria parasite, Plasmodium falciparum, were extremely unstable during a genetic cross between two different clonal genotypes. Many progeny of the heterologous cross displayed telomere-homologous restriction fragments found in neither parent. A significant number of the new fragments resulted from rearrangements at chromosome-internal locations which were bounded by more complex tracts of DNA sequence. The same instability was not seen to arise during an inbreeding cross, nor during mitotic replication of parasites. Thus, a form of genetic hypervariability results from molecular events which occur during meiotic reduction and is apparent only in a cross between heterologous strains of parasite. Since other sequences were entirely stable under the same conditions, it appears that chromosome-internal blocks of telomeric sequences in the P. falciparum genome may designate conditionally unstable chromosomal domains. We discuss some potential implications of these findings for the population biology of P. falciparum.     

Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 function

The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles. In two of these revertants, V-C8-Rev 1 and V-C8-Rev 6, the reversions lead to the wild-type Brca2 sequence. The V-C8 revertants did not gain the entire wild-type phenotype and still show a 2.5-fold increased sensitivity to mitomycin C (MMC), higher levels of spontaneous and MMC-induced chromosomal aberrations, as well as abnormal centrosomes when compared to wild-type cells. Our results suggest that Brca2 heterozygosity in hamster cells primarily gives rise to sensitivity to DNA cross-linking agents, especially chromosomal instability, a feature that might also be displayed in BRCA2 heterozygous mutation carriers.     

Origin of thymidine kinase deficient (TK-) haploid frog cells via an intermediate thymidine transport deficient (TT-) phenotype

Wild-type cultured cells of the frog cell line ICR 2A give rise to 5-bromodeoxyridine (BUdR)-resistant colonies only when the selecting concentration of the drug is 5 × 10^?5 M or lower. The progeny of these colonies multiply in 10^?4 M BUdR; resistance is correlated with the absence of a thymidine (TdR)-specific transport reaction with a K^m in the range of 2–7 × 10^?4 M. All of the TdR transport-deficient (TT-) isolates examined (25) had TdR kinase activity (4% to 100% of wild-type). Variants deficient in TdR kinase activity (5% of wild-type) were obtained by exposing TT-cultures to 10^?3 M BUdR. The TK - variants multply continuously in 10^?3 M BUdR and retain the phenotype after prolonged culture in the absence of the drug. The frequency with which they occur is increased 20 to 50 fold by prior treatment of the culture with ICR 191, an acridine mustard mutagen. In haploid cells, it would be expected that TK- variants would arise in equal numbers from wild-type and TT- cultures if loss TdR kinase occurred independently of loss of the transport reaction. However, wild-type cells give no colonies resistant to 10^?3 M BUdR under conditions the give 1 to 50 colonies per million TT- cells. The TT- phenotype seems to be a required intermediate state in the origin of the TK- phenotype. Therefore, the TK- clones described above are unlikely to be products of mutation at a single genetic locus.     

Infectious and noninfectious recombinant clones of the provirus of SNV differ in cellular DNA and are apparently the same in viral DNA

Ten clones of Charon 4A containing proviruses of spleen necrosis virus, an avian retrovirus, and flanking chicken DNA sequences were isolated and characterized. Some clones gave rise to progeny with viral DNA sequences deleted or duplicated, probably as a result of crossing-over in the 600 bp terminal redundancy in viral DNA. The cellular sequences are different in each clone, indicating that all the proviruses are integrated in different sites in cellular DNA. Six clones are infectious and four are not. All the infectious molecules containing a provirus are of a similar size and are smaller than the noninfectious molecules containing a provirus. The viral DNA is not apparently different in eight clones, but two clones, one infectious and one noninfectious, lack two restriction sites each. Large changes in proviral DNA therefore do not seem responsible for the lack of infectivity of some clones. These results are consistent with the hypothesis that neighboring cellular DNA sequences control proviral expression (infectivity).     

Unstable Lysogeny and Pseudolysogeny in Vibrio harveyi Siphovirus-Like Phage 1

Exposure of Vibrio harveyi (strain VH1114) to V. harveyi siphovirus-like phage 1 (VHS1) resulted in the production of a low percentage of lysogenized clones of variable stability. These were retrieved most easily as small colonies within dot plaques. Analysis revealed that VHS1 prophage was most likely carried by VH1114 as an episome rather than integrated into the host chromosome. In the late exponential growth phase, lysogenized VH1114 continuously produced VHS1 but also gave rise to a large number of cured progeny. The absence of phage DNA in the cured progeny was confirmed by the absence of VHS1 DNA in Southern blot and PCR assays. Curiously, these very stable, cured subclones did not show the parental phenotype of clear plaques with VHS1 but instead showed turbid plaques, both in overlaid lawns and in dot plaque assays. This phenotypic difference from the original parental isolate suggested that transient lysogeny by VHS1 had resulted in a stable genetic change in the cured clones. Such clones may be called pseudolysogens (i.e., false lysogens), since they have undergone transient lysogeny and have retained some resistance to full lytic phage development, despite the loss of viable or detectable prophage.     

Derivation of Tk- Clones from Revertant Tk+ Mammalian Cells

In order to obtain a large collection of Chinese hamster cell clones defective in thymidine kinase (TK(-)), BrdU(r) selection experiments have been performed on wild-type and revertant TK(+) cell lines. No clones (< 10(-9)) were obtained from the wild-type TK(+) cell line by single-step selection. In contrast, revertant TK(+) clones readily gave rise to stable TK(-) derivatives (1 - 2 x 10(-4)). Both wild-type and revertant TK(+) clones spontaneously yielded 8-AG(r) colonies with the same frequency (1 - 5 x 10(-6)), suggesting that the differences between wild-type and revertant cell lines specifically affected selection of the TK(-) phenotype. The increased frequency of TK(-) clones reflects perhaps the number (ploidy) or character of the autosomal TK loci in TK(+) revertants, or perhaps the mechanisms which regulate expression of the TK genes. Several mutagens, EMS, MNNG and UV, stimulated the TK(+) revertants' frequency of TK(-) subclones only slightly (< 3-fold). Biochemical and genetic data indicated that the TK(-) clones derived from one revertant are phenotypically different. The phenotypes displayed by these cell lines are stable and do not depend upon the continued presence of the selective agent.     

Karyotypic instability and centrosome aberrations in the progeny of finite life-span human mammary epithelial cells exposed to sparsely or densely ionizing radiation

The human breast is sensitive to radiation carcinogenesis, and genomic instability occurs early in breast cancer development. This study tests the hypothesis that ionizing radiation elicits genomic instability in finite life-span human mammary epithelial cells (HMEC) and asks whether densely ionizing radiation is a more potent inducer of instability. HMEC in a non-proliferative state were exposed to X rays or 1 GeV/nucleon iron ions followed by delayed plating. Karyotypic instability and centrosome aberrations were monitored in expanded clonal isolates. Severe karyotypic instability was common in the progeny of cells that survived X-ray or iron-ion exposure. There was a lower dose threshold for severe karyotypic instability after iron-ion exposure. More than 90% of X-irradiated colonies and >60% of iron-ion-irradiated colonies showed supernumerary centrosomes at levels above the 95% upper confidence limit of the mean for unirradiated clones. A dose response was observed for centrosome aberrations for each radiation type. There was a statistically significant association between the incidence of karyotypic instability and supernumerary centrosomes for iron-ion-exposed colonies and a weaker association for X-irradiated colonies. Thus genomic instability occurs frequently in finite life-span HMEC exposed to sparsely or densely ionizing radiation and may contribute to radiation-induced breast cancer.     

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.     

Implication of stringent response in the increase of mutability of the whiG and whiH genes during Streptomyces coelicolor development

In Streptomyces ambofaciens, genetic instability occurring during aerial mycelium development gives rise to white mutant papillae on colonies. Pig-pap mutants deriving from such papillae are unable to sporulate and devoid of the large genome rearrangement usually observed in the other Streptomyces mutants that genetic instability generated. Pig-pap mutants frequently harboured discrete mutations affecting the whiG gene. Furthermore, it has been established that the production of papillae dramatically increased when S. ambofaciens was grown under an amino acid limitation. In this work, we tested the implication of the stringent response, induced during an amino acid limitation, in the production of white papillae in Streptomyces coelicolor, a species which is phylogenetically close to S. ambofaciens. First, we showed that S. coelicolor produced mutant papillae and that this production was increased under an amino acid limitation. Secondly, we showed that the Pig-pap mutants generated both with and without amino acid limitation frequently exhibited mutations in whiH or whiG genes. Finally, we observed that a relA mutant of S. coelicolor, which was unable to elicit the stringent response under an amino acid limitation, was also unable to produce papillae. The restoration of the ability to elicit the stringent response also restored the papillae production. These papillae gave rise to Pig-pap mutants displaying the same characteristics as Pig-pap mutants spontaneously appearing on wild-type colonies. Altogether, these results show that whatever the underlying mechanism, the stringent response is involved in the production of white papillae in S. coelicolor.     

Is period gene causally involved in the photoperiodic regulation of reproductive diapause in the linden bug, Pyrrhocoris apterus?

Earlier experiments demonstrated a strong up-regulation of per mRNA in wild-type (Wt) females of Pyrrhocoris apterus reared under diapause-inducing short days, while per mRNA levels were low in females of two non-diapause mutant strains (Nd), irrespective of photoperiod. In the present study, different sequences of per DNA in two strains of geographically different origin enabled us to analyse genetic linkage between the per gene and the Nd phenotype. Crosses between Wt females originating from C. Budejovice (Czech Republic) and Nd males originating from Lyon (France) resulted in F(2) progeny where 411 females entered diapause under short days and 120 females were reproducing. Thus, the segregation was very close to the 3:1 ratio in favour of diapause females, suggesting that the Nd trait behaves as a single autosomal recessive. Analysis of DNA in 20 females of the F(2) progeny revealed that their phenotype was not linked to the per genotype. We conclude that the per gene is not primarily responsible for the block to diapause photoresponsiveness in Nd mutants and its role, if any, is downstream from other gene(s) controlling diapause. This is the first attempt at genetic linkage analysis between a bona fide circadian clock gene and photoperiodism in a "non-drosophilid" species.     

Genetic control of sporulation in the blue-green alga Anabaena doliolum Bharadwaja

Summary Two genetically distinct, non-sporulating mutant strains of Anabaena doliolum were isolated following exposure of the alga to ultraviolet radiation. Among the progeny of a cross between the two non-sporulating strains, some filaments were able to sporulate. The spores of such filaments were of two types. Type 1 upon germination produced the parental phenotype, type 2 gave rise to wild phenotype. This suggests the involvement of heterozygosity in genetic recombination in A. doliolum. The results further indicate that the formation of spores in this alga is under the control of more than one genetic determinant, and that nuclear segregation occurs during sporulation.     

Cause for maintaining high instability at the yellow gene in Drosophila melanogaster lines, isolated during the "mode for mutation" period in Uman populations

Mobile genetic elements are responsible for most spontaneous mutations in Drosophila melenogaster. The discovered in the 1980s phenomenon of frequent change of the wild-type yellow phenotype for a mutant one, and vice-versa, in strains of Drosophila melanogaster isolated from the Uman' natural population can be, according to our data, explained by repeated inversions and reinversions of the gene regulatory region located between the two copies of the hobo transport. However, most molecular genetic events accompanying the process can occur without the phenotype change. After several generations, the strains, remaining phenotypically unchanged, can possess different molecular genetic properties with respect to yellow. Using genetically homogenous or isogenic strains for the genetic analysis or for production of the new plant cultivars or animal breeds, geneticists and breeders often face the problem of stability of the strains. In the present study, the mechanism underlying the generation of instability at the yellow locus of D. melanogaster determined by the hobo-induced genome instability is described.     

Microsatellite instability induced by hydrogen peroxide in Escherichia coli

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using a selective assay for microsatellite instability in E. coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability. Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers. We demonstrate that exposure of E. coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences. Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats. All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences. We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.     

Radiation-induced genomic instability in tandem repeat sequences is not predictive of unique sequence instability

Tandem repeat sequences, classified as minisatellite sequences or partially duplicated genes, are inherently unstable. Radiation exposure can increase the instability of such repeat sequences, but the biological consequences of this elevated instability are not well characterized. To learn more about the characteristics of the instability at different sequences in the genome, we created mutant HT1080 cells bearing 8.4 kb of partially duplicated allele at the HPRT locus by gene targeting. The cells were then tested to determine whether repeat-sequence instability (assessed by elevated reversion rate caused by loss of one duplicated segment) accompanied increased forward mutation rates at the restored wild-type HPRT allele. After a 4-Gy X irradiation, 32 clones were selected (out of 500 clones, 6%) that showed elevated reversion rates even after many cell generations. These clones also showed general increases in the forward mutation rate, whereas the paired individual mutation rates did not correlate with each other. Furthermore, levels of intracellular reactive oxygen species (ROS) and nuclear gamma-H2AX foci, which are hallmarks for DNA damage responses, were also generally elevated, although the levels did not correlate with the individual reversion rates. It was concluded that repeat sequence instability is not predictive of unique sequence instability, probably because the instability is generated by multiple mechanisms after radiation exposure.     

Bystander effects in radiation-induced genomic instability

Exposure of GM10115 hamster-human hybrid cells to X-rays can result in the induction of chromosomal instability in the progeny of surviving cells. This instability manifests as the dynamic production of novel sub-populations of cells with unique cytogenetic rearrangements involving the "marker" human chromosome. We have used the comet assay to investigate whether there was an elevated level of endogenous DNA breaks in chromosomally unstable clones that could provide a source for the chromosomal rearrangements and thus account for the persistent instability observed. Our results indicate no significant difference in comet tail measurement between non-irradiated and radiation-induced chromosomally unstable clones. Using two-color fluorescence in situ hybridization we also investigated whether recombinational events involving the interstitial telomere repeat-like sequences in GM10115 cells were involved at frequencies higher than random processes would otherwise predict. Nine of 11 clones demonstrated a significantly higher than expected involvement of these interstitial telomere repeat-like sequences at the recombination junction between the human and hamster chromosomes. Since elevated levels of endogenous breaks were not detected in unstable clones we propose that epigenetic or bystander effects (BSEs) lead to the activation of recombinational pathways that perpetuate the unstable phenotype. Specifically, we expand upon the hypothesis that radiation induces conditions and/or factors that stimulate the production of reactive oxygen species (ROS). These reactive intermediates then contribute to a chronic pro-oxidant environment that cycles over multiple generations, promoting chromosomal recombination and other phenotypes associated with genomic instability.