Gene amplification induces mucoid phenotype in rec-2 Pseudomonas aeruginosa exposed to kanamycin.

Gene amplification in the chromosome of rec-2 Pseudomonas aeruginosa PAO2003 upon growth on kanamycin-supplemented media led to a stable mucoid phenotype. The chromosomal region controlling alginate biosynthesis was shown to be amplified four to six times as a direct tandem repeat of at least 16.8 kilobase pairs. This amplification was deduced from Southern DNA-DNA hybridization patterns of the chromosomal DNA digested with restriction endonucleases BglII and EcoRI and probed with a cloned DNA segment complementing the alg-22 mutation. The part of the amplified unit carrying the novel DNA joint was cloned. The EcoRI junction fragment was further subcloned and used to probe chromosomes of parental strain PAO2003 and mucoid variant VD2003M. As predicted, the EcoRI junction fragment hybridized to the two chromosomal fragments required to produce the novel junction. Though the mucoid phenotype caused by gene amplification was stable, nonmucoid revertants were obtained at a low frequency on tetracycline-containing media. Southern hybridization of chromosomal DNA from a nonmucoid revertant revealed a reduction in the copy number of amplified DNA. These results suggest a direct relationship between amplification of this chromosomal segment and the induction of mucoidy.     

Mutations in Pol1 Increase the Mitotic Instability of Tandem Inverted Repeats in Saccharomyces Cerevisiae

Tandem inverted repeats (TIRs or hairpins) of 30 and 80 base-pair unit lengths are unstable mitotically in yeast (Saccharomyces cerevisiae). TIR instability results from deletions that remove part or all of the presumed hairpin structure from the chromosome. At least one deletion endpoint is always at or near the base of the hairpin, and almost all of the repaired junctions occur within short direct sequence repeats of 4 to 9 base pairs. The frequency of this event, which we call ``hairpin excision,' is influenced by chromosomal position, length of the inverted repeats, and the distance separating the repeat units; increasing the distance between the inverted repeats as little as 25 base pairs increases their chromosomal stability. The frequency of excision is not affected by representative rad mutations, but is influenced by mutations in certain genes affecting DNA synthesis. In particular, mutations in POL1/CDC17, the gene that encodes the large subunit of DNA polymerase I, increase the frequency of hairpin deletions significantly, implicating this protein in the normal maintainance of genomic TIRs.     

Studies on the Effects of Intermittent Irradiation on Nuclear Aberrations of Root Tip Cells in Vicia faba

This experiment aims to compare the effect of intermittent irradiationand that of continuous irradiation on dry seeds of Vicia faba by 60Co-γ-rays. Experimental results show that intermittent irradiation possess lower damage, higher frequency of chromosomal exchange than continual irradiation. It implied that, living cellsstill have some ability to repair radiant damage, when exposed intermittently to radiation in 0.5-2 hours. The repair ability declines as the dosage increases and tile interval of radiation shortened.     

Frequency of chromosomal aberrations after exposure to γ-radiation of human chorionic villi

We investigated the chromosomal damage induced by in vitro exposure to γ-rays of uncultured first trimester chorionic villi. Frequency and types of chromosomal aberrations at increasing doses of radiation have been evaluated on cytotrophoblast spontaneous metaphases obtained after a short term incubation. Our results indicate a direct correlation between radiation dose and aberration frequency.     

Recombination between Chromosomal Is200 Elements Supports Frequent Duplication Formation in Salmonella Typhimurium

Spontaneous tandem chromosomal duplications are common in populations of Escherichia coli and Salmonella typhimurium. They range in frequency for a given locus from 10(-2) to 10(-4) and probably form by RecA-dependent unequal sister strand exchanges between repetitive sequences in direct order. Certain duplications have been observed previously to confer a growth advantage under specific selective conditions. Tandem chromosomal duplications are unstable and are lost at high frequencies, representing a readily reversible source of genomic variation. Six copies of a small mobile genetic element IS200 are evenly distributed around the chromosome of S. typhimurium strain LT2. A survey of 120 independent chromosomal duplications (20 for each of six loci) revealed that recombination between IS200 elements accounted for the majority of the duplications isolated for three of the loci tested. Duplications of the his operon were almost exclusively due to recombination between repeated IS200 elements. These data add further support to the idea that mobile genetic elements provide sequence repeats that play an important role in recombinational chromosome rearrangements, which may contribute to adaptation of bacteria to stressful conditions.     

A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast

Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5′ and 3′ gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of ~0.5%; the positive clones are identified by PCR or colony hybridization. This paper describes a novel TAR cloning procedure that selects positive clones by positive and negative genetic selection. This system utilizes a TAR vector with two targeting hooks, HIS3 as a positive selectable marker, URA3 as a negative selectable marker and a gene-specific sequence called a loop sequence. The loop sequence lies distal to a targeting hook sequence in the chromosomal target, but proximal to the targeting hook and URA3 in the TAR vector. When this vector recombines with chromosomal DNA at the gene-specific targeting hook, the recombinant YAC product carries two copies of the loop sequence, therefore, the URA3 negative selectable marker becomes mitotically unstable and is lost at high frequency by direct repeat recombination involving the loop sequence. Positive clones are identified by selecting against URA3. This method produces positive YAC recombinants at a frequency of ~40%. This novel TAR cloning method provides a powerful tool for structural and functional analysis of complex genomes.     

Possible mechanisms for occurrence of chromosomal restructuring. Interchromosomal contacts during metaphase and their role in chromosomal restructuring

The influence of colchicine-hypotonic treatment on interchromosomal aberrations at metaphase was studied in bone marrow cells of BALB mice irradiated by X-rays within the dose range from 0.25 to 1.50 Gy. In was found that after 30 min treatment with 0.002% colchicine of cells dividing 10 h following irradiation, the frequency both of chromosomal exchanges and interchromosomal contacts decrease about 3.5 times, the amount of chromosomal breaks increasing. It is calculated from the data of this experiment that two breaks induced by irradiation, which were scored at the same K metaphase as independent ones, appeared to be associated with each other at high frequency through exchange in the absence of colchicine or hypotonic treatment. It is assumed that regions of interchromosomal contacts at native metaphase are the most radiation-sensitive zones of the genome preferentially involved in chromosomal aberrations of X-irradiated cells.     

Studies on chemically induced dominant lethality. II. Cytogenetic studies of MMS-induced dominant lethality in maturing dictyate mouse oocytes.

Young adult female mice were injected intravenously with either 50- or 100- mg/kg doses of methyl methanesulfonate. The females were superovulated and mated to untreated males at intervals ranging from 0.5 to 14.5 days after treatment. The fertilized ova were collected and cultured to the first cleavage mitosis, at which time the female chromosome complement was analyzed for structural chromosomal damage. Chromatid-type aberrations were observed, but at a much lower frequency than previously reported for treatment of post-meiotic male germ cells. The time after treatment at which chromosomal damage was observed and the frequency of affected cells agree, qualitatively, with existing dominant-lethal data derived from treatment of maturing oocytes. Parallel experiments in which metaphase I oocytes were analyzed indicate a lack of MMS-induced chromosomal damage in the meiotic stages. This observation is consistent with the suggestion that an intervening round of DNA synthesis is necessary for MMS-induced lesions to be translated into chromosomal damage. The low yield of chromosomal damage is consistent with the idea that maturing oocytes, unlike later spermatids and spermatozoa, are capable of performing macromolecular repair of premutational lesions.     

Effects of X-irradiation on cell-cycle progression, induction of chromosomal aberrations and cell killing in ataxia telangiectasia (AT) fibroblasts

Survival, cumulative labeling indices, chromosomal aberrations and cell-cycle distribution by flow microfluorometry (FMF) were studied in fibroblasts from normal and three ataxia telangiectasia (AT) families after X-irradiation during density-inhibition of growth and immediate release by subculture to low density. Homozygotic AT (proband) fibroblasts were very hypersensitive to cell killing by X-irradiation (D0 = 40-45 rad). Fibroblasts from AT heterozygotes (parents) were minimally hypersensitive, with D0's (100-110 rad) slightly lower than those for normal fibroblasts (D0 = 120-140 rad). There were three different response groups for a G1 phase block induced by 400 rad of X-rays: (1) minimal or no G1 block was observed in AT homozygote cell strains; (2) 10-20% of the cells were blocked in G1 in normal cell strains; and (3) 50% or more of the cells were blocked in AT heterozygote strains. FMF profiles and cumulative labeling indices showed that homozygotic AT cells irradiated in plateau phase moved into the S-phase following subculture with no additional delay over non-irradiated controls. Homozygotic AT cells showed not only a 4-5 times higher frequency of X-ray-induced chromosomal aberrations than normal strains, but approximately 30% of these were of the chromatid-type. There were no differences in the frequency or type of X-ray-induced chromosomal aberrations between normal and heterozygotic AT cells.     

Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium

The bacterium Deinococcus radiodurans is one of the most radioresistant organisms known. It is able to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Our work aims to highlight the genes involved in recombination between 438 bp direct repeats separated by intervening sequences of various lengths ranging from 1,479 bp to 10,500 bp to restore a functional tetA gene in the presence or absence of radiation-induced DNA double strand breaks. The frequency of spontaneous deletion events between the chromosomal direct repeats were the same in recA+ and in ΔrecA, ΔrecF, and ΔrecO bacteria, whereas recombination between chromosomal and plasmid DNA was shown to be strictly dependent on the RecA and RecF proteins. The presence of mutations in one of the repeated sequence reduced, in a MutS-dependent manner, the frequency of the deletion events. The distance between the repeats did not influence the frequencies of deletion events in recA ⁺ as well in ΔrecA bacteria. The absence of the UvrD protein stimulated the recombination between the direct repeats whereas the absence of the DdrB protein, previously shown to be involved in DNA double strand break repair through a single strand annealing (SSA) pathway, strongly reduces the frequency of RecA- (and RecO-) independent deletions events. The absence of the DdrB protein also increased the lethal sectoring of cells devoid of RecA or RecO protein. γ-irradiation of recA ⁺ cells increased about 10-fold the frequencies of the deletion events, but at a lesser extend in cells devoid of the DdrB protein. Altogether, our results suggest a major role of single strand annealing in DNA repeat deletion events in bacteria devoid of the RecA protein, and also in recA ⁺ bacteria exposed to ionizing radiation.     

Natural hybridization between extremely divergent chromosomal races of the common shrew (Sorex araneus, Soricidae, Soricomorpha): hybrid zone in European Russia

The Moscow and Seliger chromosomal races of the common shrew differ by Robertsonian fusions and possibly whole‐arm reciprocal translocations (WARTs) such that their F₁ hybrids produce a chain‐of‐eleven configuration at meiosis I and are expected to suffer substantial infertility. Of numerous hybrid zones that have been described in the common shrew, those between the Moscow and Seliger races involve the greatest chromosomal difference. We collected 211 individuals from this zone to generate a total dataset of 298 individuals from 187 unique global positioning system (GPS) locations within the vicinity of interracial contact. We used a geographic information system (GIS) to map the location of the hybrid zone, which follows a direct route between two lakes, as would be anticipated from tension zone theory. Even within the central area of the hybrid zone, there is a much higher frequency of pure race individuals than hybrid, making this a clear example of a bimodal zone in the sense of Jiggins & Mallet (2000) . The zone runs through good habitat for common shrews, but nevertheless it is very narrow (standard cline widths: 3–4 km), as would be anticipated from low hybrid fitness. There is clear potential for an interruption to gene flow and build‐up of reproductive isolation. As found in some other hybrid zones, there is a high frequency of novel genetic variants, in this case, new chromosomal rearrangements. Here, we report a de novo Robertsonian fission and a de novo reciprocal translocation, both for the first time in the common shrew. There is an extraordinarily high frequency of de novo mutations recorded in F₁ hybrids in the zone and we discuss how chromosomal instability may be associated with such hybrids. The occurrence of a de novo Robertsonian fission is of considerable significance because it provides missing evidence that fissions are the basis of the novel acrocentric forms found and apparently selected for in certain common shrew hybrid zones.     

Camphor Plasmid-Mediated Chromosomal Transfer in Pseudomonas putida

Camphor-utilizing strains of Pseudomonas putida have been shown to carry the genetic information required for camphor degradation on a plasmid. The plasmid-carrying strains can serve as donors of both plasmid-borne and chromosomal genes. As recipients, plasmid-deleted strains are much superior to those carrying the camphor pathway genes. The transfer frequency of chromosomal, but not plasmid-borne, genes is markedly enhanced if the donor cells are irradiated with ultraviolet light followed by 3-h of growth on a rich medium in the dark. Recombinants selected for prototrophy are stable and most acquire the camphor (CAM) plasmid concomitantly; only a few of the Cam(+) recombinants inherit the donor's ability to transfer chromosomal genes at a high frequency. Transfer-defective mutations occur on the CAM plasmid, affecting both CAM and chromosomal gene transfer.     

Mitochondrial DNA synthesis in mouse L cells temperature sensitive in nuclear DNA replication

Temperature-sensitive (ts) A 1S9 mouse L cells continue to synthesize double-stranded covalently closed mitochondrial (mt) DNA at a temperature (38.5 degrees C) which is nonpermissive for chromosomal DNA replication. The amount of mt DNA made appears to be quantitatively linked to nuclear DNA synthesis. Nuclear DNA replication proceeds normally for 6-8 h after the cells are shifted to 38.5 degrees C, and then declines to reach a minimum at 20-24 h. The level of mt DNA synthesis remains high during this period and decreases once the ts lesion has been established.     

Catastrophic events and recovery from low densities in populations of otariids: implications for risk of extinction

Two key factors in a population's risk of extinction are major population declines induced by natural or anthropogenic events (catastrophes) and whether the population's rate of growth increases or decreases at very low abundance levels. These two elements should be included in any population viability analysis (PVA), but estimates of the frequency and intensity of catastrophic events and data on the dynamics of low population densities are difficult to obtain. We examined the literature on population dynamics of otariids (fur seals and sea lions), to determine how frequently populations are subjected to major population declines, and to what extent depleted populations recover from low population size. We present frequency distributions for percentage declines for otariid life‐stages (pup, juvenile, adult female and male), and describe eight examples of events leading to a population decline of 50% or greater among otariids. We found that numerous otariid populations have been reduced to very low densities by exploitation (low enough to be thought extinct) and have recovered to levels where they are no longer at risk of extinction. This suggests that the reduction in population rate of increase at low densities in otariid populations may not be strong.     

A moving DNA replication factory in Caulobacter crescentus

The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.     

Gene reorganization during serial divisions of normal human cells

We have followed during serial divisions of human fibroblasts the presence in chromosomal and extrachromosomal DNA, of two genes that are expressed in fibroblasts, actin and interferon, and of one that is not expressed, globin. The intensity of the blot hybridization of the actin and globin probes with chromosomal DNA diminished during serial divisions of diploid fibroblasts. The interferon gene remained constant throughout the human fibroblast life span. Chromosomal DNA sequences were present in extrachromosomal circular DNA which appeared at the end of the fibroblast life span. The results could explain some functional changes that occur in these cell populations when their division potential declines.     

The majority of G0 transgenic mice are derived from mosaic embryos

Most transgenic mice are generated by the direct microinjection of DNA fragments into the pronuclei of fertilized eggs. It has been generally assumed that the majority of integration events occur prior to the first round of chromosomal DNA replication (Palmiter and Brinster, 1986). In this study we have determined by comparison of PCR, Southern blot and transmission frequencies that at least 62% of integration events generate a mosaic (somatic and/or germline) G₀ transgenic mouse. Furthermore, the statistical probability of transgene-containing cells segregating to the various early embryo lineages implies that this is probably an underestimate of the true mosaic frequency. Thus, the majority of DNA injected into fertilized mouse eggs integrates after the first round of chromosomal DNA replication, therefore most G₀ transgenic mice are derived from a mosaic embryo.     

Treatment of mice with a herbal preparation (Liv. 52) reduces the frequency of radiation-induced chromosome damage in bone marrow

Induction of chromosomal aberrations was studied from 1/4 to 14 days post irradiation in the bone marrow of mice treated or not with Liv. 52, a herbal preparation, prior to 4.5 Gy exposure. The frequency of chromatid and chromosomal aberrations started increasing at day 1/4 in the irradiation and Liv. 52 + irradiated groups. The highest frequency of aberrations was recorded at day 1/2 post exposure which declined after day 1 in both groups. The frequency of both types of aberrations was significantly lower in the Liv. 52 + irradiated group than in the irradiated group.