Role of activated cellular c-Ha-ras-1 oncogene in the mutagenic effect of the plasmid pEJ6.6]

The role of the activated oncogene c-Ha-ras-1 from human bladder carcinoma integrated into the pEJ6.6 plasmid in the mutagenic effect of the plasmid was studied in Chinese hamster cells. The frequency of hypoxanthine-phosphoribosyltransferase defective (HPRT-) mutants after treatment with pEJ6.6 containing an active c-Ha-ras-1 exceeded that in control dishes treated with a derivative of pEJ6.6 plasmid with an inactivated oncogene. The inactivation was achieved by introducing a deletion into the coding region of the oncogene. The mutagenic effect was rather weak but statistically significant. Thus, the data obtained show that the mutagenic activity of pEJ6.6 plasmid is determined by its oncogene. The role of mutagenic effects of activated cellular oncogenes in malignant transformation is discussed.     

Gene mutations, chromosome aberrations and survival after the x-ray irradiation of a Chinese hamster cell culture protected by cysteamine

The protective effect of cysteamine against 6-thioguanine-resistance (TCr) mutations, chromosome aberrations and inactivation caused by X-ray in cultured cells of Chinese hamster (clone 431) has been studied. The dose-effect curves have been obtained under irradiation condition without protector and with it. Dose-modifying factor of 2 was calculated for chromosome aberrations and cells inactivation and 2,8 for TCr mutations. It is supposed that the cysteamine acts on the general mechanisms involved in damages realization which results in gene mutations, chromosome aberrations and cell inactivation.     

Mechanisms in metal genotoxicity: the significance of in vitro approaches

A survey of the literature published on the ability of metal salts to produce, in vitro, gene mutations, structural chromosome aberrations, sister-chromatid exchanges, to interfere with the chromosome distribution or to induce mammalian cell transformation demonstrates that the carcinogenicity of inorganic compounds is clearly associated with their genotoxicity. The induction of structural aberrations, SCEs and forward gene mutations represents the most sensitive and specific assay to assess the carcinogenic potential of metal salts.     

Aneuploidies, chromosome aberrations and dominant gene mutations detected in 113,913 consecutive newborn children in Mexico

Data on 113,913 liveborn children from a hospital in Guadalajara, Jalisco (Mexico), were analysed for birth defects (BD); mutation rates were calculated for sporadic aneuploidy, chromosome aberrations and dominant gene mutations. The results showed a general incidence of 13.92 BD cases per 1000 liveborns, of which 1.64% were chromosomal abnormalities, 1.50% were aneuploid, 0.14% were structural chromosome aberrations and 3.23% were dominant gene mutations. The mutation rates were 8.20 x 10(-4) chromosomal abnormalities, 7.5 x 10(-4) aneuploidies, 7.0 x 10(-5) chromosome aberrations and 1.61 x 10(-3) dominant gene mutations/gamete/generation, respectively. The lethality rate was 15.32% of the liveborns with BD. The described findings estimate the incidence of new human mutants detected at birth in a sample of the Mexican population. They show that the rate for some aneuploidies are similar to those found in other populations previously reported in the literature but the rates of chromosome and dominant gene mutations were different.     

High Rates of Occurrence of Spontaneous Chromosome Aberrations in DROSOPHILA MELANOGASTER

Spontaneous mutations were accumulated for 40 generations in 140 unrelated second chromosomes with the standard gene arrangement. These were extracted from the same population by using the marked inversion technique, and the following findings were obtained: (1) In 42 out of the 140 chromosome lines, chromosome aberrations were detected by examining the salivary gland chromosomes: 40 paracentric and 15 pericentric inversions, 2 reciprocal translocations between the second and the third chromosomes, and 6 transpositions. (2) In 63 out of the 90 originally lethal-free lines, recessive lethal mutations occurred. (3) There were only 3 lines that acquired chromosome aberrations (inversions) with no lethal effects in the homozygous condition. (4) In a comparison of these results with those of the (CH), (PQ), and (RT) chromosomes in which no chromosome aberrations occurred after accumulating mutations for 22058 chromosome.generations (Yamaguchi and Mukai 1974), it was concluded that some of these 140 chromosomes carried a kind of mutator. (5) The frequency of mutator-carrying chromosome lines was estimated to be 0.66 on the basis of the distribution of the break-points on the chromosome lines and the frequency of lines that acquired neither recessive lethal mutations nor chromosome aberrations. Thus, the average number of breaks per mutator-carrying chromosome was estimated to be about 0.19/generation.On the basis of these estimates, the nature of the mutator factor was discussed.     

The effect of 0-methylhydroxylamine on the chromosomes of Chinese hamster cells]

0-methylhydroxylamine increases the frequency of all kinds of aberrations in Chinese hamster cells, except chromatid exchange. The percentage of aberrant metaphases and the average number of breakages per cell increases by 5,5-6 times as compared with the control. OMHA proves to be very effective inducer of chromosome aberrations. Since OMHA induces, except gene mutations, also chromosome aberrations, there are reasons to suppose that its effect is connected not only with transitions, but also with some other molecular mechanisms.     

Mutagenesis on cloned yeast genes. The mutation of the yeast gene comprising the plasmid and chromosome

The cells of Saccharomyces cerevisiae were transformed by plasmid pYG-007 treated in vitro with o-methylhydroxylamine. The plasmid consists of a portion of the bacterial plasmid with genes of resistance to ampicillin, chloramphenicol and tetracycline, 2 mkm yeast DNA and yeast genes ADE2 and LEU2. The collection of mutants containing a mutant allele of ADE2 gene within the plasmid was obtained. Interallelic complementation and that induced by suppression were studied in these ade 2 mutants. It was shown that all these induced ade 2 mutations were base-pair substitutions. Using the mechanism of conversion we managed to transfer the plasmid ade 2 mutations into the chromosome. Three pairs of strains carrying similar mutation in plasmid and chromosome were created. Analysis of frequency of reversions induced by UV-light and hydroxylaminopurine in the mutant ade2 locus comprised in the plasmid and chromosome showed that the former induced reversions in plasmid alleles less effectively than the latter.     

Identification of the phoM gene product and its regulation in Escherichia coli K-12.

Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.     

Concurrent detection of gene mutations and chromosome aberrations induced by five chemicals in a CHL/IU cell line incorporating a gpt shuttle vector

We previously established a transgenic Chinese hamster CHL/IU cell line, designated as KN63, for concurrent analysis of gene mutations and chromosome aberrations. The KN63 cell line contains copies of a shuttle vector with the Escherichia coli gpt gene as a mutational target in its chromosome. To evaluate the sensitivity of the cell line to various types of mutagens, methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), mitomycin C (MMC), vincristine sulfate (VIN) and C.I. basic red 9 hydrochloride (CIB) were assayed. KN63 cells were treated with each test chemical and gene mutations were detected in the gpt gene of the shuttle vector rescued from the KN63 cell genome into an E. coli host. Chromosome aberrations were concurrently evaluated by conventional metaphase analysis. MMS, ENU and MMC induced both gene mutations and structural chromosome aberrations in KN63 cells, with more efficient induction of the latter. VIN, a well-known aneugen, produced only numerical changes to chromosomes, while CIB was negative for both types of alteration. KN63 cells were as sensitive to MMS, ENU, MMC and VIN as Chinese hamster cell lines such as CHL, CHO and V79 cells. The characteristics of test chemicals indicated by this system should be useful for understanding endpoints in chemical mutagenesis.     

Inducible expression and cytogenetic effects of the EcoRI restriction endonuclease in Chinese hamster ovary cells.

The cytogenetic endpoints sister chromatid exchange (SCE) and chromosome aberrations are widely used as indicators of DNA damage induced by mutagenic carcinogens. Chromosome aberrations appear to result directly from DNA double-strand breaks, but the lesion(s) giving rise to SCE formation remains unknown. Most compounds that induce SCEs induce a spectrum of lesions in DNA. To investigate the role of double-strand breakage in SCE formation, we constructed a plasmid that gives rise to one specific lesion, a staggered-end ("cohesive") DNA double-strand break. This plasmid, designated pMENs, contains a selectable marker, neo, which is a bacterial gene for neomycin resistance, and the coding sequence for the bacterial restriction endonuclease EcoRI attached to the mouse metallothionein gene promoter. EcoRI recognizes G decreases AATTC sequences in DNA and makes DNA double-strand breaks with four nucleotides overhanging as staggered ends. Cells transfected with pMENS were resistant to the antibiotic G418 and contained an integrated copy of the EcoRI gene, detectable by DNA filter hybridization. The addition of the heavy metal CdSO4 resulted in the intracellular production of EcoRI, as measured by an anti-EcoRI antibody. Cytogenetic analysis after the addition of CdSO4 indicated a dramatic increase in the frequency of chromosome aberrations but very little effect on SCE frequency. Although there was some intercellular heterogeneity, these results confirm that DNA double-strand breaks do result in chromosome aberrations but that these breaks are not sufficient to give rise to SCE formation.     

Risk assessment: the importance of genetic polymorphisms in man

Spontaneous mutation was greatly increased in a localized region of the chromosome of Haemophilus influenzae, but not at other loci, by a nov gene mutation called novC that increased DNA supercoiling. Another nov gene mutation, called novD, decreased spontaneous mutation in the same localized region and depressed DNA supercoiling. Both mutations, which code for the gyrase B subunit, have been cloned, and the cloned versions also altered mutagenesis and supercoiling in a similar fashion as the two mutations on the chromosome, although novC on the plasmid caused somewhat less mutation than on the chromosome. We postulate that the effects of the gyrase B mutations on the chromosome result from their effects on supercoiling because of increased gyrase near its site of production. The fact that the novC on a plasmid does not cause mutagenesis except in the same localized region that is altered by this mutation on the chromosome, is difficult to explain. One possibility is that there is a complex of proteins in this region which is necessary for the effects on supercoiling and thus, also on mutagenesis.     

Molecular cloning of a gene affecting the autolysin level and flagellation in Bacillus subtilis

A 2.8 kb PstI fragment of Bacillus subtilis 168W DNA has been cloned into Escherichia coli HB101 and B. subtilis AG5 using pAC3 as a shuttle plasmid. The new plasmid (pBRG1), of 10.2 kb, complemented flaD mutations which show reduced production of autolysin(s), filamentation and non-motility (deficiency of flagella). Deletion experiments showed that the suppressive gene is located between the HindIII and XbaI sites (1.0 kb apart) in pBRG1. The integration of a plasmid having chloramphenicol resistance closely linked to the flaD gene into the B. subtilis AC703 chromosome and its genetic analysis indicated that the cloned fragment contained the flaD gene itself. A high-copy-number plasmid carrying the cloned gene did not lead to an increase in autolysin production above the wild-type level, but it changed the colony morphology from smooth to rough. Among several autolysin-deficient mutations, lyt-151 was suppressed only by the high-copy-number plasmid carrying the cloned gene.     

Mutations affecting gyrase in Haemophilus influenzae.

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch.     

Transvection and silencing of the Scr homeotic gene of Drosophila melanogaster

The Sex combs reduced (Scr) gene specifies the identities of the labial and first thoracic segments in Drosophila melanogaster. In imaginal cells, some Scr mutations allow cis-regulatory elements on one chromosome to stimulate expression of the promoter on the homolog, a phenomenon that was named transvection by Ed Lewis in 1954. Transvection at the Scr gene is blocked by rearrangements that disrupt pairing, but is zeste independent. Silencing of the Scr gene in the second and third thoracic segments, which requires the Polycomb group proteins, is disrupted by most chromosomal aberrations within the Scr gene. Some chromosomal aberrations completely derepress Scr even in the presence of normal levels of all Polycomb group proteins. On the basis of the pattern of chromosomal aberrations that disrupt Scr gene silencing, we propose a model in which two cis-regulatory elements interact to stabilize silencing of any promoter or cis-regulatory element physically between them. This model also explains the anomalous behavior of the Scx allele of the flanking homeotic gene, Antennapedia. This allele, which is associated with an insertion near the Antennapedia P1 promoter, inactivates the Antennapedia P1 and P2 promoters in cis and derepresses the Scr promoters both in cis and on the homologous chromosome.     

Cytogenetic analysis of chromosome segments containing radiosensitivity genes in Drosophila. Radiation mutagenesis in the 44-45 region of chromosome 2 of Drosophila melanogaster

The first step of cytogenetic analysis of Drosophila melanogaster chromosome 2 44F-45D containing the radiosensitivity gene rad(2)201 is described. Using various mutation selection systems as well as lines of different origin and two kinds of ionizing radiation--gamma-rays and neutrons--the mutagenesis in the region of interest is characterized at the cytogenetic level. 85 gamma-induced mutations affecting viability were isolated in the 44F 2-4; 45C6-7 interval, 27% of mutations being chromosomal aberrations. 15 radiation-induced aberrations were obtained by selecting mutations at the white gene inserted into the 45D region by P-mediated transformation. The 44F-45D region is characterized by relatively low frequency of deficiency formation and by significant predomination of heterochromatic aberrations in the spectrum of rearrangements. In these regions, the existence of hot spots for heterochromatic aberrations was discovered. As low deletion frequency is not connected with the presence of haplolethal and haplosterile loci in the region studied, the unusual character of radiation mutagenesis reflects possibly the peculiarities in sequence organization of the chromosomal region mentioned or the packaging in the sperm nuclei.     

Mutations in the Escherichia coli RNA-polymerase beta-subunit gene cloned in a multicopy plasmid

A multicopy plasmid pLMN1 expressing a wild type rpoB gene encoding Escherichia coli RNA polymerase beta subunit gene was constructed. Introduction of this plasmid into rifampicin-resistant RpoB mutants makes them rifampicin-sensitive. Rifampicin-resistant clones appear in such strains with frequencies up to 10(-3), due to recombinational (recA-dependent) transfer of rif-r mutations from chromosome to pLMN1. This provides a simple selection procedure for transfer of any rpoB mutation, together with a rif-r mutation from a chromosome to pLMN1. In this way, we transferred rpoB22 amber mutation to pLMN1 for localization of the mutant codon by DNA sequencing.     

Characterization of the progeny of X-ray irradiated males from two Drosophila virilis strains differing in genetic instability.

Significant effects of X-ray treatment on the increase in the number of phenotypic variations, two visible mutations, and chromosome aberrations were found in the progeny of irradiated males from the D. virilis laboratory stock that is capable of hybrid dysgenesis syndrome induction. This effect is much more pronounced than in the progeny of irradiated males from strong wild-type strains studied. A correlation between genetic instability and chromosome radiosensitivity was outlined. The mechanism of this phenomenon and the possibilities of using the property of genome instability for the productive induction of gene and chromosome damage in radiation mutagenesis experiments are discussed.     

The genes coding for enterocin EJ97 production by Enterococcus faecalis EJ97 are located on a conjugative plasmid

Enterococcus faecalis EJ97 produces a cationic bacteriocin (enterocin EJ97) of low molecular mass (5,327.7 Da). The complete amino acid sequence of enterocin EJ97 was elucidated after automated microsequencing of oligopeptides generated by endoproteinase GluC digestion and cyanogen bromide treatment. Transfer of the 60-kb conjugative plasmid pEJ97 from the bacteriocinogenic strain E. faecalis EJ97 to E. faecalis OG1X conferred bacteriocin production and resistance on the recipient. The genetic determinants of enterocin EJ97 were located in an 11.3-kb EcoRI-BglII DNA fragment of pEJ97. This region was cloned and sequenced. It contains the ej97A structural gene plus three open reading frames (ORFs) (ej97B, ej97C, and ej97D) and three putative ORFs transcribed in the opposite direction (orfA, orfB, and orfC). The gene ej97A translated as a 44-amino-acid residue mature protein lacking a leader peptide with no homology to other bacteriocins described so far. The product of ej97B (Ej97B) shows strong homology in its C-terminal domain to the superfamily of bacterial ATP-binding cassette transporters. The products of ej97C (Ej97C) and ej97D (Ej97D) could be proteins with 71 and 64 residues, respectively, of unknown functions and with no significant similarity to known proteins. There are two additional ORFs (ORF1 and ORF6) flanking the ej97 module, which have been identified as a transposon-like structure (tnp). ORF1 shows similarities to transposase of the Lactococcus lactis element ISS1 and is up to 50% identical to IS1216. This is flanked by two 18-bp inverted repeats (IRs) that are almost identical to those of ISS1 and IS1216. ORF6 (resEJ97) shows strong homology to the resolvase of plasmid pAM373 and up to 40 to 50% homology with the recombinase of several multiresistant plasmids and transposons from Staphylococcus aureus and E. faecalis. These data suggest that EJ97 could represent a new class of bacteriocins with a novel secretion mechanism and that the whole structure could be a composite transposon. Furthermore, two additional gene clusters were found: one cluster is probably related to the region responsible for the replication of plasmid pEJ97, and the second cluster is related to the sex pheromone response. These regions showed a high homology to the corresponding regions of the conjugative plasmids pAM373, pPD1, and pAD1 of E. faecalis, suggesting that they have a common origin.     

Molecular cloning into Tn5 and integration in the Pseudomonas aeruginosa chromosome: a tool for heterologous gene expression

The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in Escherichia coli. The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to Pseudomonas aeruginosa strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the P. aeruginosa chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed P. aeruginosa strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from P. aeruginosa to P. stutzeri or to Acinetobacter calcoaceticus due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.     

High rates of "unselected" aneuploidy and chromosome rearrangements in tel1 mec1 haploid yeast strains

The yeast TEL1 and MEC1 genes (homologous to the mammalian ATM and ATR genes, respectively) serve partially redundant roles in the detection of DNA damage and in the regulation of telomere length. Haploid yeast tel1 mec1 strains were subcultured nonselectively for approximately 200 cell divisions. The subcultured strains had very high rates of chromosome aberrations: duplications, deletions, and translocations. The breakpoints of the rearranged chromosomes were within retrotransposons (Ty or delta-repeats), and these chromosome aberrations nonrandomly involved chromosome III. In addition, we showed that strains with the hypomorphic mec1-21 allele often became disomic for chromosome VIII. This property of the mec1-21 strains is suppressed by a plasmid containing the DNA2 gene (located on chromosome VIII) that encodes an essential nuclease/helicase involved in DNA replication and DNA repair.