Mutation induction in Haemophilus influenzae by ICR-191 II. Role of DNA replication and repair

Evidence is presented to show that presumptive frameshift mutations induced in Haemophilus influenzae by ICR-191 are fixed very rapidly, essentially at the time of treatment. DNA synthesis during treatment is essential for fixation, but DNA synthesis after treatment has no effect. The conclusion is drawn that the mutagen acts at the replication fork, possibly to stabilize misannealings arising in association with the discontinuities in the newly synthesized DNA. These results agree with earlier results on Escherichia coli showing that ICR-191 produces peak mutation frequencies in synchronized cultures at times when the replication fork has reached the locus being studied. They are in sharp contrast to the earlier results in H. influenzae with nitroso compounds and hydrazine that suggest these agents produce randomly distributed, reparable pre-mutational damage that still can be fixed (converted to final mutation) for some time after treatment when the replication fork reaches them. No evidence for such persistent pre-mutational lesions was found with ICR-191. A defect in incision appeared to have very little influence on mutation induction by ICR-191 though it caused much more lethality. The interpretation of the mutation data was made somewhat uncertain, however, by an unexplained plating-density effect on the expression of the mutants in this strain. In contrast, incision deficiencies in E. coli and Salmonella typhimurium have been reported to cause a large increase in mutation induction and to allow lesions at some distance from the replication fork to produce mutations.     

New shuttle vectors for Haemophilus influenzae and Escherichia coli: P15A-derived plasmids replicate in H. influenzae Rd

With the aim of identifying new plasmids useful for molecular cloning in Haemophilus influenzae, several P15A-derived plasmids were tested for their ability to transform H. influenzae Rd. The four plasmids tested, pACYC177, pACYC184, pSU2718, and pSU2719 were all able to establish in H. influenzae Rd. The plasmids were stable, could be purified by standard protocols, and were compatible with a plasmid carrying the RSF0885 origin of replication.     

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.     

Use of a rapid DNA sequencing system to demonstrate the induction of frameshift mutations by bleomycin

The rapid DNA sequencing system based on the single-stranded bacteriophage M13 and the chain-terminator method has been used to look directly for mutational alterations. A small DNA fragment that primes DNA synthesis through the N-terminal 200 base pairs of the beta-galactosidase gene was prepared, and used to detect changes in base sequence among phages that give white plaques after treatment of the host cells with bleomycin. Bleomycin treatment of E. coli in which M13 mp2 was growing gave an increase in white plaque frequency. DNA sequence analysis of phage from 7 independent mutant plaques showed them all to have a frameshift mutation.     

Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide

Haemophilus influenzae b conjugate vaccines (Hib) are almost entirely evaluated by physico-chemical methods to ensure the consistency of manufacture of batches. As different assays are employed for the quantification of Hib capsular polysaccharide PRP (polyribosyl ribitol phosphate; 5-D-ribitol-(1-->1)-beta-D-ribose-3-phosphate) in final formulations and bulk components, there was deemed a need for an International Standard of Hib PRP polysaccharide to be made available. Ten laboratories from 8 different countries participated in a collaborative study to determine the PRP content and assess the suitability of a candidate International Standard PRP preparation (02/208). The results illustrate that a reduction in between-laboratory variability could be achieved by use of a common reference preparation and data analysis showed no significant differences in the values obtained by the different assays: ribose, phosphorus, and high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), suggesting the suitability of the proposed reference for use across these assays for quantification of PRP content in Hib vaccines. On the basis of the results of this study, the First International Standard for PRP, NIBSC Code 02/208, has been established by the Expert Committee of Biological Standards of the World Health Organisation, with a content of 4.933+/-0.267mg/ampoule, as determined by the ribose assays carried out by 7 of the participating laboratories.     

Effect of N2 on the mutagenic and lethal activities of ICR-170 in Neurospora crassa

The nature of the N₂ effect for ICR-170, i.e., the greater mutagenic and lethal activities of this agent in the presence of N₂ than O₂, has been studied at the ad-3 region of Neurospora crassa. The characteristics of the N₂ effect for ICR-170 were that (1) the N₂ effect with ICR-170 was displayed in conidia when N₂ was administered during, but not before or after, ICR-170 treatment, (2) the highly increased mutagenic and lethal activities of ICR-170 in the presence of N₂ were due to an anoxic condition rather than to the presence of N₂ per se, (3) the high killing activity of ICR-170 under N₂ was due largely to increased cytoplasmic inactivation, (4) the N₂ effect was a general phenomenon at the ad-3 region of N. crassa, and (5) the highly ICR-170-induced mutation in conidia under N₂ was attributable to an actual enhancement in the mutagenic activity of ICR-170 rather than to selective killing. With regard to the mechanisms of the N₂ effect with ICR-170, results indicate that this effect (1) was not due to more extracellular oxidative degradation of ICR-170 molecules in the presence of O₂, or to a greater uptake of ICR-170 by conidia under N₂, but (2) was due to the inhibition of conidial respiration under an anoxic environment.     

Mutation induction by thymidine deprivation in Escherichia coli B/r. I. Influence of caffeine.

The addition of caffeine to the plating medium after thymine deprivation of E. coli WP2 uvr+ thyA or WP2 uvrA thyA had no influence on survival. Caffeine, however, reduced the frequency of mutants. The hypothesis is presented that the reduced mutagenesis is due to the sensitivity to caffeine of an inducible error-prone repair mechanism operating during thymine deprivation and after the re-addition of thymine.     

Mutation induction by different types of radiation at the Hprt locus

Mutation induction at the Hprt locus in Chinese hamster cells was studied after exposure to ultraviolet light, X-rays and alpha particles. While mutant frequency as a function of dose or fluence followed a linear–quadratic relationship with UV and X-rays, it showed a linear dependence for alpha particles. If mutant frequency is plotted vs. the logarithm of surviving fraction, a linear relationship is found in all cases although with different slopes. These are about equal with the two types of ionising radiations but about 10 times larger for UV. They can be used as a measure of mutagenic potential and are termed “mutagenicity”. It is shown that this parameter is correlated with the maximum of mutant yield, i.e., the number of mutants per cell at risk. It is concluded from this analysis that the maximum mutant yield is always found at doses or fluences which lead to 37% survival irrespective of the kind of radiation. If mutation induction is measured in X-irradiated cells after pre-exposure to UV, mutant frequency is higher than expected on the basis of independent action of the two radiations. Deletion spectra were determined by using multiplex polymerase chain reaction. It was found that the background of spontaneous mutants varied considerably and showed frequently repetitive patterns, presumably because of clonal expansion of pre-formed mutants. UV-induced mutants did not contain any deletions, while those with both X-rays and alpha particles the majority displayed partial and total deletions. Based on a total number of 134 X-ray- and 192 alpha-induced mutants, it is concluded that the total fraction of mutant clones without deletions (partial or total) is about 40% for X-rays and only about 20% for alpha-particles.     

Mutation induction by thymine deprivation in Escherichia coli B/R : II. Mutation in the repressed and derpressed tryptophan operon

True Trp⁺ reversions are induced by thymine deprivation in cells with repressed trp operons as efficiently as in derepressed cells. At least part of the mutations are fixed during thymine starvation, i.e. in the absence of net DNA synthesis. The hypothesis is put forward that thymineless mutagenesis is due to repair-replication under limited concentrations of 5′-dTTP, performed by an inducible error-prone “DNA-polymerizing activity” on single-strand gaps.     

Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. IV. Comparison of dose-response curves for MNNG, 4NQO and ICR-170 induced inactivation and mutation-induction

The genetic effects of MNNG, 4NQO and ICR-170 have been compared on 5 different UV-sensitive strains and a standard wild-type strain of Neurospora crassa with regard to inactivation and the induction of forward-mutations at the ad-3A and ad-3B loci. Whereas all UV-sensitive strains (upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) are more sensitive to inactivation by MNNG and ICR-170 than wild-type, only uvs-5 shows survival comparable to wild-type after 4NQO treatment, all other strains are more sensitive to 4NQO. In contrast to the effects on inactivation, a wide variety of effects were found for the induction of ad-3A and ad-3B mutations: higher forward-mutation frequencies than were found in wild-type were obtained after treatment with MNNG or 4NQO for upr-1 and uvs-2, no significant increase over the spontaneous mutation frequency was found with uvs-3 after MNNG, 4NQO or ICR-170 treatment; mutation frequencies comparable to that found in wild-type were obtained with uvs-6 after MNNG, 4NQO or ICR-170 treatment and with upr-1 after ICR-170 treatment. Lower forward-mutation frequencies than were found in wild-type were obtained with uvs-2 after ICR-170 treatment and with uvs-5 after MNNG, 4NQO or ICR-170 treatment. These data clearly show that the process of forward-mutation at the ad-3A and ad-3B loci is under genetic control by mutations at other loci (e.g. upr-1, uvs-2, uvs-3, uvs-5 and uvs-6) and that the effect is markedly mutagen-dependent.     

The early detection of frameshift mutations induced by a food-borne carcinogen in rats: a new tool for molecular epidemiology

The accumulation of genetic changes is considered as the main factor that determines the development of cancer. Recent progresses in genetics and molecular biology led to the discovery of many new molecular markers and to the development of techniques able to monitor these markers. As a consequence, molecular epidemiology has emerged as a powerful approach to study the ternary relationship between the environment, the behaviour and the genetic predisposition of each individual. Susceptibility to cancer is determined at different levels such as the genetic polymorphism of enzymes involved in the activation and detoxification of carcinogens, the polymorphism of genes that maintains the genome stability, like those involved in DNA repair or recombination processes, and finally the polymorphism in oncogenes or tumour suppressor genes. Consequently, the full assessment of each individual's genetic predisposition is a long and difficult task. As the accumulation of mutations in somatic cells integrates all these parameters, its measurement would facilitate the evaluation of the individual predisposition status, provided that a marker common to a large spectrum of carcinogens could be found. Our current studies on the molecular mechanisms of carcinogen-induced mutagenesis has revealed that G-rich repetitive sequences are mutational hot spots for several major classes of environmental genotoxins such as aromatic and heterocyclic amines, polycyclic hydrocarbons and oxidative agents. We thus consider the possibility that these sequences form a new class of biomarkers for carcinogen exposure. In order to validate this hypothesis, we designed a sensitive PCR-based assay able to detect specific mutations induced by a common food-borne carcinogen in the colon epithelium of rats exposed for a short period to this carcinogen. This assay is sensitive enough to allow early detection of induced mutations and therefore allows to differentiate between unexposed animal and those exposed for a period as short as 1 week.     

Induction of G.C to A.T transitions by the acridine half-mustard ICR-191 supports a mispairing mechanism for mutagenesis by some bulky mutagens

As the most nucleophilic atom in DNA, the guanine N7 atom is a major site of attack for a large number of chemical mutagens as well as chemotherapeutic agents. Paradoxically, while methylation of guanine N7 is believed to be largely nonmutagenic, aflatoxin B1, among the most potent mutagens, appears to exert its mutagenic activity through adduction at this site. On the basis of an analysis of the specificity of mutations induced by various adduct forms of aflatoxin B1, we have previously proposed mechanisms that can both resolve the paradox and account for the specificity of mutagenesis by aflatoxin B1. The hypothesized mechanisms specify how a bulky guanine N7 lesion can promote G.C to A.T transitions as well as frame-shift mutations. Since the proposed mechanisms are in principle lesion-independent, a simple test of the proposed mechanisms would be to examine the specificity of mutations induced by a structurally different bulky guanine N7 adduct. Toward this goal, M13 replicative form DNA was subjected to in vitro adduction with the acridine mutagen ICR-191 and transfected into Escherichia coli. Mutations in the LacZ(alpha) gene segments were scored and defined at the sequence level. The results show that ICR-191 adduction induces both base substitutions and frame shifts with near-equal efficiency. A clear majority of base substitutions were G.C to A.T transitions. On the other hand, unlike aflatoxin B1 which could induce both -1 and +1 frameshifts, ICR-191 appears to predominantly induce +1 frame shifts. This preference appears to arise by lesion-dependent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of a post-translational steroid induction system for GIGANTEA in Arabidopsis

Background  

To investigate the link between the flowering time gene GIGANTEA (GI) and downstream genes, an inducible GI system was developed in Arabidopsis thaliana L. Heynh. Transgenic Arabidopsis plant lines were generated with a steroid-inducible post-translational control system for GI. The gene expression construct consisted of the coding region of the GI protein fused to that of the ligand binding domain of the rat glucocorticoid receptor (GR). This fusion gene was expressed from the constitutive cauliflower mosaic virus 35S promoter and was introduced into plants carrying the gi-2 mutation. Application of the steroid dexamethasone (DEX) was expected to result in activation of the GI-GR protein and its relocation from the cytoplasm to the nucleus.     

recO and recR mutations delay induction of the SOS response in Escherichia coli

RecF, RecO and RecR, three of the important proteins of the RecF pathway of recombination, are also needed for repair of DNA damage due to UV irradiation. recF mutants are not proficient in cleaving LexA repressor in vivo following DNA damage; therefore they show a delay of induction of the SOS response. In this communication, by measuring the in vivo levels of LexA repressor using anti-LexA antibodies, we show that recO and recR mutant strains are also not proficient in LexA cleavage reactions. In addition, we show that recO and recR mutations delay induction of -galactosidase activity expressed from a lexA-regulated promoter following exposure of cells to UV, thus further supporting the idea that recF, recO and recR gene products are needed for induction of the SOS response.     

Mutagenesis by simian virus 40. I. detection of mutations in Chinese hamster cell lines using different resistance markers.

The mutagenic action of SV40 in permanent lines of Chinese hamster cells (CHO-K1 and V79) was investigated with the aid of different resistance markers. The markers studied had resistance to 8-azaguanine (25 and 30 mug/ml), aminopterin (3.3--5.5X10(-3) mug/ml), colchicine (6.5 and 7.0X10(-2) mug/ml) and 5-bromodeoxyuridine (50--120 mug/ml), respectively. After virus infection the mutation frequencies were increased by one (azaguanine, aminopterin) and two (colchicine) orders of magnitude as compared with spontaneous mutation frequencies. In contrast, it was not possible to enhance the frequency of mutation to BUdR resistance. On the other hand, the ability to proliferate in HAT medium was induced in three of five BUdR-resistant cell clones by infection with SV40. The resistance induced by SV40 was stable when isolated clones were cultured under non-selective conditions. Mechanisms are proposed that may be responsible for the mutagenic action of SV40.     

Y chromosome-specific mutations induced by a giant transposon in Drosophila hydei

Summary The w ^m Co duplication of Drosophila hydei (Dp (1; Y) 16B2-17B1) contains 13–16 bands in salivary gland chromosomes. The duplication resides preferentially in the X heterochromatin or on the Y chromosome. In some stocks frequent (up to 4×10^-3) exchanges of the duplication occur between different Y chromosomes (T(X; Y) and free Y) or between the X and the Y chromosome. About 60% of the T(X; Y)-Y exchanges induce mutations in the Y chromosomal male fertility genes of the recipient Y chromosome. From the mutational spectrum generated by the T(X; Y)-Y transpositions and from the variable efficiency as acceptor of different X-Y translocations it can be concluded that the exchanges show a remarkable site specificity: distal positions in the long arm of the Y chromosome are occupied preferentially. More proximal positions in the long arm of insertions into the short arm of the Y chromosome are found only with a lower frequency. No transpositions to the autosomes have been recovered. Duplications are lost with highly differing frequencies. The losses are not linked with insertions of the w ^m Co element into a new position and are more frequent than transpositions. Therefore, we regard the w ^m Co element as a giant transposon.     

Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) 1. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P₇₀₀ upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded Hinf I fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.