Characterization of mutations induced by ethyl methanesulfonate, UV, and trimethylpsoralen in the nematode Caenorhabditis elegans

The genome project of the nematode Caenorhabditis elegans is completed. It is important and useful to disrupt nematode genes to know their function. We treated wild-type animals with potential candidates for mutagens for reverse genetics, EMS (ethyl methanesulfonate), short-wavelength UV, and long-wavelength UV in the presence of TMP (trimethylpsoralen). We estimated forward mutation rates by counting the occurrence of a marker unc-22 mutation. We found that the forward mutation rate by TMP/UV could be comparable with EMS by improving the frequency one order higher than before. We next isolated mutants of another marker gene ben-1 and examined the probability for the deletion mutations by PCR and sequencing. Deletion mutations were found only by TMP/UV method, which suggested TMP/UV is the choice for deletion mutagenesis among these methods. As a pilot experiment, we could isolate actual deletion mutations at a much higher frequency than previously.     

Analysis of mutational effects at the HPRT locus in human G(0) phase lymphocytes irradiated in vitro with gamma rays

The mutational effects of ionising radiation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus were studied in human peripheral blood G(0) phase lymphocytes irradiated in vitro with gamma rays. The presence of radiation induced mutants was assessed by selecting the HPRT mutants every week on the basis of 6-thioguanine resistance up to 1 month after irradiation. A dose-related increase of 14.25x10(-6) mutants/Gy was measured after an expression time of 7 days. After 2 weeks from culture starting the fraction of clonable cells in irradiated and control cell populations decreased, limiting the measurements of mutant frequency. The mutational spectrum of the HPRT gene was determined by PCR analyses in a total of 99 mutant clones derived from irradiated lymphocytes. The independent origin of mutant clones carrying the same mutation was assessed by analysing the TCR gamma gene rearrangements. The results showed a dose-related increase of deletion mutants up to 3Gy, whereas point mutation frequency increased only up to 2Gy. Two preferentially deleted regions were identified; one involving the HPRT exon 3, and another one the 3'-terminal and the 3'-flanking region of the gene. One complex mutation involving a non-contiguous deletion of exons 2-5 and 7/8 was observed among the mutants isolated after 3Gy irradiation.     

Mutation detection in rice waxy mutants by PCR-RF-SSCP

PCR-RF-SSCP (PRS), which combines cleaved amplified polymorphic sequence (CAPS) and single-strand conformation polymorphism (SSCP), is expected to be a useful technique for DNA polymorphism analysis. We evaluated the ability of PRS to detect single nucleotide polymorphism (SNP) using the Waxy gene, Wx, of rice, and subsequently were able to identify point mutations in wx mutant lines. The approximately 6-kb Wx gene was divided into five regions for PCR amplification. Two regions, in which most of the point mutations of the wx mutants have been identified, were amplified by PCR and cloned into a vector, and those clones containing SNPs produced as a result of the inherent inaccuracy of PCR were used for the evaluation of PRS. The efficiency of PRS in the detection of SNPs of these clones was over 70%. PRS analysis of the wx genes in 18 waxy mutants was carried out in the five regions using two different restriction endonucleases and two gel conditions, with and without glycerol. Of the 18 lines tested, 17 showed band patterns different from that of the wild type. Most of the mutations identified in this study were nucleotide changes in exons, which result in amino acid changes. One mutation generated an in-frame stop codon, and another was a frame shift mutation by one-base deletion. Two mutations found at a splice site were considered to inhibit normal splicing of mRNA. These results show that PRS is a useful technique for detecting point mutations in large plant genes.     

Molecular characterisation of camptothecin-induced mutations at the hprt locus in Chinese hamster cells

The capacity of the topoisomerase I inhibitor camptothecin (CPT) to induce single locus mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and the DNA changes underlying induced mutations were analysed in Chinese hamster ovary cells. Camptothecin treatments increased hprt mutations up to 50-fold over the spontaneous levels at highly cytotoxic doses. Genomic DNA was isolated from 6-thioguanine resistant clones and subjected to multiplex PCR to screen for gross alterations in the gene structure. The molecular analysis revealed that deletion mutants represented 80% of the analysed clones, including total hprt deletion, multiple and single exon deletions. Furthermore, a fraction of the analysed clones showed deletions of more than one exon that were characterised by the absence of non-contiguous exons. These data show that single locus mutations induced by camptothecin are characterised by large deletions or complex rearrangements rather than single base substitutions and suggest that the recombinational repair of camptothecin-induced strand breaks at replication fork may be involved in the generations of these alterations at the chromatin structure level.     

Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli

We developed an efficient method for isolation of novel dnaA mutations based on PCR mutagenesis in the presence of manganese ion and shuffling of dnaA-carrying plasmids in a dnaA deletion host bacterium. Using this system, we obtained 30 cold-sensitive mutants from 4000 clones carrying plasmids with a mutagenized dnaA gene. All 27 cold-sensitive mutants analyzed were defective in DNA replication; none had a DnaAcos (over-initiation) phenotype. Nucleotide sequencing revealed that novel 15 alleles (mutations in 14 amino acid residues) are responsible for the cold-sensitive phenotype and are all located in the carboxy-terminal half of the DnaA protein.     

ndvF, a novel locus located on megaplasmid pRmeSU47b (pEXO) of Rhizobium meliloti, is required for normal nodule development.

Rhizobium meliloti strains carrying either of two overlapping deletions (delta 5408 and delta F114) of the megaplasmid pRmeSU47b form nodules on alfalfa which fail to fix N2 (Fix-). Strains carrying these deletions also fail to fluoresce on media containing calcofluor, indicating a defect in synthesis of the acidic exopolysaccharide (Exo-) of R. meliloti. We have isolated cosmid clones (pTH21 and pTH22) which complement the Fix- but not the Exo- phenotype of the strains carrying the delta 5408 and delta F114 deletions. In addition, cosmid clones which complement the Exo- phenotype fail to complement the Fix- phenotype of these deletions; thus, the Exo- phenotype is not related to the Fix- phenotype. A 5-kb region within a 7.3-kb BamHI restriction fragment was found to be required for complementation of the Fix- phenotype of the delta 5408 and delta F114 deletion strains. Tn5 insertions in the 5-kb region generated a Fix- phenotype when recombined into the wild-type genome. We have designated this locus ndvF, for nodule development. TnphoA mutagenesis of this region generated active alkaline-phosphatase gene fusions, indicating that ndvF encodes extracytoplasmic protein(s). Induction of nodules by the ndvF mutants was delayed by 2 to 3 days compared with induction by the wild-type strain. Light microscopy of nodules elicited by strains carrying the large 150-kb delta F114 deletion, a 12-kb deletion removing ndvF, or an individual ndvF::Tn5 insertion mutation demonstrated that many nodules contained few infected cortical cells, indicating that nodule development was blocked early in the infection process, before the release of bacteria from the infection threads.     

Significance of Asn-77 and Trp-78 in the catalytic function of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26

The primary structures of cis-prenyltransferases are completely different from those of trans-prenyltransferases. To obtain information about amino acid residues relating to catalytic function, random mutation of the undecaprenyl diphosphate synthase gene of Micrococcus luteus B-P 26 was carried out to construct a mutated gene library using an error-prone polymerase chain reaction. From the library, the mutants showing poor enzymatic activity were selected by the colony autoradiography method. Among 31 negative clones selected from 3,000 mutants, two clones were found to contain only one amino acid substitution at either Asn-77 or Trp-78. To determine the functional roles of these interesting residues, we prepared six mutated enzymes with substitutions at residues Asn-77 or Trp-78 by site-directed mutagenesis. Substitution of Asn-77 with Ala, Asp, or Gln resulted in a dramatic decrease in catalytic activity, but the K(m) values for both allylic and homoallylic substrates of these mutant enzymes were comparable to those of the wild-type. On the other hand, three Trp-78 mutants, W78I, W78R, and W78D, showed 5-20-fold increased K(m) values for farnesyl diphosphate but not for Z-geranylgeranyl diphosphate. However, these mutants showed moderate levels of enzymatic activity and comparable K(m) values for isopentenyl diphosphate to that of the wild-type. These results suggest that the Asn-Trp motif is involved in the binding of farnesyl diphosphate and enzymatic catalysis.     

Overproduction of lipase by<Emphasis Type="Italic"> Yarrowia lipolytica</Emphasis> mutants

Non-genetically modified mutants with increased capacities of extracellular lipase production were obtained from Yarrowia lipolytica strain CBS6303 by chemical mutagenesis. Of the 400 mutants isolated, LgX64.81 had the highest potential for the development of an industrial lipase production process. This mutant exhibits lipase production uncoupled from catabolite repression by glucose, and a 10-fold increased productivity upon addition of oleic acid. Using a LIP2- LacZ reporter gene, we demonstrate that the mutant phenotype originates from a trans-acting mutation. The glucose uptake capacity of LgX64.81 is reduced 2.5-fold compared to the wild-type-strain, and it exhibits high lipase production on glucose medium. A trans-acting mutation in a gene involved in glucose transport could thus explain this mutant phenotype.     

Characterization of Escherichia coli type 1 pilus mutants with altered binding specificities

PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.     

Tryptophan-216 is essential for the transglycosylation activity of endo-beta-N-acetylglucosaminidase A

Endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has a high level of transglycosylation activity. To determine which amino acids are involved in this activity, we employed deletion analysis, as well as random and site-directed mutagenesis. Using PCR random mutagenesis, 11 mutants with greatly decreased levels of enzyme activity were isolated. Six catalytically essential amino acids were identified by site-directed mutagenesis. Mutants E173G, E175Q, D206G, and D270N had markedly reduced hydrolysis activity, while mutants V109D, E173D, and E173Q lost all enzymatic activity, indicating that Val-109 and Glu-173 are important for the catalytic function. Moreover, we isolated a random mutation that abolished the transglycosylation activity without affecting the hydrolysis activity. The Trp-216 to Arg mutation was identified, by site-directed mutagenesis, as that responsible for the loss of transglycosylation activity. While other mutants of Trp-216 showed reduced activity, mutation to another positively charged residue (Lys) also abolished the transglycosylation activity. Sequence comparison with two other endo-beta-N-acetylglucosaminidases, that possess transglycosylation activity and that have been cloned recently, reveals a high degree of identity in the N-terminal regions of the three enzymes. These results indicate that the tryptophan residue at position 216 of Endo-A has a key role in the transglycosylation.     

Elevated incidence of loss of heterozygosity (LOH) in an sgs1 mutant of Saccharomyces cerevisiae: roles of yeast RecQ helicase in suppression of aneuploidy,interchromosomal rearrangement,and the simultaneous incidence of both events during mitotic growth

The SGS1 gene of Saccharomyces cerevisiae is a member of the RecQ helicase family, which includes the human BLM, WRN and RECQL4 genes responsible for Bloom and Werner's syndrome and Rothmund-Thomson syndrome, respectively. Cells defective in any of these genes exhibit a higher incidence of genome instability. We previously demonstrated that various genetic alterations were detectable as events leading to loss of heterozygosity (LOH) in S. cerevisiae diploid cells, utilizing a hemizygous URA3 marker placed at the center of the right arm of chromosome III. Analyses of chromosome structure in LOH clones by pulse field gel electrophoresis (PFGE) and PCR, coupled with a genetic method, allow identification of genetic alterations leading to the LOH. Such alterations include chromosome loss, chromosomal rearrangements at various locations and intragenic mutation. In this work, we have investigated the LOH events occurring in cells lacking the SGS1 gene. The frequencies of all types of LOH events, excluding intragenic mutation, were increased in sgs1 null mutants as compared to the wild-type cells. Loss of chromosome III and chromosomal rearrangements were increased 13- and 17-fold, respectively. Further classification of the chromosomal rearrangements confirmed that two kinds of events were especially increased in the sgs1 mutants: (1) ectopic recombination between chromosomes, that is, unequal crossing over and translocation (46-fold); and (2) allelic crossing over associated with chromosome loss (40-fold). These findings raise the possibility that the Sgs1 protein is involved in the processing of recombination intermediates as well as in the prevention of recombination repair during chromosome DNA replication. On the other hand, intrachromosomal deletions between MAT and HMR were increased only slightly (2.9-fold) in the sgs1 mutants. These results clearly indicate that defects in the SGS1 gene function lead to an elevated incidence of LOH in multiple ways, including chromosome loss and interchromosomal rearrangements, but not intrachromosomal deletion.     

Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells

AS52 cells are Chinese hamster ovary (CHO) cells that carry a single functional copy of the bacterial gpt gene and allow the isolation of 6-thioguanine-resistant (6TGr)mutants arising from mutation at the chromosally integrated gpt locus. The gpt locus in AS52 cells is extremely stable, giving rise to 6TGr mutants at frequencies comparable to the endogenous CHO hprt locus. In this study, we describe the spectrum of spontaneous mutations observed in AS52 cells by Southern blot and DNA sequence analyses. Using the polymerase chain reaction (PCR) and the Thermus aquaticus (Taq) polymerase, we have enzymatically amplified 6TGr mutant gpt sequences in vitro. The PCR product was then sequenced without further cloning manipulations to directly identify gpt structural gene mutations. Deletions predominant among the 62 spontaneous 6TGr-AS52 mutant clones analyzed in this study. Of these, 79% (49/62) of the mutations were identified as deletions either by Southern blotting, PCR amplification or DNA sequence analysis. Among these deletions is a predominant 3-base deletion that was observed in 31% (19/62) of the mutants. These data provide a basis for future comparisons of induced point mutational spectra derived in the AS52 cell line, and demonstrate the utility of PCR in the generation of DNA sequence spectra derived from chromosomally integrated mammalian loci.     

Cellular and molecular effects for mutation induction in normal human cells irradiated with accelerated neon ions

We investigated the linear energy transfer (LET) dependence of mutation induction on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in normal human fibroblast-like cells irradiated with accelerated neon-ion beams. The cells were irradiated with neon-ion beams at various LETs ranging from 63 to 335 keV/microm. Neon-ion beams were accelerated by the Riken Ring Cyclotron at the Institute of Physical and Chemical Research in Japan. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of mutants was analyzed using the multiplex polymerase chain reaction (PCR). The dose-response curves increased steeply up to 0.5 Gy and leveled off or decreased between 0.5 and 1.0 Gy, compared to the response to (137)Cs gamma-rays. The mutation frequency increased up to 105 keV/microm and then there was a downward trend with increasing LET values. The deletion pattern of exons was non-specific. About 75-100% of the mutants produced using LETs ranging from 63 to 335 keV/mum showed all or partial deletions of exons, while among gamma-ray-induced mutants 30% showed no deletions, 30% partial deletions and 40% complete deletions. These results suggested that the dose-response curves of neon-ion-induced mutations were dependent upon LET values, but the deletion pattern of DNA was not.     

Site-directed deletion mutagenesis using phagemid vectors and genetic selection

Oligonucleotide-directed mutagenesis was used along with the dut and ung genetic selection method of Kunkel to introduce large site-specific deletions into cDNAs cloned into phagemid vectors. We find that large deletions can be achieved with an efficiency equal to that of single point mutations, with a very low frequency of aberrent clones. To facilitate screening of clones, E. coli strain DH5 alpha was used as the recipient host cell to genetically select for deletion mutants. Comparisons were made to deletion mutagenesis without genetic selection, and to reactions utilizing two oligonucleotide primers simultaneously. The low frequency of deletion mutants observed without genetic selection renders random screening for deletion mutant clones cumbersome. The results provide representative expectations and a useful guide for those contemplating the construction of deletion mutants.     

铜绿假单胞菌对碳青霉烯类抗生素耐药相关基因的筛选

为了在基因组水平筛选获得铜绿假单胞菌PAO1对碳青霉烯类抗生素耐药相关基因,本研究通过构建PAO1转座突变体文库、筛选对亚胺培南、美罗培南和比亚培南耐药及敏感突变株;通过随机PCR、核苷酸测序及序列比对的手段,确定了突变体中转座子的插入位点及其破坏的基因,获得了48个PAO1中对碳青霉烯类抗生素耐药和敏感相关的基因,其中27个基因突变后表现为耐药性增强,21个基因突变后表现为药物敏感性增强.本实验筛选获得的48个基因中有5个与Alvarez-Ortega和Dotsch等人筛选的基因重复.通过对PA0011,PA0667及PA3901进行基因敲除及遗传互补,进一步确证这3个基因均与PAO1对碳青霉烯类的耐药性相关.本次筛选发现了38个新的与碳青霉烯类耐药相关的基因,其中包括13个class4类基因.对这些新基因的进一步研究,不仅有利于全面了解铜绿假单胞菌的耐药机制及其调控网络,而且有利于药物作用靶点的发现,为有效治疗铜绿假单胞菌的感染提供新思路.     

Random insertion and deletion of arbitrary number of bases for codon-based random mutation of DNAs.

A general method was developed for the construction of a library of mutant genes. The method, termed random insertion/deletion (RID) mutagenesis, enables deletion of an arbitrary number of consecutive bases at random positions and, at the same time, insertion of a specific sequence or random sequences of an arbitrary number into the same position. The applicability of the RID mutagenesis was demonstrated by replacing three randomly selected consecutive bases by the BglII recognition sequence (AGATCT) in the GFPUV gene. In addition, the randomly selected three bases were replaced by a mixture of 20 codons. These mutants were expressed in Escherichia coli, and those that showed fluorescence properties different from the wild-type GFP were selected. A yellow fluorescent protein and an enhanced green fluorescent protein, neither of which could be obtained by error-prone PCR mutagenesis, were found among the six mutants selected. Several mutants of the DsRed protein that show different fluorescence properties were also obtained.     

Mouse splice mutant generation from ENU-treated ES cells--a gene-driven approach

Mutant mice are important for elucidating mammalian gene functions and for modeling human disease phenotypes. In recent years, chemical mutagenesis has become an increasingly popular method to disrupt gene functions due to its high efficiency of inducing mutations throughout the genome. Mutagenesis of embryonic stem (ES) cells offers the possibility of gene-driven approaches, which, however, require efficient mutation detection procedures to screen archives of mutated samples for lesions in particular genes. We have developed an approach that focuses on the detection of splice mutations in highly pooled cDNA samples using exon-skipping PCR primers. As a proof of concept, splice mutants for the Kit gene were isolated from a library comprising approximately 40,000 ES cell clones treated with N-ethyl-N-nitrosourea followed by transmission through the mouse germ-line. The approach will be useful for the production of mouse models for human disease-related splice mutations and as a general gene disruption strategy.     

Pathways of Genetic Adaptation: Multistep Origin of Mutants Under Selection Without Induced Mutagenesis in Salmonella enterica

In several bacterial systems, mutant cell populations plated on growth-restricting medium give rise to revertant colonies that accumulate over several days. One model suggests that nongrowing parent cells mutagenize their own genome and thereby create beneficial mutations (stress-induced mutagenesis). By this model, the first-order induction of new mutations in a nongrowing parent cell population leads to the delayed accumulation of visible colonies. In an alternative model (selection only), selective conditions allow preexisting small-effect mutants to initiate clones that grow and give rise to faster-growing mutants. By the selection-only model, the delay in appearance of revertant colonies reflects (1) the time required for initial clones to reach a size sufficient to allow the second mutation plus (2) the time required for growth of the improved subclone. We previously characterized a system in which revertant colonies accumulate slowly and contain cells with two mutations, one formed before plating and one after. This left open the question of whether mutation rates increase under selection. Here we measure the unselected formation rate and the growth contribution of each mutant type. When these parameters are used in a graphic model of revertant colony development, they demonstrate that no increase in mutation rate is required to explain the number and delayed appearance of two of the revertant types.