Evidence that selected amplification of a bacterial lac frameshift allele stimulates Lac(+) reversion (adaptive mutation) with or without general hypermutability

In the genetic system of Cairns and Foster, a nongrowing population of an E. coli lac frameshift mutant appears to specifically accumulate Lac(+) revertants when starved on medium including lactose (adaptive mutation). This behavior has been attributed to stress-induced general mutagenesis in a subpopulation of starved cells (the hypermutable state model). We have suggested that, on the contrary, stress has no direct effect on mutability but favors only growth of cells that amplify their leaky mutant lac region (the amplification mutagenesis model). Selection enhances reversion primarily by increasing the mutant lac copy number within each developing clone on the selection plate. The observed general mutagenesis is attributed to a side effect of growth with an amplification-induction of SOS by DNA fragments released from a tandem array of lac copies. Here we show that the S. enterica version of the Cairns system shows SOS-dependent general mutagenesis and behaves in every way like the original E. coli system. In both systems, lac revertants are mutagenized during selection. Eliminating the 35-fold increase in mutation rate reduces revertant number only 2- to 4-fold. This discrepancy is due to continued growth of amplification cells until some clones manage to revert without mutagenesis solely by increasing their lac copy number. Reversion in the absence of mutagenesis is still dependent on RecA function, as expected if it depends on lac amplification (a recombination-dependent process). These observations support the amplification mutagenesis model.     

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.     

Histidine 454 plays an important role in polymerization of human glutamate dehydrogenase

Although previous chemical modification studies have suggested several residues to be involved in the maintenance of the quaternary structure of glutamate dehydrogenase (GDH), there are conflicting views for the polymerization process and no clear evidence has been reported yet. In the present study, cassette mutagenesis at seven putative positions (Lys333, Lys337, Lys344, Lys346, Ser445, Gly446, and His454) was performed using a synthetic human GDH gene to examine the polymerization process. Of the mutations at the seven different sites, only the mutagenesis at His454 results in depolymerization of the hexameric GDH into active trimers as determined by HPLC gel filtration analysis and native gradient polyacrylamide gel electrophoresis. The mutagenesis at His454 has no effects on expression or stability of the protein. The K_M values for NADH and 2-oxoglutarate were 1.5-fold and 2.5-fold greater, respectively, for the mutant GDH than for wild-type GDH, indicating that substitution at position 454 had appreciable effects on the affinity of the enzyme for both NADH and 2-oxoglutarate. The V_max values were similar for wild-type and mutant GDH. The k_cat/K_M value of the mutant GDH was reduced up to 2.8-fold. The decreased efficiency of the mutant, therefore, results from the increase in K_M values for NADH and 2-oxoglutarate. The results with cassette mutagenesis and HPLC gel filtration analysis suggest that His454 is involved in the polymerization process of human GDH.     

Increased expression of the Escherichia coli umuDC operon restores SOS mutagenesis in lexA41 cells

Summary The lexA41 allele of Escherichia coli encodes a semidefective mutant repressor that is also resistant to RecA facilitated cleavage. Cells harboring the lexA41 allele were found previously to repress only a subset of operons in the SOS regulon. lexA41 cells cannot promote SOS mutagenesis, presumably because one or more operons required for mutagenesis are repressed by this mutant repressor. Using the lac regulatory system to increase the expression of the umuDC operon, we were able to restore mutagenesis in the lexA41 mutant. We conclude that the products of the umuDC operon appear to be uniquely limiting in this mutant.     

An Efficient Rice Mutagenesis System Based on Suspension-Cultured Cells

Plant mutants are important bio-resources for crop breeding and gene functional studies. Conventional methods for generating mutant libraries by mutagenesis of seeds with physical or chemical agents are of low efficiency. Here, we developed a highly-efficient ethyl methanesulfonate (EMS) mutagenesis system based on suspension-cultured cells, with rice (Oryza sativa L.) as an example. We show that treatment of suspension-cultured tiny cell clusters with 0.4% EMS for 18-22h followed by differentiation and regeneration produced as high as 29.4% independent mutant lines with visible phenotypic variations, including a number of important agronomic traits such as grain size, panicle size, grain or panicle shape, tiller number and angle, heading date, male sterility, and disease sensitivity. No mosaic mutant was observed in the mutant lines tested. In this mutant library, we obtained a mutant with an abnormally elongated uppermost internode. Sequencing and functional analysis revealed that this is a new allelic mutant of eui (elongated uppermost internode) caused by two point mutations in the first exon of the EUI gene, representing a successful example of this mutagenesis system.     

piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice

Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.     

Characterization of Sleeping Beauty transposition and its application to genetic screening in mice

The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.     

Effect of growth under selection on appearance of chromosomal mutations in Salmonella enterica

Populations adapt physiologically using regulatory mechanisms and genetically by means of mutations that improve growth. During growth under selection, genetic adaptation can be rapid. In several genetic systems, the speed of adaptation has been attributed to cellular mechanisms that increase mutation rates in response to growth limitation. An alternative possibility is that growth limitation serves only as a selective agent but acts on small-effect mutations that are common under all growth conditions. The genetic systems that initially suggested stress-induced mutagenesis have been analyzed without regard for multistep adaptation and some include features that make such analysis difficult. To test the selection-only model, a simpler system is examined, whose behavior was originally attributed to stress-induced mutagenesis (Yang et al. 2001, 2006). A population with a silent chromosomal lac operon gives rise to Lac+ revertant colonies that accumulate over 6 days under selection. Each colony contains a mixture of singly and doubly mutant cells. Evidence is provided that the colonies are initiated by pre-existing single mutants with a weak Lac+ phenotype. Under selection, these cells initiate slow-growing clones, in which a second mutation arises and improves growth of the resulting double mutant. The system shows no evidence of general mutagenesis during selection. Selection alone may explain rapid adaptation in this and other systems that give the appearance of mutagenesis.     

UV-induced mutation in bacteriophage T4.

Two late gene am mutants of bacteriophage T4 that can be induced to revert by UV were crossed to a temperature-sensitive ligase mutant. In the double mutants, UV-induced reversion was eliminated at a semirestrictive temperature. When the single am mutants were irradiated and then allowed a single passage in a permissive host, the UV-induced reversion frequency was increased by 15- to 25-fold. This increased mutagenesis was also abolished by the presence of the ligase allele. When the UV-irradiated single am mutants multiply infected a permissive host, allowing multiplicity reactivation to occur, the induced reversion frequency was reduced similarly to the reduction in lethality. The mutagenesis that remained was again abolished by the presence of the ligase allele. It is concluded that UV induces mutations in phage T4 through the action of a pathway that includes polynucleotide ligase. The increase in mutation frequency after growth in a permissive host implies that mutagenesis can occur at more than one stage of the infection rather than only in an early stage before expression of the mutant genome. The process of multiplicity reactivation appears to be error-free since it overcomes lethal lesions without inducing new mutations.     

Efficient insertional mutagenesis in lactococci and other gram-positive bacteria.

In lactococci, the study of chromosomal genes and their regulation is limited by the lack of an efficient transposon mutagenesis system. We associated the insertion sequence ISS1 with the thermosensitive replicon pG+ host to generate a mutagenic tool that can be used even in poorly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococcus thermophilus. High-frequency random insertion (of about 1%) obtained with this system in Lactococcus lactis allows efficient mutagenesis, with typically one insertion per cell. After ISS1 replicative transposition, the chromosome contains duplicated ISS1 sequences flanking pG+ host. This structure allows cloning of the interrupted gene. In addition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no foreign markers. Mutants obtained by this transposition system are food grade and can thus be used in fermentation processes.     

Mutagenesis induced by 5-bromouracil and methyl methane sulfonate: role of DNA polymerase I

A polA1 mutation in the DNA polymerase I gene of E. coli results in a drastic reduction of the frequency of mutagenesis induced by 5-bromo-2'-deoxyuridine (BUdR). Comparisons of the effect of a polA1 mutation on mutagenesis induced by methyl methane sulfonate (MMS), ultraviolet irradiation (UV) and 2-aminopurine (2-AP) demonstrated that a similar effect of a polA1 mutation is observed with MMS. This effect is much less marked with UV-and-2-AP-induced mutagenesis. It follows that DNA polymerase I plays a key role in the process of mutagenesis induced by BU and MMS. Bearing in mind that mutagenesis provoked by UV, MMS and BU involves participation of the accompanying induced error-prone system, the sources of the differences in requirement for DNA polymerase I are critically examined.     

An aerobic recA-, umuC-dependent pathway of spontaneous base-pair substitution mutagenesis in Escherichia coli

Antimutator alleles indentify genes whose normal products are involved in spontaneous mutagenesis pathways. Mutant alleles of the recA and umuC genes of Escherichia coli, whose wild-type alleles are components of the inducible SOS response, were shown to cause a decrease in the level of spontaneous mutagenesis. Using a series of chromosomal mutant trp alleles, which detect point mutations, as a reversion assay, it was shown that the reduction in mutagenesis is limited to base-pair substitutions. Within the limited number of sites than could be examined, transversions at AT sites were the favored substitutions. Frameshift mutagenesis was slightly enhanced by a mutant recA allele and unchanged by a mutant umuC allele. The wild-type recA and umuC genes are involved in the same mutagenic base-pair substitution pathway, designated "SOS-dependent spontaneous mutagenesis" (SDSM), since a recAumuC strain showed the same degree and specificity of antimutator activity as either single mutant strain. The SDSM pathway is active only in the presence of oxygen, since wild-type, recA, and umuC strains all show the same levels of reduced spontaneous mutagenesis anaerobically. The SDSM pathway can function in starving/stationary cells and may, or may not, be operative in actively dividing cultures. We suggest that, in wild-type cells, SDSM results from basal levels of SOS activity during DNA synthesis. Mutations may result from synthesis past cryptic DNA lesions (targeted mutagenesis) and/or from mispairings during synthesis with a normal DNA template (untargeted mutagenesis). Since it occurs in chromosomal genes of wild-type cells, SDSM may be biologically significant for isolates of natural enteric bacterial populations where extended starvation is often a common mode of existence.     

人参多肽基因的酶法体外随机——定位诱变

本文报道了酶法体外随机——定位诱变人参多肽基因的原理和方法。该法把传统的随机诱变和现代的定位诱变融合起来,可灵活控制基因突变的随机性和定位性。我们用双脱氧末端终止法测定了三个突变株的结果证实了该法的合理性和可行性,     

Regulating general mutation rates: examination of the hypermutable state model for Cairnsian adaptive mutation

In the lac adaptive mutation system of Cairns, selected mutant colonies but not unselected mutant types appear to arise from a nongrowing population of Escherichia coli. The general mutagenesis suffered by the selected mutants has been interpreted as support for the idea that E. coli possesses an evolved (and therefore beneficial) mechanism that increases the mutation rate in response to stress (the hypermutable state model, HSM). This mechanism is proposed to allow faster genetic adaptation to stressful conditions and to explain why mutations appear directed to useful sites. Analysis of the HSM reveals that it requires implausibly intense mutagenesis (10(5) times the unselected rate) and even then cannot account for the behavior of the Cairns system. The assumptions of the HSM predict that selected revertants will carry an average of eight deleterious null mutations and thus seem unlikely to be successful in long-term evolution. The experimentally observed 35-fold increase in the level of general mutagenesis cannot account for even one Lac(+) revertant from a mutagenized subpopulation of 10(5) cells (the number proposed to enter the hypermutable state). We conclude that temporary general mutagenesis during stress is unlikely to provide a long-term selective advantage in this or any similar genetic system.     

TAMS technology for simple and efficient in vitro site-directed mutagenesis and mutant screening

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (eg. cDNA, genomic DNA and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3′ sequences of the primers match the template perfectly. When two outer mutagenic oligos are incorporated together with the desired mutagenic oligos into the newly synthesised mutant strand, they serve as anchors for PCR primers which have 3′ sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening.     

Enhancement of the chemical and antimicrobial properties of subtilin by site-directed mutagenesis.

Subtilin and nisin are gene-encoded antibiotic peptides that are ribosomally synthesized by Bacillus subtilis and Lactococcus lactis, respectively. Gene-encoded antibiotics are unique in that their structures can be manipulated by mutagenesis of their structural genes. Although subtilin and nisin share considerable structural homology, subtilin has a greater tendency than nisin to undergo spontaneous inactivation. This inactivation is a accompanied by chemical modification of the dehydroalanine at position 5 (DHA5) with a kinetic first-order t1/2 of 0.8 days. It was hypothesized that the R group carboxyl of Glu4 in subtilin participates in the chemical modification of the adjacent DHA5. Noting that nisin has Ile at position 4, site-directed mutagenesis was used to change Glu4 of subtilin to Ile, in order to eliminate this carboxyl-group participation. The DHA5 of this mutant subtilin (E4I-subtilin) underwent modification with a t1/2 of 48 days, which is 57-fold slower than natural subtilin, and the rate of loss of biological activity dropped by a like amount. These results suggest that an intact DHA5 is critical for subtilin activity against bacterial spore outgrowth. A double mutant of subtilin, in which the DHA5 residue of E4I-subtilin was mutated to Ala was devoid of detectable inhibition against spore outgrowth. The specific activity of E4I-subtilin was 3-4-fold higher than natural subtilin, suggesting that an increase in the hydrophobicity of the N-terminal end of the molecule enhances activity. These are the first mutants of subtilin that have been reported, and E4I-subtilin is the first example of any lantibiotic whose properties have been improved by mutagenesis. In order to carry out the mutagenesis, a host-vector pair was constructed that permits a deletion replacement in which the natural subtilin gene is replaced by the mutant gene at the normal location in the chromosome. This maintains normal gene dosage and regulatory responses, as well as eliminates ambiguities caused by expression of the normal and mutant genes in the same cell.     

A mutant of the cyanobacterium Anabaena variabilis ATCC 29413 lacking cyanophycin synthetase: growth properties and ultrastructural aspects

The gene cphA encoding cyanophycin synthetase was interrupted in Anabaena variabilis ATCC 29413 by insertional mutagenesis. The mutant lacked cyanophycin granules and the polar nodules of heterocysts. The mutant grew as fast as the wild-type irrespective of the nitrogen source at low light intensity whereas growth on N(2) was somewhat reduced in high light. It is concluded that cyanophycin metabolism and polar nodules are not essential for aerobic N(2) fixation.     

Different mechanisms for SOS induced alleviation of DNA restriction in Escherichia coli.

The alleviation of DNA restriction during the SOS response in Escherichia coli has been further investigated. With the EcoK DNA restriction system UV irradiated wild-type cells show a 10(4)-fold increase in ability to plate non-modified lambda phage and a 3-4 fold increase in transformation by non-modified plasmid DNA. A role for the umuDC genes of E coli in the process of SOS-induced restriction alleviation was identified by showing that a umuC122::Tn5 mutant could alleviate EcoK restriction to only 5% that of wild-type levels. Although umuDC are better characterized for their pivotal role in SOS induced mutagenesis, it is demonstrated here that umu-dependent alleviation of EcoK restriction is a transient process in which umu-dependent mutagenesis plays little part. A second form of SOS induced alleviation of DNA restriction is described in this paper involving the McrA restriction system. The mcrA gene is shown to be encoded within a defective prophage called e14 situated at the 25 min region on the Escherichia coli genetic map. e14 is known to abortively excise from the chromosome after SOS induction and it is demonstrated in this report that mcrA is lost from the genome after SOS induction as part of e14. This results in co-ordinate decrease in the level of McrA restriction within a population of cells.