The cyclization of linear DNA in Escherichia coli by site-specific recombination

The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1. Linear plasmid molecules containing directly repeated loxP sites (lox² plasmids) are cyclized in Cre⁺ E. coli strains after introduction either by transformation or by mini-Mu transduction, Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox² plasmids after transformation. By use of E. coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox² plasmids identical to those obtained with covalently closed circular plasmid DNA. Moreover, Cre⁺ E. coli recBC strains allow the efficient recovery of lox² plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.     

The effect of three rad genes on survival, inter- and intragenic mitotic recombination in Saccharomyces. I. UV irradiation without photoreactivation or liquid-holding post-treatment

The effect of UV irradiation on the survival, inter- and intragenic mitotic recombination of 3 diploid UV sensitive Saccharomyces mutants was studied and compared with the wild type RAD. These strains, homozygous for either the RAD, r1s rad 9-4, or rad 2-20 gene, have DRF values for survival of 1:1.6:3:20.6 respectively, at LD1. Their recombination behaviour is not correlated to their survival characteristics. The RAD, r1s, and rad 2-20 strains showed UV induced mitotic inter- and intragenic recombinants; the induction in the r1s diploid is ca. 100 times greater for both the inter- and intragenic recombinants than in the RAD strain. The rad 9-4 diploid produced no UV induced mitotic recombinants whatsoever, and is therefore considered to be a rec- mutation.     

UV-Induced mitotic recombination and its dependence on photoreactivation and liquid holding in the rad6-1 mutant of Saccharomyces cerevisiae

Summary Spontaneous and UV-induced mitotic recombination was compared in diploids homozygous for rad6-1 mutation and in the wild-type strain carrying heterozygous markers for detecting gene conversion (hom2-1, hom2-2) and crossing over (adel, ade2). Diploids homozygous for rad6-1 mutation were characterised by an elevated level of spontaneous and UV-induced mitotic recombination, particularly the intergenic events. Exposure of UV-irradiated strains to visible light resulted in an increased survival and decreased level of mitotic recombination. Liquid holding (LH) differentially affected frequency of mitotic intergenic and intragenic recombination in mutant and wild-type strains, being without any significant effect on cell survival. In a mutant strain intragenic recombination is significantly increased, intergenic only slightly. In the wild-type strain intragenic recombination is slightly decreased but intergenic is not changed by LH. Visible light applied after LH had no effect on survival and mitotic recombination in the wild type, while in the mutant strain photoreactivability of survival was fully preserved and accompanied by a decrease in the frequency of intragenic and intergenic recombination. The results suggest that metabolic pathways responsible for restoring cell survival are independent of or only partly overlapping with those concerning recombination events.     

Induction of homologous recombination in Saccharomyces cerevisiae

Summary We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, of the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of, homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.     

Selection for the transfer of phenotypically nonselectable chromosomal mutations to recombinant plasmids through introduction of an altered restriction site

We describe a simple method to select for transfer of mutant alleles from the Escherichia coli chromosome to a plasmid which formerly carried the wild-type (wt) allele. The wt allele on the plasmid is modified by introduction of a unique restriction site (e.g., XhoI) and transformed into a rec ⁺ strain carrying the mutant allele on the chromosome. Upon homogenotization, the efficiency of which was increased by UV irradiation of the transforming plasmid [Chattoraj et al., Gene 27 (1982) 213–222], plasmids carrying the mutant allele are formed which are resistant to XhoI. These plasmids are selected from the population by resistance to XhoI digestion coupled with the low transformation efficiency of linear DNA molecules in recA⁻ strain. The method is efficient and rapid and has particular advantages in situations where the mutant allele is difficult to detect by its phenotype.     

Inhibition of respiration in yeast by light

Irradiation of starved cultures of Saccharomyces cerevisiae with blue light under aerobic conditions inhibited the capacity of the yeast cells to respire added substrates (e.g., ethanol) and stimulated endogenous respiration. Spectroscopic examination of the cells showed that the irradiation destroyed both cytochrome a and a₃ components of cytochrome oxidase and a part of the cytochrome b. Irradiation under anaerobic conditions had no effect on the respiratory capacity or the cytochrome content of the cells. Under aerobic conditions cytochrome a₃ was protected against photodestruction when complexed with cyanide and cytochrome a was protected when complexed with azide.     

Mating type regulates the radiation-associated stimulation of reciprocal translocation events in Saccharomyces cerevisiae

Both ultraviolet (UV) and ionizing radiation were observed to stimulate mitotic, ectopic recombination between his3 recombinational substrates, generating reciprocal translocations in Saccharomyces cervisiae (yeast). The stimulation was greatest in diploid strains competent for sporulation and depends upon both the ploidy of the strain and heterozygosity at the MAT locus. The difference in levels of stimulation between MATa/MATα diploid and MATα haploid strains increases when cells are exposed to higher levels of UV radiation (sevenfold at 150 J/m²), whereas when cells are exposed to higher levels of ionizing radiation (23.4 krad), only a twofold difference is observed. When the MATα gene was introduced by DNA transformation into a MATa/matα::LEU2 ⁺ diploid, the levels of radiation-induced ectopic recombination approach those obtained in a strain that is heterozygous at MAT. Conversely, when the MATA gene was introduced by DNA transformation into a MATα haploid, no enhanced stimulation of ectopic recombination was observed when cells were irradiated with ionizing radiation but a threefold enhancement was observed when cells were irradiated with UV The increase in radiation-stimulated ectopic recombination resulting from heterozygosity at MAT correlated with greater spontaneous ectopic recombination and higher levels of viability after irradiation. We suggest that MAT functions that have been previously shown to control the level of mitotic, allelic recombination (homolog recombination) also control the level of mitotic, radiation-stimulated ectopic recombination between short dispersed repetitive sequences on non-homologous chromosomes.     

DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle

A CAN1/can1Δ heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.     

Recombination between irradiated shuttle vector DNA and chromosomal DNA in African green monkey kidney cells.

An autonomously replicating shuttle vector was used to investigate enhancement of plasmid-chromosome recombination in mammalian host cells by gamma irradiation and UV light. Sequences homologous to the shuttle vector were stably inserted into the genome of African green monkey kidney cells to act as the target substrate for these recombination events. The shuttle vector molecules were irradiated at various doses before transfection into the mammalian host cells that contained the stable insertions. The homologous transfer of the bacterial ampicillin resistance gene from the inserted sequences to replace a mutant ampicillin sensitivity gene on the shuttle vector was identified by the recovery of ampicillin-resistant plasmids after Hirt extraction and transformation into Escherichia coli host cells. Gamma irradiation increased homologous shuttle vector-chromosome recombination, whereas UV light did not increase the frequency of recombinant plasmids detected. Introducing specific double-strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was increased by UV irradiation of the plasmid but did not change with time. The ampicillin-resistant recombinant plasmid molecules analyzed appeared to rise mostly from nonconservative exchanges that involved both homologous and possibly nonhomologous interactions with the host chromosome. The observation that similar recombinant structures were obtained from all the plasmid treatments and host cells used suggests a common mechanism for plasmid-chromosome recombination in these mammalian cells.     

Homologous recombination is elevated in some Werner-like syndromes but not during normal in vitro or in vivo senescence of mammalian cells

Werner syndrome (WS) is a recessive genetic condition associated with markedly reduced replicative lifespans of cells in culture, high chromosomal instability in vivo and in vitro, and premature appearance of many characteristics of normal aging, including an increased incidence of cancer. We have monitored plasmid homologous recombination frequencies in diploid fibroblasts from 6 Werner or Werner-like syndrome patients, following transfection with a plasmid substrate containing 2 overlapping fragments of the TN5 Neor gene. Plasmid DNA recovered from these cells was then assayed for homologous recombination by (a) transformation of recA- bacteria to Ampr (indicating total viable plasmid) or Neor (indicating viable recombinant plasmid), and (b) by limited-cycle polymerase chain reaction (PCR) to co-amplify a recombinant fragment containing the overlap region, and a control region of the same plasmid, without bacterial transformation. Bacterial assay data indicated that recombination rates in 3 of the 6 WS strains were significantly elevated above normal controls; 4 of 6 appeared elevated by PCR assay. The highest-recombination WS strain showed evidence of reduced degradation of transfected plasmid DNA. For this small sample of WS strains, clinical severity of WS was not well correlated with recombination rate as determined by either assay (Pearson r = 0.78, not significant, for PCR assay); elevated recombination may, however, define a subset of WS at greatest risk for cancer and/or atherosclerosis. PCR assay of a hyperoxia-resistant HeLa cell line, displaying substantially increased chromosome breakage, indicated increased recombination between direct-repeat fragments. Nevertheless, elevated recombination in WS strains is unlikely to be secondary to impaired replicative capacity characteristic of WS cells, or to defective repair of chromosome damage which is increased in WS, since recombination in non-WS strains was unaffected by passage level or repeated UV irradiation.     

基于游离质粒的木糖诱导型枯草芽孢杆菌转化体系的建立及其应用

枯草芽孢杆菌（Bacillus subtilis）作为食品级安全菌株,因其具有理化特征清晰、培养发酵方便等特点,广泛应用于异源蛋白质的高效表达以及高附加值物质的合成。传统的B. subtilis遗传转化方法存在操作流程繁琐、效率低等缺点,因此,开发方便高效的遗传转化系统具有重要意义。转录因子ComK被证实能调控B. subtilis感受态的形成,并在B. subtilis高效转化中有重要作用。构建1个含有木糖诱导启动子P_xyl调控comK表达的穿梭质粒pUBC01?P_xyl?comK的菌株B. subtilis K1,经木糖诱导条件优化后,质粒pHY300?p43?egfp的转化效率达到4.8×10³ CFU·μg^-1。此外,质粒pUBC01?P_xyl?comK可在无胁迫条件下连续培养及消除。木糖诱导感受态体系及质粒消除极大地提高了芽孢杆菌基因编辑和菌株改造的便捷性,同时增强了菌株尤其是生产菌株的性状稳定性。     

羊肚菌不同单孢及不同萌发孔菌丝间的遗传多样性

本研究对来自不同单孢和不同萌发孔菌丝的166份羊肚菌菌株进行形态研究、交配型基因检测、基于ISSR的遗传多样性分析和菌株杂交选育。结果表明,来自不同单孢及其不同萌发孔菌丝培养的菌株间均存在不同程度的形态和遗传差异。4条ISSR引物共扩增出条带清晰的22条多态性谱带,多态性比率为88%。基于遗传相似性系数（GS）、不加权成对群算术平均法（UPGMA）构建的系统树,可将供试菌株分为梯棱羊肚菌Morchella importuna和六妹羊肚菌M. sextelata两大类群。聚类分析发现不同物种间的单孢菌株和单丝菌株的遗传差异显著;同一单孢不同萌发孔菌丝的菌株间存在较大的遗传分化,遗传多样性丰富。交配型基因检测证实羊肚菌是异宗结合型真菌,同一单孢的不同萌发孔菌丝体含相同的交配型基因（MAT 1-1-1或MAT 1-2-1）。遗传多样性结果表明：梯棱羊肚菌比六妹羊肚菌的遗传背景更广泛。此外,ISSR分子标记也表明仅依靠菌丝和菌核的形态特征并不能准确衡量羊肚菌菌株间的亲缘关系。研究羊肚菌单孢菌株、单丝菌株间的形态和遗传特征,将为羊肚菌优质菌种的精准选育提供理论参考。     

Variation of spontaneous and induced mitotic recombination in different Drosophila populations: a pilot study on the effects of polyaromatic hydrocarbons in six newly constructed tester strains

A set of six Drosophila strains was developed, by inducing by chemical treatment with N-ethyl-N-nitrosourea (ENU) new white and, in some strains, yellow mutations in 3 wild-type (WT) and 3 insecticide-resistant (IR) populations. These strains were previously shown to vary with regard to contents and inducibility of microsomal oxidative enzymes (Zijlstra et al., 1984). In this pilot study results from a first evaluation of these strains in somatic mutation experiments are reported, using as genotoxins an aromatic amine (2-naphthylamine, 2-NA), one substituted (9,10-dimethylanthracene, DA) and one non-substituted (benzo[a]pyrene, BP) polycyclic aromatic hydrocarbon. Developing larvae heterozygous for white were chronically exposed to three different exposure doses of each carcinogen. Adult females were inspected for the occurrence of mosaic light clones in their eyes, using the somatic mutation and recombination test (SMART).

Evidence is presented indicating strong genotype-dependent variation in both spontaneous and chemically induced mutational and recombinational events in somatic cells of Drosophila. The spontaneous frequencies varied from 3.5% (Hikone-R), 4.3% (Berlin-K), 6.3% (Oregon-K), 9.1% (91-C), 20.5% (Haag-79) to 49.1% (91-R), corresponding to a 14-fold difference in spot frequencies between the two extremes. BP, DA and 2-NA were readily detectable in both Hikone-R (IR) and Oregon-K (WT), less so in 91-C (WT) and Haag-79 (IR), whereas the performance of strain Berlin-K (WT) was rather poor. The special problem with strain 91-R was the high frequency with which mosaic light spots occur not only in female genotypes heterozygous for white, but also in homozygous condition in the original stock.

The up to 20-fold variation in induced spot frequencies between different genotypes poses questions for further investigations with respect to the genetic constitution of the various strains and the role of enzyme induction on somatic cell mutagenicity, which in this system is predominantly the result of mitotic recombination.     

Cell-cycle variation in the induction of lethality and mitotic recombination after treatment with UV and nitrous acid in the yeast, Saccharomyces cerevisiae.

Exponentially growing yeast cultures separated into discrete periods of the cell cycle by zonal rotor centrifugation show cyclic variation in both UV and nitrous acid induced cell lethality, mitotic gene conversion and mitotic crossing-over. Maximum cell survival after UV treatment was observed in the S and G2 phases of the cell cycle at a time when UV induction of both types of mitotic recombination was at a minimum. In contrast, cell inactivation by the chemical mutagen nitrous acid showed a single discrete period of sensitivity which occurred in S phase cells which are undergoing DNA synthesis. Mitotic gene conversion and mitotic crossing-over were induced by nitrous acid in cells at all stages of the cell cycle with a peak of induction of both events occurring at the time of maximum cell lethality. The lack of correlation observed between maximum cell and the maximum induction of mitotic intragenic recombination suggest that other DNA-repair mechanisms besides DNA-recombination repair are involved in the recovery of inactivated yeast cells during the cell cycle.     

Isolation and analysis of mutants of the methylotrophic actinomycete Amycolatopsis methanolica blocked in aromatic amino acid biosynthesis

Abstract Mutants of the actinomycete Amycolatopsis methanolica blocked in aromatic amino acid biosynthesis were isolated using brief ultrasonic treatments to obtain single cells. After UV irradiation, auxotrophic mutants were selected as pinpoint colonies on mineral agar with only 1 mg 1⁻¹ of amino acid supplements. Mutant characterization provided unambiguous evidence that l-tyrosine is synthesized via arogenate and that l-phenylalanine is synthesized via phenylpyruvate. The efficiency of chromosomal DNA marker exchange was highest in matings with mutant strains that lacked the previously characterized 13.3-kb integrative plasmid pMEA300.     

Protective action of bacterial melanin against DNA damage in full UV spectrums by a sensitive plasmid-based noncellular system

The purpose of this study was to introduce a simple and sensitive plasmid-based noncellular system to evaluate the photoprotection of bacterial melanin on DNA damage against ultraviolet (UV) radiation. Plasmid DNA was used to assess the role of melanin in different ranges of UV using a series of in vitro assays. Fluorometric measurements suggested that melanin could efficiently scavenge reactive oxygen species (ROS) generated by UVA irradiation in solution, and the scavenging capability was proportional to the pigment concentration. The protective effect of melanin on plasmid DNA under UVB irradiation was confirmed by the transformation efficiency of the protected DNA, which was at least 10-fold higher than that of the non melanin protected DNA. After the UVC irradiation, the DNA damage of strand breaks was quantified by laser-induced fluorescence capillary electrophoresis. The percentage of supercoiled plasmid was reduced from 80% to less than 5% without melanin protection. In contrast, the percentage of supercoiled DNA only decreased to about 40% in the presence of melanin under the same radiation conditions. All these results demonstrated that bacterial melanin did protect DNA from being damaged throughout full UV irradiation. This system, avoiding the potential interference by cellular DNA repair machinery and intracellular substances, may provide a sensitive in vitro means to evaluate the functions of melanin and other photoprotective compounds from different sources.     

Inhibition of respiration and destruction of cytochrome a3 by light in mitochondria and cytochrome oxidase from beef heart

Irradiation of beef-heart mitochondria and of cytochrome oxidase purified from beef-heart mitochondria with blue light inhibited electron transport from substrate (succinate for the mitochondria and reduced cytochrome c for the cytochrome oxidase) to O₂. The irradiation treatment also destroyed cytochrome a₃ as assayed by the absorption band for the reduced cyanide-cytochrome a₃ complex at 587 nm in the low-temperature absorption spectrum. Irradiation under anaerobic conditions was not inhibitory. Cytochrome a₃ was protected against photodestruction if cyanide was present during the irradiation.     

Stable transformation of the cyanobacterium Synechocystis sp. PCC 6803 induced by UV irradiation.

Irradiation of the photoheterotrophic cyanobacterium Synechocystis sp. PCC 6803 with low levels of UV light allows for stable, integrative transformation of these cells by heterologous DNA. In this system, transformation does not rely on an autonomously replicating plasmid and is independent of homologous recombination. Cells treated with UV light in the absence of DNA and cells given DNA but not exposed to UV do not yield antibiotic-resistant colonies in platings of up to 2 X 10(8) cells. Optimal conditions for this UV-induced transformation are described. Analysis of the transformants indicates that (i) only a segment of the introduced plasmid is found in the DNA of the transformed cells; (ii) in independently isolated clones, DNA insertion apparently occurs at different sites in the chromosome; and (iii) hybridization data suggest that insertion in one of the transformants may have occurred into a region of the chromosome that is repeated or that integration of plasmid DNA may have been accompanied by a rearrangement or duplication of DNA sequences near the insertion site. DNA isolated from the primary transformants as well as a cloned fragment containing the UV-inserted plasmid sequence and flanking cyanobacterial DNA transform wild-type cells at a high frequency (5.0 X 10(-4) and 1.5 X 10(-5), respectively). Possible mechanisms of this transformation system are discussed, as are the potential uses of this system as an integrative cloning-complementation vector and as a mutagenic agent in which the genetic lesion is already tagged with a selectable marker.