抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.

/pdf/one-step-inactivation-of-chromosomal-genes-in-escherichia-25avsrekpc.pdf

One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products

The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975b). The present paper is a revision of the methods. Two new tester strains, a frameshift strain (TA97) and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set. TA97 replaces TA1537. TA1535 and TA1538 are removed from the recommended set but can be retained at the option of the investigator. TA98 and TA100 are retained. We discuss other special purpose strains and present some minor changes in procedure, principally in the growth, storage, and preservation of the tester strains. Two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine. Some test modifications are discussed.

Revised methods for the salmonella mutagenicity test

DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139

Roles moleculaires des proteines de choc thermique dans le fonctionnement des organismes a des temperatures normales et suite a des chocs thermiques; differents genes impliques

The heat-shock proteins

Damage to DNA poses a serious impediment to replication and can cause genomic instability. To combat insults to the DNA, cells enact a checkpoint response to delay cell cycle progression late during the G2/M phase. This checkpoint ensures adequate time for the damage to the DNA to be repaired or tolerated by various cellular mechanisms. One of the DNA damage tolerance (DDT) mechanisms involves restoring the damaged DNA to its original form by engaging the recombinational apparatus (Homology Directed Repair or HDR), while another utilizes Translesion DNA synthesis (TLS), the process by which adducts and gaps in the DNA are replicated via the action of specialized TLS DNA polymerases. TLS is frequently highly error-prone and is required for the vast majority of eukaryotic mutagenesis. The error-prone branch of TLS is carried out by the TLS polymerases Rev1 and Polz (Rev3/Rev7), whose genes were originally identified in genetic screens for non mutability. The discovery that the REV1, 3 and 7 gene products encode TLS polymerases led to speculation that their action occurs during the S phase of the cell cycle. Several observations challenging this notion prompted Callegari and Kelly to investigate whether cells temporally partition accurate and error-prone modes of DNA damage tolerance and whether mutagenic TLS occurs during a specific phase of the cell cycle. These include evidence that the role of mutagenic TLS may be limited to a subset of daughter-strand gaps produced late during S phase, the large increase in Rev1 expression during late S phase in budding yeast and the increase in Polz late in the cell cycle in human cells. The authors use live-cell imaging to observe the response of individual fission yeast Schizosaccharomyces pombe cells to UV irradiation. Although the S. pombe cell cycle is similar to that of most eukaryotes, it differs in the respect that cells cleave soon after S phase rather than immediately after mitosis. Taking advantage of this property of the S. pombe cell cycle, Callegari and Kelly assessed the cleavage time to determine the stage at which cells incur DNA damage and to examine the contributions of the non-mutagenic and mutagenic DDT pathways after UV treatment. Surprisingly, they find that S. pombe employs primarily non-mutagenic DDT pathways prior to the G2/M checkpoint to repair the damaged DNA. One of the genes in this pathway, the Rad51 recombinase, elicits error-free repair via HDR prior to the G2/M checkpoint. These findings are significant because they provide the first evidence that repair of daughter-strand gaps occurs in a predominantly error-free manner, confining the action of the mutagenic polymerases Rev1 and Polz to after the G2/M checkpoint. DNA Pol h, a TLS polymerase that can replicate quite accurately over UV-induced cyclobutane pyrimidine dimers, also predominantly acts prior to the G2/M checkpoint. This temporal separation of non-mutagenic and mutagenic pathways in the cell cycle suggests a sequential order of events, which ensures that most of the filling in of residual gaps in the DNA occurs accurately and limits mutagenesis to a small window of time. Prior studies have highlighted the importance of PCNA monoubiquitination, which occurs when replication is blocked by DNA damage, in controlling the action of TLS polymerases. These studies suggest a role for Rad51 in preventing mutagenesis while error-free methods of DDT are in play, providing a new cellular strategy to limit mutagenesis. An important future direction will be to elucidate the mechanism by which this process occurs and the types of mutations that persist after the G2/M checkpoint in response to various DNA-damaging agents including UV. Coming on the heels of the 2015 Lasker Awards to Evelyn M. Witkin and Stephen J. Elledge for their discoveries of the DNA damage response, these crucial observations represent a major advance in our understanding of how cells maximize their survival after DNA damage while minimizing their mutational load.

https://www.tandfonline.com/doi/pdf/10.1080/15384101.2015.1132909

Non mutagenic and mutagenic DNA damage tolerance.

A non-cleavable UmuD variant that acts as a UmuD′ mimic. VOLUME 281 (2006) PAGES 9633-9640

The analysis of DNA repair and mutagenesis in organisms that have high resolution genetic and molecular biological systems has led, and will continue to lead, to major advances in our understanding of these processes. It is striking that almost all of the major insights highlighted in this chapter have been gained within the last 10 years and, in fact, several of the discoveries described here occurred during the actual writing of this report. It is worth reflecting that in the mid 1970s there was a widely held view that the essential elements of DNA repair in E. coli were well understood and, at that time, a considerable number of investigators shifted their focus to systems that did not offer the high resolution genetic and molecular biologic tools of E. coli and S. cerevisiae. The remarkable progress achieved over the past ten years underscores the limitations of that viewpoint. It is difficult, and probably unwise, to pinpoint specific issues in DNA repair in E. coli and S. cerevisiae as special targets for future research since so much remains to be learned. Most of the striking discoveries outlined in this chapter have come from skilled investigators using the full range of high resolution genetic, molecular biologic, and biochemical tools to address fundamental problems in DNA repair and mutagenesis. In most cases, it would have been impossible to have predicted the critical discoveries that have been made. For example, UV and chemical mutagenesis require the induction of a pair of genes, mediated by DNA-damage-stimulated proteolytic cleavage of a repressor and then the subsequent activation of one of these gene products by a mechanistically related proteolytic cleavage. In 1975 an hypothesis proposing such a mechanism for UV and chemical mutagenesis would have been dismissed as being utterly fanciful. Yet this is the current reality. As in the cases of the other findings detailed here, the key insights came not from the testing of fanciful hypotheses but rather from systematic scientific studies of fundamental problems. Funding in this area should then support inquiries of fundamental issues in DNA repair and mutagenesis by investigators who are skilled at using the full range of sophisticated experimental tools available for these organisms. It is worth noting that supporting this type of research will shed light not only on mechanisms of DNA repair and mutagenesis but also on more fundamental issues in the biology of living organisms.(ABSTRACT TRUNCATED AT 400 WORDS)

General principles of DNA repair in microorganisms and implications for future research.

Mutagenesis by some alkylating agents and base analogs in Escherichia coli appears to involve a simple mispairing during replication. In contrast, mutagenesis by ultraviolet (UV) light and many chemicals in E. coli requires the participation of an inducible process that is dependent on the products of the umuD, umuC, and recA genes. The requirement for induced cellular functions for UV mutagenesis was first noted by Weigle (37), who found that UV was not mutagenic for bacteriophage lambda unless the bacteria had been preirradiated with UV. The induction of this process was later shown by Defais et al. (7) to be blocked by recA(Def) or lexA(Ind) mutations. The fact that the same recA(Def) and lexA(Ind) mutations rendered E. coli nonmutable by UV and many chemicals (16,24,39) indicated that mutageneses of the bacterial chromosome required the participation of the same recA + lexA + -controlled process, and the inducibility of the system was demonstrated by subsequent physiological experiments (40).

Genetic analyses of the roles of umuDC and mucAB in mutagenesis.

A chimeric plasmid that serves as a shuttle vector for at least two species of bacteria is transformed into the bacteria cells while incubating the cells in the presence of glycine and in the absence of a chelating agent. A representative suitable plasmid is formed from the DNA of Corynebacterium and Escherichia bacteria.

Graham C. Walker

Papers

Non mutagenic and mutagenic DNA damage tolerance.

A non-cleavable UmuD variant that acts as a UmuD′ mimic. VOLUME 281 (2006) PAGES 9633-9640

General principles of DNA repair in microorganisms and implications for future research.

Genetic analyses of the roles of umuDC and mucAB in mutagenesis.

Cloned plasmids of corynebacterium