SummaryMicrosporidia are recognized as a major aetiological agent in chronic diarrhoea of immunocompromised patients. Their detection by light microscopy is hampered by the small size of the spores. A simple and rapid DNA extraction method has been developed for the detection of microsporidian DNA by PCR directly from stool specimens. It can be performed at room temperature in a I.g-ml microcentrifuge tube format in less than I hour. The subsequent nested polymerase chain reaction permits the detection of 3-100 spores in a 0.1-g stool sample. The amplification products can be verified and the species Enterocytozoon bieneusi, Encephalitozoon cuniculi and Encephalitozoon (Septata) intestinalis distinguished by a simple restriction endonuclease digest.keywords microsporidia, DNA, PCR

In crosses under rec+, red+, gam+ conditions, mutation am6 in the cI (repressor) gene of bacteriophage lambda recombines with other cI mutations much more frequently than predicted by the physical distances involved. In four-factor crosses of am6 with mutations located 22-60 base pairs to the left, cI+ recombinants that are expected to require three crossovers (triple recombinants) are more frequent than recombinants that require only one crossover. However, when am6 is crossed with large insertions in cI, which may be expected to interfere with the formation of heteroduplexes by branch migration, the frequency of cI+ triple recombinants is very low. In addition, cI+ recombinants in crosses between am6 and adjacent mutations have a high probability of retaining the flanking markers of the am6 parent. These findings suggest that am6 is particularly susceptible to mismatch repair in heteroduplexes spanning cI. A large fraction of such heteroduplexes are presumed to be the result of branch migration from crossovers occurring at some distance from am6. The absence of co-repair when am6 is crossed with adjacent cI mutations indicates that most repair tracts extend no farther than about 20 bp to either side of the mismatch. The am6 mutation arose in the glutamine codon in a CCAGG sequence, in which the central cytosines are methylated in K12 strains. Their location in methylated sequences may make certain amber mutations susceptible to a specific very short patch (VSP) repair.

Deamination of 5-methylcytosine in DNA results in T/G mismatches. If unrepaired, these mistches can lead to C-to-T transition mutations. The very short patch (VSP) repair process in Escherichia coli counteracts the mutagenic process by repairing the mismatches in favor of the G-containing strand. Previosly we have shown that a plasmid containing an l1-kilobase fragment from the E. coli chromosome can complement a chromosomal mutation defective in both cytosine methylation and VSP repair. We have now mapped the regions essential for the two phenotypes. (24). The resulting uracil/guanine mismatches are repaired by a process which, in the first step, involves the removal of uracil (23). 5-Methylcytosine is more unstable than cytosine (10, 24), and its deamination results in the creation of thymine/guanine (T/G) mismatches. If not correctly repaired prior to replication, C-to-T transitions occur. Cytosine methylation sites in DNA have been found to be "hotspots" for such mutations in Escherichia coli (7; M. Lieb, unpublished results).The only DNA cytosine methylase in E. coli K-12, Dcm, transfers a methyl group to position 5 of the internal cytosine in the sequence 5'-CCWGG-3', where W is A or T (29). E. coli also contains a base mismatch correction process that corrects T/G mismatches in sequence contexts such as Cl AGG/GjTCC and CITGG/GGACC to C/G (21). This process, called very short patch (VSP) repair, reduces the mutagenic effect of cytosine methylation (M. Lieb, unpublished results). It is likely that this process arose in E. coli to reduce the mutagenicity of 5-methylcytosine.Marinus and Morris used a chemical mutagen to obtain a series of E. coli mutations, dcm-1 through dcm-11, that were defective in the methylation of cytosines in DNA (28). One of these mutations, dcm-6, was later shown to lead to the loss of VSP repair in the cell as well (14,20,40). This led to the conclusion that Dcm was required for VSP repair and might be a bifunctional protein. We recently showed that a plasmid containing an 11-kilobase fragment of chromosomal * Corresponding author.DNA from wild-type E. coli complements both the methylation and repair defects of dcm-6 (22). This is consistent with the suggestion that Dcm plays a direct role in VSP repair. In that study, we also tested the gene for another methylase, EcoRII, for its ability to complement dcm-6 for its repair defect. EcoRII methylase is part of the EcoRII restrictionmodification system and has sequence specificity identical to that of Dcm (29,35). Hence, the gene that codes for the EcoRII methylase complements dcm-6 for methylation. Interestingly, it did not restore VSP repair in the cell (22 [F-thr-l ara-14 leuB6 A(gptproA)62 lac Yl tsx-33 supE44 galK2 hisG4 rflhbD mgl-S1 rpsL31 kdgKSJ xyl-S mtl-l metBI thi-1]; its derivatives carrying various dcm alleles; GM30 (thr-J hisG4 leuB6 rpsL ara-14 supE44 lacYl tonA31 tsx-78 galK2 galE2 xylS thi-l mtl-i) and GM31 (i.e., GM30 dcm-6) were kindly provided by M. G. Marinus (University of Massachusetts School of Medicine, Amh...

Certain amber mutations in the cI gene of bacteriophage lambda appear to recombine very frequently with nearby mutations. The aberrant mutations included C-to-T transitions at the second cytosine in 5'CC(A/T)GG sequences (which are subject to methylation by bacterial cytosine methylase) and in 5'CCAG and 5'CAGG sequences. Excess cI+ recombinants arising in crosses that utilize these mutations are attributable to the correction of mismatches by a bacterial very-short-patch (VSP) mismatch repair system. In the present study I found that two genes required for methyladenine-directed (long-patch) mismatch repair, mutL and mutS, also functioned in VSP mismatch repair; mutH and mutU (uvrD) were dispensable. VSP mismatch repair was greatly reduced in a dem Escherichia coli mutant, in which 5-methylcytosine was not methylated. However, mismatches in heteroduplexes prepared from lambda DNA lacking 5-methylcytosine were repaired in dcm' bacteria. These results indicate that the product of gene dcm has a repair function in addition to its methylase activity.

Summary In Escherichia coli and related enteric bacteria, repair of base‐base mismatches is performed by two overlapping biochemical processes, methyl‐directed mismatch repair (MMR) and very short‐patch (VSP) repair. While MMR repairs replication errors, VSP repair corrects to C•G mispairs created by 5‐methylcytosine deamination to T. The efficiency of the two pathways changes during the bacterial life cycle; MMR is more efficient during exponential growth and VSP repair is more efficient during the stationary phase. VSP repair and MMR share two proteins, MutS and MutL, and although the two repair pathways are not equally dependent on these proteins, their dual use creates a competition within the cells between the repair processes. The structural and biochemical data on the endonuclease that initiates VSP repair, Vsr, suggest that this protein plays a role similar to MutH (also an endonuclease) in MMR. Biochemical and genetic studies of the two repair pathways have helped eliminate certain models for MMR and put restrictions on models that can be developed regarding either repair process. We review here recent information about the biochemistry of both repair processes and describe the balancing act performed by cells to optimize the competing processes during different phases of the bacterial life cycle.