A novel DNA structure, sticky DNA, is described for lengths of (GAA.TTC)n found in intron 1 of the frataxin gene of Friedreich's ataxia patients. Sticky DNA is formed by the association of two purine.purine.pyrimidine (R.R.Y) triplexes in negatively supercoiled plasmids at neutral pH. An excellent correlation was found between the lengths of (GAA.TTC) (> 59 repeats): first, in FRDA patients, second, required to inhibit transcription in vivo and in vitro, and third, required to adopt the sticky conformation. Fourth, (GAAGGA.TCCTTC)65, also found in intron 1, does not form sticky DNA, inhibit transcription, or associate with the disease. Hence, R.R.Y triplexes and/or sticky DNA may be involved in the etiology of FRDA.

The influence of nucleotide excision repair (NER), the principal in vivo repair system for DNA damages, was investigated in Escherichia coli with uvrA, uvrB and uvrAuvrB mutants with the triplet repeat sequences (TRS) involved in myotonic dystrophy, the fragile X syndrome and Friedreich's ataxia. (CTG*CAG)175was more stable when the (CTG) strand was transcribed than when the (CAG) strand was transcribed in the alternate orientation. A lack of the UvrA protein dramatically increases the instability of this TRS in vivo as compared with the stability of the same sequence in uvrB mutant, which produces an intact UvrA protein. We propose that transcription transiently dissociates the triplet repeat complementary strands enabling the non-transcribed strand to fold into a hairpin conformation which is then sufficiently stable that replication bypasses the hairpin to give large deletions. If the TRS was not transcribed, fewer deletions were observed. Alternatively, in the uvrA-mutant, the hairpins existing on the lagging strand will suffer bypass DNA synthesis to generate deleted molecules. Hence, NER, functionally similar in both prokaryotes and eukaryotes, is an important factor in the genetic instabilities of long transcribed TRS implicated in human hereditary neuro-logical diseases.

Induction of transcription into long CTG.CAG repeats contained on plasmids in Escherichia coli is shown to increase the frequency of deletions within the repeat sequences. This elevated genetic instability was detected because active transcription into the triplet repeat influenced the growth transitions of the host cell, allowing advantageous growth for cells harboring plasmids with deleted repeat sequences. The variety of deletion products observed in separate cultures suggests that transcription altered the metabolism of the DNA in a manner that produced random length changes in the repeat sequence. For cultures containing plasmids without active transcription into the triplet repeat, or those maintained in exponential growth, deletions occurred within the repeat at a lower frequency (5-20-fold lower). In these incubations the extent of deletions was proportional to the number of cell divisions and many repeat lengths were observed within each culture, suggesting that the decrease in average repeat length at long incubation times was due to multiple small deletions. These observations show that deletions within long CTG.CAG repeats contained on plasmids in E.coli occur via more than one pathway and their level of genetic instability is altered by the enzymatic processes occurring upon the DNA.

Urinary tract infections are one of the most frequent bacterial diseases in humans, and Escherichia coli is most often the relevant pathogen. A specific pathotype of E. coli, known as uropathogenic E. coli (UPEC), often causes serious and difficult-to-treat infections of the urinary tract. We propose a new single-tube screening tool that uses an (N) 6 (CGG) 4 primer to generate fingerprint profiles that allow rapid discrimination and epidemiology of this group of bacteria. We found 71 different CGG-PCR profiles among 127 E. coli strains, while enterobacterial repetitive intergenic consensus (ERIC)-PCR of the same group yielded only 28 profiles. Additionally, the (CGG) 4 -based PCR test turned out to be very effective for clustering UPEC strains exhibiting multiple virulence genes and usually belonging to the B2 phylogenetic group, and it separated these strains from E. coli strains lacking most of the UPEC-specific virulence factors. Since the reproducibility of the CGG-PCR screen is higher than that of ERIC-PCR, our test should be a valuable means of increasing the discriminatory power of current UPEC typing schemes.Gram-negative rods are the major etiological agents in urinary tract infections (UTIs) in humans, and Escherichia coli comprises most of these agents (20,30,32,34,38,42). In some cases, UTI treatment is difficult because of persistent recurrences. Furthermore, UTIs are often asymptomatic at the beginning of the infection process. Particular phenotypic features of uropathogenic E. coli (UPEC) strains facilitate their persistence in urinary tracts and differentiate them from the other pathogenic and commensal E. coli strains (7,29,31). UPECspecific virulence factors (VFs), which are mostly adhesins (P and S fimbriae), toxins (cytotoxic necrotizing factor type 1, ␣-hemolysin), bacteriocin (uropathogenic-specific protein), and siderophores (aerobactin and yersiniabactin), are important for colonization of the urinary tract (7,8,27). Also, type 1 fimbriae and afimbrial adhesin I are beneficial in this type of infection. Additionally, phylogenetic analyses have revealed that UPEC strains differ substantially from other E. coli strains (2, 10, 43). Pathogenic E. coli strains, including UPEC strains, belong mainly to groups B2 and D (2,5,14).In the case of E. coli, 16S rRNA gene sequence analysis, phylogenetic studies, and VF profiles are valuable for detailed genetic identification (4,5,11,35). PCR-based methods are very efficient, inexpensive, and rapid (44). Previously, two distinct prokaryotic repetitive elements were used for gram-negative enterobacterial strain discrimination: repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus (ERIC) sequences (16,37,40). Because the ERIC-PCR band patterns were less complex than the REP-PCR band patterns, differences within the analyzed species were easier to distinguish with ERIC-PCR.The goals of this work were to develop a novel genetic test (termed CGG-PCR) for the differentiation and epidemiological investigation o...