Microarray analysis of microbial virulence factors.

Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.

Fidelity of replication of repetitive DNA in mutS and repair proficient Escherichia coli.

Replication fidelity is not constant among strains within a species or at all genetic loci within a genome. Altered fidelity of replication may affect patterns of pathogenesis and the evolution of these strains. We have been studying replication fidelity in Escherichia coli, both in laboratory attenuated strains and in food-borne pathogens. To understand the altered patterns of mutagenesis at the molecular level, we used a shuttle vector plasmid with a tRNA mutational marker gene which had been altered to include homopolymeric runs of five, seven and nine [G:C] pairs, as well as non-repetitive DNA. Replication of the plasmid in mutS strains resulted in a 20-fold increase in mutant progeny plasmids. The mutations were almost all (>90%) frameshift mutations, while base substitution mutations were rare. Most mutations were insertions or deletions of one or two [G:C] pairs in the longest homopolymeric runs. Larger deletions (5 to >70bp), also targeted to the repetitive sequence, were likewise common. Mutations increased exponentially with the length of the homopolymeric run. These patterns of mutation, including unexpectedly high levels in repair proficient strains, led to an examination of the E. coli K-12 genome for homopolymeric DNA. This sequence motif was found to be rare, particularly in genes and open reading frames. Amino acid homotrimers were found to avoid usage of homopolymeric codons, even when they are preferred among synonymous codons in E. coli. There appears to be active selection against tandem direct nucleotide repeats in the E. coli genome, correlated with the inability of the organism to accurately replicate such sequence.

Sequence-dependent mutations in a shuttle vector plasmid replicated in a mismatch repair deficient human cell line.

We utilized a shuttle vector plasmid (pLSC) to assess the role of DNA sequence and mismatch repair on mutagenesis in human cells. pLSC contains an interrupted 29 bp mononucleotide poly(G) run within a bacterial suppressor tRNA gene, which acts as a highly sensitive mutagenic target for detection of base substitution and frameshift mutations. The frequency of spontaneous mutations in pLSC was found to be similar after replication in either the hMSH6 (GT binding protein) mismatch repair-deficient MT1 line or its parental, mismatch repair-proficient line, TK6. However, the classes of plasmid mutations showed distinct differences in the two cell lines. Single base deletions comprised 48% of the mutations in the 56 independent pLSC plasmids sequenced from MT1 cells while these represented only 18% of the 40 independent pLSC mutants sequenced from the wild-type TK6 cells (P = 0.001). Virtually all the deletions included the mononucleotide run. In contrast, in pSP189, which contains the unmodified supF tRNA without the mononucleotide sequence, no single base deletions were observed for either cell line (P < 0.001). UV treatment of pLSC and pSP189 resulted in a 12-140-fold increase in mutations in TK6 and MT1 cells. These were predominately single base substitution mutations without a large increase in deletion mutations in the mononucleotide run in pLSC. These data indicate that a mononucleotide poly(G) run promotes single base deletion mutations. This effect is enhanced in a hMSH6 mismatch repair-deficient cell line and is independent of UV-induced mutagenesis.

Triplex formation by oligonucleotides containing 5-(1-propynyl)-2'-deoxyuridine: decreased magnesium dependence and improved intracellular gene targeting.

Oligonucleotides capable of sequence-specific triple helix formation have been proposed as DNA binding ligands useful for modulation of gene expression and for directed genome modification. However, the effectiveness of such triplex-forming oligonucleotides (TFOs) depends on their ability to bind to their target sites within cells, and this can be limited under physiologic conditions. In particular, triplex formation in the pyrimidine motif is favored by unphysiologically low pH and high magnesium concentrations. To address these limitations, a series of pyrimidine TFOs were tested for third-strand binding under a variety of conditions. Those containing 5-(1-propynyl)-2'-deoxyuridine (pdU) and 5-methyl-2'-deoxycytidine (5meC) showed superior binding characteristics at neutral pH and at low magnesium concentrations, as determined by gel mobility shift assays and thermal dissociation profiles. Over a range of Mg2+ concentrations, pdU-modified TFOs formed more stable triplexes than did TFOs containing 2'-deoxythymidine. At 1 mM Mg2+, a DeltaTm of 30 degreesC was observed for pdU- versus T-containing 15-mers (of generic sequence 5' TTTTCTTTTTTCTTTTCT 3') binding to the cognate A:T bp rich site, indicating that pdU-containing TFOs are capable of substantial binding even at physiologically low Mg2+ concentrations. In addition, the pdU-containing TFOs were superior in gene targeting experiments in mammalian cells, yielding 4-fold higher mutation frequencies in a shuttle vector-based mutagenesis assay designed to detect mutations induced by third-strand-directed psoralen adducts. These results suggest the utility of the pdU substitution in the pyrimidine motif for triplex-based gene targeting experiments.

Single nucleotide positions have proximal and distal influence on UV mutation hotspots and coldspots.

Base substitution mutation frequency is influenced by the sequence context surrounding lesions in the DNA. We have been studying ultraviolet mutagenesis in human repair-deficient cells in the supF marker gene carried in a shuttle vector plasmid. There are prominent hotspots, on opposite strands, at the 5' TC sites in the eight base palindrome 5' CTTCGAAG. Recently, we developed a reporter system which permits sequence manipulation in the vicinity of mutational hotspots. We have used the system to characterize the influence of individual positions in the palindrome on the frequency of mutagenesis at the two UV hotspots. In this paper we have determined the contribution of bases at the second and third positions in the palindrome. Changes in bases that were in the primer template duplex when the replication complex encountered the photoproducts at one of the hotspot sites significantly increased or decreased the probability of mutations at the site. We also observed modulation of hotspot activity at other sites as a function of single base changes as much as 80 bases away from the hotspots. In these instances, the site of the changed base was in the unreplicated template ahead of the primer terminus when the polymerase encountered the relevant photoproduct. Our results indicate that sequence context has both proximal and distal consequences for mutagenesis.

The influence of single base changes on UV mutational activity at two translocated hotspots.

Mutation hotspots have been a staple of mutation spectra since the introduction of fine structure mutation mapping almost 40 years ago. It has been well established that sequence context is an important determinant of mutational activity at mutagen induced hotspots and coldspots. However, our understanding of the sequence effectors of base substitution hotspots is quite limited. This is because manipulation of the sequence about a hotspot site in a marker gene is restricted by the need to maintain a functional marker. In this work, we describe a generalizable system for studying sequence context effects on mutagenesis. We have prepared a variant of the supF tRNA gene (a marker used by us in previous studies) in which an eight-base palindrome, the site of two UV hotspots in the interior of the gene, was copied into the acceptor stem and pre-tRNA region. The variant tRNA was active. The UV mutation spectrum of this variant showed that the new copy of the palindrome generated two hotspots which were as intense as the original sites in the interior of the gene. Variant genes were constructed with all possible bases at the first position in the palindrome in the pre-tRNA sequence, which does not affect tRNA function. The mutation analysis showed that activity at one of the hotspots could be reduced or enhanced by the changes, while activity at the other site was not significantly affected. The base changes did not influence the frequency of cyclobutane dimer or (6-4) photoproduct formation at the two hotspot sites. Thus, the changes in mutational activity were due to the influence of sequence context on the efficiency of mutation formation at the sites of UV lesions.

Expression of a transfected DNA repair gene (XPA) in xeroderma pigmentosum group A cells restores normal DNA repair and mutagenesis of UV-treated plasmids.

The XPA gene was initially cloned based on the ability of its cDNA to improve survival of cells from xeroderma pigmentosum complementation group A (XP-A) patients following irradiation of the cells with UV. We used plasmid host cell reactivation assays to compare UV mutagenesis and the proficiency of DNA repair in a cell line from an XP-A patient, XP2OS(SV40), two derivative cell lines stably expressing XPA cDNAs and in a DNA repair proficient human cell line. Expression of XPA protein in XP2OS cells allowed them to repair UV-treated plasmid pRSVCAT, increasing activity of the damaged CAT marker gene > 100-fold to levels produced by similarly damaged plasmids in normal cells. Expression of the XPA protein in XP2OS cells improved replication of the UV-treated shuttle vector pSP189, increasing plasmid survival and decreasing plasmid mutation frequency to the levels measured in normal cells. The sequence locations of most mutation hotspots in the plasmid marker gene were similar for the three cell lines and the differences did not correlate with the DNA repair status of the cells. This suggests that the location of mutation hotspots is not directly influenced by DNA repair. Expression of the XPA protein did cause a shift in the types of mutations seen in the plasmid gene. In the XP2OS cells > 95% of the plasmid mutations were G:C-->A:T transition mutations. In contrast, XP2OS cells expressing XPA produced other types of mutations: three times as many transversion mutations and a 12-fold increase in mutations at A:T base pairs. Furthermore, the distribution of these types of mutations was similar to the proportions measured in normal cells. Strikingly similar patterns of transition and transversion mutations were found by examination of reports of XP and non-XP skin carcinomas containing mutations in the p53 tumor suppressor gene, suggesting that the repair status of the cells influenced mutagenesis associated with these skin cancers. Our data suggest that loss of XPA gene function may be sufficient to effect the quantitative and qualitative changes in mutagenesis associated with the large increase in skin cancers seen in XP-A patients.

Targeted mutagenesis in mammalian cells mediated by intracellular triple helix formation.

As an alternative to standard gene transfer techniques for genetic manipulation, we have investigated the use of triple helix-forming oligonucleotides to target mutations to selected genes within mammalian cells. By treating monkey COS cells with oligonucleotides linked to psoralen, we have generated targeted mutations in a simian virus 40 (SV40) vector contained within the cells via intracellular triple helix formation. Oligonucleotide entry into the cells and sequence-specific triplex formation within the SV40 DNA deliver the psoralen to the targeted site. Photoactivation of the psoralen by long-wavelength UV light yields adducts and thereby mutations at that site. We engineered into the SV40 vector novel supF mutation reporter genes containing modified polypurine sites amenable to triplex formation. By comparing the abilities of a series of oligonucleotides to target these new sites, we show that targeted mutagenesis in vivo depends on the strength and specificity of the third-strand binding. Oligonucleotides with weak target site binding affinity or with only partial target site homology were ineffective at inducing mutations in the SV40 vectors within the COS cells. We also show that the targeted mutagenesis is dependent on the oligonucleotide concentration and is influenced by the timing of the oligonucleotide treatment and of the UV irradiation of the cells. Frequencies of intracellular targeted mutagenesis in the range of 1 to 2% were observed, depending upon the conditions of the experiment. DNA sequence analysis revealed that most of the mutations were T.A-to-A.T transversions precisely at the targeted psoralen intercalation site. Several deletions encompassing that site were also seen. The ability to target mutations to selected sites within mammalian cells by using modified triplex-forming oligonucleotides may provide a new research tool and may eventually lead to therapeutic applications.

Xeroderma pigmentosum and related disorders: examining the linkage between defective DNA repair and cancer.

Xeroderma pigmentosum, Cockayne syndrome, the xeroderma pigmentosum-Cockayne syndrome complex, and trichothiodystrophy cells have defects in DNA repair and are associated with clinical and cellular hypersensitivity to ultraviolet radiation (UV). Familial dysplastic nevus syndrome cells have UV hypermutability. Although xeroderma pigmentosum and dysplastic nevus syndrome have markedly increased cancer risk. Cockayne syndrome and trichothiodystrophy do not. At the molecular level, these disorders are associated with several different genetic defects as evidenced by the existence of multiple overlapping complementation groups. Recent progress has been made in identifying the chromosomal location and cloning the defective genes in these disorders. Using plasmid shuttle vectors we have shown abnormal repair and mutagenesis of DNA damaged by 254-nm (UVC) or 295-nm (UVB) radiation or the chemical carcinogen aflatoxin in cells from patients with xeroderma pigmentosum. Although xeroderma pigmentosum cells are defective in repair of all photoproducts, Cockayne syndrome cells appear to be defective in repair of cyclobutane dimers and have normal repair of nondimer photoproducts. DNS cells have post UV plasmid hypermutability. These diseases may serve as models for examining molecular mechanisms of carcinogenesis in humans.

Proximal and distal effects of sequence context on ultraviolet mutational hotspots in a shuttle vector replicated in xeroderma cells.

Hotspots are a standard feature of mutational spectra induced by mutagens in a variety of marker genes. While it is generally believed that sequence context exerts an important influence on hotspot location, direct experimental evidence is quite limited. We have studied ultraviolet mutagenesis in a suppressor tRNA marker gene (supF) carried in a mammalian shuttle vector and replicated in Xeroderma pigmentosum cells in culture. We have now constructed a small family of functional variant suppressor tRNA marker gene which differ from one another by one or two nucleotide changes. UV mutational spectra were generated for each variant gene. We found that the change of a dipyrimidine from 5' TC to 5' CC eliminated a strong mutational hotspot. In addition a single base change in the supF gene was accompanied by the appearance of a new hotspot eight bases away. Finally, another single base change suppressed a major hotspot 48 bases away. Polymerase stop assays on the UV modified marker genes were used to measure the frequency and distribution of photoproducts. The differences in hotspot patterns could not be explained by differences in modification patterns. These results indicate that a change in sequence context can directly influence the probability of mutagenesis at specific sites.

Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells.

The mutagenic spectrum induced by aflatoxin-DNA lesions in DNA repair deficient and repair proficient human cells was investigated. The reactive metabolite aflatoxin B1-8,9-epoxide was synthesized and reacted in vitro with the shuttle vector plasmid pS189. Plasmids were transfected into human fibroblasts and allowed to replicate, and the recovered plasmids were screened in indicator bacteria for plasmid survival and mutations in the supF marker gene. Sequence data were obtained from 71 independently arising mutants recovered from DNA repair deficient xeroderma pigmentosum (XP) cells [XP12BE(SV40)] and 60 mutants recovered from a DNA repair proficient cell line (GM0637). Plasmid survival was lower and mutation frequency higher with the XP cells, and the mutation hotspots differed substantially for the 2 cell lines. Most mutations (> 90%) were base substitutions at G:C pairs, only about one-half of which were G:C-->T:A transversions, the expected predominant mutation. One-third of the mutations at GG sites and none of those at isolated Gs were G:C-->A:T transitions. Tandem base substitutions also occurred only at GG sites and were found only with XP cells. The location of mutation hotspots with either cell line did not correlate with the level of modification within the sequence as assessed by a DNA polymerase stop assay. These results suggest that the DNA repair deficiency associated with XP can influence not only the overall frequency of mutations but also the distribution of mutations within a gene. The finding of transition mutations exclusively at GG sites may be of predictive value in attempts to link dietary aflatoxin exposure to cancers associated with specific mutations in the c-ras oncogene and the p53 tumor suppressor gene.

Site directed substitution of 5-hydroxymethyluracil for thymine in replicating phi X-174am3 DNA via synthesis of 5-hydroxymethyl-2'-deoxyuridine-5'-triphosphate.

5-hydroxymethyluracil (HmUra) is formed in DNA as a product of oxidative attack on the methyl group of Thy. It is removed from DNA by HmUra-DNA glycosylase. To determine whether the replacement of Thy by HmUra is mutagenic, which might explain the repairability of HmUra, a HmUra residue was substituted for Thy in a target (amber) codon by in vitro extension of an oligonucleotide primer annealed to phi X-174am3 virion DNA. This was accomplished by synthesizing HmdUTP and using DNA polymerase to effect primer extension. E. coli spheroplasts were transfected with the HmUra-containing DNA and the yield of revertant phage determined following replication in the bacterial host. Since E. coli do not express HmUra-DNA glycosylase activity, mutagenesis could be assessed in the absence of repair. chi 2c analysis showed that replacing Thy with HmUra did not result in an increase in revertant phage. These data indicate that the oxidation of Thy to HmUra in cellular DNA probably does not result in substantial mutagenesis.

Toxicity of 3-aminobenzamide to Chinese hamster cells containing 5-hydroxymethyluracil in their DNA.

V79 cells incorporated 5-hydroxymethyl-2'-deoxyuridine (HmdUrd) into their DNA linearly over a wide range of concentrations and time. Cells grew normally when 0.03% of thymidine residues were replaced with HmdUrd. At this level of substitution, 5-hydroxymethyluracil (HmUra) was removed from DNA at a rate of 30-40%/24 h. Concentrations of HmdUrd in the growth medium which produced higher levels of substitution reduced survival and caused cells to delay their transit through S phase. However, the treatment of HmdUrd-containing cells with 3-aminobenzamide caused extensive cell death. At levels of HmdUrd substitution compatible with near 90% survival, the addition of 3-aminobenzamide, an inhibitor of poly (adenosine diphosphoribose) synthesis, killed over 90% of the cells. This toxicity was not due to inhibition of the removal of HmUra from DNA. Cells killed by this combination of agents arrested in the G2 phase of the cell cycle. We conclude that the toxicity of HmdUrd resulted primarily from the repair of the HmUra residue in DNA and not from any intrinsic toxicity of the HmUra residue itself. We also conclude that the cytotoxicity of 3-aminobenzamide resulted from interference with the completion of DNA repair following base (HmUra) excision. Since HmUra is also formed in DNA through the action of ionizing radiation, it may be among the components of radiation-induced DNA damage which sensitizes cells to 3-aminobenzamide.

5-Hydroxymethyluracil-DNA glycosylase activity may be a differentiated mammalian function.

To determine the prevalence of the repair enzyme HMU-DNA glycosylase we assayed its activity in whole cell extracts of several bacterial species, the eukaryotic yeast Saccharomyces cerevisiae, mammalian cell lines and murine tissue. Enzyme activity was constitutively present in murine, hamster and human cell lines. It was not inducible by exposing cells to oxidative stress from ionizing radiation or by incubating cells with the 2'-deoxynucleoside of HMU, HMdU. In murine tissue, enzyme activity was highest in brain and thymus. HMU-DNA glycosylase activity was not detectable in bacteria or yeast nor could activity be detected after exposure of cells to H2O2. These results suggest that, in contrast to other DNA-repair enzymes, HMU-DNA glycosylase is a differentiated function limited to higher eukaryotic organisms.

Serologic procedures performed in hydrophilized polystyrene cuvettes and borate glass tubes: a comparative study.

Serologic procedures were performed in parallel using hydrophilized polystyrene cuvettes and conventional glass test tubes. Consistent and comparable results were obtained with both containers. No significant differences in antibody titer, specificity, neutralization, or elution were observed. The effect of a proteolytic enzyme on red blood cell antigen was not altered by the hydrophilized plastic tubes. There was no difference in the stability of immune sera stored in glass or plastic at 4 degrees C and--30 degrees C. However, hemolysis of stored red blood cells was significantly slower in the hydrophilized plastic cuvettes.