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Comparative Gene Expression Profiles Following UV Exposure In Copyright 2001 by the Genetics Society of America Comparative Gene Expression Pro®les Following UV Exposure in Wild-Type and SOS-De®cient Escherichia coli Justin Courcelle,*,1 Arkady Khodursky,²,1 Brian Peter,³ Patrick O. Brown² and Philip C. Hanawalt§ ²Department of Biochemistry, Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, ³Department of MCB, UC-Berkeley, Berkeley, California 94720, *Department of Biological Science, Mississippi State University, Mississippi State, Mississippi 39762 and §Department of Biological Sciences, Stanford University, Stanford, California 94305 Manuscript received October 4, 2000 Accepted for publication January 29, 2001 ABSTRACT The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor. Using DNA microarrays containing ampli®ed DNA fragments from 95.5% of all open reading frames identi®ed on the E. coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control. We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E. coli chromosome. We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold. In addition, several transcripts were either downregulated or degraded following UV irradiation. These newly identi®ed UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation. RRADIATION of growing Escherichia coli cultures the genes normally suppressed by LexA are more fre- I with ultraviolet light (UV) produces DNA lesions quently transcribed (Sassanfar and Roberts 1990 and that at least transiently block the essential processes of references therein; Friedberg et al. 1995). An interest- replication and transcription. A large amount of work ing feature of the LexA/RecA regulatory circuit is that has demonstrated that the cell responds to this stress the timing, duration, and level of induction can vary by upregulating the expression of several genes that for each LexA-regulated gene, depending upon the lo- function to repair the DNA lesions, restore replication, cation and binding af®nity of the LexA box(es) relative and prevent premature cell division. A number of other to the strength of the promoter. As a result of these genes are known to be upregulated, yet remain function- properties, some genes may be partially induced in re- ally uncharacterized. The changes in gene expression sponse to even endogenous levels of DNA damage, while in response to DNA damage produced by UV and some other genes appear to be induced only when high or other environmental agents have been collectively ter- persistent DNA damage is present in the cell. In fact, med the SOS response, after the international distress the SOS response may represent a continuum in the signal (Radman 1974 and reviewed in Friedberg et al. monitoring of environmental stress, rather than simply 1995; Koch and Woodgate 1998). operating as an emergency switch following acute injury. Many of the DNA damage-induced genes are nega- The ®rst systematic search for damage-inducible (din) tively regulated by the LexA repressor protein, which genes was carried out by Kenyon and Walker (1980) binds to a 20-bp consensus sequence in the operator by randomly inserting a lac reporter gene into the E. region of the genes, suppressing their expression (Brent coli chromosome to identify promoters that were upreg- and Ptashne 1981; Little et al. 1981). Derepression ulated following DNA damage in a recA/lexA-dependent of these genes occurs when the RecA protein binds to fashion. Using this same technique, subsequent studies single-stranded regions of DNA created at replication identi®ed additional din genes and in some cases identi- forks when they are blocked by DNA damage. RecA ®ed genes previously characterized to be involved in the bound to single-strand DNA becomes conformationally recovery from DNA damage (Bagg et al. 1981; Fogliano active, serving as a coprotease to cleave the LexA repres- and Schendel 1981; Huisman and D'Ari 1981; Kenyon sor. As the cellular concentration of LexA diminishes, and Walker 1981; Shurvinton and Lloyd 1982; Lloyd et al. 1983; Siegel 1983; Bonner et al. 1990; Iwasaki et al. 1990; Ohmori et al. 1995b). Analysis of the known din genes revealed a 20-bp consensus LexA- Corresponding author: Justin Courcelle, Department of Biological Science, P.O. Box GY, Mississippi State University, Mississippi State, binding motif, or ªSOS box,º shared by these genes MS 39762. E-mail: [email protected] in their promoter/operator regions (Walker 1984), 1 These authors contributed equally to this work. which has been used in more recent studies to systemati- Genetics 158: 41±64 (May 2001) 42 J. Courcelle et al. cally search and identify additional lexA-regulated genes from a fresh overnight culture into 200 ml Davis media and (Lewis et al. 1994; Ohmori et al. 1995a; Fernandez De incubated in a 1-liter Erlenmeyer ¯ask at 37Њ in a New Bruns- wick Scienti®c (Edison, NJ) model G76 gyrotory water bath Henestrosa et al. 2000). These studies in total have 8 ϫ 10 cells/ml). A 15-W 2ف ,at 220 rpm to midlog (OD600 0.4 identi®ed 31 genes under lexA/recA control. germicidal lamp (254 nm, 0.66 J/m2/sec at the sample posi- Other genes have been reported to be upregulated tion) provided the UV irradiation. A total of 70 ml of culture following DNA damage but are believed to be indepen- was placed into a 15-cm-diameter glass petri dish and irradi- dent of the lexA regulon. In some cases, the induction ated for 60 sec with gentle agitation. Two 65-ml unirradiated samples were also agitated in a 15-cm petri dish but were not is thought to be dependent on recA, but independent exposed to UV. A total of 70 ml of irradiated culture (in a from the LexA repressor. In other cases, genes have 500-ml Erlenmeyer ¯ask) and 30 ml of unirradiated culture been shown to be upregulated independently from both (in a 250-ml Erlenmeyer ¯ask) were then returned to the recA and lexA (Friedberg et al. 1995; Koch and Wood- shaking water bath for the duration of the time course. At gate 1998). The mechanism of regulation in these cases the appropriate times, 10-ml samples were placed into 20 ml of ice-cold NET (100 mm NaCl, 10 mm Tris, 10 mm EDTA), is not understood. An additional, although as yet unex- pelleted by centrifugation, washed with 1 ml cold NET, re- plored, possibility is that some genes may be repressed pelleted, and frozen at Ϫ80Њ. The limited availability of mi- or their transcripts may be degraded in response to croarray chips constrained this experiment to a single time DNA damage. course containing seven samples (®ve irradiated, two unirradi- It was of interest to us not only to learn whether ated) for each strain. Microarray procedures: Relative mRNA levels were deter- additional genes can be regulated in a LexA-dependent mined by parallel two-color hybridization to cDNA microarrays manner but also to determine whether other cellular representing 4101 open reading frames (ORFs) representing responses to UV irradiation exist that are lexA indepen- 95.5% of E. coli ORFs according to Blattner et al. (1997). dent. The lexA1 allele encodes an amino acid change cDNA arrays were manufactured as described in MGuide at at a position that is essential for the cleavage and inacti- http://cmgm.stanford.edu/pbrown/mguide/index.html. To- tal mRNA was extracted from 2±5 ϫ 109 cells using QIAGEN vation of LexA (Slilaty and Little 1987). Thus, in (Chatsworth, CA) RNeasy spin columns. A total of 25±30 ␮g lexA1 mutants, the LexA1 concentration remains high of total RNA was labeled with Cy-3-dUTP (or Cy-5-dUTP) in and LexA-regulated genes are not induced, even in the a standard reverse transcriptase (RT) reaction by Superscript presence of high levels of activated RecA. Therefore II (ϩ) (GIBCO BRL, Gaithersburg, MD) with 1 ␮g of random this system should allow an analysis of gene expression hexamer (Pharmacia, Piscataway, NJ) primers. Following puri- ®cation through Microcon-30 (Millipore, Bedford, MA) that occurs independent of the LexA repressor. (MGuide), Cy-3- and Cy-5-labeled cDNA were combined with The changes in gene expression in the entire genome SSC (2.5ϫ ®nal), SDS (0.25%), and 40 ␮gofE. coli rRNA can be measured simultaneously using high-density (Boehringer Mannheim, Indianapolis) in a ®nal volume of cDNA microarrays (Schena et al. 1995). DNA microar- 16 ml and hybridized to a DNA microarray for 5 hr at 65Њ. rays contain PCR-ampli®ed DNA fragments of known Slides were washed as described in MGuide and scanned using and predicted genetic sequences that are printed on an AxonScanner (Axon Instruments, Foster City, CA; GenPix 1.0) at 10 mm per pixel resolution. Acquired 16-bit TIFF im- the surface of a glass slide. Through the comparative ages were analyzed using ScanAlyze software, which is publicly hybridization of two cellular RNA preparations, the rela- available at http://rana.stanford.edu/software/. tive difference between transcript levels of any gene Comparative measurements of transcript abundance: Time in these preparations can be determined. Using the course samples were analyzed directly by comparing the abun- complete sequence of the E. coli genome (Blattner et dance of each gene's transcripts relative to the t0 sample. RNA samples taken during the time course were labeled with Cy-5, al.
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