A newly isolated strain of Escher ichia coli 1K12 regularly produces a large number of unusually small anucleate cells during the logarithmic phase of growth. These small cells do not divide. They may be isolated from the normal, rod-shaped cells by density gradient centrifugation and have properties that may make them useful in a variety of biological studies. In this report we communicate information regarding some of the basic properties of these miiinicells. A preliminary report of this work has been made.1 Materials and Methods. (1) Organism)is and culture methods: E. coli K12 P678 was obtained from Dr. F. Jacob of the Institut Pasteur, Paris, approximately six years ago and has been maintained on nutrient agar slants. The minicell-producing strain, P678-54, was derived from P678 after treatment of a log-phase nutrient-broth culture with triethylenemelamine (0.5 mg/ml). Both organisms are F-strains that require threonine and leucine. They are unable to utilize lactose, galactose, xylose, maltose, and mannitol as sole carbon sources.For most experiments, these organisms were cultivated in a nutrient broth prepared as described previously.2 In some experiments, appropriately supplemented synthetic medium was used.3(2) Separation of miiinicells: Cultures of P678-54 were centrifuged for 20 minutes at 10,000 X g. The pellet from 1 liter of culture medium was resuspended in approximately 20 ml of 0.067 11 potassium phosphate buffer, pH 6.8.Both minicells and normal cells were separated on sucrose gradients by centrifugation for 45 minutes at 2000 rpm (1000 X g) in a no. 253 swinging bucket rotor in an International model IPRII centrifuge. A 2.0-ml sample of the cell suspension was layered over a 40-ml linear gradient ranging from 5 to 20 per cent sucrose buffered with 0.067 Jf phosphate at pH 6.8S. The sample zones were withdrawn from the gradient, pelleted by centrifugation at 15,000 X g for 15 minutes, and resuspended in the phosphate buffer. All separations were done at approximately 40C.(3) RNA, DNA, and protein analysis: Samples of 1010 cells or 1011 to 1012 minicells were extracted in 5 ml of cold 10 per cent trichloroacetic acid for 30 minutes. After centrifugation the precipitate was resuspended in 3-5 ml of a per cent TCA and hydrolyzed at 100°for 30 minutes. The supernatant was collected after centrifugation, and the pellet digested in 2 ml of 1 N NaOH for 30 nmin. DNA was determined on the supernatant by Burton's modification of the diphenylamine reaction,4 RNA on a 5-10X dilution of the supernatant by the orcinol reaction,5 and protein on a 10-20X dilution of pellet digest by the Lowry method.6 (4) Respiration and enzyme induction: Oxygen consumption was determined in a Warburg apparatus at 370C;7 f-galactosidase was induced by methyl-fl-D-thiogalactopyranoside (TAILG) and assayed by measuring the rate of hydrolysis of 0-nitrophenol f-D-galactoside at 28S0C.8Results.(1) Isolation of mutant, general properties, and morphology: The 321

The REV) gene of Saccharomyces cerevisiae is required for nora induction of mutatio by pysical ad chemical agents. We (23,24).The REV) gene product is thought to constitute part of a DNA repair process that is "error prone" or "'mutagenic" in its function (14,20,25). Just as SOS processing by umuDC is distinguished from other Rec functions and from uvrABCcontrolled excision repair in Escherichia coli (56), mutagenic repair is distinct from the other major repair pathways in yeasts (14,20,25 In this paper we describe the isolation of the REV) gene and the mutant allele rev)-) and report their nucleotide and deduced amino acid sequences. Deletion analysis was used to determine the functional limits of the REVI coding sequence and its 5'-and 3'-flanking sequences. A preliminary characterization of the response of REVl::lacZ fusions to heat shock and DNA-damaging agents is presented. Similarity analysis suggests that a segment of the REVI protein is homologous to UMUC protein. MATERIALS AND METHODSMedia. LB medium (29) (with antibiotic supplements as appropriate) was used to propagate E. coli strains and plasmid-bearing derivatives. YEPD and synthetic complete (SC) yeast media have been described before (24). SC-URA is SC without uracil, SC-LYS is SC without lysine, and SC-LEU is SC without leucine.Yeast strains. S. cerevisiae S288C a gal2 mel mal SUC2 CUP) was used as the source of wild-type DNA for library construction. Strains XL68-3D a rev)-) lys)-) leu2-3 his4-38 trpl-289 ura3-52, XL69-5A a REVI lys)-) leu2-3 trpl-289 his3M, and XL7O-1B a RPV) lysl-l leu2-3 trp)-289 ura3-52 were constructed in this laboratory, using standard techniques for mating, sporulation, and tetrad analysis (52).Bacterial strains. E. coli BNN45 (F metB thi hsdR lacY supE44 supF), derived from ED8654 (31), was used routinely as a cloning host. E. coli DH1 recAl (F-Alac-pro, endA) gyrA96 thi-) hsdR) 7 supE44 relAI) (13) was used as a cosmid host. E. coli JM107 (Alac-pro endAl gyrA96 thi-) hsdR)7 supE44 relA) (F' traD36 proAB+ lacIZAM)5]) (57) was used as the host for M13 vectors. E. coli GM215 (F-dam-3 endAl rna-) thi-) supE44) (30) Was used to produce Damplasmid DNA for BclI subcloning.Plasmids, enzymes, and biochemicals. Plasmids YCp5O, YIp5, YEp13, and pHC79 have been described previously (6,15, 19,50

ADl.ER, HOWARD I. (Oak lRidge National Labotatory, Oak Itidge, Tenn.), AND ALICE A. HARDI-CGREE. Analysis of a genetic locus controlling cell division and sensitivity to radiation in Escherichia (o0i. J. Bacteriol. 87:720-726. 1964.-Radiation senisitivity in Eseherichia (oli is under control of

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