A mouse glutathione S-transferase gene encoding the Ya subunit was isolated and sequenced. The gene spans about 11 kb, contains seven exons, and encodes an mRNA of 841 nucleotides. Promoter elements, TATA and CAAT box sequences, were located 32 and 70 nucleotides upstream from the initiation of transcription site. The mRNA coding sequences of the mouse gene were highly homologous to a rat liver Ya mRNA species detected by cDNA cloning. The mouse Ya gene produces a 223-amino-acid polypeptide that differs from the 222-amino-acid rat Ya by 10 amino acid substitutions and a carboxyl terminus Phe-Lys-Ile-Gln instead of Phe-Lys-Phe. A genomic clone containing the last three exons of the rat Ya gene was also isolated, sequenced, and compared with the mouse Ya gene. An extensive sequence conservation (70-80%) in the 50 to 200 bases of introns at the exon-intron junctions as well as in the region beyond the cleavage-polyadenylation site of pre-mRNA was observed.

Genetic analysis and molecular characterization of plasmid deoxyribonucleic acid (DNA) was performed in a toxigenic isolate ofStaphylococcus aureus strain DU4916. Elimination, transduction, and transformation experiments provided us with a series of derivatives similar except for the presence or absence of genes mediating resistance to penicillin (penr), methicillin (mecr), and tetracycline (tetr) and enterotoxin type B (SEB) production (entB+). The derivatives were examined for the presence of a plasmid species which encodes for SEB production. Two distinct species of covalently closed circular DNA of about 2.8 x 106 and 0.75 x 106 daltons were identified in an ethidium bromide-cured, penicillinase-negative (pens) isolate, SN109 (mecr tetr emtB+). Further segregation of either methicillin resistance or tetracycline resistance or of both together resulted in the loss of SEB production and the disappearance of both plasmids. Transduction from strain SN109 showed that determinants for tetracycline resistance are carried by the 2.8 x 106f-dalton plasmid. Transformation with covalently closed circular DNA from strain SN109 yielded mecs tetr entBtransformants harboring the tetracycline resistance plasmid alone and mecr tetr entB+ transformants harboring both the tetracycline resistance and the 0.75 x 106dalton plasmid. Further segregation of methicillin resistance in transformants was not associated with any change in plasmid DNA. The results indicate that a genetic determinant for SEB production is carried by the 0.75 x 106-dalton plasmid. It is possible, however, that this plasmid cannot be maintained in the host independently from the tetracycline resistance plasmid. Methicillin resistance in the strains examined could not be ascribed to any of the covalently closed circular DNA components resolved in strain DU4916.

iDuring the last decade many thorough investigations were carried out on the mechanism of transcription and translation in the living organism. These have clarified the main features of nucleic acid replication and of protein biosynthesis in the cell, and have enabled the study of the early stages of differentiation at a molecular level.'-' Such studies are at present mainly concerned with the structure, function, and changes in the molecular pattern of animal eggs before and after fertilization,4-and of spores and seeds before and after germination.7 Seed germination transfers the embryo from the latent stage into an active developmental stage, and thus represents an interesting phase in the life cycle of the plant. Marcus et al.8 9 have shown that protein synthesis is inactivated both in vivo and in vitro in the ungerminated wheat embryo, which was found to contain ribosomes that could be activated'1 and yielded soluble RNA (sRNA) and supernatant capable of supporting amino acid incorporation. It was thus suggested that messenger RNA (mRNA) is the limiting factor.In the present work, evidence is forwarded for the presence of conserved mRNA in the dry wheat embryo. It is suggested that the masked mRNA is activated upon germination, and supports early protein biosynthesis. Transcription of new mRNA starts at a later stage of development.Materials and Mlethods.-Germination procedure: Wheat embryos (Triticum durumt var. Nursit) were prepared according to Johnston and Stern,1' and stored over CaCl2 at 4°. Viable embryos (500 mg) were germinated in the dark at 230 in a Petri dish containing 5 ml of a germinating medium (GM) containing 0.01 11 tris(hydroxymethyl)aminomethaiie (Tris) buffer, pH 7.6, 0.02 M KCl, sucrose (10 mg/ml), chloramphenicol (50 Ag/ml), and mycostatin (200 ug/ml). Fresh germinating medium was added after 6, 24, 48, and 72 hr germination.Preparation of DNA: DNA was prepared from wheat embryos (3 gm) germinated for 24 hr according to the procedure of Marmur.'2 Preparation of RNA: Wheat embryos were suspended in 0.05 M1 KCl (50 ml) for 2 min at room temperature. Homogenization was performed at 00 in 50 ml T1\I solution (0.01 3l Tris buffer, pH 7.8, 0.01 M1 MgCl2, and 0.023ll KCl), and the RNA was extracted by the phenol method.'3 The RNA obtained was dissolved in TM solution and digested for 4 hr at 280 with deoxyribonuclease (30 ,g/ml Worthington Biochemical Corp., electrophoretically purified). Predigested pronase was then added (200 tg/ml, Calbiochem) and incubation prolonged for 12 hr at 230 in a dialysis bag against 1 liter of TM solution.The final RNA digest was extracted and dissolved in TM solution as above. The DNase and pronase treatments were repeated in the dialysis bag to ensure removal of any DNA and protein contaminants. Final purification of the RNA preparation obtained was effected by methylated albumin kieselguhr (1IAK) column chromatography'4 and filtration through a membrane filter to remove traces of DNA. The purified RNA obtained was 99% sensitive to RNase digestion and insensit...

Germinating wheat embryos (Triticum vulgare var. Florence) synthesize proteins before the onset of DNA synthesis. The onset of DNA replication occurs at about 15 hours of germination and was shown to depend on proteins synthesized before 9 hours of germination with the use of blasticidin S, a specific inhibitor of protein synthesis. A 10-fold increase in the activity of DNA-dependent DNA polymerase was found in extracts derived from germinated embryos, as compared to the activity found in extracts from ungerminated embryos.Water imbibition by the dry seed triggers an array of molecular events which cause the ungerminated seed to change from the quiescent stage to an active growing stage. Marcus et al. (14) showed that protein synthesis is activated in germinating wheat embryos after 30 min of water imbibition. On the other hand active DNA synthesis cannot be observed in such embryos before 15 hr of germination (3). This sequence of events suggests that the early synthesized proteins may be prerequisites for the subsequent DNA replication. Kogoma and Lark (11) suggested that protein synthesis is necessary to initiate, but not to sustain, DNA replication in Escherichia coli. Evidence for the presence of structural and initiating proteins was reported by Lark and Lark (12) for DNA synthesis in E. coli. Yoshikawa (20) showed that germinating spores of Bacillus subtilis which have entered the replication cycle can complete the cycle in the presence of chloramphenicol. On the other hand, continuous protein synthesis is required for DNA replication in Chlorella pyrenoidosa (18), and simultaneous synthesis of nuclear proteins is required for DNA synthesis in lily (8). Most of the evidence for the dependence of replication on protein synthesis shown by Jakob and Bovey (9) in Vicia and Harris (7) in mammalian cells was obtained using inhibitors of protein synthesis and the exact nature of the proteins is unknown. The temporal relation between protein synthesis and DNA replication in the germinating wheat embryo offers an opportunity to determine how early synthesized proteins control both the initiation and continuation of DNA synthesis.In the present work germinating wheat embryos were exposed to blasticidin S, a specific protein synthesis inhibitor, and the effects of the drug on DNA replication were studied.

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