Mating type switching in Saccharomyces cerevisiae initiates when Ho endonuclease makes a doublestranded DNA break at the yeast MAT locus. In this report, we characterize the fundamental biochemical properties of Ho. Using an assay that monitors cleavage of a MAT plasmid, we define an optimal in vitro reaction, showing in particular that the enzyme has a stringent requirement for zinc ions. This suggests that zinc finger motifs present in Ho are important for cleavage. The most unexpected feature of Ho, however, is its extreme inefficiency. Maximal cleavage occurs when Ho is present at a concentration of 1 molecule/3 base pairs of substrate DNA. Even under these conditions, complete digestion requires >2 h. This inefficiency results from two characteristics of Ho. First, Ho recycles slowly from cleaved product to new substrate, in part because the enzyme has an affinity for one end of its double strand break product. Second, high levels of cleavage in the in vitro reaction correlate with the appearance of large protein-DNA aggregates. At optimal Ho concentrations, these latter aggregates, referred to as "florettes," have an ordered structure consisting of a densely staining central region and loops of radiating DNA. These unusual properties may indicate that Ho plays a role in other aspects of mating type switching subsequent to double strand break formation.Genetic recombination is the intracellular process that moves DNA sequence information from one genomic location to another. A plethora of genetic and biochemical experiments, conducted primarily in prokaryotic and fungal systems, has led to a detailed understanding of the DNA intermediates that arise during this process. Examples of such intermediates include broken duplexes, heteroduplex DNA joints, and Holliday junctions. The first of these intermediates (broken duplexes or double-stranded breaks (DSBs) 1 ) are thought to initiate recombination. Thus, in the yeast Saccharomyces cerevisiae, an organism in which DSB formation has been intensively analyzed, the following lines of evidence indicate that DSBs initiate meiotic recombination. First, DSBs are repaired using gene products required for meiotic recombination (reviewed in Ref. 1). Second, certain meiotic segregation patterns of yeast genes are consistent with molecular models of recombination that posit a DSB initiation event (2). Third, DSBs occur at regions of the genome defined genetically as initiators of recombination (3-11). Fourth, DSBs introduced into DNA during meiosis stimulate nearby meiotic recombination (12). Finally, meiotic DSBs precede and are independent of pairing between homologs, implying that DSBs initiate chromosomal pairing (13,14 (Fig. 1A). The W, X, Z1, and Z2 blocks are identical in a and ␣ cells, but each mating type contains a unique MAT Y sequence: Ya in a cells and Y␣ in ␣ cells. Roughly once per cell generation, new Ya or Y␣ information transposes into MAT from two unlinked and transcriptionally inactive loci known as HML␣ and HMRa, thereby switching the cell's mating ty...

The cloned bacteriophage T4 pin gene functions to stabilize several different kinds of proteins in Escherichia coli bacteria. Incomplete proteins such as puromycyl polypeptides, abnormal but complete proteins such as the A phage tsO protein, and labile eukaryotic proteins encoded by genes cloned in E. coli such as mature human fibroblast interferon are stabilized in cells in which the T4 pin gene is expressed. The cloned T4 pin gene does not seem to affect the turnover of normal E. coli proteins.The introduction of cloned genes into Escherichia coli bacteria for the purpose of producing large amounts of specific proteins not normally present in E. coli cells has led to an examination of various problems associated with the expression of such cloned genes. Difficulties in obtaining expected levels of expression from genes cloned in bacterial cells may be encountered at the levels of stability of the gene and its vector within the host cell (1, 2), transcription of the cloned gene (3-7), and translation of its mRNA into protein (5,6,8 Difco agar, maltose/L bottom agar is identical to maltose/L top agar. Bacteria that were to be assayed for interferon activity were grown in the presence of the appropriate antibiotic(s) in either M9, which contains, per liter: NH4Cl, 1.0 g; KH2PO4, 3.0 g; Na2HPO4, 6.0 g; NaCl, 5.0 g; MgSO4 7H2O, 0.25 g; CaCl2, 0.015 g; glucose, 2.0 g; supplemented with vitamin BI and Casamino acids to final concentrations of 0.001% and 0.5%, respectively, 2059The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.