Characterization of double-strand break-induced recombination: homology requirements and single-stranded DNA formation.

In the yeast Saccharomyces cerevisiae, a double-strand chromosome break created by the HO endonuclease is frequently repaired in mitotically growing cells by recombination between flanking homologous regions, producing a deletion. We showed that single-stranded regions were formed on both sides of the double-strand break prior to the formation of the product. The kinetics of the single-stranded DNA were monitored in strains with the recombination-deficient mutations rad52 and rad50 as well as in the wild-type strain. In rad50 mutants, single-stranded DNA was generated at a slower rate than in the wild type, whereas rad52 mutants generated single-stranded DNA at a faster rate. Product formation was largely blocked in the rad52 mutant. In the rad50 rad52 double mutant, the effects were superimposed in that the exonucleolytic activity was slowed but product formation was blocked. rad50 appears to act before or at the same stage as rad52. We constructed strains containing two ura3 segments on one side of the HO cut site and one ura3 region on the other side to characterize how flanking repeats find each other. Deletions formed preterentially between the homologous regions closest to the double-strand break. By varying the size of the middle ura3 segment, we determined that recombination initiated by a double-strand break requires a minimum homologous length between 63 and 89 bp. In these competition experiments, the frequency of recombination was dependent on the length of homology in an approximately linear manner.     

Selective phagocytosis of gram-positive bacteria and interleukin 1-like factor production by a subpopulation of large granular lymphocytes

There has been a consensus that a large granular lymphocyte (LGL) population with natural killer (NK) function is nonadherent and nonphagocytic. However, a significant proportion of the nonadherent cells purified by the two-step depletion of adherent cells with a plastic surface and nylon wool columns engulfed Sta. aureus into their cytoplasm. These cells were morphologically identified as LGL in light and electron microscopies. Two-color immunofluorescence tests, furthermore, demonstrated that Leu-11+ LGL, Leu-11+7-, and Leu-11+7+, but not Leu-11-7+, phagocytosed Sta. aureus. Among the particles tested here, only Gram(+) bacteria were preferentially phagocytosed, whereas Gram(-) bacteria, other large-sized microbes (e.g., baker's yeast and Candida albicans), latex, silica, and carbonyl iron were not. LGL exhibited a substantial level of bactericidal activity against Sta. aureus, although the level was one third of that mediated by monocytes. When Gram(+) bacteria were incubated with nonadherent cells for 18 hr, significant amounts of interleukin 1 (IL 1)-like factors (or IL 1 itself) as well as interferon were detected in the supernatants. On the other hand, this incubation did not induce interleukin 2 (IL 2). The IL 1-like factor producer cells were demonstrated to be the low-density lymphocytes on Percoll separation and to have the Leu-11+ phenotype. The phagocytosis was suggested to be an important stimulus in producing IL 1-like factors from LGL. Thus, the treatment of cells with cytochalasin B, a microfilament disrupting agent, completely abrogated both phagocytosis and IL 1-like factor production. Some cell wall components of Gram(+) bacteria might be important to a recognition process of the phagocytosis, since the protoplasts of Sta. aureus, when prepared by the treatment of bacteria with lysostaphin, were no longer phagocytosed by LGL. The present results therefore identify an additional unique characteristic similar to, but not identical with, the myelomonocytic nature of Leu-11+ LGL.     

Homeostatic Regulation of Membrane Potential by an Electrogenic Ion Pump against Change in the K+ Concentration of the Extra- and Intra-Organ Perfusion Solutions

The dependence of membrane potentials on changes in the extra-cellularK⁺ concentration [K⁺]_e was investigated in potato tuber sliceswith dripping perfusion, and in growing Vigna hypocotyl segmentswith pressurized intra-organ perfusion methods. Only under anoxiawere the membrane potential of potato tuber slices and the electricpotential difference between the parenchyma symplast and xylem(V_px) of Vigna hypocotyl segments depolarized markedly (46 mVand 42 mV/log[K⁺]_e unit, respectively) with increasing [K⁺]_eabove the critical values. The electric potential differencebetween the parenchyma symplast and organ surface (V_ps of thehypocotyl segments remained nearly unchanged up to 30 mEq [K⁺]_e.Under highly aerobic conditions the membrane potentials wererelatively independent of [K⁺]_e except at very high K⁺ concentrations.V_ps showed even hyperpolarization with the increasing KCl concentrationin the perfusion solution that is not in direct contact withthe surface membrane of the parenchyma symplast. The respiration-dependentelectrogenic components of the membrane potentials regularlyincreased with the increasing [K⁺]_e. A voltage-dependent homeostaticcontrol of membrane potential is discussed. (Received August 13, 1984; Accepted December 21, 1984)     

Correlation between X-chromosome inactivation and cell differentiation in female preimplantation mouse embryos

By means of a cytological method involving BrdU incorporation and acridine orange fluorescence staining in combination with embryo manipulation, we studied X-chromosome activity in female preimplantation mouse embryos with special reference to the correlation between X-chromosome inactivation and cell differentiation. There was no sign of asynchronous replication between the two X chromosomes from the one-cell to intermediate blastocyst stage. The allocyclic X chromosome, first detected in late blastocysts, was paternal in origin, mostly replicating early in the S phase and limited to the trophectoderm. Subsequent X-chromosome inactivation occurring in the primary endoderm was also characterized by the involvement of the paternal X and early replication. Both X chromosomes continued to replicate synchronously in the embryonic ectoderm or epiblast at this stage. It was evident that overt cell differentiation preceded the appearance of the asynchronously replicating X chromosome in the trophectoderm and primary endoderm. This finding seems to support the view that cell differentiation is an important correlate of X-chromosome inactivation.     

Intra uterine survival and growth of Wistar and Wistar x Brown Norway rat embryos

Embryos of the Wistar strain and its F(1) cross (Wistar females mated with Brown Norway males) of rats were transferred nonsurgically to 48 Wistar, 17 F(1) cross and 20 Wistar-Imamichi recipients. The two types of embryos were transferred together to each recipient to compare the viability of the embryos. Pregnancy rate was 78.8% (67 85 ). The survival rate of fetuses to term was 11.5% (20 174 ) and 25.1% (42 168 ) for the Wistar and F(1) embryos, respectively. Placental weight differed significantly (P<0.05) between embryo types and among recipient types while fetus weight differed (P<0.01) only among recipient types, with a significant interaction between recipient and embryo types (P<0.01). It was concluded that the F(1) embryos (Wistar x Brown Norway) were twice as viable as Wistar embryos under the conditions provided.     

Actions of amino-beta-carbolines on induction of sister-chromatid exchanges

Synthetic 3-aminoharman and 3-aminonorharman (amino-beta-carbolines) caused slight but definite induction of sister-chromatid exchanges (SCEs) in human lymphoblastoid cells NL3 and Chinese hamster cells CHO-K1. These amino-beta-carbolines are ranked between 2-amino-alpha-carboline and 2-amino-6-methyl-9a-aza-delta-carboline (Glu-P-2) and much lower than 3-amino-gamma-carbolines (Trp-P-1 and 2) in inductive activity. 1-Amino-beta-carboline, harman and norharman had very weak, if any, SCE-inducer activity. Norharman had a synergistic effect with aromatic amines such as Trp-P-2 and aniline on SCE induction, while 3-aminoharman suppressed SCE induction by more potent inducers such as Trp-P-2 and benzo[a]pyrene.     

Reversal of X-inactivation in female mouse somatic cells hybridized with murine teratocarcinoma stem cells in vitro

A series of near-diploid embryonal carcinoma-like hybrid cells were obtained from polyethylene glycol mediated cell fusion between murine embryonal carcinoma cells (PSA-6TG1 or OTF9-63) having one X chromosome and thymocytes or bone marrow cells from female mice carrying Cattanach's or Searle's translocation. Prior to fusion with EC cells the somatic cells are presumed to contain only one active X chromosome. Following hybrid formation, the chronology of X chromosome replication and the expression of X-linked gene Pgk-1 indicated that all X chromosomes contributed by both parents were active in these hybrids. Experiments were performed to rule out the possibility that the hybrids were formed by fusion of EC cells with rare somatic cells in which both X chromosomes were active. Taken together the data indicate that within four days of fusion there is reactivation of the entire inactive X chromosome.     

Measurement of turbulence intensity in the center of the canine ascending aorta with a hot-film anemometer

The blood flow velocity near the central axis of the canine ascending aorta was measured with a hot-film anemometer. The cardiac output and the heart rate were controlled at will by means of an extracorporeal circulation and by atrial pacing. The turbulent component of the blood flow velocity was calculated using an ensemble average technique. Ensemble average turbulent intensity was also calculated to show the time course of turbulence in the aorta. The ratio of the mean turbulence intensity to the time mean sectional average velocity in the aorta was constant in most animals regardless of the changes in fluid mechanical parameters. The correlation between the frequency parameter and the relative mean turbulence intensity was weakly positive. The power spectrum of the turbulence was also calculated.