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Studies on the isolation and characterization of DNA from yeast mitochondria (Mt-DNA)4 have been extended to mitochondria from respiratory deficient cells (ϱ− petites). Under appropriate conditions assuring the absence of gluocse repression such cells have been found to contain mitochondrial DNA in amounts comparable to those of the wild type. Mt-DNA from a neutral petitite exhibits a buoyant density in CsCl and a thermal transition midpoint (Tm) very close to that of its isogenic wild type. Mt-DNA's from two other isogenic ϱ− strains, known to be 10 and 50% suppressive respectively, show a very sharp thermal transition with a Tm of 66.5 ° in 0.15 m NaCl—0.015 m sodium citrate. Upon quick cooling almost complete renaturation is observed, as evidenced by second-cycle heating, again with a sharp transition and a Tm of 66.5 °. The average value of the buoyant density of this DNA in CsCl (at 20 °) is 1.6685 which is 0.003 g/ml lower than that reported for synthetic or crab d(A — T). There is virtually no change in the density of this Mt-DNA upon heating and quenching, with or without prior exposure to proteases, again confirming the great ease of its renaturation. In alkaline CsCl its density increases by 0.076 g/ml, indicating strand separation, but upon reneutralization the density returns to that of native Mt-DNA. Ready renaturation, as measured by thermal profiles, is found even for partially degraded Mt-DNA molecules. All these observations suggest that ease of renaturation is due to compositional homogeneity and therefore does not require these DNA's to exist as covalently continuous circles. The composition and homogeneity of the Mt-DNA from one of these suppressive strains (D310-2A-184) was confirmed by direct analysis of base composition: It was found to contain an equimolar amount of G and C as well as of A and T with the former two bases accounting for no more than four mole percent. The implications of these findings with regard to the mutagenic events reponsible for the formation of these aberrant DNA's and their possible genetic capabilities are discussed.

Repression of respiratory activity in Saccharomyces cerevisiae by glucose has been studied. Experiments were designed so that repression was maximal within 4 hours after their start and substantially relieved within an additional 2 hours. Various parameters were studied as a function of this repression/de-repression cycle. These included alterations in a variety of respiratory enzymes; in their distribution in several particulate as well as the soluble fractions; in the number and morphology of mitochondria and other membranous elements; in mitochondrial cytochromes; in their ability to be subjected to respiratory control; and the ability of various fractions to incorporate radioactive amino acids into protein in a pulse-chase experiment. Based on these experiments a model is presented which postulates that, during repression there takes place, at least in part, a disassembly of functional mitochondria to nonfunctional, probably particulate, entities and that new functional mitochondria arise from them during de-repression.

Properties of the Escherichia coli protoplast-infective agent (π) derived from T2 are compared with those of T2, as well as with the published properties of pneumococcus transforming principle (TP). π is somewhat more resistant to heat inactivation than T2, much less stable than TP. The destruction of π is a complex process characterized by at least three different reactions. Ultraviolet light inactivates π at a rate closely comparable to that observed for T2 and qualitatively similar to that for TP. The data indicate that the π mixture contains many damaged particles incapable of producing intact virus but capable of contributing to multiplicity reactivation. Unlike T2 and TP, π shows a remarkable instability in solutions of high osmolarity and in solutions containing surface-active agents. The pH stability of all three infective agents is roughly similar. Antiserum against T2 inactivates π, but at a slower rate and to a lesser extent than T2. Protein-inactivating reagents (especially those affecting sulfhydryl groups) have a greater effect on T2. On the other hand, agents primarily affecting DNA and presumed mutagens have parallel effects on T2, on π, and, qualitatively, on TP. DNAase and trypsin have no effect on T2 but destroy π rapidly. TP is destroyed more rapidly by DNAase, but is unaffected by trypsin. It is concluded that π is a highly organized structure consisting of other components in addition to DNA. In size and physical properties π is similar to a phage head, but its sensitivity to various reagents indicates that at least some of the physically protecting protein envelope is lacking or altered. More than one component of the π mixture may contribute to the infectivity for protoplasts; these components seem to differ markedly in their stability to various treatments.

Deoxycytidylic deaminase (dCMP deaminase) was found in Escherichia coli after infection with T2 bacteriophage. This enzyme could not be detected in uninfected cells but appears 3–5 min. after infection. Enzyme synthesis could be stopped at any stage by addition of chloramphenicol (20 μg./ml.) to the medium. The enzyme activity reached a maximum approximately 12 min. after infection and declined thereafter. Ultraviolet irradiation of the phage prior to infection resulted in only little loss in the capacity to induce the synthesis of dCMP deaminase under conditions where the replication of the phage was strongly impaired.

1. 1. A method has been described to test hyaluronidase activity of saliva. 2. 2. Hyaluronidase-producing microorganisms have been isolated from 60 saliva samples. The predominant enzyme producer, an alpha hemolytic streptococcus, occuring in 55 per cent of the cases, was identified as Strepto-coccus mitis. Micrococcus pyogenes var. aureus was isolated from 18 per cent of the samples. Alpha and beta hemolytic streptococci of Lancefield groups A and K occurred in 11 per cent of the cases. 3. 3. Upper respiratory and dental diseases caused a marked increase in salivary hyaluronidase titer.

In a previous paper of this series (1) we have demonstrated that preparations obtained from chick embryos are capable of catalyzing all the individual reactions of the citric acid cycle, and that the activities of the various enzymes involved appeared to undergo changes in the course of embryonic development. This latter finding, for at least two of the enzymes studied, is in agreement with work from other laboratories (2, 3). The oxidative reactions of the cycle, except for the reaction catalyzed by succinic dehydrogenase, are linked to oxygen through the pyridine nucleotides. Thus it became imperative to study the aerobic oxidation systems for reduced diphosphopyridine nucleotide in order to obtain some understanding of the respiratory capabilities of embryonic tissues. Among work done by others on this subject, we may mention that Philips (4) measured the over-all oxygen consumption of the early chick embryo at various stages of development. He found, with unhomogenized tissue, and by means of Cartesian diver techniques, that the oxygen uptake with glucose as substrate, did not increase markedly between the first and third day, although he reported a 100 per cent increase in the Qo, during the first 4 hours of incubation. In later work Philips (5) showed that under his conditions there was no variation in the oxygen uptake of different regions of chick blastoderms between zero and 37 hours of incubation. A detailed discussion of the determination of Qo, values in the chick embryo has been given by Romanoff (6). Sippel (7) measured the increase of succinoxidase activity in the heart and heart forming tissue of the chick embryo with Cartesian diver and Warburg techniques. He reported an increase in the specific activity, relative to nitrogen, with age from the second day onward. Albaum et al. (8) also measured cytochrome oxidase manometrically in water homogenates of chick embryos and found an increase in activity per embryo which exceeded the corresponding increase in succinoxidase. More recently Davidson (9) assayed for cytochrome oxidase spectrophotometrically in buffered homogenates of embryonic organs. He found an increase in the specific activity of the heart and the liver from the fourth day on while the activity in the nervous system remained constant throughout the period of study. Scevola and De Barbieri (3) subjected homogenates of 5- and lo-day-old embryos to differential centrifugation. They report that the distribution of succinic oxidase and cytochrome oxidase in these fractions is not unlike that which might be expected with adult tissue. * Supported by Grant-in-aid H-2177 from the National Heart Institute, National Institutes of Health, United States Public Health Service, and a program grant of the

1. 1. The study of the effects of aliphatic diamines on the thermal stability of polynucleotides has been extended to an investigation of the interaction of these cations with the helical duplexes poly-A-poly-U and poly-I-poly-C. In both instances there is stabilization which decreases in the order Mg2+≅ diaminoethane>diaminopentane>diaminooctane, i.e. an “abnormal” order as compared to DNA. 2. 2. In aqueous mixtures of organic solvents diamines produce a profound stabilization of DNA with respect to thermal helix → coil transition. The absolute stabilization decreases in the order H2O > H2O-glycol >H2O-dimethylsulfoxide >H2O-dimethylformamide for any one cation, while in any one solvent the order of effectiveness for different cations is Mg2+ ⩽diaminoethane diaminooctane (the “usual” order, observed previously). 3. 3. Diamines also protect DNA against denaturation by acid. This effect is, however, chain length independent and does not alter any of the spectrophotometrically observable changes produced in DNA as a result of protonation. 4. 4. The addition of amines to DNA produces a relatively insignificant change in viscosity while lowering the sedimentation rate of high-molecular-weight DNA by about 50%. 5. 5. These and other results previously reported have been interpreted in terms of a model which postulates strong and specific interactions between two binding sites on the two antiparallel strands of a DNA helix and the added diamines, leading to a profound alteration in the “atmosphere” in the immediate vicinity of the stacked base pairs.

A variety of inhibitors has been shown to have pronounced effects on the replication of T2 bacteriophage in protoplasts of E. coli B. Ribonuclease applied in 5 or 10 min pulses exerts a maximum effect (40% reduction of burst size) when applied between 5 and 10 min after infection. p -chloromercury benzoate shows a sharp maximum of effectiveness and inhibits the burst completely when added 15 to 20 min after infection. Of a variety of purine and pyrimidine antagonists, 5-fluorouridine and 2′-deoxy-5-fluorouridine showed the most striking effects. The latter compound appears to have a specific inhibitory action on DNA synthesis and the inhibition (some 98%) is essentially completely reversed by thymidine. The action of 5-fluorouridine appears to be considerably more complex. In part its effect is also directly on DNA synthesis and is reversible by thymidine. During the first 10 min of infection, however, this inhibitor, especially when tested in the presence of thymidine, appears to exert its primary effect of RNA synthesis with resulting effects on the synthesis of “early protein” and hence DNA. These effects can be overcome by uridine. A combination of thymidine and uridine completely abolishes the inhibition by 5-fluorouridine even at quite high concentrations. These conclusions have been reached on the basis of measurements of phage yield and also of incorporation of 35 SO 4 and 32 PO 4 into the appropriate macromolecules.

The time courses of the radioactive labels in subcellular fractions from cerebral cortex of mature rats following intraventricular injection of 14C-orotate and 3H-leucine were followed from 4 hours to 12 weeks. Special attention was directed to fractions obtained from nerve endings by sucrose gradient centrifugation. After initial transients, the declines in radioactive labels usually followed first order kinetics from 2 to 12 weeks. Within experimental error, the τ values for RNA in all fractions except the nuclear were the same, i.e. 12 ± 1 days. The value of τ in days for protein labels included: mitochondria, 20; synaptic vesicles, 21; synaptic membranes, 21; nerve ending soluble protein, 22; cell body soluble, 16; ribosomes, 12. The nuclei included long lived components (τ g 40 days) of both RNA and protein. These results indicate that both synaptic vesicles and the adjacent membranes are in a dynamic steady state with respect to protein synthesis, but there is no evidence that vesicles are appreciably broken down as a consequence of electrical activity of the neurons.

The incorporation of l-[4,5-3H]leucine into slices of rat cerebral cortex has been measured, with emphasis on the time course of the distribution of label in subcellular fractions prepared by zonal isopycnic centrifugation in continuous sucrose gradients. Rapidly labeled proteins are found in both the soluble and membranous components of the nerve end particles. Except for a minor part of the mitochondrial protein, the synthesis of all membrane-bound proteins is inhibited drastically by cycloheximide. When synthesis is blocked by cycloheximide, the supply of label continues for some minutes to certain membrane fractions derived from synaptic structures, apparently by a rapid transport of newly synthesized proteins from cytoplasmic ribosomes in the neuronal perikaryon. An increased extracellular concentration of potassium ion inhibits and ouabain accelerates protein synthesis in some fractions. These effects suggest a coupling of the protein synthesizing system with the (Na+ + K+)-activated ouabain-sensitive adenosine triphosphatase in the plasma membranes.

A method is described for the purification of cytochrome c oxidase (EC 1.9.3.1) from a particulate fraction of Red Star yeast. The enzyme as isolated contains up to 7.2 nmoles of heme a per mg of protein and is free from contamination by hemes other than heme a. The spectrum of the reduced enzyme equilibrated with carbon monoxide is apparently dependent on the aggregation state of the enzyme. The fact that the enzyme is capable of assuming both a monomeric and a dimeric state of aggregation is demonstrated by sucrose density gradient centrifugation and susceptibility to dithionite reduction. After dissociation at 100° in sodium dodecyl sulfate-urea-mercaptoethanol, the enzyme shows four major polypeptide components when subjected to electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate and 6 m urea.

A number of biochemical properties of mitochondria from a cytoplasmic petite mutant ofSaccharomyces cerevisiae with an extremely high adenine plus thymine content have been studied. When such particles are isolated by means of standard procedures developed for use with wild-type yeasts they are grossly contaminated by non-mitochondrial membrane fragments. Further enrichment of mitochondria is achieved by non-equilibrium centrifugation in sucrose gradients. Throughout this purification procedure the particles can be shown to retain an outer limiting, as well as a non-cristate inner membrane. In many of their morphological and physical features (size, shape, buoyant density) they resemble mitochondria isolated from the wild type. Although enzymes of the respiratory chain are absent from the mutant particles, their content ofl-malate dehydrogenase, NADP-dependent isocitrate dehydrogenase, and ATPase is comparable to that found in the wild type. The mitochondrial ATPase in this mutant strain is cold stable, oligomycin insensitive, Dio-9 sensitive, and susceptible to inhibition by the F1 inhibitor of beef heart. The enzyme can be rendered cold labile by its detachment from the membrane, followed by fractionation with protamine sulfate and ammonium sulfate. The existence of mutant particles that are incapable of function in oxidation and phosphorylation but resemble their functional homologues in many other ways raises the possibility that mitochondria are required in the cellular economy for purposes not directly linked to oxidative phosphorylation and electron transport. This hypothesis has led us to suggest that, contrary to models currently under discussion, mitochondria did not evolve as a consequence of endosymbiosis. We propose as an alternative that the mitochondrial organelle evolved as a means of improvement of existing subcellular structures in the primordial (perhaps eukaryotic) cell. Partial autonomy may thus constitute a relatively recent modification; the present-day mitochondrial genome had its origin in nuclear DNA and may have been amplified in a manner not unlike the amplification of ribosomal RNA cistrons in developing oocytes ofXenopus.

Two experiments were conducted in the west side of Kern County to evaluate and develop appropriate reclamation procedures for salt-affected high boron soils. The experiments consisted of sprinkler irrigation applied at two rates with straw, gypsum, and slip-plowing. The reclamation was primarily in response to depth of water percolating through the soil profile and that neither amendments nor slip-plowing facilitated reclamation. Soluble salts were reduced to safe levels throughout 5-foot soil profiles by application of about 5 acre-feet of water. Reduction of boron and exchangeable sodium to safe levels required approximately 15 acre-feet of water. No resalinization occurred upon fallowing leached soil profiles for 12 months.

The isolation, purification, physical and chemical properties of a mitochondrial DNA of Saccharomyces cerevisiae are described and compared with the nuclear DNA of the same organism. The two entities are clearly distinct in absorption spectra at various pH values, thermal transition temperature in two different solvent systems, buoyant density in CsCl and base composition, all of which are consistent with a GC content of 35 % for nuclear and 21 % for mitochondrial DNA. The latter, a normal double helical molecule, occurs integrated into respiratory particles which renders it DNase resistant. It is present to the extent of 0.71 μg per mg of particle protein in highly aerobic cells of the wild type, decreases during glucose repression and is greatly reduced (⩽ 0.06 μg/mg protein) in a cytoplasmic respiratory-deficient mutant (vegetative petite, ϱ−). The So20,w of the isolated DNA equals 33 s, hence its molecular weight is approximately 2 × 107. Evidence is also presented for its preferential interaction with acridines. The implications of all these findings in the problems of mitochondrial autonomy and self-duplication are discussed.

Emetine (at 100 μg/ml) blocks the incorporation of 3 H-leucine into the total protein of slices from rat cerebral cortex by > 95%; the block on the synthesis of cytosol proteins is quantitative. Under these conditions protein synthesis resistant to emetine is localized in a mitochondrial fraction that is sensitive to chloramphenicol at 100 μg/ml to the extent of ∼ 70%. We infer that some 15% of the proteins of cerebral cortex mitochondria formed during a 60 min period have been provided by their own protein synthesizing system.

Ribonucleic acid isolated from cerebral cortex of mature male and female rats was fractionated by chromatography on methylated albumin Kieselguhr columns or by sucrose gradient centrifugation. Three principal fractions were obtained with analytical ultracentrifuge s20,w0 values of 4, 17, and 28. By comparison with ribonucleic acid prepared from purified rat brain ribosomes, the 17 S and 28 S fractions were shown to be predominantly ribosomal ribonucleic acid. Differences in base analyses between the three fractions were small but significant. Compositions of all fractions were markedly complementary with G ≈ C and A ≈ U.

— The incorporation of l-[4,5-3H]leucine into the primary visual cells of lateral eyes of Xiphusura polyphemus (Limulus) was followed by electron microscopic radio-autography, while protein synthesis in the tissue surrounding the primary cells was measured by scintillation counting of the extracted protein. In the primary cells, the incorporation of radioactivity during 24 h into the rhabdomal membranes was 10–20 times greater in dark than in light, and was especially high in eyes that had been exposed to light before a period of incorporation in the dark. In tissue adjacent to the primary cells, protein synthesis was about 50 per cent greater in eyes exposed to light. These results are interpreted in terms of the photochemistry of Limulus rhodopsin and the competition for ATP between sodium-pump and protein-synthetic mechanisms.

Inhalt: Bei der Tiefgefrierung von 36 Hengstejakulaten nach der Pelletmethode lag die Vorwartsbewegung der Samenzellen nach dern Auftauen zwischen 15 % und 70 %, im Durchschnitt bei knapp 50 %. Sowohl zwischen den Ejakulaten verschiedener Hengste als auch denen desselben Hengstes bestanden in den Auftauergebnissen teil-weise erhebliche Unterschiede. Derartige Differenzen ergaben sich auch hinsichtlich der Lebensdauer der Samenzellen in aufgetautem und bei +5° C gelagertem Sperma. Von 21 Stuten, die mit in Pelletform tiefgefrorenem Sperma besamt wurden, konzipierten 10; 9 Stuten blieben gust, bei 2 anderen ist das Ergebnis nicht bekannt. Von den tragenden Stuten nahmen 8 in der ersten genutzten Rosse und je eine in der dritten bzw. funften Rosse auf. Die Trachtigkeiten wurden mit 3 Wochen bis 14 Monate altem, bei—196° C in flussigem Stickstoff gelagertem Sperma erzielt. Contents: Thirtysix ejaculates were collected from various stallions and the semen preserved by deep-freezing using the pelleting technique. After thawing these pellets in sterile-milk at +40 centigrade approximately 50 % (15–70 %) of the spermatozoa resumed full vigorous motility. There were marked differences in freezing-ability of the semen of the various males used and also in between ejaculates of the same semen donor. The same applied to thawed semen stored at 5 Centigrade, which showed different degrees of viability. Twentyone mares were inseminated; ten conceived, nine did not conceive, the result in two mares is unknown. Eight mares were settled during first oestrus, one during the third and one during the fifth. Pellets were stored at —196 centigrade in liquid nitrogen ower periods ranging from three weeks to fourteen month.

— Synaptic plasma membranes from the cortices of adult rat brain were isolated from synaptosomes prepared by flotation of a washed mitochondrial pellet (P2) in a discontinuous Ficoll-sucrose gradient. Contamination of the synaptosome fraction by microsomes was estimated by enzymic and chemical analysis to be less than 15 per cent. (2) The purified synaptosome fraction was subjected to osmotic shock, subfractionated on a discontinuous sucrose gradient and the distribution of enzymic and chemical markers for synaptic plasma membranes, microsomal membranes and mitochondria was determined. (3) Comparison of synaptosome subfractions prepared in the presence and absence of 1 mM NaH2 PO4/0.1 mM EDTA buffer pH 7.5, indicated that the ionic composition of the isolation medium markedly affected the distribution and enzymic composition of the subfractions. (4) Synaptic plasma membranes prepared in the presence of PO4/EDTA exhibited a 10-fold enrichment in [Na++ K+] ATPase and were characterized by less than 15 and 10 per cent contamination by microsomes and mitochondria respectively. (5) The polypeptide composition of the purified synaptic plasma membranes was compared with the microsomes and mitochondria by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. No differences between the protein and glycoprotein composition of the synaptic plasma membranes and microsomes were detected. The mitochondria, in contrast, possessed a unique protein composition.

In ts - 136, a temperature-sensitive mutant of Saccharomyces cerevisiae , nuclear and mitochondrial RNA production can be inhibited selectively by exposure to 36° and ethidium bromide, respectively. Using the programming of mitochondrial polysomes, as measured by their ability to form nascent polypeptide chains, as an assay for functional messenger RNA, we have determined its response to temperature shifts and ethidium bromide. Only ethidium bromide produced a measurable effect; in contrast the cell-sap system responded exclusively to temperature shifts. We conclude that transcription of mitochondrial DNA is sufficient and that import of messenger RNA transcribed from nuclear chromosomes makes no measurable contribution to intramitochondrial protein synthesis.

When two strains of phage T5 (heat-susceptible form T5 st + and its heat-resistant mutant T5 st ) were placed in solutions containing various high concentrations of chelating agents (sodium citrate and ethylenediaminetetraacetic acid) at room temperature, they could be effectively inactivated by rapid dilution in distilled water of relatively low temperatures (2 to 37 C). This phenomenon has been termed “chelating agent shock” (CAS). The susceptibility of phage T5 to CAS increased with an increase in the concentration of chelating agents and with an increase in temperature of the water used for rapid dilution. Under any given condition, T5 st + was much more sensitive to CAS than was T5 st . Phage T5 was protected against inactivation by the addition of monovalent or divalent metal salts, but not by the addition of nonionic solutes, to the shocking water prior to CAS treatment. This finding is compatible with the view that cations combined with the phage protein are removed by the chelating agent, although no metal ion has been identified in the phage protein. Alternatively, since the chelating agents used are polyanions, they may bind relatively tightly to the protein subunits in the head of T5, thereby distorting the structure of the phage head. Rapid dilution of these distorted particles could lead to loss of phage DNA. No evidence for recovery of phage activity could be obtained by the addition of metal salts to the inactivated phage after CAS. The morphological properties of phage inactivated by CAS are similar to those of heat-inactivated T5 phage. Electron micrographs showed that most of the phage particles consisted of empty head membranes; some of the particles had lost their tails. Both heritable and nonheritable resistance to heat was accompanied by resistance to CAS in phage T5. The sensitive element detected by each test seemed to be the same.

Nitrous acid has been found to be an effective mutagenic and inactivating agent for bacteriophage T2 in the vegetative phase. The mutagenic efficiency for infected 2- and 5-minute complexes of Escherichia coli B is at least as high as that observed with extracellular phage, both with respect to r and ht mutations. The kinetics of inactivation of infected complexes by nitrous acid has been shown to be sensitive to the stage of development and to the presence of chloramphenicol.