Search

Feasible bioprocessing of lignocellulosic biomass requires the use of microbial strains with tolerance to inhibitor compounds and osmotic pressure, able to provide high product yield and productivity. In this sense, this study evaluated the potential of two non-conventional yeasts, Hansenula polymorpha CBS 4732 and Debaryomyces hansenii CBS 767, for use on biomass conversion in a biorefinery perspective. The ability of the strains to consume pentose and hexose sugars, to resist the toxic compounds present in hydrolysates, as well as to produce sugar alcohols and ethanol, was investigated. H. polymorpha showed highlighted resistance to toxic compounds and relevant ability to consume xylose and produce xylitol and ethanol under these conditions, at 37 °C. D. hansenii was a great producer of arabitol from glucose. The implications for sustainability due to the use of these yeasts in biorefineries was discussed. These results open up new perspectives for the development of the biorefinery sector.

The sexually dimorphic cremaster nucleus contains motoneurons that project via the genitofemoral nerve and theoretically direct androgen dependent testicular descent. The effects of flutamide on descent and masculinization of the cremaster nucleus were studied in the rat and pig.Flutamide was given to pregnant rats and pigs on days 16 to 22 and 65 to 113 of gestation, respectively. Tissues were perfused and examined at birth (pigs) or at age 30 days (rats). Spinal cords were removed, sectioned and immunohistochemically stained for serotonin (rats) or substance P (pigs) to demarcate the position of the cremaster nucleus and allow the determination of cremaster motoneuron number.After exposure to flutamide testes were undescended in 6 of 9 rats and 7 of 10 pigs. Cremaster motoneuron number per nucleus were 288 +/- 22 in control versus 250 +/- 27 in flutamide treated rats, and 165 +/- 28 in control versus 148 +/- 24 in flutamide treated pigs. The decrease in motoneuron number by flutamide was significant in both species (p0.02) but it did not approach the levels in female rats (93 +/- 11) and pigs (57 +/- 12). Cremaster motoneuron number did not correlate with testicular position. Porcine undescended testes were associated with a significant increase in mean gubernacular volume.Unlike other sexually dimorphic spinal cord nuclei masculinization of the cremaster nucleus appears to be largely androgen independent and it does not correlate with ipsilateral testicular descent. These data suggest that androgens do not mediate descent of the testes via the efferent limb of the genitofemoral nerve.

We have tested the hypothesis that the glycoproteins in the cell membrane of axonal terminals are involved in high affinity uptake of neurotransmitters by studying the effects of lectin binding and trypsin treatment on this process in synaptosomes. Binding of two lectins, Concanavalin A and a lectin isolated from the lentil Lens culinaris, to synaptosomes does not change the uptake of six putative transmitters: L-glutamate, norepinephrine (NE), 5-hydroxytryptamine (5-HT), dopamine, choline (Ch), and γ-aminobutyrate (GABA). While trypsin digestion of surface proteins of synaptosomes has no effect on the uptake of NE, 5-HT, dopamine, Ch and GABA, it reduces the rate of uptake of L-glutamate. This reduction is not due to synaptosomal lysis or a profound conformational change of the synaptic plasma membrane since the maximal velocity of high affinity uptake is reduced drastically with little attendant change in Km.

The preparation of a four-subunit enzyme from yeast, capable of catalyzing the oxidation of ferrocytochrome c, is described. It is derived from proteins containing seven or five subunits by means of recycling exclusion chromatography in the presence of 0.1% sodium dodecyl sulfate. Its catalytic properties are similar to those of the parent enzyme. Gel electrophoresis of this preparation in the presence of sodium dodecyl sulfate reveals three bands, migrating with RF values that correspond to molecular weights of 14.6, 12.3, and 10.6 X 10(3), with the largest exhibiting an apparent 2:1 stoichiometry relative to the other two. Visible spectra in the region of 390 to 630 nm do not show any detectable difference from that of the parent cytochrome oxidase, while its heme a and copper content are raised to values around 20 nmol or ng atoms/mg of protein, respectively, corresponding to minimal molecular weights of 50 X 10(3). The molecular weight determined by physical means equals 107 X 10(3). Thus the enzyme probably contains two copies of each subunit. After extensive dialysis to remove as much as possible of the sodium dodecyl sulfate used in its preparation, this enzyme remains in solution in phosphate buffer in the absence of any added detergent, while under similar conditions the seven-subunit complex precipitates completely. A similar preparation can also be obtained from beef heart. The significance of these findings is discussed with respect to the role of the large subunits in the function as well as the biogenesis of the mitochondrial cytochrome oxidase complex.

Highly active, essentially homogeneous, preparations of ferrocytochrome c oxidase (EC 1.9.3.1) have been obtained from both yeast and beef heart by extraction with cholate, fractionation with ammonium sulfate, and replacement of cholate by Tween 20. The molecular weights of the resultant proteins equal 260 +/- 23 X 10(3) and 205 +/- 10(3); they contain seven and six different polypeptide subunits, respectively, all in equimolar amounts, with apparent molecular weights of 42.4, 34.1, 24.7, 14.6, 14.6, 12.3, 10.6 X 10(3), and 47.5, 20.4, 14.5, 14.5, 13.0, 11.0 X 10(3), respectively. By means of apolar chromatography on L-leucine coupled to agarose these enzymes can be stripped of their largest subunit(s) resulting in preparations with molecular weights of 170 +/- 17 X 10(3) and 124 +/- 20 X 10(3), and containing only five polypeptides, with the largest remaining one (molecular weight congruent to 20 X 10(3)) present in less than stoichiometric amounts. This interconversion and subunit removal has been monitored by exclusion chromatography, four systems of acrylamide gel electrophoresis--some with the protein labeled with 125I under denaturing conditions--isoelectric focusing, and hydrodynamic methods. It has virtually no effect on heme a and copper content and on the catalytic parameters of the enzymes. We conclude that subunits I and II in enzymes from fungal, and subunit I in those from animal, sources are dispensable for the catalysis of the oxidation of ferrocytochrome c by, and are probably not essential for the attatchment of prosthetic groups to, these proteins.

Protein synthesis was studied in slices from rat cerebral cortex and localized in various purified subcellular membrane fractions isolated after incubation with L-leucine. A synaptosomal fraction isolated from a several times washed crude mitochondrial pellet showed very little contamination by free membranes, and the synaptic membrane fraction isolated from it was estimated to c +ain about 50% of this component. Leucine incorporation into all fractions was highly sensitive (greater than 95%) to emetine except for the cell and synaptic mitochondrial subfractions. They were only 60 to 70% inhibited by emetine and showed 10 to 20% inhibition by chloramphenicol which probably was due to those proteins synthesized in situ by the mitochondria. The net rate of incorporation of labeled protein into the synaptosomal and synaptic membrane subfractions was low, and axonal flow was excluded as a significant source of this label. On the basis of autoradiography it was concluded that the majority of the protein label was contributed by free and membrane-enclosed ribosome-containing contaminants in these fractions. Unlabeled and labeled subcellular fractions were also analyzed by electrophoresis in the presence of sodium dodecyl sulfate. The staining profiles of the microsomal and synaptic membrane subfractions were nearly identical, whereas those of synaptic mitochondria and the soluble proteins of the cell bodies were unique. The labeling of all proteins was blocked by emetine, except for the synaptic mitochondria (cell mitochondria were not examined). These products of mitochondrial protein synthesis exhibited three labeling peaks, the major one with a molecular weight of approximately 38,000. In the absence of emetine, slices incorporated amino acids into soluble and microsomal proteins with high specific activity. The labeled proteins of the soluble fraction were more highly concentrated in the range of molecular weights smaller than 50,000 than were those in the microsomal fraction, and it was concluded that a considerable portion of the protein-synthesizing machinery of the brain is geared to form soluble proteins. The gel patterns of labeled microsomal and synaptic membranes were consistent with contamination by the former contributing most of the protein label in the latter.

When starved, exponential phase cells of Saccharomyces cerevisiae are exposed to 10–25 μM ethidium bromide in buffer, the proportion of respiration deficient mutants in the population exhibits a rapid increase, followed by a pronounced decrease. This “self-rescue” by the mutagen can be dissociated from and studied independently of its mutagenic action and is shown to exhibit different requirements. These and related observations have been used to formulate a consistent model for mutagenesis by ethidium bromide and its modulation under a variety of conditions.

When isolated, respiring mitochondria of Saccharomyces cerevisiae are exposed to ethidium bromide (EtdBr) they are capable of catalyzing the following discrete reactions. mtDNA + mEtdBr (Me2+)/→ [EtdBrm·DNA'] (1) [Etdbrm·DNA'] + (ATP) (nuclease(s))→ fragments + ... (2) (Reactions 1 + 2) mtDNA (3) + mtEtdBr + (ATP) → fragments + ... nATP ([EtdBrm·DNA'])/→ nADP + nPi (4) In this scheme (EtdBrm·DNA'] represents a novel, stable derivative of EtdBr, linked (probably covalently) to mitochondrial DNA fragments (DNA') which, as isolated, have a mean mass of 12.5 x 106 daltons. This intermediate had previously been shown to be formed in vivo and in vitro with kinetics of appearance and degradation consistent with it being the first product in the mutagenic sequence known to be initiated by EtdBr. Experiments using respiratory inhibitors and uncouplers suggest that Reaction 1 depends on an energized state of the inner membrane, while Reactions 2 and 3 are driven by ATP, either added externally or generated by oxidative phosphorylation. Simultaneously there is an enhancement of the hydrolysis of added ATP to ADP plus Pi (Reaction 4). Experiments with inhibitors, plus the use of mutants with lesions either in their mitochondrial ATPase complex or in enzyme(s) involved in the repair and recombination of their DNA, have shown that Reactions 2 (and hence 3) and 4 are tightly linked. Thus the DNAse (Reaction 3) depends on an intact coupling device and the EtdBr-induced uncoupling (Reaction 4) is dependent on the presence of mtDNA and appropriate enzyme(s) for its degradation. The addition of uncouplers, such as chlorocarbonylphenylhydrazone or colicin K, disrupts this link by providing an alternate pathway for the activation of ATPase and leads to an immediate cessation of Reaction 2. Euflavine, a known antagonist to EtdBr in vivo has no effect on any of the reactions by itself, but blocks Reactions 2, 3, and 4 when added simultaneously with or subsequent to EtdBr. Strains of S. cerevisiae known to be resistant to mutagenesis by EtdBr all exhibit a lowered rate of Reaction 3 but for different reasons: some exhibit a lowered capacity to form [EtdBr·DNA'], others to bring about its degradation. Obligately aerobic, petite negative yeasts are known not to be affected by EtdBr in a permanent fashion: mitochondria of all these strains show a complete inability to catalyze Reaction 1.

1. The subcellular distribution of binding sites for 125I-labeled alpha-bungarotoxin was studied in rat cerebral cortex. Primary fractions showing higher specific activity than homogenate were P2 (crude mitochondria and nerve endings) and P3-P2 was subfractionated on a Ficoll gradient with the P2B (nerve ending) subfraction exhibiting the greatest recovery (65%) and enrichment of toxin binding. Toxin binding showed a distribution similar to that of acetylcholinesterase, choline acetyltransferase, and sodium and potassium ion-activated ATPase. 2. P2B and P3 were subfractionated on five-step discontinuous sucrose gradients. The highest specific activity of toxin binding and acetylcholinesterase was associated with fractions of relatively low buoyant density, while choline acetyltransferase activity was associated with fractions of higher density. 3. Toxin binding, acetylcholinesterase, and choline acetyltransferase activities were relatively high in olfactory lobes, cerebral cortex, thalamic region, caudate nucleus, and brain stem; intermediate in hippocampus; low in cerebellum. 4. The relationship of toxin binding to the putative acetylcholine receptor in brain is discussed.

—Acetyl-CoA:choline-O-acetyltransferase (ChAc, EC 2.3.1.6) was purified from rat cerebral cortex and its half-life determined. The molecular weight of the enzyme under non-denaturing conditions was estimated by gel filtration to be in the range of 60,000–65,000. On SDS acrylamide gels, the purified enzyme migrated as a single band with a molecular weight estimated as 62,000. The turnover rate of ChAc in the mature rat was determined by the double label method, employing l-[1-14C]leucine and l-[4,5-3H]leucine. Its half-life under steady-state conditions was estimated to be 5.2 days. As a control, tubulin was isolated from the same preparation and its half-life measured. Under these conditions tubulin exhibited a half-life of 3.8 days.

— The subcellular distribution of binding sites for tritiated quinuclidinyl benzylate ([3H]QNB) was studied in cow retina. Primary fractions showing higher specific activity than homogenate were P2 (synaptosomal-mitochondrial) and P3 (microsomal). P2 was subfractionated on a Ficoll gradient with the P2B subfraction exhibiting the greatest enrichment in [3H]QNB binding. Similar subfractionation of P2 on a discontinuous sucrose gradient showed that fractions of particles banding in 0.8m and in the 0.8-1.0 m-sucrose interface also exhibit the greatest enrichment of [3H]QNB binding. When subjected to Scatchard analysis, this reaction shows a density of sites equal to 0.212-0.294 pmol per mg of protein. By plotting the apparent dissociation constant (KD) values vs protein concentration a‘true’KD value of 0.73 nM was obtained. Only one set of binding sites was found using three different concentrations of protein. The reaction was specificially antagonized by atropine (DI50= 7 nM) and scopolamine (DI50= 9 nM) whereas carbamylcholine and d-tubocurarine exhibited DI50's of 0.4 and 0.15 mM, respectively. For P3 the binding of [3H]QNB is characterized by one set of binding sites with ni= 0.250 pmol per mg of protein and an apparent KD of 8.2 nM, and a DI50 for atropine of 15 nM. The [3H]QNB binding sites showed a subcellular distribution similar to that of acetylcholinesterase and choline acetyltransferase. P1 fractions accounted for 40–60% of the total activity of the three cholinergic markers. Purification of the crude P1 yielded an additional fraction in which the cholinergic markers showed an enrichment with respect to homogenate and P1. Synaptosomes isolated from this fraction exhibited the unusual ultrastructure expected from nerve endings in the outer synaptic layer of retina. The possible location of the muscarinic cholinergic transmitter system in the vertebrate retina is discussed.

A novel class of respiration-deficient (RD) yeast mutants has been isolated by a procedure designed to enrich for mitochondrial mutations. Four such independently isolated mutants exhibit a phenotype that differs from the classical petite mutant and does not show complementation with a DNA-less (p0) member of the latter class. One of these mutants, strain 73/1 was subjected to further analysis and found to exhibit mitochondrial inheritance on the basis of: (a) lack of this complementation; (b) non-Mendelian segregation of the RD trait after meiosis of diploids formed by mating with standard auxotrophic testers; (c) segregation on mitosis of these diploids, with the RD trait exhibiting vegetative dominance; (d) elimination of the trait and of c upon conversion to a p0 mutant by prolonged growth in the presence of ethidium bromide. Examination of the phenotype disclosed the absence in cells and mitochondria of cytochrome oxidase, cytochrome aa3, and of NADH- and succinate-cytochrome c reductases, and the presence of significant amounts of cytochromes b and c1. These two cytochromes appear reducible by succinate to a small and by NADH to a considerable extent. The mitochondrial ATPase in the mutant is sensitive to the specific inhibitor Dio-9 but resistant to oligomycin at concentrations that inhibit the wild type enzyme ≥50%. Unlike cytoplasmic petites, mitochondria of mutant 73/1 contain a functional protein-synthesizing system. Its cells appear capable not only of mitochondrial polypeptide chain initiation as measured by the formation of formylmethionyl puromycin, but also of incorporating labeled amino acids into mitochondrial proteins in the presence of cycloheximide, a process inhibited by chloramphenicol and acriflavin. The presence of a functional mitochondrial system of protein synthesis is confirmed by the elimination of the cytochrome b peak at 560 nm upon prolonged growth (six generations) in the presence of chloramphenicol. This conversion to a petite phenocopy is reversed completely upon removal of the inhibitor.

An experimental model was explored for the study of the effect of thyroid hormone on lipid transport and metabolism in the rat. The development of the hypothyroid state was accompanied by an early decrease in free fatty acids (FFA) followed by an increase in the level of cholesterol and a decrease in triglyceride concentration. Simultaneously, the adipose tissue lipoprotein lipase activity increased while the lecithin-cholesterol acyltransferase was unchanged. The hypothyroid state was also accompanied by a decrease in the concentrations of tryglycerides and cholesterol in very low density lipoproteins (VLDL) while the 2 lipids increased in the low density lipoprotein (LDL) fraction. Administration of triiodothyronine markedly reduced the amount of cholesterol in both the LDL and high density lipoprotein (HDL) fractions. The animal model has been shown to have similarities with findings in human subjects.

A mutant of Saccharomyces cerevisiae has been isolated which, though exhibiting a normal response to nuclear genetic damage by ultraviolet light (UV), is more sensitive than its wild type specifically in the production of the cytoplasmic (rho-) mutation by this agent. Some of the features of this mutation which has been designated uvsrho 5 are: i) The mutation is recessive, it exhibits a Mendelian, and hence presumably nuclear, pattern of segregation, but manifests its effects specifically and pleiotropically on mitochondrial functions. ii) Mutant cells resemble their wild type parents in a) growth characteristics on glucose; b) in their UV induced dose response to lethality or nuclear mutation and c) the ability of their mitochondrial genome, upon mating with appropriate testers, of transmitting and recombining various markers, albeit with enhanced efficiency. Similarly, d) they are able to modulate the expression of mitochondrial mutagenesis by ethidium bromide. Thus their mitochondrial DNA appears genetically as competent as that of the wild type. iii) Mutant cells differ from their wild type parents in a) growth characteristics on glycerol; b) susceptibility to induction of the mitochondrial (rho-) mutation by various mutagens, in that the rate of spontaneous mutation is slightly and that by UV is significantly enhanced, whild that by ethidium bromide is greatly diminished. Conversely, c) modulating influences resulting in the repair of initial damage are diminished fro UV and stimulated in the case of Berenil. iv) The amount of mitochondrial DNA per cell appears elevated in the mutant, relative to wild type, and its rate of degradation subsequent to a mutagenic exposure to either UV or ethidium bromide is diminished. v) A self-consistent scheme to account for this and all other information so far available for the induction and modulation of the (rho-) mutation is presented. In a previous study it was shown that some nuclear mutants of Saccharomyces cerevisiae, more sensitive to lethal damage induced by ultraviolet light (rad) than their parent wild type (RAD), also exhibit a concomitant modification in sensitivity to both nuclear and cytoplasmic genetic damage (Moustacchi, 1971). However, another class of rad mutants respond to the induction of the cytoplasmic "petite" also designated as rho- (or rho-) mutation by UV in a manner indistinguishable from that of the RAD strain. One possible interpretation of this last observation is that some of the steps in the expression of the UV damage on mitochondrial (mt)DNA may be governed by other nuclear and cytoplasmic genetic determinants, the products of which may then act specifically on mitochondrial lesions. If this assumption is correct, it should be possible to find mutants with a normal response to nuclear damage but specifically UV-sensitive towards induction of (rho-)...

— A system was devised for the study of protein turnover in isolated lateral eyes of Limulus. In eyes incubated in a medium containing L-[3H]leucine, radioactivity of the fraction soluble in trichloroacetic acid increased steadily during 8 h. Amino-acid incorporation into proteins was investigated by scintillation counting of trichloroacetic-acid precipitates and by radioautography. At least 89% of the incorporation was inhibited by puromycin and 86% by emetine, an inhibitor of cytoplasmic protein synthesis. Label accumulated preferentially in the rhabdomes, which contain the visual pigment. Radioautography indicated that labeled protein, probably synthesized in the cytoplasm, was distributed in the microvilli of the photoreceptor cells in a diffuse pattern. In contrast to in vivo results, the photoreceptor cells in isolated eyes were labeled to a greater extent in light than in the dark. These different kinetics of labeling were both explained by the hypothesis that light increased the rate of degradation of proteins (particularly opsin) in the visual cells.

Possible photoaffinity probes for muscarinic acetylcholine receptors have been explored for the first time: Specific [ 3 H]-quinuclidinyl benzylate binding sites of several fractions from rat brain can be irreversibly inactivated by photoaffinity labeling with two p-azidophenylacetate esters of tropine. Inactivation of these sites depends on formation of a reversible complex with the azides prior to their photolytic conversion to the highly reactive nitrenes; it is dependent on ligand concentration and length of photolysis. Atropine and oxotremorine, but not d-tubocurarine, afford protection against photoinactivation. These findings suggest the utility of these and related azido derivatives as potent, selective photoaffinity ligands directed against binding sites for muscarinic antagonists.

— RNA labelling patterns in nuclei from rat cerebral cortex were investigated subsequent to intracerebral injection of [3H]uridine. Although there was a rapid uptake of label into the ‘heavy’ regions when nuclear RNA was analysed in density gradients, it was not possible to show conclusive evidence for 455 ribosomal precursor RNA. Methyl-ation of 18S and 28S nuclear RNA became evident only after 2 hr and did not appear to involve the intermediacy of RNA species of higher molecular weight.

The characteristics of DNA-dependent RNA polymerase activity in intact rat cerebral cortex nuclei tested at low ionic strength are presented. The system was most dependent on the presence of spermidine and an ATP-generating system and to a lesser extent on Mg2+ and K+ for maximal incorporation. Substitution of Mg2+ by Mn2+ or of K+ by Na+ resulted in substantially less activity than under the optimum conditions described. Maximal incorporation was about 10 per cent that of brain nuclear systems of high ionic strength. In addition, the labelling patterns of the in vitro RNA products were shown to be very similar to those found in vivo. The stability of isolated nuclei toward degradation of RNA synthetic capacity and products formed was much greater than that of a similar liver system.

1. 1. The interaction of nucleic acids with aliphatic diaminns (H 2 N(CH 2 ) n NH 2 ; 2≦ n ≦10) has been investigated, largely by means of helix → coil transition (absorbancy-temperature) profiles. In general the most pronounced effects effects produced by amines were those on T m , the transition midpoint, which is increased (rise ≡ ΔT m ) in the presence of diamines depending on the following parameters. (a) The ionic strength: for any one DNA and amine a decrease in ionic strength leads to an increase in ΔT m and in the strength of the interaction. (b) The concentration of amine: a typical saturation behavior is observed with ΔT m proportional to the amine concentration at low values of the latter and reaching a limiting value ΔT m (max.) at high concentration. Half saturation with diaminopentane is reached at 10 −2 M amine at high, and 10 −4 M amine at low ionic strength. (c) The chain length of amine: the effect is optimal when n = 5 and decreases on either side of the optimum. (d) The conformation of the nucleic acid: the effect is exhibited by double-stranded DNA but not by RNA or single-stranded DNA, nor by “acid” polyadenylic acid. (e) The base composition of the DNA: ΔT m is related to mole % (adenine plus thymine) or, more rigorously, proportional to T m of the DNA used. Teh proportioonality constants are −0.13 and −0.47° per degree in high- and low-ionic-strength media, respectively. 2. 2. These observations have been interpreted in terms of a model which postulates m independent binding sites for diamines on a DNA helix, where m ⪡ total number of nucleotide pairs. These sites involve bridging between the phhosphate residues of one strand and the adenine and/or thymine residues of the other, with the most stable interaction occuring for n = 5.

The oxidation of N -benzyl-1,4-dihydronicotinamide (BzNH) by 2,5-dihydroxy-benzoquinone (Q(OH) 2 ) in the presence of air has been studied as a possible enzyme model. The rate of disappearance of the dihydropyridine was found to depend on both the concentration of quinone and the concentration and nature of the buffer added. The latter effect appeared to be primarily exerted on the spontaneous oxidation of the dihydropyridine by air. In phosphate buffer at 37° and a constant ionic strength, the empirical rate law was shown to be − d [BzNH]/ dt =[BzNH] ( k 1 ′[phosphate]/( K + [phosphate]) + k 2 ′[Q(OH) 2 ]). The experimentally determined values for the various constants in this expression were: k 1 ′ = 25 ± 5 × 10 −5 sec. −1 ; k 2 ′ = 5.0 ± .5 × 10 −2 M −1 sec. −1 ; K = 0.12 ± 0.2 M . A mechanism which postulated the reversible formation of a stoichiometric complex of BzNH and phosphate was presented and shown to give rise to the observed rate law.

A method is described for the synthesis of labeled ethidium (3,8-diamino-5-ethyl-6-phenyl phenanthridinium) bromide. Based on benzoic acid, the radioactive precursor used, the yield is 15% of a compound that is indistinguishable from authentic ethidium bromide in its absorption spectra (uv, visible, ir), Chromatographie behavior, and mutagenic effectiveness in the induction of respiration-deficient cell lines in baker's yeast.

The incorporation of labeled precursors into ribonucleic acid with cell-free preparations of animal tissues has been observed in a number of laboratories (l-lo). Further studies with partially purified preparations indicate that the proximate precursors are nucleoside trirather than diphosphates (24). These enzyme preparations, unlike deoxyribonucleic acid polymerase (11-13) or polynucleotide phosphorylase (14)) incorporate adenine nucleotide into RNA almost exclusively at the terminal position of the polyribonucleotide chain adjacent to a cytosine monophosphate residue (2). The final product, adenosineCMP-CMP-RNA is the amino acid acceptor required for the transfer of aminoacyl residues to the site of their ultimate incorporation into proteins (15-18). Edmonds and Abrams (3), however, have provided some evidence for a separate enzyme, responsible for the incorporation of the adenine nucleotide moiety of ATP into internucleotide linkages in RNA. Herbert (10) has reported that this type of reaction is enhanced if cytoplasmic enzyme preparations are supplemented by nuclei.’ In the course of studies on the possible occurrence of polynucleotide phosphorylase in extracts of tissues of chick embryos, we investigated the exchange of labeled inorganic phosphate and pyrophosphate with ribonucleoside diand triphosphates. Although the rate of incorporation of phosphate into ribonucleoside diphosphates was rather low, that of inorganic pyrophosphate into ribonucleoside triphosphates appeared relatively high and independent of the presence of amino acids, but dependent on the presence of polynucleotide (19-21). The present communication examines the reactions involved in more detail. When the soluble fraction of homogenates of 14-day-old chick embryonic livers or hearts was purified by means of ammonium sulfate and calcium phosphate gel, an enzyme preparation was obtained which catalyzed the incorporation of the AMP moiety of ATP into RNA. The incorporated adenine nucleotide was found almost entirely at the terminal position of the polynucleotide chain. The reaction did not take place when ADP was substituted for ATP. The incorporation of adenine nucleotide into the interior of the polynucleotide chain could be shown to take place in the presence of a cruder enzyme preparation. This system required ATP as a substrate and was dependent on the presence of Mg++. Addi

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.

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.

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 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.

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.

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.

Initial steady-state velocities using highly purified deuterated substrates and coenzymes as well as an isoenzymatically homogeneous enzyme preparation were measured for liver alcohol dehydrogenase at 25 deg in 20 mm phosphate (pH 7.0) or 20 mm pyrophosphate (pH 8.8). Significant isotope effects of v/sub 0,H//v/sub 0,D/ were observed. In the reduction of acetaldehyde by A- NADD the isotope effects were found to decrease with increasing acetaldehyde concentration, a result consistent with an ordered Theorell-- Chance mechanism for the reduction of acetaldehyde only at saturating substrate concentrations. No kinetically significant interactions were observed between the enzyme and the untransferred hydrogen of NADH. (auth)

Isolation of synaptosomes and synaptic membranes from the cerebral cortex of the rat, monkey, cat and pig was achieved by centrifugation of the crude mitochondrial fraction through a continuous sucrose gradient using high-speed zonal rotors. Electron microscopy and the activity of certain marker enzymes, such as choline acetyltransferase, acetylcholinesterase and (Na + -K + )-activated ouabin-sensitive ATPase, were used to characterize the various gradient fractions. In all four species studied the synaptosomes were found in the region from 31 to 37% sucrose of the gradient of the crude mitochondrial fraction (P 2 ) and a synaptic plasma membrane fraction was found in the region from 24 to 31% sucrose of the gradient of the lysed crude mitochondrial fraction (P 2 L). In the case of the rat and monkey there appeared to be some resolution of noncholinergic nerve endings from cholinergic endings.

1. 1. The thermal helix → coil transition of 3 different DNAs and 4 different RNAs in H 2 O and D 2 O solutions has been studied by means of absorbancy-temperature profiles. There is no significant difference in total hyperchromicity, the midpoint or the width of the transition, even after extensive preincubation of the nucleic acid at elevated temperature in the fully deuterated medium. This absence of any isotope effect for denaturatiion is also independent of base composition, which varied from 35.2 to 65 and 40 to 64 mole % (guanine plus cytosine) for DNA and RNA, respectively. 2. 2. In a medium containing 1.5·10 −3 M NaCl, 1.5·10 −4 M sodium citrate and 1.0·10 −3 M MgCl 2 there is a significant difference in T m , the transition temperature, of three transfer RNA's which appears to be correlated with their guanine plus cytosine content: for alanine, tyrosine and valine RNA the values are 73°, 52.5° and 49.5°, respectively.

The ultraviolet absorption spectra of a number of DNA's of natural origin, and varying in base composition between 73 and 37 mole per cent (guanine plus cytosine) have been obtained in a methanolic medium of low ionic strength both at room temperature and at a temperature 10°C above the midpoint for the helix → coil transition. In addition, the spectra of the stoicheiometric mixtures of deoxynucleotides corresponding to the identical base composition have been computed from published data. Similar sets of spectra have also been obtained for the synthetic DNA's, dAT and dG : dC. From these spectra we have calculated two sets of difference spectra (coils-helix) and (nucleotides-helix), as well as the corresponding wavelength-dependent hypochromicities as a function of base composition. The results have been interpreted in terms of a linear combination of difference spectra for isolated “average” A-T and G-C pairs in the helix, which differ from those of the corresponding synthetic, helical polydeoxyribonucleotides.

Tritiated reserpine binds to synaptic vesicles from bovine caudate with high affinity (Kappd = 1.25 nM, Bmax = 3.3 pmol/mg protein). This interaction is both ATP-dependent and sensitive to the protonophores CCCP and nigericin, suggesting that a proton electrochemical gradient is required for binding. Dopamine, epinephrine, norepinephrine and serotonin all inhibit reserpine binding at concentrations similar to those required for inhibition of dopamine uptake. Treatment with saponin to release vesicle contents results in complete loss of accumulated dopamine but retention of bound reserpine. these results indicate that reserpine binds to the catecholamine transport system of synaptic vesicles with high affinity and specificity.

Determination of dissociation constants by two different methods yield the following mean values in 20 millimolar phosphate, pH 7.0, 25 degrees C: 0.27 micromolar for reduced nicotinamide adenine dinucleotide (NADH); 0.29 micromolar for NADH with deuterium in the nicotinamide 4-B position (B-NADD); and 0.46 micromolar for NADH with deuterium in the nicotinamide 4-A position (A-NADD). These results indicate that dehydrogenases are capable of recognizing and distinguishing the appropriate hydrogen in the coenzyme already in the initial binding reaction.