The Male Sterility-Associated Pcf Gene and the Normal Atp9-1 Gene in Petunia Are Located on Different Mitochondrial DNA Molecules

A mitochondrial DNA (mtDNA) region termed the S-pcf locus has previously been correlated with cytoplasmic male sterility (CMS) in Petunia. In order to understand the relationship of the S-pcf locus to homologous sequences found elsewhere in mtDNAs of both CMS and fertile lines, the structure of the mitochondrial genome of CMS Petunia line 3688 was determined by cosmid walking. The S-pcf locus, which includes the only copies of genes for NADH dehydrogenase subunit 3 (nad3) and small ribosomal subunit protein 12 (rps12) was found to be located on a circular map of 396 kb, while a second almost identical circular map of 407 kb carries the only copies of the genes for 18S and 5S rRNA (rrn18 and rrn5), the only copy of a conserved unidentified gene (orf25), and the only known functional copy of atp9. Three different copies of a recombination repeat were found in six genomic environments, predicting sub-genomic circles of 277, 266 and 130 kb. The ratio of atp9 to S-pcf mtDNA sequences was approximately 1.5 to 1, indicating that sub-genomic molecules carrying these genes differ in abundance. Comparison of the mtDNA organization of the CMS line with that of the master circle of fertile Petunia line 3704 reveals numerous changes in order and orientation of ten different sectors.     

Preservation of RNA for in situ hybridization: Carnoy's versus formaldehyde fixation.

Tissues fixed with organic solvent fixatives such as Carnoy's solution are known to give poor and erratic results with in situ hybridization, whereas those fixed with paraformaldehyde produce more consistent results. To understand this difference and to improve the utility of Carnoy's-fixed tissue for in situ hybridization, we explored several parameters of RNA integrity and preservation. Carnoy's-fixed, paraffin-embedded livers and paraformaldehyde-fixed, paraffin-embedded livers of mice were compared for RNA extractability, degradation, and hybridizability. In addition, retention of RNA in tissue sections after sequential in situ hybridization treatments was compared. RNA was found to be easily extractable from Carnoy's-fixed liver and was well preserved, with only slight degradation of high molecular weight RNA. Conversely, only a small percentage of the RNA was extractable from paraformaldehyde-fixed liver unless the tissue was digested with protease. The extracted RNA was well preserved, without detectable degradation. Sections of tissue fixed in Carnoy's solution subjected to in situ hybridization retained only about 10% of their original RNA content and gave correspondingly weak in situ hybridization signals. Formaldehyde-fixed tissues retained much more of the RNA (about 45%) and produced strong in situ hybridization signals. Treatment of Carnoy's-fixed tissue sections with vaporous formaldehyde increased retention of RNA and provided in situ hybridization signals comparable with those of paraformaldehyde-fixed tissues.     

Transformation of Nε-CBZ-l-lysine to CBZ-l-oxylysine using l-amino acid oxidase from Providencia alcalifaciens and l-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus

Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O₂ oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O₂ oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H₂O₂:H₂O₂ oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions. Correspondence to: R. L. Hanson     

Citrate transport in corn mitochondria

Citrate uptake by corn mitochondria (Zea mays L. B73 × Mol9) was investigated by osmotic swelling and [¹⁴C]citrate accumulation. Uptake driven by passive influx, ammonium gradients, and respiration was followed. There was no requirement for phosphate and/or malate to secure citrate uptake, although under some conditions these additives were promotive. Inhibition of the phosphate and dicarboxylate carriers did not eliminate citrate uptake. Citrate_in/malate_out exchange occurs, but at a rate too slow to account for observed citrate uptake, and depletion of endogenous malate only reduced citrate uptake by 38%. It was concluded that citrate can be rapidly accumulated by a mechanism other than by exchange for dicarboxylates. The effect of uncoupler on respiration-driven [¹⁴C]citrate accumulation, and studies of passive swelling using ionophores and uncouplers indicated that the major avenue of citrate uptake is by H⁺/citrate co-transport with a pH optimum near 4.5. The in vivo role of this mechanism is not yet understood.     

Adenine nucleotide content of corn roots as affected by injury and subsequent washing

The adenine nucleotide content of the 2-centimeter segments excised from tray-grown corn (Zea mays L., WF9 × Mo17) roots declines for the first hour after excision. Concomitant with the loss of adenine nucleotides is a decline in respiration and a leakage of K⁺. With continued washing, these parameters partially or completely recover and increased phosphate influx develops. Increasing the wound effect by cutting 0.5-centimeter segments gives a more rapid and pronounced degradation of adenine nucleotides and slower recovery. Conversely, the mild injury caused by submerging intact roots induces less degradation and produces greater net adenine nucleotide synthesis during recovery; adding auxin to the washing medium produces a similar result. With all treatments, there is stabilization of energy charge at about 0.85.     

Real space refinement of neutron diffraction data from sperm whale carbonmonoxymyoglobin

Neutron diffraction data from crystals of sperm whale carbonmonoxymyoglobin have been refined by the real space refinement technique. Estimates of the neutron occupancies at the end of the refinement show that the mean for each atom type (including hydrogen and deuterium) is close to the expected value and has a standard deviation from the mean of about 5%. Mean neutron occupancies of main-chain atoms involved in deuterium bonds versus those not involved in deuterium bonds demonstrate that the hydrogen/deuterium exchange of the latter group is higher. The oxygen and deuterium co-ordinates for 40 water molecules have been determined: 27 of these water molecules were involved in bridges between protein atoms, and nine were involved in deuterium bonds with main-chain atoms. The deuterium-bond angles in helical regions show significant deviations from linearity. The mean ND … O angle was 154(3) °² and the mean CO … D angle was 145(3) °.     

Action of protein synthesis inhibitors in blocking electrogenic h efflux from corn roots

The block in the electrogenic H⁺ efflux produced by protein synthesis inhibitors in corn root tissue can be released or by-passed by addition of fusicoccin or nigericin. The inhibition also lowers cell potential, and the release repolarizes. Associated with the inhibition of H⁺ efflux is inhibition of K⁺ influx and the growth of the root tip; fusicoccin partially relieves these inhibitions, but nigericin does not. The inhibition of H⁺ efflux which arises from blocking the proton channel of the ATPase by oligomycin or N,N′-dicyclohexylcarbodiimide can also be partially relieved by fusicoccin, but not by nigericin; the inhibition produced by diethylstilbestrol is not relieved by fusicoccin.     

Photoaddition of trimethylpsoralen as a probe for the intracellular organization of Escherchia coli DNA.

It has been previously demonstrated that photoreaction with 4,5',8-tri-methylpsoralen (trioxsalen) can be used as a probe for the in vivo structure of eucaryotic chromatin. We have used this probe to analyze the organization of intracellular Escherichia coli DNA. In contrast to eucaryotic DNA, bacterial DNA within the intact cell is not protected from saturating doses of trioxsalen photoaddition. The final level of covalently bound trioxsalen upon saturation is identical to that found with purified DNA. In addition, the distribution of interstrand DNA cross-links formed by low doses of trioxsalen photoadducts does not exhibit the repeating pattern that has been observed with eucaryotic nucleosomes.     

Carbamyl phosphate synthetase A of Neurospora crassa.

Carbamyl phosphate synthetase A of Neurospora crassa was partially purified from mitochondrial extracts. It is an extremely unstable enzyme (t 1/2 = 45 min at 25 detrees C) made up of two unequal subunits. The native enzyme has a molecular weight of approximately 175,000, and the large subunit has a molecular weight of about 125,000. Both the native enzyme and its large subunit are quite asymmetric, as revealed by slow sedimentation in sucrose gradents (7.3S and 6.6S, respectively). The small subunit has not been identified physically as a separate entity. The denaturation of the native, glutamine-dependent activity is correlated with dissociation of subunits, the larger of which retains a more stable, ammonia-dependent activity. Neither substrates nor any other agents except glycerol or polyethylene glycol appreciably stabilized the glutamine-dependent activity. Kinetic studies showed the native enzyme to have a Km for glutamine of about 0.16 mM, and a Km for NH4Cl of about 16 mM, at the optimal pH, 8.0. The enzyme, using either N donor, has a K+ requirement for activity, for which NH4+ can substitute. The glutamine leads to glutamate reaction, which requires the small subunit, also requires the large subunit and all reaction substrates for optimal activity. Other evidences of subunit interaction are the greater activity of the native enzyme, as opposed to the large subunit, with low concentrations of adenosine 5'-triphosphate-Mg2+, and in the stimulation of the ammonia-dependent activity of the native enzyme by glycine. Curiously, although the enzyme's role in biosynthesis is confined to the arginine pathway, it is completely indifferent to arginine or its precursors as feedback effectors or activators. The enzyme is compared with carbamyl phosphate synthetases of other organisms.     

Inositol-Limited Growth, Repair, and Translocation in an Inositol-Requiring Mutant of Neurospora crassa

The biochemical consequences of inositol limitation in an inositol auxotroph of Neurospora crassa have been examined as a means of disclosing the cellular role of inositol. The cellular levels of inositol in the inl mutant were proportional to the concentration of inositol in the growth medium whereas inositol phosphate levels remained relatively constant at about 0.1 mumol/g (dry weight). After 72 h of growth, about 57-fold more protein per milligram (dry weight) was released by the mutant grown on limiting inositol than by the inositol-supplemented control. When the inositol-limited growth medium was osmotically buffered with 1% NaCl, 3% NaCl, or 6% sorbitol, there was about 33, 74, or 54%, respectively, less protein released by the mutant. These results are consistent with cell lysis occurring in the mutant grown on limiting inositol because of a structurally weakened cell wall and membrane deterioration. When sufficient inositol for normal mycelial growth was supplied to an inositol-deficient mycelium, there was within 2 h a rapid incorporation of inositol to 85% of control levels. This incorporation occurred without significant growth by any area of the mycelium. About 10 to 15% of the total cell inositol was translocated forward from the older mycelial areas to the growing tips; only 2 to 5% of the total cell inositol was translocated backward toward the older mycelial areas. Possible mechanisms of translocation are discussed.