Selective allele loss and interference between cauliflower mosaic virus DNAs

Summary Some cauliflower mosaic virus (CaMV) alleles are selectively lost during growth of the virus in mixedly infected turnip plants. Viral DNA from plants co-inoculated with DNA of the cabbage S isolate and infectious cabbage S DNA with an extra EcoRI restriciion site lacked the extra site. The EcoRI allele was also lost in most plants co-inoculated with a non-infectious mutant of cabbage S DNA while little selective allele loss was observed with two other non-infectious mutant DNAs. Plants co-inoculated with DNAs of closely-related isolates (CM4-184 and W) contained both parental viral DNAs and some DNAs with characteristics of both parents. Interference, scored as a reduced frequency of infection or a delay in symptom appearance relative to plants inoculated with wild-type DNA, occurred when plants were inoculated with wild-type and mutant DNAs covalently attached to one another in partial dimer plasmid DNAs. Similarities in the conditions leading to selective allele loss and those leading to interference suggest that both may have been due to active gene conversion between CaMV DNA molecules.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Correlation of 3,4-dihydroxybutyl 1-phosphonate resistance with a defect in cardiolipin synthesis in Escherichia coli.

Escherichia coli treated for 1 h with 100 microM rac-3,4-dihydroxybutyl 1-phosphonate (DBP), a glycerol-3-phosphate analog, die when sorted at 5 degrees C, whereas the viability of untreated cells is relatively unaffected. This observation formed the basis of a selection procedure that was used to isolate mutants that are partially resistant to DBP. One such mutant, strain 6204, is constitutive for DBP transport, exhibits a particularly high degree of cold resistance, has the same doubling time as the parent, and is similar to the parent strain in terms of incorporation of DBP into the lipid fraction. Glycerol-3-phosphate and phosphatidylglycerol phosphate synthetases obtained from strain 6204 and its parent were identical in terms of DBP recognition. The parent strain is killed when incubated in the presence of a combination of 70 microM rac-DBP and 0.25% deoxycholate, whereas strain 6204 continues to grow, albeit more slowly, in the presence of this combination. Strain 6204 can be distinguished from the parent strain on agar plates (low phosphate minimal medium with glucuronate as the sole carbon source) containing 15 microM rac-DBP. The insertion of Tn10 near the 6204 mutation has facilitated genetic manipulations. All phenotypic effects attributed to strain 6204 appear to be due to a single mutation. Genetic analysis indicates that Tn10, inserted near the gene responsible for DBP resistance, maps in the vicinity of 27 min. Three-factor crosses reveal a gene order of hemA-Dbpr-Tn10(zch)-trp. The only gene for phosphoglyceride metabolism known to map in this region is the gene associated with cardiolipin synthetase, cls. Genetic results suggest that the mutation responsible for DBP resistance maps in or very near cls. Analysis of the lipids isolated from untreated strain 6204 (and from each of the transductants prepared by P1 vir-mediated transfer of DBP resistance of wild-type strains) reveals that cardiolipin synthesis is defective. These results strongly suggest that the mutation responsible for DBP resistance has its primary effect on cardiolipin synthesis. To further test this hypothesis, strains with an authentic cls mutation were constructed and examined for resistance to DBP. These strains had growth properties that were identical with those of strain 6204. Wild-type strains and mutants defective in cardiolipin synthesis were treated with DBP and 20 mM magnesium or calcium chloride. Simultaneous treatment of either cell type with DBP and divalent cation not only failed to stimulate growth but, quite the contrary, had a marked synergistic growth inhibitory effect.     

Characterization of the subunit structure of gonadotropin receptor in luteinized rat ovary

Gonadotropin receptors with specificity, high affinity and low capacity for luteinizing hormone and human chorionic gonadotropin (hCG) have been identified in rat luteal cells. To investigate the nature of the receptor, we have employed disuccinimidyl suberate, a cross-linker noncleavable by reducing agents, and dithiobis(succinimidyl propionate), a cleavable cross-linker, to covalently cross-link the 125I-hCG . receptor complex. The molecular weight of 125I-hCG-linked receptor complex and the receptor subunit structure were determined by electrophoresis in either 10 or 4.5% acrylamide in the presence of 0.1% sodium dodecyl sulfate with or without reducing agents. Autoradiographic analysis of the 125I-hCG-linked receptor separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing condition revealed a single labeled band corresponding to Mr = 305,000 +/- 15,000. However, electrophoresis performed in the presence of 50 mM dithiothreitol and 2% beta-mercaptoethanol resulted in the appearance of four labeled bands corresponding to Mr = 105,000 +/- 4,000, 96,000 +/- 5,000, 74,000 +/- 4,000, and 62,000 +/- 4,000 concomitant with the loss of the labeled band in the Mr = 305,000 region. Further experiments demonstrated that these four labeled bands were derived from the same molecular species. In addition, the 125I-hCG-linked receptor in the absence of reducing agent was not dissociated into subunits even by treatment with strong denaturing agent (8 M urea). The appearance of the cross-linked 125I-hCG . receptor was effectively inhibited by the unlabeled beta-subunit of hCG, intact hCG, and luteinizing hormone and partially inhibited by the alpha-subunit of hCG but not by choleratoxin, gonadotropin-releasing hormone, insulin or bovine serum albumin. These data suggest that 1) the hCG/luteinizing hormone receptor is an oligomeric complex linked by disulfide bonds and 2) that under reducing conditions, the oligomeric receptor dissociates into four nonidentical subunits.     

Influence of pulmonary inflations on discharge patterns of phrenic motoneurons

The purpose of this study was to assess the influence of pulmonary inflations on activities of single phrenic motoneurons. Studies were performed in decerebrate and paralyzed cats; activities of phrenic nerve and single phrenic motoneurons were recorded. Animals were ventilated with a servo-respirator which produced alterations in tracheal pressure in parallel with changes in integrated activity of the phrenic nerve. At end-tidal fractional concentrations of CO2 of 0.05, phrenic motoneurons were distributed into "early" and "late" populations, depending on time of onset of activity. During the late stages of neural inspiration, differences in levels of integrated activity of the phrenic nerve became evident between cycles with and without lung inflations. At a time approximating 90% of the inspiratory duration during inflations, integrated phrenic activity was higher for cycles with inflation. Concomitantly, with lung inflations, the discharge frequencies of early phrenic motoneurons were lower, and late motoneurons began to discharge sooner than when inflations were withheld. Similar results were obtained in hypercapnia. We conclude that reflexes activated by pulmonary inflations may produce augmentation, as well as inhibition of phrenic motoneuronal activities. Factors responsible for eliciting these reflex augmentations and inhibitions are discussed.     

Effect of parasympathetic denervation on acetylcholine levels in the rat parotid gland. Is there an extraneuronal pool of acetylcholine?

Parasympathetic denervation of the rat parotid gland by avulsion of the auriculotemporal nerve caused a marked and lasting decrease in gland weight. Parasympathectomy did not change the levels of choline in the gland but decreased by 60% the levels of acetylcholine (ACh) ten days after surgery and 65% at 28 days. It is puzzling that relatively high levels of ACh remained after parasympathetic denervation. The presence of additional cholinergic nerves that innervate the gland, or pass through it en route to other structures may account for some of the remaining ACh. Also, Schwann cells from denervated nerves might have contributed to some of the ACh. The existence of an extraneuronal source of ACh is considered.     

Potentiation by the Tetraphenylboron Anion of the Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and Its Pyridinium Metabolite

The 1-methyl-4-phenylpyridinium species (MPP+) is the four-electron oxidation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is widely assumed to be the actual neurotoxic species responsible for the MPTP-induced destruction of dopaminergic neurons. MPTP is oxidized by the enzyme monoamine oxidase-B to a dihydropyridinium intermediate which is oxidized further to MPP+, an effective inhibitor of the oxidation of the Complex I substrates glutamate/malate in isolated mitochondrial preparations. In the present study, the tetraphenylboron anion (TPB) greatly potentiated the inhibitory effects of MPP+ and other selected pyridinium species on glutamate/malate respiration in isolated mouse liver mitochondria. At 10 microM TPB, the potentiation ranged from approximately 50-fold to greater than 1,000-fold for the several pyridinium species tested. In other experiments, TPB greatly enhanced the accumulation of [3H]MPP+ by isolated mitochondrial preparations. This facilitation by TPB of MPP+ accumulation into mitochondria explains, at least in part, the potentiation by TPB of the above-mentioned inhibition of mitochondrial respiration. Moreover, TPB addition increased the amount of lactate formed during the incubation of mouse neostriatal tissue slices with MPTP and other tetrahydropyridines. The administration of TPB also potentiated the dopaminergic neurotoxicity of MPTP in male Swiss-Webster mice. All of these observations, taken together, are consistent with the premise that the inhibitory effect of MPP+ on mitochondrial respiration within dopaminergic neurons is the ultimate mechanism to explain MPTP-induced neurotoxicity.     

Engineered herpes simplex virus DNA polymerase point mutants: the most highly conserved region shared among alpha-like DNA polymerases is involved in substrate recognition.

Eucaryotic, viral, and bacteriophage DNA polymerases of the alpha-like family share blocks of sequence similarity, the most conserved of which has been designated region I. Region I includes a YGDTDS motif that is almost invariant within the alpha-like family and that is similar to a motif conserved among RNA-directed polymerases and also includes adjacent amino acids that are more moderately conserved. To study the function of these conserved amino acids in vivo, site-specific mutagenesis was used to generate herpes simplex virus region I mutants. A recombinant virus constructed to contain a mutation within the nearly invariant YGDTDS motif was severely impaired for growth on Vero cells which do not contain a viral polymerase gene. However, three recombinants constructed to contain mutations altering more moderately conserved residues grew on Vero cells and exhibited altered sensitivities to nucleoside and PPi analogs and to aphidicolin. Marker rescue and DNA sequencing of one such recombinant demonstrated that the region I alteration confers the altered drug sensitivity phenotype. These results indicate that this region has an essential role in polymerase function in vivo and is involved directly or indirectly in drug and substrate recognition.