Random mutant generation and its utility in uncovering structural and functional features of cytochromeb inSaccharomyces cerevisiae

The generation of random mutations in the mitochondrial cytochromeb gene ofSaccharomyces cerevisiae has been used as a most fruitful means of identifying subregions that play a key role in thebc ₁ complex mechanism, best explained by the protonmotive Q cycle originally proposed by Peter Mitchell. Selection for center i and center o inhibitor resistance mutants, in particular, has yielded much information. The combined approaches of genetics and structural predictions have led to a two-dimensional folding model for cytochromeb that is most compatible with current knowledge of the protonmotive Q cycle. A three-dimensional model is emerging from studies of distant reversions of deficient mutants. Finally, interactions between cytochromeb and the other subunits of thebc ₁ complex, such as the iron-sulfur protein, can be affected by a single amino acid change.     

Isolation of mutants of Penicillium purpurogenum with enhanced xylanase and β-xylosidase production

Penicillium purpurogenum was mutated with u.v. light to increase xylanase production. The best mutant, UV-64, was treated with N-methyl-N'-nitro-N-nitrosoguanidine and a second generation of mutants was obtained (NG-188 and NG-737). NG-737 produced 125 U of xylanase/ml when grown on oat spelts xylan supplemented with wheat bran compared with 69 U/ml for the wild-type strain. The mutants also showed a 2.2-fold increase in -xylosidase as compared with the wild-type.     

Vegetative regeneration on sexual organs inPhycomyces blakesleeanus

Phycomyces blakesleeanus produced an abundance of sexual organs when two mating types met on solid medium, but only about 14.7% of the sexual organs developed to the final stages. On the sexual organs showing arrested development, vegetative hyphae or dwarf sporangiophores (microphores) often regenerated. This vegetative regeneration was accelerated when the paired and looped progametangia were isolated from mycelia, when the counterparts of the progametangial cells constructing the loop were surgically incised, and whenPhycomyces was mated at high temperature (25–27°C). A leaky-carotenogenic mutant, whose sexual reaction was imperfect and arrested at an intermediate stage even when mated with the wild type, also regenerated hyphae with a high frequency on these arrested intermediate organs. The vegetative regeneration seems to result from interruption of a cell-to-cell recognition system between cells of different mating type, which is believed to be essential for the mating process of this fungus in addition to the pheromonal actions.     

The herbicide-resistant D1 mutant L275F of Chlamydomonas reinhardtii fails to show the bicarbonate-reversible formate effect on chlorophyll a fluorescence transients

Herbicide-resistant mutants of the eukaryotic green alga Chlamydomonas reinhardtii, that are altered in specific amino acids in their D-1 protein, show differential bicarbonate-reversible formate effects. These results suggest the involvement of D1 protein in the bicarbonate effect. A 25 mM formate treatment of mixotrophically or photoautotrophically grown wild type cells results in a slower rise of chlorophyll a fluorescence transient followed by a dramatically slowed decline during measurements in continuous light. These effects are fully reversed upon addition of 10 mM bicarbonate. The mutant BR-202 [L275F] is, however, highly insensitive to 25 mM formate suggesting that a significant change in formate (bicarbonate) binding has occurred in helix V of the D1 protein near histidine involved in Fe binding. With the exception of DCMU-4 [S264A], which is considerably more sensitive to formate than the wild type, five other different [V219I, A25IV, F255Y, G256D and cell-wall deficient CW-15] mutants display a relatively similar response to formate as wild type. Absence of formate effect on a PS II-lacking [FuD-7] mutant confirms the sole involvement of PS II in the bicarbonate effect.     

The cytochromec reductase/oxidase respiratory pathway ofParacoccus denitrificans: Genetic and functional studies

Data are presented on three components of the quinol oxidation branch of theParacoccus respiratory chain: cytochromec reductase, cytochromec ₅₅₂, and thea-type terminal oxidase. Deletion mutants in thebc ₁ and theaa ₃ complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochromec ₅₅₂ in electron transport between complex III and IV is presented.     

Functions of the white and topaz loci of Lucilia cuprina in the production of the eye pigment xanthommatin

The white and topaz eye color mutants of L. cuprina are defective in the production of the brown screening pigment xanthommatin. Both white and topaz mutants were found to be unable to accumulate xanthommatin precursors in the larval malpighian tubules, correlating with their reduced early pupal level of this metabolite. In addition, white mutants showed reduced rates of accumulation of kynurenine and 3-hydroxykynurenine in the adult eyes. Another mutant strain, grape, was also defective in its ability to accumulate these xanthommatin precursors in the eyes, although accumulation was normal in the larval tubules. In contrast, the topaz mutants were found to be normal in eye accumulation, although tubule accumulation was markedly abnormal. These properties of the white and topaz mutants of L. cuprina are compared with those of the white and scarlet mutants of D. melanogaster, and it seems likely that in the two species these genes are involved with the uptake or storage of xanthommatin precursors in specific tissues.This work was supported by Grant D2 75/15248 from the Australian Research Grants Committee.     

Adh-negative mutants: Detection of an altered tryptic peptide in a mutant enzyme of Drosophila

Adh^nll is an ethyl methanesulfonate induced mutant that lacks detectable alcohol dehydrogenase activity. A number of indirect lines of evidence have indicated that the mutation responsible for this loss in enzyme activity is locoalized in the Adh structural gene. We present more direct evidence for this hypothesis here. Utilizing a newly developed procedure for comparing tryptic peptides of Drosophila alcohol dehydrogenase obtained from different strains, we show that the alcohol dehydrogenase-like protein isolated from Adh ^nll exhibits an altered peptide profile when compared to that of wild type. The implications of this finding as well as the utility of the method for attacking other genetic and developmental problems are discussed.This work was supported by grants from the NIH (GM-18254 and ES-01527) and by a contract from the Department of Energy (EY-76-S-02-2965).Contribution No. 1050 from the Department of Biology, John Hopkins University, Baltimore, Maryland 21218.     

Dimorphism of Benjaminiella poitrasii: Isolation and biochemical studies of morphological mutants

The yeast-mycelium dimorphims of the genus Benjaminiella poitrasii has been investigated. To understand the mechanism of dimorphism two stable yeast-phase mutants (Y-1 & Y-2) and one slow growing mycelial mutant (M-1) of B. poitrasii were isolated after NTG treatment of parent strain spores and studied for their biochemical characteristics. Effects of (i) kind and concentration of carbon source, (ii) presence of complex organic nitrogen and (iii) C:N ratio in the growth medium on the morphology of parent and mutant strains were carried out at 28°C under shaking conditions. Ethanol induced morphological change and its reversal were studied in all the strains in order to elucidate the possible mechanism of morphogenesis.     

Nickel-dependent reconstitution of hydrogenase apoprotein in Bradyrhizobium japonicum Hupc mutants and direct evidence for a nickel metabolism locus involved in nickel incorporation into the enzyme

A double mutant (JH103K10) was created from hydrogenase constitutive mutant (JH103) by replacement of a chromosomal 0.60 kb nickel metabolism related locus with a kanamycin resistance gene. The double mutant required 10 to 20 times more nickel (Ni) to achieve near parental strain levels of hydrogenase activity. In the absence of nickel, both JH103K10 and JH103 synthesized high levels of (inactive) hydrogenase apoprotein (large subunit, 65 kDa). With nickel, the double mutant JH103K10 synthesized the same level of hydrogenase apoenzyme (65-kDa subunit) as the JH103 parent strain; however, whole cell hydrogenase activity in JH103K10 was less than half of that in JH103, and the CPM (due to ⁶³Ni in hydrogenase) of membranes and the calculated ratio of nickel per unit of hydrogenase enzyme of the double mutant were 40% of that in JH103. Therefore, the difference in hydrogenase activities between the double mutant and the Hup^ck strain can be accounted for by different abilities of the strains to incorporate nickel into the hydrogenase apoenzyme. The addition of nickel ions to previously Ni-starved and then chloramphenicol-treated Bradyrhizobium japonicum whole cells (JH103 and JH103K10) resulted in (an in vivo) restoration of hydrogenase activity, suggesting that the apoprotein synthesized in the Ni-free cultures could be activated by addition of nickel even in the absence of protein synthesis. The extent of reconstitution of active hydrogenase by nickel was greater in the absence of chloramphenicol. Hydrogenase apoprotein could not be activated by nickel in vitro even with the addition of ATP. The successful in vivo but not in vitro results suggest that enzymatic but cell-disruption labile factors are required for Ni incorporation into hydrogenase.