A Neurospora crassa mutant which overaccumulates carotenoid pigments

A Neurospora crassa mutant which overaccumulates carotenoid pigments Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol31/iss1/6 Results: Using this' system the incorporation of [35S]methionine into acid-insoluble material increased linearly for 40-60 min. in the endogenous system. The N. crassa lysate subjected to micrococcal nuclease treatment did not support any translation; no increase in incorporation of the label into protein fraction was observed as a function of incubation time. ceeded for about 60 min. On priming this lysate with Neurospora RNA translation proAt optimal levels of exogenously added RNA, the incorporation of the label approached the level of endogenous translation observed in untreated lysates. Translation of both poly (A)containing and poly (A-) RNA fractions of Neurospora was supported by this system. Analysis of in vitro translation products of endogenous messengers and exogenously supplied Neurospora mRNA on SDS-polyacrylamide gels revealed a protein profile identical to that demonstrated in extracts of cells pulse-labelled in vivo with [35S]methionine. Polypeptides of up to 200,000 daltons were synthesized in vitro. Pyruvate kinase was detected in the translation products by immunoprecipitation using monospecific antibodies as well as by immunoadsorption on Affigel columns coupled to anti-PK antibody. It is necessary to establish the optimum Kand Mg-acetate concentration for each type of mRNA as efficiency of translation and its dependence on these salts may vary from message to message. Whereas optimal Mg-acetate requirement for both endogenous and exogenous translation was 0.35 to 0.40 mM, endogenous protein synthesis exhibited a sharp K-acetate optimum at 80 mM but the exogenous PK-RNA enriched fraction was translated more efficiently with 20-50 mM. Another factor that influenced translation was GTP concentration. With 0.125 mM GTP both endogenous exogenously primed protein synthesis proceeded efficiently for only ~ 1 0 min. On the other hand with 0.25 mM GTP it was observed to proceed linearly for at least 40 min. This could be due to the stabilization of initiation factors by higher levels of GTP. Heterologous RNA, such as globin mRNA (BRL) was translated efficiently in this system, the optimum Kand Mg-acetate concentrations being 250 mM and 4 mM, respectively. The translation of exogenous RNA was comparable to that su ported by the commercial rabbit reticulocyte lysate (BRL) as witnessed by a similar incorporation of [35S]methionine. University of Calgary, Calgary, Alberta, Canada, T2N lN4. Department of Biology, Harding, R.W., D.Q. Philip*, B.Z. Drozdowicz**, and N.P. Williams A Neurospora crassa mutant which overaccumulates carotenoid

. --Absorption spectra of neutral and acidic carotenoid extracts (in hexane) obtained from dark or light treated wild-type and ovc (S20-16) strains.
Treatments were given at 6 vs. 25° C as described in the text. The Em5297a wild-type strain is designated as Em in the figure. The volume of each extract was adjusted to 10 ml hexane/g dry weight of mycelia extracted before absorption spectra were determined.
Previously it was demonstrated that photoinduced carotenoid biosynthesis in Neurospora crassa mycelia shows an unusual temperature dependence (R.W. Harding. 1974 Plant Physiol. 54: 142-147). The primary light reaction is independent of temperature as expected, but the amount of pigment which subsequently accumulates in the dark is temperature dependent, and surprisingly the optimum temperature is 6°C. We have isolated mutants which produce more pigment than the wild-type strain at temperatures above 6° C but about the same amount at 6° C. We have designated this type of mutant as ovc (overaccumulator of carotenoids).
The o v c locus was then put into a wild-type background by a series of four backcrosses with Em5297a (FGSC #352). Corresponding dark controls were carried out for each of these treatments. After a light treatment and dark incubation at 25° C, the ovc mutant had accumulated more neutral and acidic carotenoid pigment than the wild-type, but both strains produced less pigment at 25° C than at 6° C. The ovc strain also produced significantly more pigment than the wild-type in the dark at both 6° and 25°C. This dark production of pigment can be readily observed visually. however, the mutant is in the dark, since light still produces a dramatic increase in carotenoid production.

not fully induced
To determine which neutral pigment the ovc mutant overaccumulates at 25° C, alumina chromatography (6% water deactivated alumina) of neutral carotenoid extracts was carried out.
In Table I, it is shown that the light treated ovc mutant produces higher levels of every neutral carotenoid at 25° C except phytoene.
In addition, more neurosporaxanthin (the major acidic pigment) is produced by the ovc strain. At 6° C, the accumulation of each pigment following irradiation (with the exception of α-carotene and 3,4-dehydrolycopene) is comparable in the two strains. The ovc mutant did not produce any carotenoids not previously identified in wild-type Neurospora.

Mapping of the ovc locus was carried out.
From backcrosses of o v c , col-4 double mutants with wild-type, the ovc and col-4 loci were found to be linked. In a subsequent cross of ovc, col-4 + , X ovc + col-4, a recombination frequency of 14% (133 progeny scored) was determined.
Subsequently ovc was-shown to be linked to met-5 (9666, FGSC #141) as expected, since the met-5 locus is about 23 map units to the right of col-4 on linkage group IVR (A. Radford, 1972 Neurospora Newsletter 19: 25-26).
The order of these loci was determined by the cross shown in Table II.
These results show that the gene order is col-4, ovc , met-5 with a recombination frequency between col-4 and ovc of approximately 10% and between ovc and met-5 of about 14%.