A negative regulator of light-inducible carotenogenesis in Mucor circinelloides.

Mucor circinelloides responds to blue light by activating carotene biosynthesis. Wild-type strains grown in darkness contain minimal amounts of beta-carotene because of the low levels of transcription of the structural genes for carotenogenesis. When exposed to a light pulse, the level of transcription of these genes increases strongly, leading to the formation of high concentrations of beta-carotene. The crgA gene is involved in the regulation of light-induced carotenoid biosynthesis. This gene, originally identified as a 3'-truncated ORF which causes carotene over-accumulation in the dark, encodes a protein with a cysteine-rich, zinc-binding, RING-finger motif, as found in diverse groups of regulatory proteins. The expression of the crgA gene is activated by a light pulse, with a time course similar to that of the structural genes for carotenogenesis. To understand the regulatory role of the crgA gene in carotenogenesis, we have used a genetic approach based on the construction of crgA null mutants by gene replacement. Lack of the crgA function provokes the over-accumulation of carotenoids both in the dark and the light. Introduction of the wild-type crgA allele into these mutants restores the wild-type phenotype for carotenogenesis. The high levels of carotenoid accumulation shown by the null crgA mutants are correlated with an increase in the expression of carotenogenic structural genes. These results strongly indicate that crgA acts as a negative regulator of light-inducible carotenogenesis in M. circinelloides.

Overexpression of the crgA gene abolishes light requirement for carotenoid biosynthesis in Mucor circinelloides.

This work describes the isolation and characterization of crgA, a Mucor circinelloides gene, which has a dominant-positive effect on light-regulated carotenogenesis. The crgA gene was originally identified in a transformation experiment as a 3'-truncated open reading frame which caused carotenoid overaccumulation in the dark. The complete cloning and sequencing of crgA revealed that its putative product presented several recognizable structural domains: a RING-finger zinc binding domain near the N-terminus, a putative nuclear localization signal, two stretches of acidic amino acids, glutamine-rich regions and a putative isoprenylation motif. The expression of exogenous copies of the complete crgA gene or two different 3'-truncated versions, produced a similar dominant-positive effect on the light-inducible carotenogenesis of M. circinelloides. The presence of these exogenous sequences also caused a missregulation of the endogenous crgA gene, resulting in its overexpression. Collectively, these observations suggest that crgA is involved in the regulation of carotenoid biosynthesis by light.

Prt1, an unusual retrotransposon-like sequence in the fungus Phycomyces blakesleeanus.

This work reports the isolation and structural characterization of Prt1, a 4.7 kb retrotransposon-like sequence from the filamentous fungus Phycomyces blakesleeanus. Two open reading frames are found within Prt1. The first shows no similarity with known genes. The second encodes peptide stretches similar to the reverse transcriptase and RNaseH domains of the Ty3/gypsy family of LTR-retrotransposons. Prt1 lacks long terminal repeats, having instead short (54 bp) terminal inverted repeats. No target site duplication has been found. A single copy of Prt1 was detected in the genome of P. blakesleeanus. Adjacent to this sole copy of Prt1, a cluster of various short sequence repeats, both direct and inverted, is found. These sequences, which are reminiscent of defective, non-retroviral transposable elements, are also represented in other regions of the P. blakesleeanus genome.

Mutants of Phycomyces blakesleeanus Defective in Acetyl-CoA Synthetase

Nine mutants of the filamentous fungus Phycomyces blakesleeanus have been isolated on the basis of their resistance to fluoroacetate. None of the isolates uses acetate as the sole carbon source. Genetic complementation experiments revealed that all the mutants belong to the same complementation group. Biochemical analysis indicated that the acetate-induced acetyl-CoA synthetase activity is abolished in all nine mutants, thus suggesting that they are affected in the gene coding for acetyl-CoA synthetase (facA).

PkpA, a novel Phycomyces blakesleeanus serine/threonine protein kinase.

This work reports the cloning and sequencing of pkpA, a gene of the filamentous fungus Phycomyces blakesleeanus, whose expression seems to be coupled to vegetative growth. This gene encodes a putative serine/threonine-specific protein kinase, whose sequence is related to that of the yeast protein STE20, involved in pheromone-response pathways, and to a number of MAPK kinase proteins. However, detailed analysis of the kinase sequence suggests that PkpA is a novel serine/threonine protein kinase that probably participates as an intermediate in an intracellular system controlling nuclear proliferation in P. blakesleeanus.

Isolation of the facA (acetyl-CoA synthetase) gene of Phycomyces blakesleeanus.

A 5.6 kb DNA fragment from the fungus Phycomyces blakesleeanus has been cloned and sequenced. The fragment contains a gene that probably codes for the enzyme acetyl-coenzyme A synthetase (facA). The amino acid sequence deduced for the P. blakesleeanus protein is highly homologous to those of acetyl-coA-synthetases from other organisms. When placed under the control of a constitutive promoter from Aspergillus nidulans, the cloned gene complemented a facA- mutation of this organism. In P. blakesleeanus, the expression of facA is induced by acetate.

A cytoplasmically inherited mutation in the fungus Phycomyces blakesleeanus.

Fourteen mutants of the fungus Phycomyces blakesleeanus, showing high levels of resistance to copper, were isolated. In all the mutants, copper resistance behaved as a very variable and unstable trait. In the mutant strain MU102, the mutation was demonstrated to be cytoplasmically inherited. In addition, this mutant strain differed from the wild-type in growth, respiration rate, and shape and viability of spores.

Expression of Tn5-derived kanamycin resistance in the fungus Phycomyces blakesleeanus.

A plasmid, carrying the Tn5 gene for kanamycin resistance lacking its own promoter, has successfully been used in the selection of DNA sequences of the fungus Phycomyces blakesleeanus having promoter activity in Escherichia coli. Many of these sequences were also effective in promoting resistance to kanamycin when the corresponding chimeric plasmids were introduced in the fungus via spheroplast transformation. The selected phenotype was easily propagated through vegetative spores and behaved as a stable character since it was not appreciably lost in the absence of selection.

Nucleosomes containing histones H1 or H5 are closely interspersed in chromatin.

The distribution of histones H1 and H5 along chromatin fibers has been examined in the nucleated hen erythrocyte. Nucleosome oligomers, produced by micrococcal nuclease digestion of nuclei, were sequentially reacted with affinity-chromatography purified rabbit anti-H5 and sheep anti-rabbit antibodies. Quantitation of the relative amounts of H1 and H5 in the precipitated and supernatant fractions as a function of the oligomer number was consistent with a close interspersion of both types of histones, probably a random one. This conclusion was supported by the immunoprecipitation of longer chromatin fibers. This pattern of distribution appears to apply both to bulk chromatin and to chromatin inactivated during the maturation of the erythrocyte.

Substrate transfer in carotene biosynthesis in Phycomyces.

Lycopene cyclization to gamma-carotene and then to beta-carotene is partially blocked in Phycomyces by the presence of car R mutations in heterokaryosis or by the addition of 2-(4-chlorophenyl)thiotriethylamine . HCl to the medium. We have quantitatively determined the carotenes synthesized by Phycomyces under conditions of partially blocked cyclization and in the presence of either car A mutations in heterokaryosis or polyoxyethylenesorbitan monooleate (Tween-80) in the medium. We conclude that transfer of intermediate substrates between the enzyme aggregates involved in carotenogenesis does occur in the wild type but not in heterokaryons for car A mutations, and is facilitated by Tween-80.