Mukti Ojha

Characterization of Allomyces genome

Allomyces arbuscula DNA isolated from whole cells (bulk DNA) is composed of a major (alpha) and two minor components (beta & gamma) with buoyant densities in neutral CsCl corresponding to 1.721, 1.710 and 1.702 g/cm3, respectively. The DNA obtained from purified nuclei contains alpha component only. The beta component corresponds to mitochondrial DNA. The gamma component is also extra-nuclear but has not been characterized. The reassociation kinetics of sheared, bulk and nuclear DNA show that (i) 25 % bulk and 10% of nuclear DNA reanneal very rapidly and contain highly repeated sequences; (ii) moderately repeated sequences, accounting for 15% of both bulk and nuclear DNA, have a sequence complexity of approximately 7.2-10(6) daltons and are repeated about 320 times; (iii) the slow reannealing fraction accounts for about 60% of the genome and has kinetic properties similar to single copy sequences. The sequence complexity of this fraction was determined in relation to that of Escherichia coli. After a correction for the size of the repeated sequences the genome size of A. arbuscula was calculated to be 1.7-10(10) daltons.

In vitro and in vivo phosphorylation of calpain-like protease of Allomyces arbuscula

Abstract The calpain-like protease from Allomyces arbuscula was phosphorylated in vitro by a serine/threonine protein kinase from Neurospora crassa typical of casein kinase II. The protease contained covalently bound phosphate when the culture was labelled in vivo with 32Pi. The phosphoryl linkage was sensitive to alkaline phosphatase. Phosphoserine was the major phosphohydroxylamino acid detected after acid hydrolysis of the in vitro and in vivo phosphorylated enzyme. Phosphopeptide maps of the protease phosphorylated either in vitro or in vivo were similar. These data suggest that in vivo the protease may be regulated by phosphorylation-dephosphorylation mechanisms.

Allomyces Ca2+-activated neutral protease: interaction with phospholipids and plasma membranes

Abstract The cytosolic Ca 2+ -activated neutral protease bound to plasma-membranes and microsomal membranes when micromolar Ca 2+ was added to the homogenate. When purified preparations of enzyme and plasma membranes were reconstituted, 80% of the enzyme was recovered in the particulate fraction at a Ca 2+ concentration as low as 50 μM. The binding was reversible since chelation of Ca 2+ liberated the membrane-bound enzyme into the soluble fraction. Plasma membranes, particularly of inside out configuration, enhanced the maximum activable enzyme activity. Phospholipids particularly phosphatidylethanolamine and to a lesser extent phosphatidylserine, diacylglycerol and phorbol esters stimulated the enzyme activity. However, contrary to vertebrate Ca 2+ -activated neutral protease neither plasma membranes nor phospholipids significantly altered the Ca 2+ requirement for its activation.

Developmentally regulated proteases in Allomyces arbuscula

Abstract A calcium-requiring neutral protease has been detected in the vegetative mycelia of Allomyces arbuscula . The half maximum activation of the enzyme required 0.7 mM and 2.8 mM Ca 2+ in the crude and partially-purified preparation, respectively. Coinciding with differentiation of zoosporangia, there is a massive induction of another neutral protease which does not require Ca 2+ for its activity and is of the serine type.

DNA nucleotide sequence homologies between some zoosporic fungi

SummaryRepresentative species from the monoflagellate Blastocladiales and the biflagellate Saprolegniales were studied for their DNA base composition, heterogeneity, nucleotide sequence homology and divergence. Intergeneric, intrageneric and interstrain DNAs of Blastocladiales were heterogenous. The G+C values for their main component (average 64 percent) and two minor ones (average 52 and 44 percent) were found to be significantly higher than the corresponding values from the biflagellate Saprolegnia ferax (55, 46 and 36 percent respectively). In Allomyces species, the two hybrid, male and female strains were found to have closer homology with their parental types than these last between themselves. Among Blastocladiales, interspecific similarities between the epigynous A. macrogynus and the hypogynous A. arbuscula were higher (average 75 percent) than intergeneric similarities between Allomyces and Blastocladiella (average 58 percent). The biflagellate S. ferax was found to be distantly related to the uniflagellate Allomyces (average 48 percent similarity). The nucleotide sequence divergences obtained from thermal elution data correlated the hybridization values.

Purification, properties and developmental regubdion of a Ca2+-independent serine—cysteine protease from Allomyces arbuscula

Reproductive differentiation in Allomyces takes place against the background of substrate limitation, a sharp increase in intracellular proteolysine and the induction of at least one specific protease. The aim of this report is to describe the purification, properties and developmental regulation of this enzyme. The enzyme has been partially purified by a combination of ion exchange chromatography, ultrogel filtration and affinity chromatography. The purified enzyme in SDS-PAGE appeared as a doublet of M(r) 40-43 kDa. Two bands corresponding to a relative molecular mass of 40-43 kDa were also apparent in activity gels. The protein has an apparent molecular mass in the region of 43 kDa as estimated by gel filtration. The enzyme therefore, seems to be a monomer of 43 kDa. The second band in SDS-PAGE and activity gels is probably the proteolyzed form of the enzyme. The protease recognized alanine and to a lesser extent phenylalanine in the P1 position when assayed with a range of synthetic peptides. The active site of the enzyme contains a reactive serine residue, as shown by its inhibition with PMSF and soya bean trypsin inhibitor. There is probably a reactive cysteine residue as well since the enzyme activity is strongly inhibited by HgCl2, a thiol group binding reagent. The enzyme is present in zoospores but disappears progressively during germination and hyphal growth. It reappears when actively growing cultures are transferred to dilute salt solution. In conclusion, we have purified a serine-cysteine protease of M(r) 43 kDa. This enzyme has a very restricted substrate specificity and appears to be developmentally regulated.

DNA Synthesis during Zoosporangial Differentiation in Allomyces arbuscula

The rate of DNA synthesis in Allomyces arbuscula during differentiation of zoosporangia depends on the stage of development. The first stage of cytoplasmic reorganization in the hyphal apex is accompanied by a rapid decrease in DNA synthesis. The appearance of septa and the development of young zoosporangia are paralleled by a renewal of DNA synthesis which then declines as the zoosporangia mature. The transfer of an actively growing mycelium to the induction medium results in a transient accumulation (up to 1 h post-transfer) of ATP in the cell, which then starts to diminish. Analysis of the pulse-labelled native DNA by neutral CsCl equilibrium density gradient centrifugation indicates that in these early stages, all the DNA is being synthesized at a uniform rate. The amount of rRNA cistrons varies with the developmental stage and, on reorganization of the hyphal apex, the synthesis of such genes is inhibited and subsequently stimulated as zoosporangia mature.

Structure, expression and function of Allomyces arbuscula CDP II (metacaspase) gene ☆

Allomyces arbuscula, a primitive chytridiomycete fungus, has two Ca(2+)-dependent cysteine proteases, the CDP I and CDP II. We have cloned and analyzed the nucleotide sequence of CDP II gene and domain structure of the protein. Blast analysis of the sequence has shown that the protein belongs to a newly described member of caspase superfamily protein, the metacaspase, a CD clan of C14 family cysteine protease, we hence-forth name it as AMca 2 (Allomyces metacaspase 2). Southern hybridization studies have shown that the gene exists in a single copy per genome. The transcriptional analysis by Northern hybridization has confirmed our previous results that the protein is developmentally regulated, i.e. present in active growth phase but disappears during nutritional stress which also induces reproductive differentiation, indicating that the protein promotes cell growth, not death. The recombinant gene product expressed in Escherichiacoli has all the catalytic properties of native enzyme, i.e. sensitivity to protease inhibitors and substrate specificity. There is an absolute requirement of Ca(2+) for the activation of catalytic activity and the presence of R residue at the cleavage site (P1 position) in the substrate. The presence of a second basic residue, either R or K, in the P2 position strongly inhibits the catalytic activity which is stimulated by the presence of P and to a lesser extent G at this site. Peptide substrates with D at the cleavage site are not recognised and therefore not cleaved. The enzyme activity is inhibited by EDTA-EGTA, cysteine protease inhibitors and a specific peptide inhibitor Ac GVRCHCL TFA, but not by E64, although a potent inhibitor of cysteine proteases.

Immunocytochemical localization of Ca2+-dependent protease from Allomyces arbuscula

The Ca2+‐dependent protease antisera and the purified specific antibodies from Allomyces arbuscula have shown very specific recognition when blotted against the total protein extract or the purified 43–40 kDa Ca2+‐dependent protease from this aquatic fungus. By immunoblotting and immunofluorescence techniques using specific antibodies, we have shown that the enzyme activity is developmentally regulated and is related to the presence of antigen and not to any specific inhibitor. The immunofluorescence was absent in zoospores but appeared in polarized forms in germinating spores. In elongating hyphae the protease was mainly localized along the cytoplasmic membrane and in the cytoplasm, with predominance at the apex.

Oxidative deficiencies and some ultrastructural features of acridine-induced male strains of Allomyces arbusculus

SummaryA high proportion of small, frequently TTC- and highly (some 100%) masculinized gametophytic colonies have been produced from meiospores of Allomyces arbusculus plated on acridine-containing media.Endogenous O2-consumption as well as succinate dehydrogenase and cytochrome oxidase activities are reduced in the acridine-treated masculinized strains studied. Such sharp deficiencies in the oxidative metabolism are paralleled in an acridine-male strain by structural defects such as highly reduced number of cristae in the mitochondria.