James S. Lovett

RNA and protein synthesis during zoospore differentiation in synchronized cultures of Blastocladiella

Abstract Techniques are described for producing the highly synchronized differentiation of Blastocladiella zoospores in liquid culture. Under these conditions, the plants form discharge papillae at 17 1 2 hours, nuclear cap formation and zoospore cleavage occur just before 19 hours, and zoospore discharge is complete by 19 1 2 hours. The patterns of change in dry weight, RNA, DNA, and protein were examined before and after the induction of differentiation at the end of the exponential growth phase. After changing from the rich growth medium to a dilute salts medium, all synthetic rates decrease, and net increase in DNA, RNA, dry weight, and protein ceases at 16 1 2 , 16 1 2 , 17, and 17 1 2 hours, respectively. Significant RNA degradation occurs after 16 1 2 hours resulting in a final 35% loss in the total amount. Incorporation of externally added C14-uracil into RNA falls to a very low value by 17 hours, while C14-leucine incorporation into protein reaches a maximum between 16 1 2 and 17 1 2 hours, followed by a steady decline. Pulse labeling and density gradient analysis of the whole-cell RNA demonstrated that before 16 1 2 hours transfer-RNA and a “heavy” RNA fraction of high specific activity were rapidly labeled, followed by labeling in the ribosomal peaks. No uracil entered RNA during a 10-minute pulse of plants at, or after, 17 hours. The cessation of major RNA synthesis after 16 1 2 hours was accompanied by a significant decrease in apparent uracil pool size. In an experiment where the pools were heavily labeled before 16 hours, a small amount of uracil incorporation could be detected after 17 hours, but the extensive RNA degradation and failure of exogenous uracil to enter the pools prevented a reliable estimate of the actual quantity of new RNA produced. However, when the whole-cell RNA was randomly labeled during exponential growth and examined during differentiation, no evidence could be obtained for significant turnover of this RNA after 16 1 2 hours, despite a large loss in the total amount. These results provided direct evidence for the conservation of preexisting, exponential-phase ribosomes by aggregation to form the zoospore nuclear cap. Indirect evidence for the requirement of messenger-RNA synthesis during spore differentiation was obtained by the use of actinomycin D, puromycin, and p-fluorophenylalanine (PFP). Puromycin at 100 μg/ml was not effective in reducing leucine incorporation and did not inhibit papilla formation, but did prevent zoospore cleavage. The papillae formed in the presence of puromycin were, however, multiple and abnormal. Actinomycin D (25 μg/ml) and the amino acid analog PFP (0.005 M) were effective inhibitors for the incorporation of uracil and leucine, respectively. Both caused inhibition of papillae only if added to plants 30 minutes or more before these were formed. Both also prevented spore cleavage only when added to plants 1 hour before this event. From their identical patterns of inhibition, it was concluded that papilla formation and cleavage probably require the production of one or more short-lived messenger-RNAs 30 minutes and 1 hour, respectively, before they occur.

Molecular weights of the ribosomal ribonucleic acid of fungi

SummaryThe molecular weights of the 18s and 25s ribosomal RNA components of fungi from all major classes were determined by electrophoresis in polyacrylamide gels. The molecular weight of the 18s RNA was found to be very similar for all fungi (range 0.71–0.75 million) and about 4–5% larger than the 18s RNA of HeLa cells and soybean. The molecular weight of the 25s RNA ranged between 1.45 million in the Myxomycetes and 1.30–1.31 million in the Ascomycetes and Basidiomycetes. The differences in the 25s RNA molecular weights between various classes of fungi were interpreted as being in agreement with a monophyletic origin of the Chytridiomycetes, Zygomycetes, Ascomycetes and Basidiomycetes, and independent origins for the Myxomycetes and the Oomycetes. The Hyphochytridiomycete examined could not be placed unequivocally in any group on the basis of its 25s RNA. Fungal RNA extracted with a p-aminosalicylate-triisopropylnaphthalene sulfonate-phenol mixture at 40–60°C contained a high molecular weight aggregate of the 18s and 25s ribosomal RNA; this suggested significant base sequence homology between the two ribosomal RNA species in fungi.

Isolation of the cellulase enzymes from the thermophilic fungus Thermoascus aurantiacus and regulation of enzyme production

Thermoascus aurantiacus is capable of good growth and cellulase enzyme production at 50°C. The enzyme system was produced when grown on a variety of cellulosic and non-cellulosic substrates and is stable at 70°C for at least 24 h. In time course studies on 1.0% glucose, CM-cellulase production began when the glucose concentration was reduced to a non-repressive level (ca. 2 mg ml−1). β-Glucosidase and α-amylase, enzymes considered constitutive in other organisms, were not produced until much later when autolysis became evident. The cellulase enzyme components were identified on non-denaturing (native) polyacrylamide gels by direct assay procedures. When these proteins were eluted and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they were found to have molecular weights of: β-glucosidase, 85 kDa; exoglucanase, 40 kDa; endoglucanase, 32 kDa; and α-amylase 52 kDa. SDS-polyacrylamide gels of filtrates from time-course fermentations showed good correlation between protein production and the detection of enzyme activity. The ability of substrates to act as repressors or inducers of enzyme production was also studied. Constitutive enzyme levels were produced on all of the substrates tested, although glucose in high concentrations was a strong repressor. Only cellulosic materials were found to act as inducers. Neither sophorose nor gentiobiose caused induction.

Apical vesicles and microtubules in rhizoids ofBlastocladiella emersonii: Effects of actinomycin D and cycloheximide on development during germination

SummaryThe ultrastructure of untreated germinating cells ofBlastocladiella emersonii was compared with that of cells inhibited by Actinomycin D and cycloheximide. The rhizoids and germ tubes of the fungus contained longitudinally oriented microtubules and apical clusters of cytoplasmic vesicles. The vesicles, apparently derived from Golgi apparatus equivalents also described in this paper, were involved in the tip growth of the germ tubes and rhizoids. Zoospores germinated in Actinomycin D encysted and developed short germ tubes containing microtubules and apical vesicles before further development was arrested. Zoospores treated with cycloheximide encysted but did not develop germ tubes.

Gene expression during development of Blastocladiella emersonii

Abstract To evaluate gene expression during sporulation and early development of the aquatic fungus Blastocladiella emersonii, the nucleotide sequence complexity of the polysomal RNA has been measured at different stages. To assess the effect of medium composition on gene expression, similar experiments were completed during early development in a range of simple to complex media. The polysomal RNA sequence complexity was measured by hybridization with single-copy tracer DNA and with a complex class-enriched cDNA fraction copied from the stored zoospore poly(A+)RNA. Forty-four to eighty-six percent (8.2 × 106 to 16 × 106 nucleotides) of the single-copy DNA sequence complexity was found on polysomes, depending upon the stage examined or the medium used, compared to 42.5% (8 × 106 nucleotides) in the stored RNA pool of zoospores. The highest levels of complexity occurred during the two periods of active differentiation, sporulation and germination. During starvation-induced sporulation, and average of 82% of the total asymmetrically transcribed complexity was expressed; half of this complexity was lost prior to the completion of zoospore differentiation and was missing from the zoospore-stored RNA pool. During the first 30 min of zoospore germination the level of sequence complexity increased by 46 to 66% over the zoospore level, depending upon the medium used. The polysomal RNA complexity then decreased by a nearly equal amount between 30 and 60 min when the cells entered the growth phase. An inverse relationship was found between the richness of the medium and the level of sequence complexity found on polysomes. The data indicate that sequences representative of most of the zoospore-stored poly(A+)RNA were expressed at all other stages and maintained by turnover and resynthesis. In addition, significant numbers of new sequences were also expressed, particularly during stages of active differentiation. Cells that germinated and completed early development in an inorganic starvation medium showed a marked loss of the middle and high abundance classes of poly(A+)RNA and slight enrichment for the low abundance class.

Regulation of protein synthesis in Blastocladiella zoospores: Factors for synthesis in nonsynthetic spores

An improved procedure for the isolation and enrichment of Blastocladiella emersonii polysomes is described. The enriched polysomes are stable to dialysis and storage at 0–4°C for up to 16 h. A small fraction (≤10%) of polysomes has now been identified in the nonsynthetic zoospores. Although a small amount of amino acid uptake can be detected with zoospores, no hot trichloroacetic acid-insoluble polypeptides are associated with the ribosomes, indicating a complete lack of function by this minor fraction of stored polysomes in motile cells. If the enriched zoospore polysome fractions are washed with, or dialyzed against, high-salt buffer to remove an inhibitor, they will support amino acid incorporation in vitro with dialyzed zoospore high-speed supernatants. The initiation inhibitor aurintricarboxylic acid inhibits incorporation by growth-phase polysomes with zoospore supernatant enzymes and by whole-zoospore low-speed supernatants after dialysis to remove inhibitor. The results show that, despite the absence of protein synthesis in Blastocladiella zoospores, these cells contain the factors for both initiation and elongation of polypeptide chains. Taken together with earlier evidence for the presence of messenger RNA, aminoacyl-tRNA, and aminoacyl-tRNA synthetases in zoospores, the results further indicate the presence of a potentially functional but inactive system for synthesis which must therefore be controlled at the translational level.

Ultrastructure of a reduced developmental cycle (minicycle) in Blastocladiella emersonii

Binucleate germlings of Blastocladiella emersonii were induced to differentiate into zoosporangia containing only two zoospores by shifting them from growth medium to dilute phosphate buffer. Samples for both light and electron microscopy were taken every 30 minutes from the time of induction at 3 h after inoculation through zoospore discharge at 7.5 h. The sequence and timing of intracellular structural changes during sporangium formation and zoospore differentiation were estimated from thin sections prepared from each 0.5-h sample. The intrinsic sequence of intracellular change in such “minicycle” cells was found to be similar to that of late log phase cells. It differed from cells induced at the late log phase in that mitosis did not occur after induction, the abundant macrotubular elements characteristics of late log phase cells were absent, and the timing of developmental events was different. Thus, although mitosis may occur, it is not a necessary event in sporulation. Furthermore, the contribution of macrotubular membranes to γ -particle formation, although a normal part of differentiation, is not essential. The absence of the macrotubules may contribute to the longer time required for zoosporangial differentiation in minicycle cells when compared with the “normal” 19-h zoosporangial cycle.

Synthesis of ribosomal protein without de, novo ribosome production during differentiation in Blastocladiella, emersonii☆

Abstract The synthesis of ribosomal protein was found to occur without de , novo ribosome synthesis during zoosporangium development in Blastocladiella , emersonii . The amount of ribosomal protein synthesized was greatly reduced, compared to the growth phase level of synthesis. The production of novel proteins was not detected, but turnover was found to occur for all the ribosomal proteins analyzed. There was considerable heterogeneity in the relative rates of turnover for different proteins, with the smallest proteins turning over most rapidly.

Base sequence complexity of the polyadenylated and nonpolyadenylated stored RNA in Blastocladiella zoospores

The sequence complexity of Blastocladiella emersonii DNA and zoospore stored RNA was measured using DNADNA reassociation and RNA excess/tracer DNA hybridization. The base sequence complexity of the single-copy DNA (scDNA) was estimated to be 1.86 × 107 nucleotide pairs. The sequence complexity of the zoospore total RNA represented 8 × 106 nucleotides (NT), or 42% of the scDNA sequence complexity. The zoospore poly(A+)RNA fraction was found to contain base sequence complexity equivalent to 4.7 × 106 NT by poly(A+)RNA excess/scDNA hybridization and 5.7−7.6 × 106 NT by poly(A+)RNAcDNA hybridization. The average poly(A+)RNA base sequence complexity of 5.2–6.2 × 106 NT represented 65–78% of the zoospore total RNA complexity. The poly(A+)RNA fraction was made up of at least three frequency classes; of these, greater than 95% of the sequence complexity was represented by only 32% of the poly(A+)RNA. A complex nonpoly(A)RNA fraction was also found in zoospores. This RNA fraction represented 4 × 106 NT of base sequence complexity, or about 50% of the zoospore total RNA sequence complexity; however, only 106 NT or 25% of its sequence complexity was held in common with the zoospore poly(A+)RNA fraction. When corrected for this overlap, the sum of the poly(A)-containing and nonpoly(A)RNA fractions (8–9 × 106 NT) agreed well with the total RNA complexity (8 × 106 NT).

Cell wall composition of the aquatic fungusBlastocladiella emersonii

Abstract Cell walls fromBlastocladiella emersonii were isolated by repeated washing and centrifugation. Purity and uniformity of cell wall preparations were assessed by light and electron microscopy and chemical reproducibility. Electron microscopy showed the cell walls to consist of an inner microfibrillar network and an outer amorphous layer. Analyses by X-ray and infrared spectroscopy were consistent with chitin as the major wall component. Gross chemical analysis indicated that the cell walls were composed of 74.7% amino sugar (as anhydroN-acetylhexosamine), 10.7% neutral sugar (as anhydro hexose), 10.6% protein, and 4.2% lipid. Analysis of the neutral sugars showed that isolated cell walls contain 1.5% mannose, 3.0% galactose, and 3.0% glucose. Isolated cell walls were fractionated using a hot sodium dodecyl sulfate (SDS) extraction followed by either Pronase digestion or hot KOH extraction. The hot SDS extract was found to contain two polymer types, galactose- and/or glucose-containing polymers and glycoprotein. However, the residue from the hot SDS extraction still contained most of the neutral sugars and protein present in the isolated walls. Both Pronase digestion and the hot potassium hydroxide extraction removed all of the neutral sugars except glucose. The cell wall fractionation results indicate that the major wall component is microfibrillar chitin. The results further suggest that the SDS-solubilized glycoproteins and neutral sugar polymers may represent an outer amorphous layer.