William E. Timberlake

Developmental gene regulation in Aspergillus nidulans.

Abstract The Ascomycete fungus Aspergillus nidulans reproduces asexually by differentiating conidiophores and conidia. Gene regulation during asexual reproduction was investigated by comparing poly(A) RNA populations derived from somatic hyphae, conidiating cultures and purified conidia. Single-copy and complementary DNA hybridization experiments showed that vegetative cells contained 5600–6000 diverse, average-sized poly(A) RNA sequences distributed into three prevalence classes. cDNA hybridization experiments indicated that a significant proportion of the poly(A) RNA derived from either conidiating cultures or spores consisted of sequences absent from somatic hyphae. To assess accurately the degree to which the poly(A) RNA populations differed, cDNA preparations were isolated which were complementary to sequences present only in conidia or in conidiating cultures. Hybridization of these cDNAs with poly(A) RNA from conidiating cultures showed that approximately 18.5% of the poly(A) RNA mass comprised 1300 diverse sequences not present in somatic cells. Of these, about 300 were present only in conidia. The remainder were accumulated specifically during sporulation, but were absent from spores. Analogous experiments showed that the great majority of the poly(A) RNA sequences accumulated by vegetative hyphae were also present in conidiating cultures. Thus, cell differentiation during A. nidulans asexual reproduction involves the accumulation of many new poly(A) RNA sequences, but not the loss of preexisting ones.

Molecular Cloning and Selection of Genes Regulated in Aspergillus Development

Over 350 clones homologous to poly(A)+ RNAs that are significantly more prevalent in conidiating cultures of Aspergillus nidulans than in somatic cells have been selected from a recombinant DNA library formed between nuclear DNA and lambda Charon 4A. The procedure used for this selection involved in situ hybridization to a cDNA probe which had been selectively depleted of sequences represented in somatic cells by complement hybridization. Five of these clones have been characterized further. All but one encoded poly(A)+ RNAs that were at least ten times more prevalent in conidiating cultures than in somatic cells. One clone hybridized to a single, developmentally regulated RNA. The three others were complementary to several RNAs having different molecular weights, each of which was more prevalent in condiating cultures than in vegetative cells. These results and quantitative aspects of the selection procedure suggest that developmentally controlled poly(A)+ RNA coding regions may not be distributed randomly in the Aspergillus genome.

Sequence complexity of nuclear and polysomal RNA in leaves of the tobacco plant

The first measurements are reported of the sequence complexity of nuclear and polysomal RNA contained within the cells of a higher plant. Polysomal RNA from tobacco leaves was prepared by a procedure which minimized contamination with nuclear RNA. Hybridization of 3H-cDNA complementary to polysomal poly(A) RNA with an excess of tobacco DNA indicated that greater than 95% of the poly(A) mRNA was transcribed from single-copy sequences. RNA excess hybridization reactions with polysomal poly(A) RNA and 3H-cDNA revealed the presence of three abundance classes in the poly(A) mRNA. The best least-squares solution indicated that these classes comprise 9, 52, and 39% of the poly (A) mRNA and contain sequences present an average of 4500, 340 and 17 times per cell. Hybridization reactions containing an excess of nuclear or total polysomal RNA and 3H-single-copy DNA indicated the complexity of these RNA populations to be 1.19 X 10(8) nucleotides (nuclear) and 3.33 X 10(7) nucleotides (polysomal). Thus only 28% of the nuclear RNA sequence diversity (27,000 average-sized mRNA sequences) is respresented in leaf polysomes. These results suggest that there is a general similarity in the basic transcriptional processes of metaphytan and metazoan cells.

catA, a new Aspergillus nidulans gene encoding a developmentally regulated catalase

Abstract  Aspergillus nidulans asexual sporulation (conidiation) is a model system for studying gene regulation and development. The CAN5 cDNA is one of several clones isolated based on transcript induction during conidiation. Here we present the molecular characterization of its corresponding gene, demonstrating that it encodes a developmentally regulated catalase, designated catA. The catA 744-amino-acid-residue polypeptide shows significant identity to other catalases. Its similarity to prokaryotic catalases is greater than to other fungal catalases. catA mRNA is barely detectable in growing mycelia, highly induced during sporulation, and present in isolated spores. However, catA expression is not dependent on the developmental regulatory genes brlA, abaA and wetA. Direct catalase activity determination in native gels revealed the existence of two bands of activity. One of these bands represented the major activity during vegetative growth and was induced during sporulation. The second catalase activity appeared after the induction of sporulation and was the predominant activity in spores. Disruption of catA abolished the major spore catalase without eliminating the vegetative activity, indicating the existence of at least two catalase genes in A. nidulans. catA-disrupted mutants produced spores that were sensitive to H2O2, as compared to wild-type spores. The increase in the activity of the vegetative catalase and the appearance of a second catalase during asexual sporulation is consistent with the occurrence of an oxidative stress during development.

Alterations in RNA and protein synthesis associated with steroid hormone-induced sexual morphogenesis in the water mold Achlya☆

Abstract The addition of physiological concentrations of the steroid hormone antheridiol to cultures of a strong male strain of the water mold Achlya ambisexualis results in the synchronous development of antheridial branches. Development is transiently dependent on continued transcription and translation and is accompanied by increased rates of synthesis of ribosomal RNA, polyadenylic acid-containing RNA, and protein. However, the quantitative changes in RNA and protein synthesis are most likely secondary effects of the hormone which are the result of a process sensitive to inhibitors of both RNA and protein synthesis.

Relationship between nuclear and polysomal RNA populations of Achlya: a simple eucaryotic system

The relationship between hnRNA and mRNA in the water mold Achlya has been investigated in several ways. Analysis of the nuclear and polysomal poly(A) RNA by sucrose density gradient centrifugation under denaturing and nondenaturing conditions showed that the populations have indistinguishable size distributions. The number average sizes were calculated to be 1150 nucleotides for nuclear and 1140 nucleotides for polysomal poly(A) RNA. Selective inhibition of rRNA synthesis was used to investigate the size distribution of hnRNA without regard to poly(A) content. Very little hnRNA was observed which sedimented more rapidly than polysomal poly(A) RNA. Hybridization experiments in which an excess of nuclear DNA was reacted with 3H-poly(A) hnRNA or 3H-poly(A) mRNA showed that both populations contain repetitive transcripts (9-10%) as well as single-copy transcripts (44%). Analysis of hybrids on hydroxyapatite in the presence of 8 M urea demonstrated that the poly(A) RNA complementary to repetitive DNA sequence components represented a population of molecules distinct from the population complementary to single-copy DNA. The complexity of whole cell, nuclear and polysomal RNA was determined by saturation hypbridization to single-copy 3H-DNA. All three populations were complementary to essentially the same fraction of the DNA. Terminal hybridization values were 3.84, 3.76 and 3.76% for whole cell, nuclear and polysomal RNA, respectively, representing a complexity of 2.1 X 10(6) nucleotides. These data suggest that the composition of the hnRNA and mRNA populations are essentially identical. No evidence for selective turnover of any sequence component or size class within the nucleus was observed.

Absence of evidence for changes in messenger RNA populations during steroid hormone-induced cell differentiation inAchlya

The addition of the steroid hormone antheridiol to male strains of the aquatic fungusAchlya ambisexualis induces the differentiation of numerous, morphologically distinct reproductive structures called antheridial branches. We have investigated mRNA populations derived from control cells and from cells treated for various times with antheridiol to determine if and to what extent the hormone causes the accumulation of new mRNA sequences or the loss of preexisting ones. The experimental approaches which we employed had a wide range of sensitivities and were potentially capable of detecting even very small changes in the mRNA populations. These procedures failed to reveal any alterations in the spectrum of mRNA sequences transcribed and accumulated byAchlya males during antheridial branch differentiation. We conclude that any qualitative changes in gene activity induced by antheridiol are quite subtle.

Polyadenylate metabolism in Achlya ambisexualis

The polyadenylate sequences of the polyadenylated RNA of Achlya ambisexualis were found to be heterogeneous in size. Under steady-state labeling conditions, three classes of polyadenylate were identified having modal sizes of approximately 50, 30, and 15 nucleotides and representing about 30, 50, and 20% of the radioactivity, respectively. However, polyadenylate sequences, isolated after pulse-labeling for 10 min with [ 3 H]adenosine, contained most of the label in the 50-nucleotide size class. With increasing labeling intervals, the proportion of the isotope in the large sized class decreased, while that in the two smaller sized classes increased. In experiments in which a 10-minute pulse was followed by an unlabeled adenosine chase, the amount of label in the 50-nucleotide component was rapidly reduced. However, most of the label lost from the large sized class could not be accounted for by appearance in the small sized classes. Cell fractionation following labeling with [ 3 H]adenosine showed that the polyadenylate size classes are differentially distributed within the cell. Poly-adenylate sequences within the nucleus are predominantly in the 50-nucleotide size class. On the other hand, those present in mRNA active in translation are predominantly in the small sized classes. Thus, in Achlya , there exist classes of polyadenylate, linked to nuclear pre-messenger RNA and polysomal messenger RNA, which differ in size and in rates of synthesis and turnover.

Optimum conditions for synchronous oospore production in crosses of Achlya ambisexualis

Conditions are described for the rapid and synchronous production of oospores in crosses of Achlya ambisexualis (J. R. Raper) under conditions which facilitate oospore isolation. The temperature range 18 to 22°C yielded optimum numbers of oogonia which contained 8 to 20 oospores. Lower temperatures resulted in slower development of equivalent numbers of oogonia whereas higher temperatures resulted in more rapid development of reduced numbers of oogonia, an increased proportion of which were abnormal. A ratio of seven female to three male oospores yielded maximum numbers of oogonia-containing oospores. Lower ratios of females to males reduced the number of oogonia. Higher ratios resulted in increased numbers of oogonia; however, the proportion of oogonia which failed to delimit oospores increased significantly. Under optimum conditions 5 × 10 7 oospores were produced per 10-cm petri dish in 8 days. Oospores could be freed from other cellular material by a simple grinding procedure followed by centrifugation in colloidal silica step gradients.