GENOME EXPRESSION DURING GERMINATION AND EARLY DEVELOPMENT OF BLASTOCLADIELLA EMERSONII

STEVEN ANDREW JOHNSON, Purdue University

Abstract

Stored or cryptic mRNA is found in the dormant stages of diverse eukaryotic organisms and plays an important role in development following the dormant stage. The amount of genomic information, or nucleotide sequence complexity, represented by stored mRNA has been measured for a number of animal oocytes. The utilization of stored mRNA sequence complexity for directing protein synthesis during further development has been examined for only a few higher eukaryotes, the sea urchin being the most extensively studied. The research presented here was designed to measure the extent to which stored zoospore and additional non-zoospore mRNA sequence complexity was used to encode protein synthesis during zoospore germination and subsequent development of Blastocladiella. The effects of growth in widely varying nutrient conditions on this utilization were also studied. The single copy sequence complexity of Blastocladiella DNA was measured and found to be 3.41 x 10('7) nucleotide pairs, a value typical of other fungal DNA measurements, but much lower than values determined for higher eukaryotes. RNA excess/single copy DNA hybridization gave a sequence complexity estimate of 14.5 x 10('6) nucleotides (NT) for the total RNA of zoospores. This value represented 42% of the asymmetrically transcribed DNA. Zoospore poly(A)+RNA contained only 65% of the spore total RNA sequence complexity, or 9.3 x 10('6) NT. This estimate was verified independently by kinetic analysis of spore poly(A)+RNA hybridization to its homologous cDNA. A non-polyadenylated spore RNA fraction was identified and found to represent 7.3 x 10('6) NT. A small portion of this sequence complexity was held in common with the poly(A)+RNA fraction. During the first half hour of spore germination in a variety of media, the sequence complexity of polysomal RNA was much higher than the level originally present in spore total RNA. This indicated that a substantial number of few transcripts were added to the polysomal RNA sequence population, regardless of the fraction of stored spore mRNA sequence complexity present. This high level decreased dramatically during the next half hour of germination to nearly that found in the spore. A similar pattern of rise and fall in polysomal RNA sequence complexity occurred again during the next five hours of development, although absolute changes were smaller. The extent of the observed changes was differentially affected by the different growth conditions used. The polysomal sequence complexity level was very high during sporulation. Throughout this stage, at least 80% of the total single copy complexity was represented in polysomal RNA. However, a selective elimination of about one-half of this RNA sequence complexity must have occurred prior to the completion of spore differentiation to result in the measured amount of stored mRNA complexity. The utilization of the moderate abundance class sequences in spore poly(A)+RNA was subtly affected by growth in different media. However, it appeared that most, if not all, of the spore poly(A)+RNA sequence complexity, as well as the total RNA complexity, was recruited into the polysome fraction during spore germination. The reults suggested that the zoospore-stored mRNA represents a program for germination that is largely insensitive to the nutritional sufficiency of the growth medium. The level of transcription appeared to be modulated during development and was responsive to growth conditions. Finally, the data show that most, if not all, of the genome is transcribed during the Blastocladiella life cycle.

Degree

Ph.D.

Subject Area

Microbiology

COinS