Studies on the regulation of ribosomal RNA synthesis in sea urchin development.

Cells of sea urchin hatching blastulae and gastrulae when reaggregated together do not influence each other with respect to the rate of rRNA synthesis. Extracts from unfertilized eggs and embryos inhibited rRNA synthesis by gastrulae. However, the inhibition was equally strong with extracts from stages that have a low rate of rRNA synthesis (eggs, cleavage embryos) as with extracts from stages that have a high rate of rRNA synthesis (oocytes, gastrulae). Synthesis of ppGpp is not detected at any of the investigated developmental stages.     

Drosophila ribosomal RNA stability increases during slow growth conditions

We have developed density labeling pulse-chase methods which, in contrast to a conventional radiolabeling approach, allow us to determine the effectiveness of our chase and to measure RNA stability in vivo without measuring precursor pool specific activities. We have used these methods to determine the stability of the embryonic ribosomal RNA inherited by either normally or slowly growing Drosophila melanogaster larvae. If larvae are raised in a rich growth medium, embryonic rRNA decays with a half-life of 48 h. However, if larvae are raised in a poor growth medium, which slows larval growth and prolongs development, the half-life of rRNA increases to 115 h. This is the only example, of which we are aware, directly showing that rRNA half-life increases during slow growth conditions. We propose that the increased stability of rRNA that we find may enable slowly growing larvae to maintain the ribosome levels necessary to continue growth and development under conditions of nutrient deprivation.     

Changes in rate of RNA synthesis and ribosomal gene number during oogenesis of Drosophila melanogaster.

A new method for separating Drosophila egg chambers into different developmental classes (Jacobs-Lorena and Crippa, 1977) made it possible to study changes in the rate of ribosomal RNA (rRNA), 5S RNA, and tRNA synthesis and the changes in ribosomal gene number during oogenesis. Synthesis of RNA was measured by [³H]uridine incorporation in vivo and subsequent analysis on sucrose gradients or gel electrophoresis. Specific radioactivity of nucleotide pools has also been determined. Ribosomal gene number has been measured by hybridization of egg chamber DNA to rRNA of high specific radioactivity. Our findings led us to conclude that in Drosophila melanogaster: (i) rRNA, 5S RNA, and tRNA are synthesized in all stages of oogenesis. (ii) In every stage, rRNA is the main RNA species synthesized. (iii) The rate of rRNA, 5S RNA, and tRNA synthesis increases greatly during oogenesis and is paralleled by a similar increase in ribosomal gene number resulting from the polyploidization of the nurse cell nuclei.     

Quantitative aspects of ribosomal RNA synthesis during ovarian development in two mutants of Drosophia melanogaster

A quantitative polyacrylamide gel electrophoresis procedure for the analysis of microgram quantities of RNA has been developed. The method was used to determine the rates of rRNA synthesis and the molar ratios of various RNA species in Drosophila females homozygous for either of two X chromosome inversions that result in sterility of the females and produce lethality in X/0 males. Evidence is presented that in these genotypes the rate of rRNA synthesis during oogenesis is unimpaired but the mature oocyte has a 10–12% reduction in rRNA content.     

The small RNA profile during Drosophila melanogaster development

Small RNAs ranging in size between 20 and 30 nucleotides are involved in different types of regulation of gene expression including mRNA degradation, translational repression, and chromatin modification. Here we describe the small RNA profile of Drosophila melanogaster as a function of development. We have cloned and sequenced over 4000 small RNAs, 560 of which have the characteristics of RNase III cleavage products. A nonredundant set of 62 miRNAs was identified. We also isolated 178 repeat-associated small interfering RNAs (rasiRNAs), which are cognate to transposable elements, satellite and microsatellite DNA, and Suppressor of Stellate repeats, suggesting that small RNAs participate in defining chromatin structure. rasiRNAs are most abundant in testes and early embryos, where regulation of transposon activity is critical and dramatic changes in heterochromatin structure occur.     

Rates of ribosomal protein and total protein synthesis during Drosophila early embryogenesis

Embryos at various stages of early development from 1.5 to 5 hr after oviposition were made permeable with octane and labeled for 1 hr with [³H]phenylalanine. Measurements of the rate of incorporation of [³H]phenylalanine into ribosomal proteins and total protein were made using these synchronized Drosophila embryos. The rate of synthesis of those ribosomal proteins incorporated into ribosomes increases until 3 to 4 hr after fertilization (550 pg/embryo-hr) then declines later in embryonic development. The rate of total protein synthesis is maximal as early during embryonic development as could be measured. During the period between 1.5 and 2.5 hr after fertilization this rate is 9.4 ng/embryo-hr and then also declines. The synthesis of ribosomal proteins accounts for a substantial portion (4.5%–8.9%) of total protein synthesis in early embryos. These results indicate that ribosome formation is a significant activity during the earliest stages of Drosophila development.