Analysis of the coding activity and stability of messenger RNA in Drosophila oocytes.

The coding activity of the messenger RNA in the ooplasm of late stage 14 (S14) oocytes of Drosophila melanogaster was analyzed by labeling the oocytes in vitro with [³⁵S]methionine and examining the labeled products by two-dimensional gel electrophoresis and fluorography. This analysis was done both with newly formed S14 oocytes from rapidly laying females and with S14 oocytes stored for about 10 days in females that were prevented from laying. Comparison of the fluorographs showed that the proteins labeled in the newly formed oocytes were also labeled in the stored oocytes. Thus, the coding activity of S14 oocyte messenger RNA appears to remain stable during prolonged storage in utero. The oocyte proteins synthesized during oogenesis and incorporated into S14 oocytes were labeled in vivo by injecting [³⁵S]methionine into newly eclosed females, and the S14 oocytes were removed 2 days later for gel electrophoresis and fluorography. Comparison of the fluorographs produced by the in vivo and in vitro labeling procedures showed that most of the oocyte proteins labeled in vivo were also labeled in vitro. The S14 oocytes, therefore, appear to contain messenger RNA for most of the oocyte proteins synthesized during oogenesis. There were also several additional proteins detected only in the fluorographs of the in vivo labeled oocytes; the most prominent of these were identified by immunoprecipitation tests as vitellogenin proteins of yolk granules, which are known to be synthesized outside the oocyte, in fat bodies. The occurrence of stable S14 oocyte messenger RNA for most of the oocyte proteins suggests that the synthesis of those proteins during oogenesis occurs in the developing oocytes, specified by a stable population of oocyte messenger RNA.     

A comparison of Xenopus laevis oocyte and embryo mRNA

Total RNA, extracted from mature oocytes and tadpoles of Xenopus laevis, was used as a template for in vitro protein synthesis. The oocyte RNA is markedly deficient in abundant mRNA species by comparison to tadpole RNA or other somatic RNAs, in agreement with previous experiments using RNA-cDNA hybridization analysis (S. Perlman and M. Rosbash, 1978, Develop. Biol.63, 197–212). Oocyte pA⁺ RNA is also larger than tadpole pA⁺ RNA or other somatic pA⁺ populations. The larger oocyte pA⁺ RNA and smaller oocyte pA⁺ RNA are equally good templates for in vitro protein synthesis, which implies that much, and perhaps all, of the large oocyte pA⁺ RNA is bona fide mRNA. We suggest that the relatively large size of the oocyte pA⁺ RNA population is due, at least in part, to the relative lack of abundant mRNA species in the population. This suggestion follows from the observation of 0. Meyuhas and R. P. Perry (1979, Cell16, 139–148) that L-cell-abundant mRNAs are preferentially small and rare mRNAs preferentially large. Most of the oocyte pA⁺ sequences are also present in tadpoles and are still adenylated at this stage. Oocyte proteins synthesized in vivo do not appear deficient in abundant proteins, suggesting that a translational control mechanism operates to select certain pA⁺ RNAs at higher frequencies than others.     

Biochemical research on oogenesis: Oocytes and liver cells of the teleost fish Tinca tinca contain different kinds of 5S RNA

Previtellogenic oocytes of Tinca tinca accumulate very large amounts of 5S RNA. We show here that 5S RNA stored in oocytes differs from liver 5S RNA in 3 out of 120 nucleotides. Liver and oocyte 5S RNAs, therefore, are produced by different genes. Both kinds of 5S genes are active in oocytes. However, only 5S RNA of the oocyte type accumulates in these cells. In Tinca tinca as in Xenopus laevis, oocyte-type and somatic-type 5S RNAs differ by three properties, ie., primary structure, conformation, and metabolic stability. Nucleotide substitutions occur in different positions in oocyte and somatic 5S RNAs of Tinca tinca and Xenopus laevis. We do not understand how different sets of nucleotide substitutions confer to 5S RNAs of both species similar properties in vivo, namely, increased metabolic stability.     

Aspects of DNA, RNA and protein synthesis during somatic embryogenesis in a carrot cell suspension culture

Changes in DNA, RNA and protein content, incorporation of ³H-thymidine, ¹⁴C-uridine and ³H-leucine and template activity of chromatin were investigated in the early process of somatic embryogenesis in a carrot (Daucus carota L. cv. Kurodagosun) cell suspension culture using a synchronous system. An embryogenetic culture in a medium containing 10^-7M zeatin was compared with a non-embryogenetic culture in a medium containing 10^-7M zeatin and 5 x 10^-7M 2,4-D. DNA was synthesized very actively prior to and during the formation of globular embryos in the embryogenetic culture. The RNA and protein content per tube increased at an almost constant rate in both cultures, while the rate of incorporation of labelled precursors of RNA and protein rose much more prior to active DNA synthesis in the embryogenetic culture than in the non-embryogenetic culture. Template activity of chromatin was high in the early stage of embryogenesis in the embryogenetic culture. The results obtained here showed that synthesis and turnover of RNA and protein became active prior to active DNA synthesis in the early stage of embryogenesis, and that these changes at macromolecular levels may play important roles in embryogenesis.     

Further studies on the synthesis of RNA in vitro by enzyme--template complexes isolated from induced lambda lysogens

RNA synthesized in vitro by enzyme-template complexes isolated from λ lysogens at early or late times following induction has been shown by competition-hybridization procedures to resemble messenger RNA transcribed in vivo at the same stage of viral development, and to differ from RNA made in vitro by purified Escherichia coli RNA polymerase. It is demonstrated here that RNA synthesis by such complexes involves elongation of chains which have been started in vivo, rather than initiation of new RNA chains in vitro.