RIBOSOMAL RIBONUCLEIC ACID SYNTHESIS IN 

 RANA PIPIENS EMBRYOS 



David E. Kohne 



Biology Department, Purdue University, Lafayette, Indiana! 



One primary reason for the difficulty in 

 studying the biochemistry of development is 

 the lack of good genetic information on the 

 developing systems which are normally used. 

 It is now possible through the study of ribo- 

 nucleic acid (RNA) synthesis to investigate the 

 direct expression of a specific class of genes, 

 ribosomal RNA (R-RNA) genes, during develop- 

 ment. By utilizing developing Rana pipiens em- 

 bryos we have attempted to get an insight into 

 the gross aspects of the regulatory processes 

 which control the synthesis of R-RNA during 

 embryogenesis. 



There were two technical problems to be 

 solved before Rana pipiens could be used for 

 the experimental animal in this study: 1) The 

 utilization of standard ribosome isolation pro- 

 cedures resulted in the ribosomes being irre- 

 versibly bound to the egg proteins. It was 

 found that the egg ribosomes could be readily 

 isolated if the frog eggs were homogenized in 

 a buffer of high ionic strength and high pH, to 

 which sodium lauryl sulphate had been added 

 (1). 2) When used onRana pipiens eggstheusual 

 methods for the isolation of undegraded high 

 molecular weight R-RNA resulted in highly 

 degraded low molecular weight R-RNA as the 

 isolation product. It was obvious that large 

 amounts of powerful nucleases existed in these 

 eggs and a method had to be devised to negate 

 the «ffect of these enzymes. This procedure 

 primarily involved maintaining a temperature as 

 low as possible during the RNA isolation pro- 

 cedure (1). 



Three experimental embryological systems 

 were used in this work to ask some simple 

 questions about the regulative phenomena in- 

 volved in the synthesis of ribosomal RNA during 

 development. 1) Hybrid embryos were utilized 

 in order to study the effect of a qualitative 

 change in the genome of Rana pipiens on R-RNA 



synthesis during development. 2) Haploid em- 

 bryos were employed to ascertain the effect 

 on R-RNA synthesis during development of a 

 quantitative change in the frog genome. 3) Em- 

 bryos reared in a medium lacking in magnesium 

 were studied to determine the effect of mag- 

 nesium deprivation on R-RNA synthesis during 

 development. 



In order to have a base line for comparison 

 of R-RNA synthesis in experimental systems to 

 that in normal development, it was necessary 

 to determine the pattern of R-RNA synthesis 

 in the normally developing iiana/)z7)zen5 embryo. 

 Figure 1 depicts the pattern of R-RNA synthesis 

 during normal development in Rana pipiens, 

 R-RNA synthesis could not be detected during 

 early development and was first detected at 

 early gastrula stage (two left peaks in gradients 

 shown). From early gastrula stage R-RNA 

 synthesis increases rapidly as development 

 proceeds. The base ratio of this newly syn- 

 thesized RNA is high in guanine + cytosine 

 which is a characteristic of all ribosomal RNA 

 (Table I). 



The first experimental system was picked 

 in order to investigate the effect of a qualitative 

 change in the Rarui pipiens genome on the pattern 

 of synthesis of R-RNA during development. 

 Hybrid embryos produced by fertilizing Rana 

 pipiens eggs with Rana catesbeiana sperm were 

 used for these experiments. These hybrids 

 developed normally until the onset of gastrula- 

 tion and at this time development ceased. Al- 

 though development ceased at the early gastrula 

 stage, the hybrid embryos continued to live for 

 several days (2). It was of interest to determine 

 the pattern of R-RNA synthesis in the hybrid 



* Present address: Department of Terrestrial Mag- 

 netisn;!, Carnegie Institution of Washington, Washington, D.C. 



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