Rana pipiens haploid embryos were next in- 

 vestigated in our search for some clue to the 

 mechanism of control of R-RNA synthesis during 

 embryogenesis. These embryos were useful for 

 studying the effects of a quantitative change in 

 the Rana pipiens genome on R-RNA synthesis 

 during development. Haploid embryos were 

 produced by fertilizing normal Rana pipiens 

 eggs with ultraviolet irradiated sperm. The 

 subsequent haploid embryos exhibited all of the 

 characteristics usually associated with the 

 "haploid syndrome." 



Rana pipiens haploid embryonic develop- 

 ment is characteristically abnormal and delayed 

 as compared to control embryos. Development 

 proceeds normally until late blastula, at which 

 time the haploid embryos begin to show develop- 

 mental retardation. Haploids continue to develop 

 for eight days at which time the majority of the 

 embryos become edematous and die (6, 7). 

 Cytological studies demonstrated that the normal 

 sized cells of the control embryos contained a 

 diploid set of chromosomes and two nucleoli. 

 The smaller cells of the haploid embryos 

 contain one nucleolus and a haploid set of 

 chromosomes. These haploid cells, as expected 

 contain one-half as much DNA as diploid cells 

 (8). 



It was possible to study the effect of quan- 

 titative changes in the gene complement of de- 

 veloping embryos on R-RNA synthesis by inves- 

 tigating the synthesis of R-RNA in haploid 

 embryos. Four- and six-day old ^^p.^^i^gled 

 control and haploid embryos were analyzed for 

 RNA, DNA and incorporation of 32p into R-RNA. 

 Developmental retardation, characteristic of 

 haploidy, necessitated still another type of 

 control. Haploid and normal embryos of the 

 same chronological age were not the same 

 developmental age since the haploids developed 

 at a slower rate. The additional control con- 

 sisted of five-day old normal embryos, which 

 closely approximated the same developmental 

 age as the six-day haploid embryos. Both 

 haploid and control embryos originated from 

 the same clutch of eggs. Tail tips of these 

 embryos were also examined cytologically to 

 determine the number of nucleoli per cell. 



Quantitative determinations demonstrated 

 that considerable RNA and DNA synthesis oc- 

 curred in both haploid and control embryos 

 between four and six days of development (Table 

 II). The RNA increase was almost directly pro- 

 portional to the DNA increase in both haploid 

 and control embryos (Table II). Sucrose density 

 gradient analysis also indicated that R-RNA was 

 being synthesized in both haploids and controls 



TABLE II 



The values in this table arise from the experiment 

 illustrated in Fig. 2. An aliquot was taken from the 

 whole homogenate of each set of embryos and assayed 

 for RNA and DNA. All values are given on a per embryo 

 basis. 



(Fig. 5). As expected, control embryos contained 

 more RNA and DNA than did haploid embryos 

 of the same chronological age (Table II). Haploid 

 embryos (6-day) contained nearly the same 

 amount of DNA and RNA as did control embryos 

 (5-day) of about the same developmental age 

 (Table II). Cytological examinations demon- 

 strated the presence of one normal sized 

 nucleolus per cell in haploid embryos while 

 the larger cells of the control embryos contained 

 two nucleoli. 



The RNA/DNA ratios of both haploid and 

 diploid embryos were approximately the same 

 at all stages checked (Talbe II). This indicated 

 that a unit of DNA produced about the same 

 amount of R-RNA whether it resided in a 

 haploid cell or a diploid cell. Since the cells 

 of haploid embryos contained only one-half as 

 much DNA as the cells of diploid embryos, the 

 cells of haploid embryos produced only one-half 

 as much R-RNA as the cells of diploid embryos. 



Haploid embryos were developmentally re- 

 tarded and it was expected that they would con- 

 tain less RNA and DNA than control embryos 

 of the same chronological age. It was, however, 

 surprising that haploid embryos contained ap- 

 proximately the same amount of RNA as control 

 embryos of the same developmental age. These 

 results implied that the amount of RNA syn- 

 thesized during development was a function of 

 the stage of development. Brown reached a 

 similar conclusion in studies on Xenopus haploid 

 embryos where the haploid embryos also con- 

 tained the same amount of RNA as control 

 embryos of a comparable developmental age (9). 



Haploid and diploid embryos of the same 

 developmental age also contained about the same 

 amount of DNA. Haploid embryos, then, had 



41 



