Studies of Structural Gene Transcripts in Sea Urchin Embryos and Adult Tissues

Citation

Wold, Barbara J. (1978) Studies of Structural Gene Transcripts in Sea Urchin Embryos and Adult Tissues. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/rc6x-ar52. https://resolver.caltech.edu/CaltechETD:etd-02272006-084615

Abstract

The RNA stored in the mature sea urchin oocyte includes sequences transcribed from about 6% of the single copy DNA or approximately 3.7 x 10(7) nucleotide pairs. It is estimated that there are about 1500 transcripts of each sequence per oocyte. A (3)H-labeled single copy DNA fraction highly enriched for sequences complementary to mature oocyte RNA was prepared. This DNA, referred to as oDNA, was reacted with excess polysomal mRNA from sea urchin embryos at 16-cell, blastula, and gastrula stages. Sixteen-cell embryo mRNA reacted with oDNA to a level which is 73% of that observed for the reaction of oDNA with oocyte RNA. This represents about 2.7 x 10(7) nucleotides of maternal sequence, or 12-15,000 different mRNAs of average size. Blastula and gastrula stage mRNA reacted with oDNA to about 56% and 53%, respectively, representing 2.1 x 10(7) and 1.9 x 10(7) nucleotides of oocyte RNA sequence. From the kinetics of hybridization, we calculate that the concentration of mRNAs belonging to the maternal sequence set is about 500 copies per embryo. This is typical of the "complex class" of embryo mRNA which contains the vast majority of diverse messenger RNA sequences but comprises only a small fraction (about 10%) of the total mRNA mass. Total cytoplasmic RNA was extracted from 16-cell, blastula, prism and pluteus stages and reacted in excess with (3)H-oDNA. The amount of hybridization of oDNA with 16-cell cytoplasmic RNA was 100% of the value obtained when oDNA was reacted with oocyte RNA. Hybridization levels observed with cytoplasmic RNAs from later stages were progressively less, but were higher than the corresponding mRNA hybridization levels through the blastula stage. To detect and quantitate embryo messenger RNA sequences which are not homologous with maternal RNAs, a labeled single copy DNA fraction entirely devoid of oocyte RNA complementary sequences was prepared. This selected DNA, termed null oDNA, was reacted with excess polysomal mRNA from 16-cell, blastula and gastrula embryos. Non-maternal sequences could not be detected in the mRNAs from 16-cell and gastrula stage embryos. Mesenchyme blastula mRNA, however, hybridized about 3.6 x 10(6) nucleotides of null-oDNA sequence which is sufficient to code for approximately 2,000 mRNAs of average size. These nonmaternal mRNAs are complex class sequences present on blastula polysomes at about the same concentration (500 copies per embryo) as are most maternal sequence mRNAs. Thus, a significant number of new, non-maternal structural genes are expressed in blastula stage embryos. Because the sea urchin displays large differences between embryo and adult tissue messenger RNA sequence sets, it is possible to test the proposition that structural gene sequences are transcribed only in cells where the transcripts are translated. A (3)H-labeled single copy DNA highly enriched for sequences complementary to blastula polysomal messenger RNA was prepared. This selected DNA fraction, referred to as mDNA, represents about 2.6 x 10(7) nucleotides of embryo messenger RNA sequence. A maximum of 16% of the blastula sequences are present in coelomocyte and intestine messenger RNAs while 40% of the blastula sequences are represented in gastrula mRNA. (3)H-mDNA was hybridized with excess nuclear RNA from coelomocytes, intestine, and gastrula embryos. Within our limits of detection, all mDNA sequences were hybridized by each of the nuclear RNAs. We conclude from this result that virtually all of the blastula mRNA sequences are transcribed in three heterologous tissues in which the transcripts are not utilized as messenger RNAs. The kinetics of hybridization show that the blastula structural gene sequences are present in each nuclear RNA at about the same concentration as are the majority of the complex single copy nuclear transcripts.

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