Donald G. Comb

Isolation, purification and properties of 5 s ribosomal RNA: a new species of cellular RNA.

The isolation procedure for a new species of ribosomal RNA is described. The name 5 s ribosomal RNA is proposed for this component. It has been found to occur in ribosome preparations from many different organisms. Approximately one molecule of 5 s rRNA is associated with the ribosome. It has a chain length of 120 in ribosomes from Escherichia coli and 148 in ribosomes from Blastocladiella emersonii . The major base composition is similar to transfer RNA but no methylated bases can be detected. We have estimated that about 0.5 residue of pseudouridine occurs per molecule. Other physical and chemical properties are described.

Characterization of RNA species synthesized during early development of sea urchins

Sea urchin embryos ( Lytechinus variegatus ) were subjected to pulse and chase experiments with 32 P 1 during cleavage, blastula, gastrula and pluteus stages of development. The nucleic acids present in the nuclear, microsomal and ribosomal-transfer RNA fractions were isolated and chromatographed on methylated albumin columns. The results indicate no detectable RNA synthesis during cleavage and only messenger RNA synthesis during the blastula stage. After gastrulation, the synthesis of transfer RNA and what appears to be ribosomal RNA is initiated. The movement of cytoplasmic ribosomes to the nucleus is reported and the stability of DNA to isolation procedures during development is discussed.

STUDIES ON THE BIOSYNTHESIS AND METHYLATION OF TRANSFER RNA.

A rapidly labeled RNA fraction of the nucleolus has been isolated and partially characterized. This RNA component appears to be a precursor of transfer RNA. It contains neither pseudouridine nor methylated bases, and is unable to form amino acyl RNA. However, it resembles transfer RNA in base composition and molecular weight. It serves as a substrate for in vitro methylation to a greater extent than transfer RNA.

Early and late histones during sea urchin development.

Abstract During cleavage of sea urchin embryos, replication of the chromosomes occurs at about hourly intervals whereas at later stages of development the cells divide at a much slower rate. A single slightly lysine-rich histone predominates in the rapidly dividing chromosomes. When cell division slows, lysine-rich and argininerich histones become the major components.

THE NUCLEAR DNA AND RNA COMPONENTS OF THE AQUATIC FUNGUS BLASTOCLADIELLA EMERSONII.

The genome of the aquatic fungus Blastocladiella emersonii has been demonstrated to consist of two distinct species of DNA. The major or α -DNA component has a guanine-cytosine content of 66%, and the minor or β -DNA component has a guanine-cytosine content of 51%. The β -DNA component is present only in the chromatin fraction of the nucleus, whereas α -DNA is present in both the chromatin and nucleolar fractions. The characterization of 32 P pulse-labeled RNA from both nuclear fractions by chromatography on methylated albumin columns has indicated the nucleolus as the site of ribosomal RNA synthesis, and the chromatin as the probable site of messenger RNA synthesis.

Studies on the attachment and release of 5 s ribosomal RNA from the large ribosomal subunit

Abstract The 80 s ribosomes of Blastocladiella emersonii or the 50 s ribosomal subunits of Escherichia coli RNase − 10 exchange their 5 s ribosomal RNA with externally added 5 s rRNA at 0.1 m m -Mg 2+ but at 10m m exchange is greatly inhibited. Very high external concentrations of 5 s rRNA are required to demonstrate exchange. When Mg 2+ is partially removed from the large ribosomal subunit of E. coli with EDTA, 5 s rRNA is released when the particle is unfolded to a 33 to 36 s particle. The 33 to 36 s particles are capable of binding 5 s rRNA at 0.1 m m -Mg 2+ but the addition of protein extracted from 50 s particles, stimulates the binding of 5 s at low ratios of 5 s per particle. Particles from which 5 s has been partially or completely removed are inactive in in vitro polyphenylalanine synthesis directed by polyuridylic acid. Reassociation with 5 s did not restore activity. Unfolded particles without 5 s can be converted to a more compact 47 s particle by heating to 50 °C for 10 minutes in 10 m m -Mg 2+ buffer.

The binding of 5 s ribosomal ribonucleic acid to ribosomal subunits

Abstract The 75 s ribosomes of the aquatic fungus, Blastocladiella emersonii dissociate at 10 −4 m -Mg 2+ into 63 s and 45 s subunits. Only the 63 s subunit contains 5 s rRNA, about one molecule per subunit being present. When these subunits are treated with EDTA, the 63 s particle is unfolded to a 47 s particle without extensive release of protein but with the release of 5 s rRNA. Using 32 P-labeled 5 s rRNA, we have demonstrated that 5 s rRNA will exchange with the 5 s rRNA on the 63 s subunit. Binding studies demonstrate that 2 molecules of 5 s rRNA will bind to ribosomal subunits or to the isolated 47 s large subunit. Binding to one of the two sites is displaced by transfer RNA.

Sea urchin embryogenesis. I. RNA synthesis by cytoplasmic and nuclear genes during development.

Abstract The cytoplasmic or nuclear origin of newly synthesized RNA has been investigated during early embryogenesis of Lytechinus variegatus . A large fraction of labeled high molecular weight RNA was recovered in an EDTA- and RNase-resistant form from the cytoplasmic particle fraction of the embryos during all stages of development studies. The labeled RNA in the particle fraction annealed to cytoplasmic DNA 50 times better than to a similar amount of nuclear DNA. Labeled messenger RNA associated with polyribosomes did not show this high affinity for cytoplasmic DNA. Approximately 50% of the labeled particle RNA banded with the mitochondria in density gradients and is probably intramitochondrial. Hybridization-competition experiments demonstrated that newly synthesized RNA associated with the polyribosomes at the blastula stage was to some extent different from that present in the unfertilized egg. A similar difference in the population of messenger RNA molecule on the polyribosomes was observed between the blastula and gastrula stages of development. RNA synthesized on cytoplasmic templates (mitochondrial DNA) was not detectably different between the blastula and gastrula stages.

Acetic acid-soluble proteins in the developing sea urchin☆

Abstract Sea urchin embryos ( Lytechinus variegatus ) were subjected to pulses of 14 C-alanine during cleavage, blastula, gastrula, and pluteus stages of development. Acetic acid-soluble proteins from nuclear, soluble, and ribosomal fractions were isolated and chromatographed on carboxymethylcellulose columns. The results indicate that one group of acetic acid-soluble proteins may be nuclear proteins which are selectively synthesized at the beginning of the blastula stage.