Elizabeth C. Travaglini

Nucleic acids and their components as affected by the Y chromosome of Drosophila melanogaster: I. Constitution and amount of the ribonucleic acids in the unfertilized egg☆

Abstract 1. 1. To evaluate the relation of the heterochromatic chromosome regions to the nucleic acids, a comparison has been made of the nature and the amount of the ribonucleic acids in two types of unfertilized Drosophila melanogaster eggs; one, the normal wild type (XX), and the other containing the Y chromosome attached to one of the Xs ( X XY sc 8 ). 2. 2. The proportions of the purine and pyrimidine bases present in purified ribonucleic acids from both types of egg has been determined. 3. 3. The X XY sc 8 type RNA contains a significantly higher proportion of adenine than does the XX RNA. 4. 4. The ultraviolet absorption curves of hydrolysates of the two types of RNA correspond to those expected for a mixture of nucleotides calculated from the analysis of the respective base compositions. Both before and after hydrolysis, the X XY sc 8 RNA has a higher specific absorption than does the XX, in conformity with its higher content of adenine. 5. 5. The quantity of RNA per egg is substantially equivalent in the XX and the X XY sc 8 types. 6. 6. In the light of the new chemical data, earlier measurements of the effect of the Y chromosome on the cytoplasm of the egg are now interpreted as indicating an effect of the Y on the composition instead of the amount of RNA in the egg cytoplasm. In this way, the hypothesis of a relation between the heterochromatic regions and the nucleic acid metabolism of the cell receives new support.

Nucleic acids and their components as affected by the Y chromosome of Drosophila melanogaster: II. Nucleosides and related compounds in the acid soluble fraction of the unfertilized egg

Abstract 1. 1. The purine and pyrimidine-containing compounds in the cold acid-soluble fraction of the unfertilized Drosophila melanogaster egg have been investigated, in order to compare the contents of these substances in the two genetic types (XX and X XY sc 8 ) in which differences in the RNA composition have already been found. 2. 2. Identification of 14 compounds has been accomplished by determination of their Rfs on paper, ultraviolet absorption spectra at 3 pHs and by tests for deoxyribose, ribose and phosphorus. The quantities found were compared with analyses of similar extracts after hydrolysis to the free bases, to gain estimates of recoveries. Although low, these were the same for the extracts from both genetic types, permitting comparisons to be made. 3. 3. No nucleotides were recovered from these extracts, the major components being ribosides and deoxyribosides, with free bases present to a lesser degree. All the usual purine and pyrimidine bases with the exception of 5-methylcytosine have been found; in addition, some unidentified compounds having absorption maxima around 235 and 290 mμ in the ultraviolet were isolated chromatographically. 4. 4. The presence of deoxyribosides, and particularly of deoxyuridine in the extracts, is noteworthy in relation to the synthesis of DNA. 5. 5. The content of purine compounds in the extracts from X XY sc 8 eggs is more than double that of the XX type; the chief difference is due to adenine containing compounds. Although the total content of pyrimidines is the same in the two types, the proportions differ: cytosine is present in only trace amounts in the X XY sc 8 type, as compared with sizable amounts in the XX, the converse being the case for thymine. 6. 6. The existence of effects of the Y chromosome on the pool size of purine compounds, on the proportions of the pyrimidines, as well as on the base composition of the RNA, may indicate the site of action of this chiefly heterochromatic chromosome to be on the synthesis of the nucleic acid bases.

Structure of Drosophila melanogaster dAT replicated in an in vitro system.

Abstract Poly dAT found in Drosophila melanogaster has a lower density than poly dAT synthesized de novo , or than any naturally occurring poly dAT. Used as a primer in an E. coli DNA polymerase system, it proved highly efficient, and yielded a product of the same density. Nearest neighbor analysis showed this product to be composed predominantly of alternating sequences of dA and dT, with about 2% dG and dC present, but with a sufficient quantity of apposed dA-dT to account for its low density. Thus D. mel . dAT appears to have a distinctive structure differing from the other members of the group of naturally occurring poly dATs.

Primary differentiation and ectoderm-specific gene expression in the animalized sea urchin embryo☆

Primary differentiation in sea urchin embryos, animalized by zinc, has been gauged by the formation of characteristic endodermal and mesodermal tissue derivatives and by the accumulation of the ectoderm-specific Spec 1 mRNA. Increasing the dosage of zinc diminishes the differentiation of secondary mesenchyme, primary mesenchyme, endoderm, and ectoderm, in decreasing order. Treatment is effective only during the blastula stages, involving successive periods of sensitivity for these tissues. Removal of zinc with chelator results in the resumption of differentiation to increasing degree for this series of tissues. The developmental initiation of Spec 1 gene expression, normally at the earliest blastula stage, can be delayed by zinc for at least 30 hr before being implemented by treatment of the animalized embryos with a chelator. We conclude (1) that those processes in the blastula which are required for differentiation and are suppressed by zinc are distinguishable from the determinative processes, which are not affected by the animalizing agent and occur earlier during midcleavage; (2) that animalization by zinc involves a graded failure of primary tissues to form; and (3) that animalization involves a pause in the schedule of differentiation, which can be reinstated by removal of the animalizing agent, thereby providing a survival value inherent in a flexible schedule of development.

Template recognition and chain elongation in DNA synthesis in vitro.

Abstract DNA polymerase from Escherichia coli (Pol I) and from avian myeloblastosis virus (AMV polymerase) were compared for the manner in which they catalyze the polymerization of deoxynucleotides upon a variety of synthetic and natural templates. It was found that the rates of nucleotide incorporation with different natural RNAs were similar. Both polymerases have an associated RNA endonuclease which hydrolyses RNA templates containing double-stranded regions. This activity depends on the presence of the complementary deoxynucleoside triphosphates, and/or polymerization. Both enzymes copy natural DNA, which has been sonicated and treated with E. coli exonuclease III, at the same rate. However, avian myeloblastosis virus DNA polymerase, which has no associated DNA exonuclease activity, is unable to copy double-stranded DNA and copies DNAase-treated DNA only 10% as well as Pol I. Pol I copied all the homopolymers investigated at a greater rate than AMV polymerase with the exception of poly(C) · oligo(dG). However, the initial rate of chain elongation, as measured by gel electrophoresis, was the same for the two polymerases, approximately 300 nucleotides incorporated per minute. Template saturation experiments show a stoichiometric relationship between template and enzyme at optimal rates of nucleotide incorporation which suggests that all enzyme molecules are potential catalysts. Enzyme saturation experiments indicate that not all enzyme molecules are “effectively” bound to a template. Fewer AMV polymerase than Pol I molecules are functionally bound to a particular template. From these data, it is concluded that the two polymerases elongate DNA chains in a similar way and that the manner in which the polymerases bind to a particular template accounts for the discrepancies found in their turnover numbers.

Chapter 5 Methods for the Extraction and Purification of Deoxyribonucleic Acids from Eukaryote Cells

Publisher Summary This chapter describes the methods that are routinely used in the extraction and purification of DNA. The choice of a method is usually determined by the type and amount of material available, whether a quantitative recovery of the DNA per cell is mandatory and in what molecular state the DNA is desired. For many analytical studies (molecular hybridization, renaturation kinetics, base sequencing, etc.), the molecular weight of the DNA need not be extremely high; however, the DNA should be in a native state, and it must be pure. Often the investigator is interested in measuring the amount of DNA per cell. Usually, he resorts to the Schmidt and Thannhauser or a similar procedure wherein DNA is precipitated by acid from an alkaline digest of a tissue homogenate and measured colorimetrically. This method is easy and very reproducible for most tissue culture cells and other eukaryote cells with a minimal amount of cytoplasm. The gradient extraction techniques are the best suited for microextractions of DNA, particularly the DNA from pure organelles. In these cases, a consideration of the manner in which the DNA is to be detected determines the methodology. If the DNA can be detected by its radioactive label, gradients in the preparative ultracentrifuge are to be preferred because the DNA can be recovered.