Thermal Melting Profiles Research Articles

Isolation and characterization of chromatin and DNA from the sperm of the spider crab, Libinia emarginata.

ABSTRACT An isolation technique is described which yields highly purified sperm from various regions of the reproductive tract of the crab Libinia emarginata. Sperm chromatin was isolated and analysed with respect to ultrastructure, ultraviolet absorption spectrum, thermal melting profile, behaviour in CsCl gradients, and chemical composition. Sperm-isolated chromatin not stabilized with uranyl acetate prior to ethanol dehydration, like prokaryotic chromatin, has aggregated fibres with a diameter in the range 30–95 nm or more. With uranyl acetate stabilization, only very fine fibres are seen. Pronase treatment of the thick fibres leads to formation of very fine fibres, showing that protein is associated with this isolated chromatin. Polyacrylamide electrophoretic analysis of the chromatin protein shows that it is not basic. Standard basic protein staining of sperm taken from testes, vas deferens, and seminal receptacles after various periods of storage there gives a negative nuclear reaction in all cases. This suggests the absence of sperm nuclear histones and protamines. Sperm chromatin protein is salt-dissociable and has a ratio of basic to acidic amino acids of 0·43–0·48. The-protein/DNA ratio is 2·3–3·2, and the ratio E280/E260, is 0·50–0·67. Thermal denaturation curves are identical for sperm chromatin and purified sperm DNA, supporting the other data showing lack of nuclear basic proteins. An (A + T)-rich satellite DNA component comprises about 7–9 % of the total sperm genome. It is suggested that the genes which specify gamete-type nuclear basic proteins are either permanently repressed or deleted in decapod Crustacea.

Studies on the secondary structure of ribosomal ribonucleic acid components of rabbit reticulocytes.

AbstractThe conformation in solution of fractionated 30 S and 19 S ribosomal RNA from rabbit reticulocytes has been studied by optical rotatory dispersion, analysis of thermal melting profiles and their derivatives, and spectrophotometric acid‐base titration. From a consideration of the limitations of these methods, it has been possible to set limiting values on the degree of base‐pairing and the lengths of the double helices: between 60 and 80% of the bases in 19 S and 30 S RNA are estimated to be paired. The paired segments are not shorter than 4 base pairs, and evidence from other sources is available which indicates that they are not longer than 8–16 base pairs. The spread of helix lengths is greater in the 30 S than in 19 S RNA; and other differences are noted. Several distinct populations of double helices, differing in their thermal stability, are present. Estimates are presented from spectrophotometric and titration data for the base compositions of the paired and unpaired regions.