Chromatography of nucleic acids on hydroxyapatite III. Chromatography of RNA and polyribonucleotides

The chromatographic behaviour on hydroxyapatite columns of ribosomal and viral RNA, of transfer RNA and of several biosynthetic polyribonucleotides has been investigated in view of obtaining information useful for our understanding of the relationship between the secondary structure of polynucleotides and their elution molarity.Single-stranded, random-coiled polynucleotides are eluted at lower phosphate molarities than double-stranded, rigid polynucleotides. All double-stranded polynucleotides are eluted at about the same phosphate molarity, 0.20–0.22 M. Triple-stranded 2 poly U-poly A is eluted at a higher molarity (about 0.45 M).The chromatographic behaviour of bases, nucleosides, nucleoside mono- and polyphosphates, and oligonucleotides has also been studied, mainly for elucidating the mechanism of adsorption of polynucleotides on hydroxyapatite.     

Fractionation of brain macromolecules. Chromatography on hydroxyapatite of soluble proteins and nucleic acids from whole rat brain and the effect of autolysis

1. A procedure for the chromatographic fractionation of soluble brain proteins on calcium hydroxyapatite is described. Chromatograms obtained are reproducible; approximately 11 protein and at least three nucleic acid components can be identified. The effects of column dimensions and flow rate on the chromatograms obtained are described. 2. One of the nucleic acid components appears to correspond to soluble RNA and another to ribosomal RNA. Treatment of homogenates by procedures that cause the removal of ribosomes from soluble extracts also cause the disappearance of the component corresponding to ribosomal RNA. Centrifugation of rat-brain homogenates prepared in 0·25m-sucrose at 105400g for 90min. causes the sedimentation of ribosomes as well as the disappearance of the component corresponding to ribosomal RNA. Extraction of brain tissue, or particulate fractions prepared from brain, with dilute buffers causes the solubilization of part of the ribosomal RNA that then can subsequently be identified in the chromatogram. 3. Autolysis under aerobic conditions has been shown to cause an increase in the nucleic acid component in the chromatogram that corresponds to the ribosomal RNA. Aerobic autolysis causes part of the ribosomal RNA to be solubilized, as evidenced by its failure to be sedimented by centrifugation at 105400g for 90min. and by its appearance in the fraction corresponding to ribosomal RNA in the chromatogram. These changes were not observed when autolysis was carried out under anaerobic conditions. Aerobic autolysis also caused changes in those proteins that are strongly adsorbed on the gel. 4. In general, the proteins of the cell sap appeared to be fairly stable to autolysis. Marked differences in the chromatograms are observed when soluble proteins from the particle fraction were autolysed and chromatographed.     

Thermal elution chromatography of nucleic acids on hydroxyapatite.

Hydroxyapatite thermal elution chromatography was studied from an empirical standpoint. The dependence of elution temperature on elution buffer concentration was determined for various types of buffer, hydroxyapatite and nucleic acid. The results are analyzed in terms of the proper design and interpretation of thermal elution experiments. The potential for serious artifacts is demonstrated and the means by which they may be avoided is described. Various commercially available hydroxyapatites were tested in conjunction with various aqueous and partially non-aqueous buffer systems. Among the materials tested, potassium phosphate and Bio-Rad HTP were found to constitute the best buffer-hydroxyapatite system for most types of thermal elution study.     

Shedding light on proteins, nucleic acids, cells, humans and fish

I was trained as a physicist in graduate school. Hence, when I decided to go into the field of biophysics, it was natural that I concentrated on the effects of light on relatively simple biological systems, such as proteins. The wavelengths absorbed by the amino acid subunits of proteins are in the ultraviolet (UV). The wavelengths that affect the biological activities, the action spectra, also are in the UV, but are not necessarily parallel to the absorption spectra. Understanding these differences led me to investigate the action spectra for affecting nucleic acids, and the effects of UV on viruses and cells. The latter studies led me to the discovery of the important molecular nature of the damages affecting DNA (cyclobutane pyrimidine dimers) and to the discovery of nucleotide excision repair. Individuals with the genetic disease xeroderma pigmentosum (XP) are extraordinarily sensitive to sunlight-induced skin cancer. The finding, by James Cleaver, that their skin cells were defective in DNA repair strongly suggested that DNA damage was a key step in carcinogenesis. Such information was important for estimating the wavelengths in sunlight responsible for human skin cancer and for predicting the effects of ozone depletion on the incidence of non-melanoma skin cancer. It took experiments with backcross hybrid fish to call attention to the probable role of the longer UV wavelengths not absorbed by DNA in the induction of melanoma. These reflections trace the biophysicist's path from molecules to melanoma.     

Self-assembly of proteins and their nucleic acids

We have developed an artificial protein scaffold, herewith called a protein vector, which allows linking of an in-vitro synthesised protein to the nucleic acid which encodes it through the process of self-assembly. This protein vector enables the direct physical linkage between a functional protein and its genetic code. The principle is demonstrated using a streptavidin-based protein vector (SAPV) as both a nucleic acid binding pocket and a protein display system. We have shown that functional proteins or protein domains can be produced in vitro and physically linked to their DNA in a single enzymatic reaction. Such self-assembled protein-DNA complexes can be used for protein cloning, the cloning of protein affinity reagents or for the production of proteins which self-assemble on a variety of solid supports. Self-assembly can be utilised for making libraries of protein-DNA complexes or for labelling the protein part of such a complex to a high specific activity by labelling the nucleic acid associated with the protein. In summary, self-assembly offers an opportunity to quickly generate cheap protein affinity reagents, which can also be efficiently labelled, for use in traditional affinity assays or for protein arrays instead of conventional antibodies.     

Stimulatory effects of certain amino acids,sugars, organic acids,nucleic acids and metals on nitrate utilization by alkalophilic Bacillus

The growth of alkalophilic Bacillus no. A-40-2 with nitrate as the nitrogen source was highly stimulated by the addition of 0.1% of certain amino acids, sugars, organic acids, nucleic acids, or Fe²⁺ or Mn²⁺ at concentrations of 10 mM or more to the medium, resulting in maximum growth after 24 h. Other alkalophilic Bacillus strains also showed the same results. A decrease in the amount of nitrate in the medium was observed. The optimum pH of nitrate reductase was 7.5.     

Vibrational Raman optical activity of proteins,nucleic acids,and viruses

Due to its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light, is a powerful probe of biomolecular structure in solution. Protein ROA spectra provide information on the secondary and tertiary structures of the polypeptide backbone, hydration, side-chain conformation, and structural elements present in denatured states. Nucleic acid ROA spectra yield information on the sugar ring conformation, the base stacking arrangement, and the mutual orientation of the sugar and base rings around the C-N glycosidic linkage. ROA is able to simultaneously probe the structures of both the protein and the nucleic acid components of intact viruses. This article gives a brief account of the theory and measurement of ROA and presents the ROA spectra of a selection of proteins, nucleic acids, and viruses which illustrate the applications of ROA spectroscopy in biomolecular research.     

Further study of hydroxyapatite high-performance liquid chromatography using both proteins and nucleic acids, and a new technique to increase chromatographic efficiency

Previously developed hydroxyapatite high-performance liquid chromatography columns were tested further by using not only proteins but also nucleic acids. In both cases it was confirmed that the column can, in fact, discriminate subtle structural differences among molecules. Especially for protein mixtures a new technique was introduced to increase the efficiency of chromatography.