M.R.V. Murthy

Ultracentrifugation of small volumes of tissue extracts without the use of tube adapters.

Abstract A method is described for high speed centrifugation of small volumes of homogenates, tissue extracts, or body fluids without the use of expensive adapters. The procedure consists of introducing the samples into light polyethylene microcentrifuge tubes and then floating them in a medium contained in standard cellulose nitrate tubes. This assembly can be centrifuged at 198,000g for 90 min with no visible distortion of the sample tubes.

Determination of protein in extracts containing interfering substances and in radioactive samples following scintillation counting

Abstract A method is described for determination of protein in biological preparations containing various interfering substances normally present in extraction media. The main steps of the procedure consist in depositing the protein solution on filter paper strips and removing all small molecular weight substances by washing with a number of aqueous and nonaqueous solvents. The protein remaining on the paper is then determined by a modification of Lowrys colorimetric procedure. The method also permits the determination of protein in radioactive samples which have been previously counted using a liquid scintillation mixture such as Apuasol or toluene-POPOP.

Rapidly reassociating DNA of rat brain.

Abstract— DNAs were isolated from brains of rats at different stages of growth and were fractionated on hydroxyapatite columns on the basis of their denaturation and reassociation properties. The results indicate that rat brain DNA had a heterogeneous population of base sequences. Approximately 10 per cent of the total DNA exhibited rates of reassociation characteristic of repetitive DNA. Differences were also observed in the pattern of denaturation of brain and liver DNAs of newborn and adult rats.

ISOACCEPTOR tRNAs FOR GLUTAMATE, GLUTAMINE, ASPARTATE AND ASPARAGINE IN CALF BRAIN

Abstract— Glutamyl, glutaminyl. aspartyl and asparaginyl tRNAs of calf brain were analysed by reverse phase chromatography for isoacceptor tRNAs. The radioactivity profiles revealed two peaks for gluta‐mate. three for glutamine, two for aspartate and one for asparagine. Comparison of brain tRNAs with tRNAs from other sources showed that glutamate and aspartate tRNAs of brain closely resembled a majority of other tRNAs in the number and relative abundance of isoacceptors. Glutamine and asparagine tRNAs from different sources exhibited more marked differences.

Effects of Cap Analogue or Cap Removal on the Translation of Rat Brain mRNA In Vitro

Abstract: The role of cap structures in the translation of brain mRNA was examined by measuring protein biosynthesis in vitro in wheat germ and reticulocyte systems programmed by mRNA that was either untreated or oxidized by periodate or from which 5′‐terminal 7‐methylguanosine (m7G) was removed by oxidation and β‐elimination. In another series of reactions, amino acid incorporation into polypeptides was measured in the absence and in the presence of varying concentrations of the cap analogue 7‐methylguanosine 5′‐triphosphate (pppm7G). The results indicated that any of the above treatments interfered with brain mRNA translation, the degree of inhibition depending on the translation system used, the concentration of mRNA, and the source of initiation factors. Homologous brain initiation factors were superior to reticulocyte factors in providing a partial relief from inhibition of translation caused by these treatments. It was also found that synthesis of the brain‐specific protein S‐100 was inhibited by β‐elimination of mRNA, by pppm7G, or by the presence of capped globin mRNA, indicating that the mRNA for this protein was probably capped.

Separation and purification of small quantities of specific RNA species by polyacrylamide gel electrophoresis using prestained RNAs as markers

Abstract A method is described for the separation and purification of different molecular species of RNA in microquantities using polyacrylamide gel electrophoresis. The experimental procedure consists of the following steps; (i) partial prestaining of RNA with methylene blue at a concentration of the dye which would not affect the electrophoretic migration of the RNA bands, but which would permit visual observation of the migrating bands during electrophoresis; (ii) use of short columns just sufficient to achieve separation of each species of RNA as a single compact band rather than a series of bands; (iii) trapping of each of the eluting RNA species from the gel on DEAE-cellulose dises in sequential order; and (iv) elution of the RNAs from the DEAE-discs by extraction with triethylammonium bicarbonate and recovery of the corresponding RNAs by lyophylization.

Recovery of microquantities of subcellular particles by ultracentrifugation in automatic pipet tips.

Abstract A method is described for centrifugation and retrieval of microquantities of sedimentable subcellular particles and macromolecules using disposable polypropylene automatic pipet tips. The procedure is based on the observation that such tips covered at the two openings by easily removable stoppers can be centrifuged at high speeds without leakage of contents. This makes available separate exits for easy removal of supernatant and sediment at the ends of the tips where the two fractions collect after centrifugation. Quantitative recovery was obtained for as little as 3 μg of RNA and for polysomes from as little as 5 mg of rat brain tissue.

MOLECULAR BIOLOGICAL MODELS IN GERIATRIC NEUROBIOLOGY

Abstract 1 Some of the theoretical and experimental approaches based on concepts of molecular biology that have been developed recently for the study of the aging phenomenon have been summarized. The importance of investigation of the aging nervous system in this context has been discussed. 2 Experimental results have been presented showing that chromatins from cerebral cortices of young and old rats differ from each other in a number of qualitative and quantitative parameters, for example, in the relative proportion of DNA, RNA, histone and nonhistone proteins, in the stability of DNA secondary structure to thermal denaturation and in the amounts of single-stranded DNA, free double-stranded DNA and protein-covered DNA. 3 Cerebral cortex from the young rat appears to possess a larger portion of DNA which is free or attached to acidic protein and a smaller portion of DNA attached to histories, as compared to the old tissue. This correlates with the greater metabolic activity of the nervous system of the young as compared to the old animal. 4 The data suggest that the stabilization of DNA in the aged tissue may be due to intercalation with protein and that both the unique and reiterated sequences may be stabilized in this manner.

Recovery of amino acids and tRNA after scintillation counting of radioactive aminoacyl-tRNA in filter paper strips

Abstract Amino acids are quantitatively recovered from aminoacyl-tRNA in filter paper strips by treatment with 0.3 m ammonium carbonate. A simple apparatus is described for deaminoacylation and separation of amino acids and tRNA. The amino acids, thus recovered, can be directly analyzed by paper chromatography since ammonium carbonate is removed by lyophilization. Nonvolatile reagents such as Tris-HCl and sodium carbonate also hydrolyze aminoacyl-tRNA, but unless the products of deaminoacylation are desalted, the presence of these reagents gives rise to chromatographic artifacts such as peak broadening, appearance of multiple peaks, displacement of R f values, etc. tRNA remaining after deaminoacylation retains only a small fraction of its original capacity to bind amino acids. Part of this loss of activity appears to be due to structural damage to tRNA in the course of washing and counting procedures and in part, due to the effect of filter paper in shifting the equilibrium of the reaction toward hydrolysis of aminoacyl ester bond.

Blocked and Methylated 5′-Terminal Cap Structures of Rat Brain Messenger Ribonucleic Acids

Abstract: The presence and identity of 5′‐terminal cap structures in rat brain polysomal mRNA were investigated by radiolabeling the mRNA by periodate oxidation and [3H]sodium borohydride reduction or by β‐elimination of 5′‐terminal nucleoside and incorporation of 32P in the presence of polynucleotide kinase. The labeled mRNAs were digested with nucleases and the cap structures were isolated and identified by chromatographic and electrophoretic procedures. The results showed that rat brain mRNAs contained cap 1 and cap 2 structures and no caps of the zero type. The proportion of cap 2 was higher than that of cap 1. Both caps had 7‐methylguanosine (m7G) as the 5′‐terminal nucleoside, which was linked to the next nucleoside by an inverted triphosphate bridge, as in other eukaryotic mRNAs. The most prominent nucleoside in the 5′‐penultimate position was 6‐methyl‐2′‐O‐methyiadenosine [m6A(m)] followed by 2′‐O‐methyladenosine [A(m)], which together contributed to nearly 70% of both cap 1 and cap 2 structures. 2′‐O‐Methylguanosine [G(m)] accounted for approximately 18%, the rest being made up of 2′‐O‐methyl‐cytidine [C(m)] and 2′‐O‐methyluridine [U(m)].