Joseph Ilan

Sampling of the leucine pool from the growing peptide chain: Difference in leucine specific activity of peptidyl-transfer RNA from free and membrane-bound polysomes☆

Abstract The specific activity of leucine in newly synthesized protein was determined by isolating the nascent polypeptides of the growing polypeptide chains. The newt, Triturus viridescens , was labeled in vivo with [ 3 H]leucine. Polysomes were prepared from the livers. Peptidyl-tRNA was released from the polysomes by EDTA, isolated by sucrose gradient and purified on hydroxylapatite. It was then hydrolyzed with HCl and the amino acids were reacted with 14 C-labeled 1fluoro-2,4-dinitrobenzene. The specific activity of [ 3 H]leucine was determined from the [ 14 C]dinitrophenyl-[ 3 H]leucine after purification by two-dimensional thin layer chromatography. By this approach we found twofold differences between leucine specific activity in the growing polypeptide chain of free polysomes and that of membrane-bound polysomes. Moreover, we recorded eight to tenfold differences between the specific activity of leucine in peptidyl-tRNA and that in the acid-soluble pool. Our results indicate and define the intracellular compartmentalization of the leucine pool available for protein synthesis.

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Abstract Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 m M MgCl 2 , which is considered very high. The same high Mg 2+ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 m M MgCl 2 . This concentration of Mg 2+ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, m M MgCl 2 . The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg 2+ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.

Presence of MHC-I and B-7 molecules in rat and human glioma cells expressing antisense IGF-I mRNA

Tumor cells frequently express the insulin-like growth factor I (IGF-I). Recently we demonstrated that rat glioma cells when transfected with a vector encoding antisense IGF-I cDNA lost tumorigenicity and induced a tumor specific immune response involving CD8+ lymphocytes. Here we show that the cultured transfected cell lines, rat C-6 glioma, human primary glioma and mouse teratocarcinoma, expressed an increased level of MHC-I and of co-signaling B-7 molecules. This increased expression of MHC-I and B-7, demonstrated by 51Cr release complement dependent cytoxicity assay and by immunostaining flow cytometry analysis, could contribute to the final immune recognition of glioma immunogenicity.

Large-scale isolation of plasmid DNA and purification of λ phage DNA using hydroxylapatite chromatography

Abstract A rapid and relatively simple procedure for purifying large quantities of plasmid DNA is described. Plasmid thus purified contains no detectable chromosomal DNA and little RNA or protein. The procedure combines alkaline denaturation and hydroxylapatite chromatography and utilizes an improved method of separating DNA from RNA. It was observed that the phosphate concentrations at which previously bound DNA as well as RNA elute from hydroxylapatite changed markedly as a function of urea concentration. In the presence of urea concentrations higher than 4 m , the ranges of phosphate concentration over which DNA and RNA elute show no overlap. This permits efficient washing of hydroxylapatite-bound DNA under conditions which should remove all bound RNA. λ Phage DNA is also easily eluted from hydroxylapatite under the conditions used.

Nerve-dependent regulation of absolute rates of protein synthesis in newt limb regenerates: Measurement of methionine specific activity in peptidyl-tRNA of the growing polypeptide chain

Abstract Limb regeneration in the newt requires the presence of nerve fibers at the amputation surface. Regeneration does not occur upon denervation. Incorporation of [ 35 S]methionine into hot TCA-precipitable material (cpm/μg of protein) increased sharply in the first few hours after denervation, then declined rapidly and plateaued at about 60% of the innervated control side at 48 hr. The specific activity of methionine was determined from hydrolyzed peptides of peptidyl-tRNA of the growing peptide chain. The hydrolyzed peptides were reacted with 3 H-labeled 1-fluoro-2,4-dinitrobenzene (FDNB), and the resulting DNP-methionine was isolated by two-dimensional thin-layer chromatography. Since the specific activity of [ 3 H]DNP is given, the ratio of 35 S to 3 H in DNP-methionine is a measure of methionine specific activity. Our results show that nervous control does not affect the specific activity of methionine in the growing polypeptide chain; the absolute rate of protein synthesis is affected. In both denervated and innervated regenerates, methionine specific activity in nascent peptides of membrane-bound polysomes was approximately half that of free polysomes, suggesting the existence of two distinct intracellular pools which are utilized for protein synthesis. Moreover, our results indicate that the specific activity of methionine in the acid soluble pool is 2–5 times lower than that found in the nascent peptides. Therefore, the acid-soluble pool cannot serve as a precursor pool for protein synthesis. We also estimated the half translation time of the nascent peptide chains. At any given moment, the nascent chain on the polysomes could be assumed to be half synthesized. Half translation time was measured as the time needed to release the labeled nascent peptides into the supernatant fluid. Denervation did not significantly affect the half translation time of average mRNA.

Purification on hydroxyapatite of liver ribosomes and polysomes from unfasted mice

Abstract Batchwise purification of liver ribosomal and polysomal preparations on hydroxyapatite removes glycogen, bound hemoglobin, ribonuclease and other proteins contaminating ribosomal preparations. This procedure eliminates mandatory fasting and thus provides pure ribosomes and polysomes from animals maintained under normal physiological conditions. Ribosomes and polysomes purified on hydroxyapaptite and eluted at low salt concentrations (0.4 M KH 2 PO 4 ) can be dissociated into subunits in the presence of puromycin and 0.3–0.5 M KCl. These subunits are active in poly(U)-directed phenylalanine synthesis. Sodium dodecylsulfate—acrylamide gel electrophoresis detected no degradation of the ribosomal RNA. The ability of polysomes to direct amino acid incorporation into hot trichloroacetic acid precipitate is not lost during purification on hydroxyapatite. In addition, the resolution of polysome profiles was greatly improved following hydroxyapatite purification. Incubation of purified murine liver polysomes in the absence of ribonuclease inhibitor resulted in no polysome breakdown. Identical treatment of unpurified polysomes resulted in a partial conversion to 80-S monomers, and suggested that ribonuclease was washed from the polysome preparations during purification. Ribosomes and polysomes from both mouse liver and rat liver can be purified batchwise on hydroxyapatite. In addition, this procedure can be used to purify ribosomes from both fed animals, with large amounts of cellular glycogen, and fasted animals with significantly less glycogen contamination.

Release factor regulating termination of complete protein in a eukaryotic organism

Abstract Polypeptide release reaction was studied using a protein release factor and a physiological substrate containing a complete polypeptide chain attached to monosomes of the insect Tenebrio molitor. The intermediate substrate used for the release reaction was synthesized using a cell-free protein synthesizing system from Tenebrio capable of polypeptide synthesis but not release of the completed chain. This system synthesized predominantly adult cuticular protein. The released product was characterized by chromatography after tryptic digestion; many of the tryptic peptides corresponded to those of cuticule labeled in vivo. The protein release factor was obtained as microsomal wash and was further purified by ammonium sulfate precipitation and column chromatography. It released about 30% of the monosome-bound peptide in the absence of GTP. The remaining 70% of peptidyl-tRNA was released as peptidyl-puromycin in the absence of release factor, but required transferase II and GTP. The peptidyl-puromycin varied in size from dipeptide to almost complete protein. The puromycin reaction was inhibited by diphtheria toxin and NAD and was dependent on GTP, while the release of completed peptide was independent of GTP and not affected by diphtheria toxin and NAD. The release factor was capable of releasing formylmethionine as formylmethionine-puromycin from ribosomes in response to poly(A3,U). Hence it is suggested that the release factor is responding to UAA as terminating codon.

Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor☆

In Tenebrio molitor, as well as in other biological systems, there are indications that differences in leucyl-tRNA synthetase activity may play a role in translational control. However, it has not been clear whether the difference in activity is due to the appearance of a multiplicity of enzymes during development or to the alteration of a single enzyme. The purification of leucyl-tRNA synthetase from day 1 and day 7 after the larval pupal molt of Tenebrio molitor is described. The enzyme from both developmental stages was purified over a 1000-fold. The two enzyme preparations are identical in molecular weight (99,000). They show the same characteristics after aging. The pH optimum, heat inactivation behavior, and dependency on divalent cations are the same for both enzymes. They also show identical kinetics with similar values of Km for leucine, ATP, Mg2+, and tRNA day 1. However, leucyl-tRNA synthetase purified from day 7 exhibits an additional function in recognizing a new species of isoaccepting tRNA in day 7 tRNA. We have tentatively concluded that the two enzymes are probably different forms of the same enzyme and the additional activity is due to alteration of the enzyme at the macromolecular level during development.

Chapter 4 Regulation of Messenger RNA Translation During Insect Development

Publisher Summary Insect metamorphosis is the differentiation at the postembryonic level. The process of cell differentiation is linked to the mechanisms that direct specific protein synthesis rather than with the ultimate appearance of protein molecules. The development of pupa into adult occurs in a differentiating system, where many changes take place, usually in one direction. In insects, growth and differentiation are regulated by several hormones. The use of juvenile hormone helps to study the mechanisms of gene expression. The major influence of juvenile hormone in promoting the synthesis of the two classes of cuticular protein-adult and pupal is at the translational level. This is facilitated by controlling the translation of cuticular messenger ribonucleic acid (mRNA). The translational control is mediated by the appearance of new rate-limiting aminoacyl-transfer ribonucleic acid (tRNA). The chapter explains the steps involved in protein synthesis. One of the characteristics of the differentiated cell is its ability, at one point in its development, to synthesize a large amount of a specific protein. This is accomplished by the production of stable mRNA. If a specific mRNA is stable, this amounts to the amplification of this specific gene. The tRNA and its activating enzymes are the limiting factors in the microsomal cell-free system for the regulation of cuticular mRNA translation. The chapter discusses the regulation of cuticular mRNA translation in cell-free systems from Tenebrio pupae . In addition, the chapter discusses the experiments on the initiation of mRNA translation during the development of Tenebrio pupae . In eukaryotic organisms, protein synthesis is initiated in a manner similar to the bacterial protein synthesis. The specificity in the formation of 80S initiation complex suggests that mRNA contains, besides the AUG codon for methionine, a specific sequence of nucleotides that is recognized by some elements of the initiation machinery.

Nerve-dependent changes in content of ribosomes, polysomes, and nascent peptides in newt limb regenerates

Abstract Regeneration of a newt limb requires a constant supply of adequate amounts of a neuronal contribution at the amputation site. Denervation during the early stages of regeneration precludes its growth and morphogenesis. It has been reported that denervation of a regenerating limb lowers the efficiency of incorporation of radioactive amino acids to 60% of contralateral control levels. To gain more insight into the mechanism responsible for this decrease, we examined the effects of denervation on the size distribution and quantity of regenerate polysomes. We characterized the [35S]methionine-labeled nascent peptidyl-tRNA from polysomes by hydroxyapatite chromatography. Moreover, we show that the labeled nascent peptides on polysomes can serve as a measure to quantitate the relative amounts of ribosomes on polysomes and the relative size of the translational machinery. Thus, we report that [35S]methionine-labeled nascent polypeptides on polysomes from denervated regenerates contain about 48% less radioactivity than those from controls. Despite decreased incorporation of [35S]methionine into nascent peptides, the relative distribution of radioactivity across linear sucrose gradients is not significantly altered by denervation. Studies of polysomes labeled with [3H]uridine prior to denervation indicate that ribosome content is depressed by denervation. Our results suggest that the nerve-dependent decrease in protein synthesis is mediated by decreasing the number of ribosomes active in protein synthesis. In addition, similarities in the ratios of free monosomes to polysomes and the relative size distribution of polypeptides between denervated and innervated regenerates indicate that in denervated regenerates the number of translatable mRNA molecules decreases in a coordinate manner with the number of ribosomes active in protein synthesis.