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J Ilan

Publications and source records attributed to J Ilan.

At least 73 records · Page 4Linked to original sources
PubMed1980

Isolation of ribonuclease-free polysomes from human placenta.

Human placenta is known to have a high level of polysome-bound ribonuclease which has hindered the isolation of intact polyribosomes from this tissue. We describe conditions for preparing polysomes devoid of apparent ribonuclease activity from both first trimester and term placenta. This stable preparation was achieved by utilizing buffer at low pH containing 300 mM LiCl and precipitating the polysomes chemically with 200 mM MgCl2. The yield of polysomes obtained by this procedure is 2-2.5 fold greater than that obtained by the conventional method of preparing placental polysomes. The polysomes are considered pure as judged by the ratios of absorbance 260/280 and 260/235. Moreover, the yield of polysomes obtained is greater than 95% of the tissue content and the profile of the polysomes is probably representative of the in vivo population. This is concluded from experiments in which a known amount of labelled chick polysomes was added to fresh placental tissue and the recovery of label and its distribution was analyzed.

Cell Fractionation↗
PubMed1980

The effect of cytosine arabinoside on the synthesis of rapidly labeled RNA during DNA replicating and non-DNA replicating periods of the cell cycle.

The effects of various concentrations of cytosine arabinoside (Ara-C) on the rates of DNA and RNA synthesis were investigated during the peak of DNA synthesis, using a naturally synchronized culture of Aedes aegypti (mosquito) cells. During this stage of the cell cycle, the synthesis of both DNA and RNA was found to be progressively inhibited with increasing concentrations of Ara-C. When the same concentrations of Ara-C were added to the culture at a time period when no DNA was being synthesized, it was found that the synthesis of RNA was not inhibited. Rapidly labeled polysomal RNA species were isolated from cultured cells in which DNA was being synthesized. The synthesis of these RNA species is inhibited completely in the presence of a concentration of Ara-C which inhibits 90% of DNA synthesis. It is suggested that during periods of DNA replication, Ara-C is a selective inhibitor of the synthesis of rapidly labeled RNA species or of the processing of these RNA species from the nucleus to the polysomes.

Aedes↗
PubMed1979

The synthesis and secretion of human chorionic gonadotropin by tissue slices from first trimester placentas.

Placental tissue slices from first trimester placentas synthesize and secrete proteins labeled by radioactive glucosamine are preferentially secreted as compared to proteins in general. One of the proteins synthesized and secreted is hCG. Processing and secretion of proteins, including hCG, by the tissue slices need a two-hour period. Both secretion and glycosylation of the protein can take place independently of protein synthesis. A method was developed for the specific determination of newly synthesized radioactive hCG in placental tissue.

Chorionic Gonadotropin↗
PubMed1979

Characterization of presumptive histone messenger RNA from a cell line of Aedes aegypti.

Four presumptive histone messenger RNAs were characterized from a cell line of Aedes aegypti, and their molecular weights were determined by electrophoresis. They were shown to be associated with polysomes during the peak of DNA synthesis, but not when DNA synthesis was inhibited by cytosine arabinoside or when DNA was not being synthesized. These mRNAs are associated with polysomes containing less than 8 ribosomes and having a high ratio of incorporation of lysine to tryptophan into their nascent peptides. The mRNAs released from these polysomes were translated in vitro and histone products were synthesized. Histones were not synthesized when the mRNAs were obtained from large polysomes or from small polysomes during the non-DNA synthetic period.

Aedes↗
PubMed1978

The ribosomes of Plasmodium berghei: isolation and ribosomal ribonucleic acid analysis.

Ribosomes and high molecular weight ribosomal ribonucleic acid (rRNA) from the blood stages of Plasmodium berghei parasites were studied in preparations free from host ribosome contamination. Purified malarial ribosomes were isolated in high yield from a population of ultrastructurally intact, viable parasites by hypertonic lysis with Triton X-100 and differential centrifugation. These ribosomes were shown to be derived from active polysomes and could be dissociated into subunits by puromycin-0.5 M KCl treatment. Malarial rRNA extracted from purified 40S and 60S ribosomal subunits was characterized by electrophoretic, sedimentation and base ratio analyses. Like certain other protozoa, the P. berghei 40S ribosomal subunit possessed an exceptionally large RNA species (mol. wt 0.9 X 10(6), while RNA isolated from the parasite's 60S subunit (mol. wt 1.5 X 10(6)) was specifically 'nicked' to produce one large component (mol.wt 1.2 X 10(6)) and one small component (mol.wt 0.3 X 10(6)) in equimolar quantities. These rRNA's migrate identically on polyacrylamide gels after heating to 63 degrees C for 5 min or under denaturing conditions in the presence of formamide, indicating an absence of aggregation and non-specific degradation of the rRNA species. Base composition studies showed P. berghei rRNA to be low in guanosine and cytosine content, as is the case for protozoa generally.

Animals↗
PubMed1977

Analysis by two-dimensional polyacrylamide gel electrophoresis of liver ribosomal subunnit proteins obtained from free and membrane-bound polysomes of unfasted animals.

Ribosomal proteins were analyzed by means of two-dimensional gel electrophoresis. To insure that the analysis included only that fraction of the ribosome actively participating in protein synthesis, only polysomal-bound ribosomes were used. This differs from previously reported analyses of liver ribosomal proteins. The ribosomal proteins were prepared from ribosomes of polysomal origin from membrane-bound and free polysomes. Membrane-bound and free liver polysomes were isolated from unfasted mice. The polysomes were purified on hydroxyapatite under conditions known to result in polysomes and ribosomes that are active in both endogenous and synthetic mRNA translation. Moreover, this is the first time that liver ribosomal protein was obtained and analyzed from animals that have not been starved prior to sacrifice. The puromycin-released ribosomes were dissociated into subunits and ribosomal proteins were analyzed by means of two-dimensional polyacrylamide gel electrophoresis. When 100-200 mug samples of the ribosomal subunit proteins were analyzed by two-dimensional electrophoresis, approximately 32 major proteins were detected for the 60 S ribosomal subunit and 25 major proteins for the 40 S ribosomal subunit. A total of 13 "fractional" ribosomal proteins was also detected in the ribosomal subunit profiles. No differences in number or mobility of the ribosomal proteins were found between the membrane-bound and free ribosome populations. We describe a system in which all ribosomal proteins are completely solubilized and quantitatively move from the first to the second dimension gel. Thus the total sample is separated and fractionated. This procedure elimates artifacts due to incomplete solubilization of ribosomal proteins, which is common for the transfer from the first- to second-dimension gel. Therefore, a more detailed and accurate analysis is achieved.

Animals↗
PubMed1977

Unwinding protein specific for mRNA translation fractionated together with rabbit reticulocyte initiation factor 3 complex.

Experiments with a rabbit reticulocyte cell-free system dependent on the addition of initiation factor 3 (eIF-3) and mRNA were carried out. In this system, using ribosomal subunits, AUG(U)(n) can direct polyphenylalanine synthesis in the absence of eIF-3 at 3 mM MgCl(2). Globin mRNA was not translated under similar conditions; its translation requires the addition of eIF-3. Moreover, the maximal rate of globin synthesis was achieved when the molar ratio of eIF-3 to ribosomes was approximately 1. This was taken to indicate that some ribosomal proteins were fractionated with eIF-3 and functioned in reconstitution of salt-washed ribosomes. In our system, almost all ribosomes were active, as evident from the fact that all were found in polysomes when analyzed at the time of linear incorporation, and the molar ratio of ribosomes to mRNA was maintained at 4:1. When AUG(U)(n) was hybridized with poly(A), it could not direct polyphenylalanine synthesis with or without eIF-3 and was a potent inhibitor of the translation of globin mRNA in the presence of eIF-3. When poly(A) containing 10% U was hybridized with AUG(U)(n) and added to the cell-free system, addition of eIF-3 promoted polyphenylalanine synthesis to about 80% of control. Moreover, eIF-3 was seen to shift significantly the melting temperature of globin and synthetic double-stranded RNA. These observations suggest that extraction of ribosomes with 0.5 M KCl may release a ribosomal protein that fractionates with eIF-3. This protein may function in unwinding or melting the secondary structure of mRNA and thus facilitate translation.

Animals↗
PubMed1977

Coordination of ribosome content and polysome formation during estradiol stimulation of vitellogenin synthesis in immature male chick livers.

To elucidate the mechanisms by which protein synthesis is affected by estradiol, we characterized cockerel liver polysomal profiles during hormone induction and withdrawal. We describe a method for isolating intact polysomes which results in preparations that are stable even after storage in solution at 10 degrees for 16 hr. In addition, our procedure eliminates the necessity for starving animals prior to experiments. Recovery of radioactive polysomes indicated that yield is about 90% and that our polysomal preparations appear to represent polysome distribution in vivo. Using this approach we show that estradiol injection stimulates ribosome content 6-fold and that formation of polysomes is coincident with the induction of vitellogenin synthesis. We also demonstrate that the size and number of polysomes increase and decrease in a coordinated fashion with the rate of vitellogenin synthesis. The kinetics of ribosome synthesis and the fact that at least 80% of the newly synthesized ribosomes are directly recruited into polysomes indicate that ribosomes might be limiting the rate of protein synthesis during the stimulatory phase of the hormone cycle.

Animals↗
PubMed1977

Influence of 9-beta-D-arabinofuranosyladenine on total protein synthesis and on differential gene expression of unique proteins in the rodent malarial parasite Plasmodium berghei.

The antibiotic 9-beta-D-arabinofuranosyladenine is a drug with a broad spectrum of activity against animal viruses, with little or no effect on mammalian cells, when administered in vivo or in vitro. Here we report that the antibiotic markedly inhibited the incorporation of [35S]methionine into malarial protein. Inhibition was apparent when the parasites were either exposed to the drug in vivo during the course of infection or incubated with the drug in vitro. Moreover, the antibiotic induced pronounced changes in the spectrum of proteins synthesized. Some proteins that are prominently apparent in the control disappear from the drug-treated parasites; others specific for drug-treated parasites appear, indicating changes in the commitment for gene expression as manifested by the appearance of the final protein product. Proteins synthesized were analyzed by two-dimensional polyacrylamide gels; the first dimension used isoelectric focusing in cylinder gels and the second dimension used electrophoresis in a lithium dodecyl sulfate slab gel. Proteins were visualized by radioautography.

Animals↗
PubMed1976

Requirement for homologous rabbit reticulocyte initiation factor 3 for initiation of alpha- and beta-globin mRNA translation in a crude protozoal cell-free system.

Experimental evidence showing specificity of rabbit reticulocyte initiation factor 3 (EIF-3) for selective initiation of mRNA translation is presented. A new cell-free system was developed from Crithidia fasciculata. The crude postmitochondrial supernatant fluid was treated with puromycin and 0.5 M KCl to dissociate mRNA from polysomes and ribosomes into subunits. The drug and salt were removed by gel filtration on Sephadex G-25. Additions of amino acids and energy source initiate protein synthesis. All synthesis starts at the initiation site. This treatment brought about a shift in MgCl2 optimum from 6 to 3 mM. Exogenously supplied rabbit reticulocyte globin mRNA is faithfully translated in this system. However, crithidial EIF-3 has a low affinity for globin mRNA as evidenced by a 6-fold increase in the rate of globin synthesis after the addition of rabbit reticulocyte EIF-3 in the range at which globin synthesis is linear to the amount of globin mRNA added to the system. It is also shown that in a reconstituted system in which ribosomal subunits are depleted from initiation factors, EIF-3 from rabbit reticulocytes has a higher affinity for globin mRNA, as measured by the formation of polysomes during the linear time of amino acid incorporation. These results are taken to indicate that initiation factor EIF-3 action should be considered as an enzyme catalyzed reaction for which various mRNAs serve as different substrate analogs. Therefore, specificity is most likely to be expressed as an affinity of enzyme to substrate and would show as rate difference rather than an all-or-none phenomenon.

Animals↗
PubMed1976

Galactosyltransferase activities during embryonic development of chick neural tissue.

Galactosyltransferase specific activities in embryonic chick retina, optic tectum, and telencephalon were found to decline during embryonic development. Incorporation of galactose from nucleotide sugar into exogenously added glycoprotein acceptor was measured in the presence of excess of glycoprotein acceptor. This ensured that the specific activity measurements were reflections of tru enzyme specific activity rather than availability of acceptor. Moreover, we have shown that the decline in specific activity is not due to degradation of the nucleotide sugar, UDP-galactose, under our in vitro assay conditions. Enzymatic specific activity declined sharply with embryonic age for all tissues tested. This decline was not affected by the presence of 5-bromodeoxyuridine during in vitro culture of embryonic chick neural retina above that caused by the culturing alone. Galactosyltransferase activity was not found to be associated with the plasma membrane fraction from homogenized tissue but rather with the microsomal fraction. Thus, the changes in galactosyltransferase specific activity detected here do not reflect changes at the cell surface.

Animals↗
PubMed1975

Analysis by two-dimensional polyacrylamide gel electrophoresis of the in vivo phosphorylation of ribosomal proteins derived from free and membrane-bound polysomes.

Analysis of in vivo phosphorylation of mouse liver ribosomal proteins was performed by two-dimensional polyacrylamide gel electrophoresis following 32P-injection. Our method is special and differs from other eukaryotic systems reported in that all proteins separated on the first dimension gel are completely solubilized, moving quantitatively to the second dimension gel. Only ribosomes from polysomes were used, ensuring analysis of ribosomes actively engaged in protein synthesis. We resolved sixty-five distinct proteins from ribosomes from membrane bound or free polysomes. In both cases radioautography revealed similar labeled patterns with one highly phosphorylated ribosomal protein and five marginally labeled spots.

Animals↗