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W F Anderson

Publications and source records attributed to W F Anderson.

At least 343 records · Page 19Linked to original sources
PubMed1975

Binding of MET-TRNAf and GTP to homogeneous initiation factor MP.

Homogeneous initiation factor MP forms a stable complex with Met-tRNAf which binds to nitrocellulose filters in the absence of ribosomal subunits. Complex formation is rapid at 0 degrees and the rate of reaction is stimulated 20-fold by GTP when freshly prepared initiation factor MP is used. Under optimal assay conditions, a 1:1:1 stoichiometry for initiation factor MP, GTP, and Met-tRNAf is indicated, based on a molecular weight for initiation factor MP of 180,000. Kinetic analysis of ternary complex formation suggests an ordered reaction sequence with binding of GTP followed by binding of Met-tRNAf. However, binding of GTP appears to produce an unstable state which leads to rapid inactivation of initiation factor MP in the absence of Met-tRNAf. Formation of a stable binary complex of initiation factor MP and Met-tRNAf occurs in the absence of GTP. The binary complex cannot subsequently bind GTP. While storage of initiation factor MP at 0 degrees for several weeks has no effect on the rate or extent of Met-tRNAf binding in the presence of GTP, the rate of binary complex formation is increased 10-fold. The binary and ternary complexes appear to bind to 40 S ribosomal subunits with equal efficiency.

Animals↗
PubMed1975

Purification and physical properties of homogeneous initiation factor MP from rabbit reticulocytes.

Initiation factor MP was purified 1570-fold with 67% recovery of total activity present in 0.5 M KCl extracts of rabbit reticulocyte ribosomes. Initiation factor MP forms a ternary complex with Met-tRNAf and GTP or a binary complex with Met-tRNAf alone, the details of which are presented in the accompanying paper (Safer, B., Adams, S. L., Anderson. W. F., and Merrick, W. C. (1975) J. Biol. Chem. 250, 9076-9082). Initiation factor MP was homogeneous by the following criteria: (a) electrophoresis as a single band in gels of 5, 6, 7, 8, 9, and 10% acrylamide; (b) equilibration as a single band during isoelectric focusing; (c) sedimentation as a single symmetrical boundary during sedimentation velocity experiments; (d) linear plots of sedimentation equilibrium data; (e) symmetrical absorbance (at 280 nm) and activity profiles during DEAE-cellulose and Sephadex G-200 chromatography, and (f) symmetrical distribution of initiation factor MP during sucrose density gradient band sedimentation. The molecular weight of the initiation factor MP monomer (0.2 mg/ml) by low speed sedimentation equilibrium was 90,800. Calculations based on the Stokes radius and sedimentation velocity show the existence of relatively stable 90,000-dalton monomers or 180,000-dalton dimers at low (0.1 mg/ml) and high (9.75 mg/ml) concentrations of initiation factor MP, respectively. Electrophoresis in sodium dodecyl sulfate gels indicates that initiation factor MP monomer is composed of two noncovalently linked subunits with molecular weights of 52,000 and 34,000. Despite a relatively normal amino acid composition and an isoelectric point of 6.4, initiation factor MP behaves as a basic protein, eluting from phosphocellulose at 650 mM KCl (pH 7.9). Both ternary complex formation and methionyl-puromycin synthesis co-purify, indicating that a single protein is required for both activities.

Amino Acids↗
PubMed1975

Eukaryotic initiation complex formation. Evidence for two distinct pathways.

Two distinct pathways have been elucidated which lead to the formation of an AUG-dependent initiation complex. One pathway involves the use of initiation factor M1 (IF-M1) to promote AUG-dependent binding of the initiator tRNA to the 40 S subunit, followed by joining of the 60 S subunit in the presence of IF-M2A, IF-M2B, and GTP. The second pathway involves the IF-MP-directed binding of initiator tRNA to the 40 S subunit via a ternary complex of IF-MP-GTP-Met-tRNAf. This reaction does not require AUG codon. However, subsequent formation of an 80 S initiation complex (as determined by methionyl-puromycin synthesis) required AUG as well as IF-M2A, IF-M2B, and GTP. Since both pathways require the same complementary initiation factors (at the same level), it would appear that the only difference is the manner in which the initiator tRNA is bound to the 40 S subunit, either by IF-M1 or IF-MP. Examination of the requirements for endogenous mRNA-directed methionyl-puromycin synthesis indicates a greater difference between IF-MP and IF-M1 in that only IF-MP was capable of forming an 80 S initiation complex which was sensitive to puromycin.

Animals↗
PubMed1975

Cell-free transcription of mammalian chromatin. Quantitative measurement of newly synthesized globin messenger RNA sequences.

Transcription of globin mRNA sequences from rabbit marrow chromatin was detected by hybridization with globin complementary DNA (cDNA). The presence of newly synthesized RNA in cDNA-globin mRNA hybrids isolated by Cs2SO4 density equilibrium centrifugation required the addition of both rabbit marrow chromatin and DNA-dependent RNA polymerase (Escherichia coli) to the transcription reaction. No globin mRNA sequences were detected in RNA transcribed from rabbit liver chromatin or from rabbit marrow DNA. Selective transcription of globin mRNA sequences was therefore tissue-specific and dependent on the presence of chromosomal proteins. Globin mRNA sequences synthesized by E. coli RNA polymerase were distinguished from those synthesized by chromatin-bound (endogenous) RNA polymerases by the use of alpha-amanitin. A typical reaction with rabbit marrow chromatin yielded 100 mug of purified RNA which contained approximately 5 ng (0.005%) of globin mRNA sequences synthesized by E. coli RNA polymerase, 1 ng (0.001%) of globin mRNA seqeences synthesized by endogenous RNA polymerases, and 4 ng (0.004%) of globin mRNA sequences derived from chromatin-associated (endogenous) RNA. Forty per cent of the globin mRNA sequences derived from endogenous RNA could be removed by poly(U)-Sepharose chromatography. The accurate measurement of globin mRNA sequences required improved conditions for the purification and hybridization of RNA transcribed from chromatin.

Animals↗
PubMed1975

Purification and characterization of homogeneous initiation factor M2A from rabbit reticulocytes.

Rabbit reticulocyte initiation factor M2A has been prepared in homogeneous form. The final preparation was purified 2,300-fold and ran as a single band on polyacrylamide gel electrophoresis in three different buffer systems: alkaline, sodium dodecyl sulfate, and acidic 6.5 M urea. IF-M2A also ran as a single band in polyacrylamide gel isoelectric focusing experiments with an apparent pI of 6.45. The molecular weight of IF-M2A was approximately 125,000 based on determinations by low speed equilibrium centrifugation (118,000), sodium dodecyl sulfate gel electrophoresis (130,000) and s20,w combined with Stokes radius (124,000). The amino acid composition of IF-M2A revealed three unusual features: a) the basic amino acids represented 19.4 mol %; b) glutamicacid (plus glutamine) constituted 18.8 mol%; c) tryptophan and cysteine residues were only 0.4 and 0.7 mol%, respectively. Homogeneous IF-M2A was tested in several initiation assays using either natural or artificial mRNAs. In each assay tested, homogeneous IF-M2A fully substituted for cruder preparations and at concentrations commensurate with its increased purity. IF-M2A was also examined for ribosome-dependent GTP hydrolysis, an assay requiring ribosomes but no other initiation factors. Analysis of the data yielded a Km for GTP of 10 muM and a Vmax for hydrolysis of 1.20pmol/mug IF-M2A/min. In addition, IF-M2A mediated GTP hydrolysis required both 40 S and 60 S subunits for maximal activity. The possibility that IF-M2A is a factor required for the joining of 40 S and 60 S subunits is discussed.

Amino Acids↗
PubMed1975

Comparison of fMet-tRNAf and Met-tRNAf from Escherichia coli and rabbit liver in initiation of hemoglobin synthesis.

A comparison has been made of the ability of the formylated and unformylated initiator tRNAs of Escherichia coli and rabbit liver to participate in a number of model reactions of protein synthesis. These reactions include: (a) formation of a ternary complex composed of the initiator tRNA, GTP, and initiation factor MP; (b) ApUpG-directed binding of the initiator tRNA to 40 S subunits with initiation factor Ml; (c) formation of the artificial dipeptide, methionylpuromycin; (d) formation of the natural initial globin dipeptide, methionylvaline; and (e) synthesis of sheep alpha and betaB-globin chains on reticulocyte polysomes from a type BB sheep. The results of these studies indicate that although the prokaryotic initiator tRNA species function efficiently in the partial reactions which involve only binding, the methionine donated by the prokaryotic tRNA is not incorporated efficiently into peptide linkage. This suggests that the initial high level of binding of the E. coli initiator tRNAs may be nonspecific, and that the structure of the tRNA itself is important for specific recognition by eukaryotic initiation factors. The effect of formylation on the effectiveness of the initiator tRNA is not clear; it reduces activity in ternary complex formation, does not affect ApUpG-directed binding to 40 S subunits, and increases the rate or extent of incorporation of methionine, or both, into methionylpuromycin and globin chains.

Animals↗
PubMed1975

alpha-and beta-Globin complementary deoxyribonucleic acids of human and rabbit. Specificity of hybridization.

The specificity of hybridization was compared between the human and rabbit alpha and beta-globin complementary DNAs (cDNAs) and the corresponding alpha and beta-globin messenger RNAs (mRNAs). The globin chain-specific mRNAs of rabbit were prepared from polysomes incubated with O-methylthreonine (alpha and beta) or from postribosomal supernatant (alpha). Enrichment for either the alpha- or beta-globin mRNA was demonstrated by cell-free protein synthesis and by RNA-cDNA hybridization. Human mRNAs, active as templates for RNA-directed DNA polymerase, were prepared from reticulocytes of patients with hemolytic anemia, alpha-thalassemia (hemoglobin H disease), and beta-thalassemia. Because there was partial cross-hybridization between human mRNA and rabbit cDNA, the rabbit alpha- and beta-globin cDNAs could be used to demonstrate that the beta-thalassemia mRNA was enriched in human alpha-globin mRNA sequences and that the alpha-thalassemia mRNA was enriched in human beta-globin mRNA sequences. These results were confirmed by preparation of thalassemia globin cDNAs and subsequent hybridization to their template mRNAs. The amount of cross-hybridization between the human and rabbit alpha-globin mRNA and the two alpha-globin cDNAs was comparable to the cross-hybridization between the two beta-globin mRNAs and the two beta-globin cDNAs, indicating a similar degree of evolutionary divergence in the nucleotide sequences of the two globin genes.

Animals↗
PubMed1975

Purification and properties of rabbit reticulocyte protein synthesis elongation factor 2.

A homogeneous preparation of elongation factor 2 (EF-2) has been obtained from rabbit reticulocytes. EF-2, purified 1,960-fold, appears to be active as a single polypeptide chain with a molecular weight of approximately 100,000 based upon the following determinations: sodium dodecyl sulfate gel electrophoresis (95,000); sedimentation equilibrium centrifugation (112,000); gel filtration (97,000); ADP-ribosylation (103,000). The amino acid composition of rabbit reticulocyte EF-2 is almost identical with that of rat liver EF-2. The unknown amino acid in rat liver EF-2 which can be ADP-ribosylated appears also to be present in rabbit reticulocyte EF-2. A comparison of the amino acid composition of rabbit reticulocyte and rat liver EF-2 with Escherichia coli EF-G shows a high degree of similarity with only four amino acids differing by more than 10% (alanine, lysine, cysteine, and leucine).

Adenosine↗
PubMed1975

Purification and characterization of homogeneous protein synthesis initiation factor M1 from rabbit reticulocytes.

An eight-step procedure has been devised for the preparation of homogeneous rabbit reticulocyte IF-M1. Molecular weight determinations based on IF-M1 activity (gel filtration and sucrose density gradient sedimentation) and based on IF-M1 protein (low speed equilibrium sedimentation and sodium dodecyl sulfate gel electrophoresis) indicate that IF-M1 is active as a single polypeptide chain of 65,000 molecular weight. The amino acid composition of IF-M1 has been determined. There appears to be no unique features in the amino acid composition of IF-M1, except perhaps an elevated proline content (6.9 mol %). The catalytic properties of purified IF-M1 were similar to those previously reported by this laboratory for crude preparations of IF-M1. The sensitivity of IF-M1 activity to N-ethylmaleimide and heat (45 degrees) inactivation was tested in two model reactions requiring minimal complementary factors: (a) AUG-directed fMet-tRNAf binding to ribosomes; and (b) poly(U)-directed polyphenylalanine synthesis at 4 mM Mg2+ (IF-M2A, IF-M2B, EF-1, and EF-2 also required). IF-M1 activity proved to be sensitive to both N-ethylmaleimide and temperature (45 degrees). In addition, a contaminant of partially purified IF-M1 preparations has been found which is capable of fMet-tRNAf binding but is inactive in poly(U)-directed polyphenylalanine synthesis at low Mg2+ concentration.

Amino Acids↗
PubMed1975

Activation and inactivation of genes determining hemoglobin types.20s.

Globin gene switching in sheep and goats has been used as a model system for examining gene expression in differentiating red blood cells. Sheep and goats switch from the synthesis of hemoglobin A to hemoglobin C in response to erythropoietin. The regulatory mechanism producing this switch in hemoglobin types could occur at the cellular, nuclear, or cytoplasmic level. Evidence is presented which suggests that regulation is occurring, in fact, at the nuclear level. Sheep and goat erythroid colonies have been grown in plasma clot culture in order to study the synthesis of individual globin chains. Erythropoietin is required for colony formation. The switch from hemoglobin A to hemoglobin C synthesis requires not only colony formation but also a higher concentration of erythropoietin than is required just for the production of colonies. A cell-free transcriptional system using bone marrow chromatin and mammalian DNA-dependent RNA polymerase has been developed in order to examine the nuclear control mechanisms in more detail.

Amino Acid Sequence↗
PubMed1975

Hemoglobin synthesis in somatic cell hybrids: globin gene expression in hybrids between mouse erythroleukemia and human marrow cells or fibroblasts.

Somatic cell hybrids were generated by fusion of mouse erythroleukemia cells either to mouse L cells (B82), human fibroblasts (W1-18 VA2), or human marrow fractions enriched in erythroblasts. The hybrid cells were examined for globin gene expression by benzidine staining to detect cytoplasmic hemoglobin, and by molecular hybridization of cellular RNA to globin complementary DNA (cDNA) to detect globin messenger RNA (MRNA). The fibroblast (human or mouse) times erythroleukemia cell hybrids grown in monolayer retained most of the chromosomes of each parent. Neither cytoplasmic hemoglobin nor globin mRNA was detected in dimethylsulfoxide-treated fibroblast times erythroleukemia hybrid cells, indicating extinction of hemoglobin synthesis prior to the formation of cytoplasmic mRNA. The human marrow times mouse erythroleukemia hybrid cells grown in suspension culture contained only a few human chromosomes and exhibited low levels of hemoglobin synthesis which were amplified by 2% dimethylsulfoxide. Mouse (but not human) globin mRNA was demonstrated in these hybrid cells. The results suggest that somatic cell hybrids may be useful in searching for genetic factors which regulate activity of the globin genes.

Bone Marrow Cells↗
PubMed1975

Inhibition of protein synthesis in rabbit reticulocyte lysates by double-stranded RNA and oxidized glutathione: indirect mode of action on polypeptide chain initiation.

In the presence of added double-stranded RNA or oxidized glutathione, protein synthesis in heminsupplemented reticulocyte lysates declines abruptly after 8-12 min of incubation at 30 degrees. The kinetics of amino-acid incorporation are very similar to those seen when lysates incorporation are very similar to those seen when lysates are incubated in the absence of added hemin. The inhibitory effects of double-stranded RNA (dsRNA) and oxidized glutathione (GSSG) are partially overcome by a homogeneous initiation factor, IF-MP, which also stimulates protein synthesis in hemin-deficient lysates. This factor is involved in the binding of Met-tRNAfmet to 40S ribosomal subunits during protein chain initiation. However, neither dsRNA alone nor GSSG alone significantly inhibits formation of [40S subunit-Met-tRNAf] complexes induced in reticulocyte lysates by dsRNA or GSSG involves one or more components present in the lysates but absent from the fractionated in vitro system. Such components may be related to the translational inhibitor that is active in hemin-deficient lysates.

Animals↗
PubMed1975

Hemoglobin synthesis in somatic cell hybrids: coexpression of mouse with human or chinese hamster globin genes in interspecific somatic cell hybrids of mouse erythroleukemia cells.

Somatic cell hybrids were derived by fusion of mouse erythroleukemia cells with fractionated human marrow enriched in erythroblasts, or with chinese hamster fetal liver erythroid cells. Such interspecific hybrid cells, when isolated in suspension culture, had retained nearly all the mouse chromosomes and had lost most of the human or chinese hamster chromosomes. However, two such hybrids (one human, the other hamster) studied 4-6 weeks after fusion, were found to contain several non-mouse chromosomes. RNA extracted from the human marrow x erythroleukemia hybrid annealed equally to both human and mouse globin complementary DNA, indicating that coexpression of the globin genes of each species had occurred in the hybrid cells. Mouse and human mRNA were found to accumulate only after incubation of the cells in 2% dimethylsulfoxide. The chinese hamster x erythroleukemia hybrid appeared to contain a double complement of mouse chromosomes in addition to several chinese hamster chromosomes. After 7 days of incubation in 2% dimethylsulfoxide, [3H]leucine was incorporated into chinese hamster beta-globin and the mouse globin chains. Thus, globin genes from differentiated cells, when introduced into spontaneously proliferating erythroleukemia cells, may be expressed after exposure of the resulting hybrid cells to an agent capable of inducing hemoglobin synthesis in the erythroleukemia cell.

Animals↗
PubMed1975

Hemoglobin switching in sheep and goats. V. Effect of erythropoietin concentration on in vitro erythroid colony growth and globin synthesis.

Erythroid colonies were generated in response to erythropoietin in plasma clot cultures of sheep and goat bone marrow cells. At low concentration erythropoietin only hemoglobin A (betaA globin) was synthesized in goat cultures, but at high concentrations 50% of the hemoglobin synthesized was hemoglobin C (betaC globin). This effect of erythropoietin on the expression of a specific beta globin gene was manifested only after 72 h in vitro and followed the development of erythroid colonies. Sheep colonies behaved differently from those of goat in that little or no betaC globin synthesis occurred even at high erythropoietin concentration. To investigate this difference, sheep marrow cells were fractionated by unit gravity sedimentation. The erythroid colony-forming cells sedimented more rapidly (3.5-6mm/h) than the hemoglobinized eththroid precursors (1-3.5 mm/h), suggesting that the colonies were formed from an early erythroid precursor, However, the colonies formed from the sheep marrow fractions synthesized only betaA globin even at concentrations of erythropoietin sufficient to stimulate betaC globin synthesis in goat colonies. Morphologically, the goat colonies were larger and more mature than those of the sheep. By 96 h in vitro three-fourths of the goat colonies contained enucleated red cells compared to only 3% of the sheep colonies. Thus, erythropoietin had an equivalent effect in stimulating erythroid colony growth from the marrow of both species although there were both biochemical and morphological differences between the colonies. Hemoglobin switching appeared to require exposure of an early precursor to high erythropoietin concentration, but the results with sheep marrow suggested that the rate of colony growth and cellular maturation might also be important.

Animals↗