David Kabat

Synthesis of erythrocyte-specific proteins in cultured friend leukemia cells

We have studied synthesis of specific proteins in two permanent lines of Friend virus-induced erythroleukemia cells (Friend line 745 and Ostertag line FSD-1, both derived from DBA/2 mice). By 96 hr following treatment with 1-2% dimethyl sulfoxide (Me2SO), up to 25% of the protein being synthesized by both these cultures is hemoglobin. At that time, hemoglobin constitutes up to 10% of the cellular soluble protein. Both lines synthesize heme and globin coordinately, and alpha and beta globin chains in a nearly balanced 1:1 ratio. However, the ratio of betaMajor:betaMinor chains synthesized by these induced Friend leukemia (FL) cells is approximately 9 in the FSD-1 line and 1.3 in the Friend Clone 745 line, whereas it is 4 in normal adult DBA/2 mouse erythrocytes. Evidence for the latter conclusion was obtained by electrophoresis of FL hemoglobins on cellulose acetate membranes, and also by chromatographic separation of alpha, betaMajor, and betaMinor globins on carboxymethylcellulose in 8 M urea at 20 degrees C. Carbonic anhydrase activity per mg protein is 3 times higher in induced than in control cultures. 2,3-diphosphoglyceric acid is not found in induced FL cells. Induced and control FL cells agglutinate strongly and equally with Phaseolus vulgaris phytohemagglutinin. The developmental process in these cultured leukemia cells appears to be an aberrant erythropoiesis.

Changes in RNA and protein metabolism preceding onset of hemoglobin synthesis in cultured friend leukemia cells

Abstract We have studied the hematopoietic process which is induced by dimethyl sulfoxide (Me2SO) in the Ostertag FSD-1 line of Friend erythroleukemia (FL) cells and have observed several changes that precede the onset of hemoglobin synthesis at 48 hr. Although cellular viability, mitotic rate, and deoxyribonucleic acid content are unaffected by our induction procedure, the induced cells become progressively smaller, and by 96 hr contain only 55% as much ribonucleic acid and 60–70% as much protein as control cells. The decline in ribonucleic acid content is significant by 24 hr and affects 4S and ribosomal ribonucleic acids in a noncoordinated manner throughout the hematopoietic process. Furthermore, incorporation of radioactive uridine into the 45S precursor of ribosomal ribonucleic acid is specifically inhibited by 1–2 hr after first treating FL cultures with 1% Me2SO. This earliest known effect of Me2SO on FL cells is followed by a decline in synthesis of protein. The basic sequence of macromolecular and cell size changes are similar to those that occur during normal erythropoiesis.

[54] Isotopic labeling and analysis of phosphoproteins from mammalian ribosomes

Publisher Summary This chapter describes the methods that are found most useful for the isotopic labeling and characterization of phosphoproteins from mammalian ribosomes. The chapter describes methods for removing contaminating phosphoproteins and for establishing the reality of this ribosomal modification. These same methods should be applicable to analysis of other phosphoproteins, especially those which occur in complex subcellular organelles. The chapter shows that at least five polypeptide chains from ribosomes of rabbit reticulocytes, rat liver cells and mouse sarcoma 180 tumor cells are phosphoproteins rather than simple proteins. The phosphoryl groups in these proteins are present in o-phosphoseryl and in o-phosphothreonyl residues. Because these phosphoryl groups turn over intracellularly, the proteins become radioactive when the cells are incubated with [32P]orthophosphate.

Presence of hemoglobin messenger ribonucleoprotein in a reticulocyte supernatant fraction

Abstract A study is made of globin messenger ribonucleic acid (mRNA) distribution in a rabbit reticulocyte lysate. Ribonucleic acid with the electrophoretic mobility of globin mRNA is found not only in polyribosomes but also in a ribonucleoprotein complex which sediments at 15 S. Such presumptive mRNA is not found in single ribosomes or in the subribosomal particle fraction. Further evidence that the 15-S ribonucleoprotein contains globin mRNA is obtained using a protein synthesizing extract from embryonic muscle. Addition of 15-S complex to the extract specifically stimulates incorporation of radioactive amino acids into material which cochromatographs with carrier hemoglobin and which coprecipitates with the carrier hemoglobin upon addition of anti-hemoglobin antiserum.

Mutant cells that abnormally process plasma membrane glycoproteins encoded by murine leukemia virus

Wild-type normal rat kidney fibroblasts infected with the Friend strain of murine leukemia virus (MuLV) contain two virus-encoded glycoproteins on the outer surfaces of their plasma membranes: an envelope glycoprotein with an apparent molecular weight of 70,000 (gp70), and a glycoprotein that reacts with antisera to the major virion internal core proteins p30, p15, p12 and p10 and has an apparent molecular weight of 93,000 (gp93gag). To analyze the functions of these glycoproteins and to develop a model system for studying genetics of membrane synthesis, we used an immunoselection method to isolate variant cell clones defective in processing these glycoproteins into their plasma membranes. Several lines of evidence, including complementation of glycoprotein processing defects by fusion with uninfected wild-type cells, indicate that the immunoselected variants have stably inherited membrane synthesis abnormalities that are encoded by cellular rather than by viral genes. The H-4 cell line, which was selected by use of antiserum to gp70, has metabolic defects that interfere with processing of both gp70 and gp93gag into its plasma membranes. Nevertheless, this cell line releases noninfectious MuLV. Furthermore, two cell lines (2 and 5), which were selected by use of antiserum to the virion core protein p30, specifically lack detectable cell surface or intracellular gp93gag but contain cell surface gp70 and release infectious MuLV. These results suggest that MuLV particles can bud efficiently from cells that lack known virus-encoded plasma membrane constituents.

Direct method of measuring transfer ribonucleic acid binding to polyribosomes and to single ribosomes in cells of higher organisms

Abstract The transfer ribonucleic acid content of ribosomal preparations is measured by a method which employs electrophoresis in polyacrylamide gels. Peptidyl tRNA is measured separately after controlled proteolytic digestion of the ribosomes. Unlike previous methods of analysis, this method does not require radioactively labeled tRNA, is generally applicable to all types of cells, and does not require use of a cell-free system. Drug- or virus-induced modifications of tRNA binding to ribosomes can now be routinely analyzed in cellular systems under physiological conditions. The method was used with reticulocytes and with embryo skeletal muscle in the absence of inhibitors of protein synthesis and in the presence of NaF and of puromycin. In agreement with previous results, native polysomal ribosomes carry approximately two tRNA molecules, one being covalently bonded to a nascent peptidyl chain. On the other hand, single ribosomes contain only one relatively weakly bound tRNA unliked to a peptidyl chain. Brief treatment of reticulocytes with puromycin caused release from polysomes of their nascent peptide chains, apparently without causing release of the previously associated tRNA molecules from the donor ribosome sites. However, it is found here that puromycin does cause a significant release of approximately 0.4 tRNA molecules from the polysomal ribosomes, and it is suggested that this loss occurs from the ribosome acceptor sites by a noncompetitive mechanism.

The thalassemias: model for analysis of quantitative gene control.

The pace at which information about the thalassemias is accumulating has quickened.* There are a number of excellent reviews21,22,24,26,55,190,249, 269,394,398,402 which cover the historical development of clinical and genetic information, together with concepts to account for the pathophysiology of this group of familial anemias. We have selected highlights of this history in order to draw comparisons with the parallel growth of information concerning control of gene activity. These efforts began for different reasons and depended at first on different experimental systems. The theme for this review is that they now appear to be converging on a common question: what determines the rate of production of a given gene product (hemoglobin) in differentiated cells (erythrocytes) of a eukaryote (man) ?

THE SWITCH FROM FETAL TO ADULT HEMOGLOBININ HUMANS: EVIDENCE SUGGESTING A ROLE FOR γ‐β GENE LINKAGE *

Recent studies of Hemoglobin Kenya have provided evidence that the human genes for fetal y chains are closely linked to those which specify the adult j3 and S chains.1. * It would thus appear that in man there are two distinct and separate gene loci that code for globins-one encodes for a chains (there is evidence for two a loci in at least one Hungarian family3) and the other is il cluster of genes that encodes the non-a chains (“7, Ay, 8, and 8). The non-a gene cluster is also interesting in that the associated genes have the same which suggests that they are transcribed from the same strand of the DNA double helix. Their arrangement in the direction of transcription is believed to be G y + Ay + S + j3, although the orientation Ay + 8 4 j3 + ”y cannot be excluded.2 The purpose of this paper is to present evidence that this gene linkage plays an important role in regulating the switch from synthesis of fetal (a,y,) to adult hemoglobins (adr plus 2% a

Phosphorylation of Ribosomal Proteins in Eukaryotes

,). Generally, blood from the umbilical cord of a newly born human contains 1 6 2 5 % adult hemoglobin, the remainder being fetal hemoglobin. Furthermore, there is substantial evidence that in addition to erythrocytes having only Hb F and others having only adult hemoglobins, some of the cord blood cells are transitional-stage t erythrocytes that contain both fetal and adult hemog l o b i n ~ . ~ ~ Although these observations demonstrate that the switching from fetal to adult hemoglobin synthesis occurs within single clones of cells, the mechanism of this switching has remained unknown. This research was designed to distinguish between two alternative models for regulation of the chromosomal region that contains the genes for human non-a globin chains: ( 1 ) Leaky Model. The chromosomes become leaky in the late fetal period. Their genes are not yet fully repressed and their j3 genes not yet fully derepressed. Transitional stage erythrocytes arise by virtue of this leakiness of the individual chromosomes. According to this model, each homologous chromosome in the diploid erythroid cells can express both its y and its B genes within a single erythrocyte. (2) Mutually Exclusive Model. This was proposed previously.g Expression of the chromosomal region is mutually exclusive and nonleaky. In other words, each chromosome can only express either its y or its j3 gene in any erythrocyte, but not both. Since

Protein A-coated erythrocyte binding to cell surface antigens: Application to quantitate retrovirus infectivity in vitro

INTRODUCTION Approximately four years ago, we first obtained evidence that several of the proteins in rabbit reticulocyte ribosomes are phosphoproteins rather than simple proteins. Their phosphoryl groups turn over intracellularly with a half-life of approximately 25 min, they become radioactive when the cells are incubated with [ 32 P]orthophosphate, and they occur in o -phosphoserine and in o -phosphothreonine residues (Kabat 1970Kabat 1972). Thus the phosphoproteins are sites of an active and continuous metabolism. In addition, phosphorylation of ribosomal proteins occurs in other tissues (Blat and Loeb 1971; Loeb and Blat 1970; Majumder and Turkington 1972; Barden and Labrie 1973; Correze, Pinell and Nunez 1972; Bitte and Kabat 1972), in higher plants (Trewavas 1973) and in yeasts (J. Warner, personal communication), and it may therefore be ubiquitous in eukaryotes. However, ribosome phosphorylation is apparently absent from bacteria (Gordon 1971). In this paper we review the evidence which indicates that ribosomal phosphoproteins exist intracellularly and describe some of the physiological variables, including cyclic AMP, that influence this metabolism. Also described are some of our preliminary attempts to analyze the functions of ribosome phosphorylation. In addition, we have briefly reviewed the studies of ribosomal protein phosphorylation in vitro which have employed [γ- 32 P]ATP and several different kinds of protein kinase. Evidence for Ribosomal Phosphoproteins When rabbit reticulocytes are incubated in a nutritionally rich medium with [ 32 P]orthophosphate at 37°C, several of their ribosomal proteins become highly radioactive. Furthermore, acid hydrolysates of these proteins contain radioactive o -phosphoserine and o -phosphothreonine (Kabat 1970Kabat 1972; Bitte and Kabat 1974). Typical electrophoretic fractionations...