Conservation of globin messenger RNA in rabbit reticulocyte monoribosomes after sodium fluoride treatment

doi:10.1016/0006-291X(72)90558-X

Biochemical and Biophysical Research Communications

Volume 47, Issue 4, 26 May 1972, Pages 766-774

https://doi.org/10.1016/0006-291X(72)90558-X Get rights and content

Abstract

Following NaF treatment of rabbit reticulocytes, polyribosomes dissociate to monoribosomes with concomitant cessation of protein synthesis. mRNA was isolated from different fractions of the cell before and after NaF treatment and quantitated by assay for globin synthesis in a cell-free system prepared from Krebs ascites tumor cells. mRNA activity present in polyribosomes of control cells was recovered quantitatively from monoribosomes following NaF treatment. On removal of NaF, polyribosomes reform with mRNA activity similar to that of control cells. During polyribosome dissociation, no significant amount of mRNA is recovered from ribosomal subunits, the supernatant fraction or is lost by degradation.

References (18)

F.M. Conconi et al.
J. Mol. Biol
(1966)
B.L. Hogan
Bioch. Biophys. Acta
(1969)
B. Lebleu et al.
Bioch. Biophys. Acta
(1967)
P.R. Gross et al.
Biochem. and Biophys. Res. Commu
(1963)
P.R. Gross et al.
Exptl. Cell Res
(1964)
F.H. Wilt
J. Mol. Biol
(1965)
S. Lin et al.
J. Mol. Biol
(1966)
B. Lebleu et al.
Bioch. Biophys. Res. Commu
(1970)
W. Hoerz et al.
Bioch. Biophys. Acta
(1971)

There are more references available in the full text version of this article.

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We have developed a method for the separation and quantitation of human α-, β-, and γ-globins utilizing cellulose acetate electrophoresis. The relative rates of synthesis of globin chains in reticulocytes in peripheral blood is determined by: (i) incubating intact cells with [³⁵S]methionine; (ii) preparing globin from the hemolysates; (iii) performing electrophoresis of the globin on cellulose acetate strips; and (iv) autoradiography or direct determination of the radioactivity incorporated into each globin chain. The method is simple and rapid, requires only small amounts of hemolysate (30 μg of globin), and provides excellent resolution and reproducible quantitation of α-, β^A-, β^S-, and γ-globin chains for up to 24 peripheral blood samples at one time. Measurements by this method in patients with thalassemia variants and sickle-cell disorders correlate well with analysis of the same samples by carboxymethyl cellulose chromatography. This methodology may permit more widespread analysis of globin synthesis in the thalassemia syndromes and may also be useful in the analysis of globins synthesized from human globin mRNA in cell-free systems.
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Free 40 S ribosome subunits have been purified in large quantities from rabbit reticulocytes and mouse Krebs ascites cells under low ionic conditions. Protein and RNA composition as well as functional aspects were compared to polysome derived 40 S subunits. Initiation factors for protein synthesis were identified and partially purified from free 40 S subunits.o
1. (1)
  The large amount of non-ribosomal protein and tRNA associated with free 40 S subunits are selectively removed by treatment with 0·5 m-KCl, giving rise to 40 S subunits very similar to polysome derived subunits.
2. (2)
  The associated proteins at least equal the mass of structural ribosomal proteins estimated from the buoyant density in CsCl (1·40 g/cm³) and from recovery of protein in the KCl-wash. Their molecular weights are generally much higher than those of the 40 S structural proteins.
3. (3)
  Sixty to eighty percent of free 40 S subunits from both cell types carry a tRNA, whereas less than five percent of the reticulocyte free 40 S subunits contain globin mRNA.
4. (4)
  The initiation factor activity that we have found associated with free 40 S subunits is recovered in the KCl-wash fraction.
5. (5)
  All six initiation factors as characterized by this laboratory are present in the KCl-wash fraction in varying amounts. The presence of all six factors in the free 40 S KCl-wash were tested with a globin synthesizing system dependent on each factor. Specific factor activities were demonstrated by initiation complex formation with ribosome subunits and Met-tRNA_f in the presence and absence of mRNA. The factor IF-E₂, which forms a GTP-dependent, stable complex with initiator tRNA in the absence of ribosomes, was partially purified from the KCl-wash. The large IF-E₃ protein complex seems to occur in almost stoichiometrical amounts on the free 40 S subunits. IF-E₃ was purified as a 17 S particle that stimulated globin synthesis in an IF-E₃ depleted system about 20-fold. IF-E₅ was specifically demonstrated by its ability to promote conversion of the 40 S initiation complex into the 80 S initiation complex.
6. (6)
  A Met-tRNA specific deacylase is also present in the free 40 S KCl-wash. We have separated this enzyme from the initiation factors by ammonium sulfate fractionation and chromatography on DEAE-cellulose, which was a prerequisite for initiation experiments involving precharged Met-tRNA_f.
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Conservation of globin messenger RNA in rabbit reticulocyte monoribosomes after sodium fluoride treatment

Abstract

J. Mol. Biol

Bioch. Biophys. Acta

Bioch. Biophys. Acta

Biochem. and Biophys. Res. Commu

Exptl. Cell Res

J. Mol. Biol

J. Mol. Biol

Bioch. Biophys. Res. Commu

Bioch. Biophys. Acta