Isolation and identification of chick lens crystallin messenger RNA

Williamson, Robert; Clayton, R. M.; Truman, D. E. S.

doi:10.1016/0006-291x(72)90073-3

Cited by 35 publications

(5 citation statements)

References 19 publications

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“…Lens messengers have been isolated [ 113, translated and identified in several heterologous systems such as the ascites cell-free system [12], the ret,iculocyte lysate derived from rabbits [13] and ducks [14] and in vivo in the system of oocytes from Xenopus Zaevis [15]. Here we shall describe the more definite identification of one of the products synthesized in a rabbit reticulocyte lysate under the direction of one of the lens messengers.…”

Section: Resultsmentioning

confidence: 99%

Synthesis of Lens Protein in vitro

Berns¹,

Schreurs²,

Kraaikamp³

et al. 1973

European Journal of Biochemistry

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Messenger ribonucleic acid was isolated from calf lens polyribosomes and fractionated. Previous identifications of the newly synthesized products have been extended in that the fidelity of the translation of the 14-5 mRNA was assayed by fingerprinting analysis. The results suggest that lens messenger is translated with a high d(%ree 0: fidelity in the cross systems even to the extent that the newly synthesized aA, c h k jontain an acetylated methionine residue in the N-terminal position like the native chains. There seems to be no requirement for cell-specific protein factors in order to obtain accurate translation. Evidence is accumulating that the acetylation is not involved in the initiation mechanism.Lens messengers appear to possess interesting aspects for a bett3er understanding of the process of protein biosynthesis in eukaryotes.The The biosynthesis of oc-crystallin polypeptides appeared to be also interesting for the elucidation of the initiation mechanism in eukaryotes, as all four chains contain an acetylated methionine residue in the N-terminal position [5]. This knowledge formed the background of our attempts to isolate mRNA4 from lens tissue and to assay its biological activity in cross systems. Isolation of mRA?A front Calf-Lens Polyssomes Calf lens polysomes were isolated as described earlier [6] except that. only the cortex was used as starting tissue. From the polysomes messenger was obtained as described for the isolation of hemoglobin messenger by Lockard and Lingrel [7]. Polysomes were disaggregated by 1 O/, sodium dodecylsulfate in 0.05 Tris-HC1 pH 7.5 for 5 min a t 37 "C, diluted twice and applied on a sucrose gradient at a concentration of about 2 mg/ml.The gradient was constructed by pumping 4001, (w/w) sucrose, containing 0.05 M Tris-HC1 pH 7.4 (freed from RNAase by boiling for 30 min with 0.020/, diethylpyrocarbonate) into a mixing chamber of 800 ml containing buffer only. The chamber was connected with the edge of a B-XXIX zonal rotor of IEC. 180 ml of the gradient was used in the overlayer. Overlayer + sample volume comprised 500 ml.After centrifugation the gradient was displaced from the edge of the rotor by pumping in a heavy sucrose solution. The absorbance was measured a t 260 nm with the aid of a Gilford speotrophotometer adapted

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Section: Resultsmentioning

confidence: 99%

Synthesis of Lens Protein in vitro

Berns¹,

Schreurs²,

Kraaikamp³

et al. 1973

European Journal of Biochemistry

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“…This system has the additional advantage that the differentiation of lens fibers can be observed in vitro when lens epithelia are cultured in the presence of serum (5)(6)(7)(8)(9). These facts, together with the success of other workers in isolating calf lens crystallin mRNAs (13)(14)(15)(16) and in demonstrating template activity of polysomal RNA from chick lens (17), encouraged us to attempt the isolation of acrystallin mRNA from embryonic chick lens as a first step toward analysis of mRNA metabolism during the in vivo and in vitro differentiation of this tissue. We report here the isolation of 5-crystallin mRNA and its translation in two heterologous cell-free systems.…”

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confidence: 95%

“…In this case the fiber masses were thawed and homogenized in 0.001 M MgCl2 (17). The supernatant fraction, after low-speed centrifugation, was centrifuged for 1 hr at 140,000 X g in a Beckman SW-65 rotor.…”

mentioning

confidence: 99%

Isolation and In Vitro Translation of δ-Crystallin mRNA from Embryonic Chick Lens Fibers

Zelenka

Piatigorsky

1974

Proc. Natl. Acad. Sci. U.S.A.

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Of the protein synthesized and accumulated during differentiation of embryonic chick lens fibers, 70-80% is the tissue specific protein b-crystallin. We have isolated and partially characterized the total cytoplasmic mRNA from purified lens fibers of 15-day-old embryos as an initial step toward understanding the regulated expression of 6-crystallin during development. Each lens fiber mass contained an average of 10 sg of cytoplasmic RNA; approximately 0.1 Isg per fiber mass was recovered in the mRNA fraction by oligo(dT)-cellulose chromatography. The mRNA electrophoresed primarily as a single peak on a polyacrylamide-agarose gel with an apparent molecular weight of about 9 X 105 estimated by comparison with 28S and 18S rRNA markers. Of the protein synthesized in response to the mRNA in cell-free systems derived from Krebs II ascites tumor cells or rabbit reticulocytes, 70-80% comigrated with 6-crystallin on sodium dodecyl sulfatepolyacrylamide-agarose gels. Comparison of the tryptic peptides of 8-crystallin with those of the in vitro products from both heterologous systems established that the lens fiber mRNA contained 5-crystallin mRNA, and that no other functional mRNAs were present in detectable quantities. Thus, the specialization of protein synthesis in embryonic chick lens fibers apparently results from an accumulation of 5-crystallin mRNA in the cytoplasm.Studies of the synthesis and utilization of specific mRNAs in differentiating tissues provide information about gene expression during differentiation. Studies of this kind have recently been conducted with the chick oviduct (1), with erythrocyte precursor cells of fetal-mouse liver (2), and with Friend leukemia cells induced by dimethylsulfoxide to initiate globin synthesis (3,4). These studies have taken advantage of tissues highly specialized in protein synthesis for the initial isolation and identification of a specific mRNA. The results in each of these cases correlate the onset of synthesis of a specific protein with the appearance and accumulation of the corresponding mRNA.Another tissue which is highly specialized in protein synthesis, and in which a specific mRNA might be studied during differentiation, is the embryonic chick lens. Of the protein synthesized in maturing chick lens fibers, 70-80% is the lensspecific protein, 6-crystallin (10-12). This system has the additional advantage that the differentiation of lens fibers can be observed in vitro when lens epithelia are cultured in the presence of serum (5-9). These facts, together with the success of other workers in isolating calf lens crystallin mRNAs (13-16) and in demonstrating template activity of polysomal RNA from chick lens (17), encouraged us to attempt the isolation of acrystallin mRNA from embryonic chick lens as a first step toward analysis of mRNA metabolism during the in vivo and in vitro differentiation of this tissue. We report here the isolation of 5-crystallin mRNA and its translation in two heterologous cell-free systems. A preliminary report of this work has been ...

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“…Messenger RNAs for embryonic chicken myosin, bovine lens a-crystallin protein, mouse hemoglobin, rabbit hemoglobin, duck hemoglobin, mouse myeloma immunoglobin light chain, hen oviduct albumin, and reovirus capid proteins have been translated in a reticulocyte cell-free system (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Messenger RNAs for rabbit hemoglobin, mouse globin, human globin, mouse myeloma light chain, bovine lens a-crystallin protein, chicken oviduct ovalbumin, HeLa cell histones, encephalomyocarditis viral coat proteins, reovirus capid proteins, QB bacteriophage, and hepatic tryptophan oxygenase have been translated in a murine cell-free system derived from Krebs II ascites tumor cells (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Messenger RNAs for reovirus, encephalomyocarditis virus, mouse Elberfeld virus, and Mengo virus have been translated in cell-free systems derived from mouse L cells, Chinese hamster ovary cells, HeLa cells, and Ehrlich ascites tumor cells (11,29,30).…”

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Cell-Free Synthesis of Rat Growth Hormone

Bancroft¹,

Wu²

1973

Proc. Natl. Acad. Sci. U.S.A.

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Growth hormone has been synthesized in a cell-free system derived from Krebs II ascites cells, under the direction of RNA prepared from rat pituitary tumor (GC) cells. Growth hormone synthesized in the cell-free system was identified by precipitation with antiserum against growth hormone developed in baboon, followed by electrophoretic analysis of the dissolved precipitate on sodium dodecyl sulfate-polyacrylamide gels. RNA from both the membrane and the post-membrane fractions of the cytoplasm of GC cells stimulated protein synthesis in the cell-free system, but only RNA from the membrane fraction was found to direct the synthesis of growth hormone.

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Isolation and identification of chick lens crystallin messenger RNA

Cited by 35 publications

References 19 publications

Synthesis of Lens Protein in vitro

Synthesis of Lens Protein in vitro

Isolation and In Vitro Translation of δ-Crystallin mRNA from Embryonic Chick Lens Fibers

Cell-Free Synthesis of Rat Growth Hormone

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