Physical and chemical characterization of purified ovalbumin messenger RNA.

Journal of Biological Chemistry (Impact Factor: 4.6). 10/1975; 250(17):7027-39.
Source: PubMed

ABSTRACT Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylaminde gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T1 ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation. Ovalbumin mRNA has a nucleotide composition of 32.3% A, 21.0% G, 25.7% U, and 20.7% C [(A+U)/(G+C) equal 1.41]. The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 +/- 63,000, corresponding to a chain length of 1890 +/- 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde. When 125I-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin. It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.

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    ABSTRACT: The differentiated chick oviduct is a target tissue for progesterone. Administration of the hormone to estrogen-primed chicks causes a rapid induction of new messenger RNA, resulting in the appearance in cytoplasm of specific mRNA species. One of these species is the mRNA coding for the egg white protein avidin, which is produced only in response to progesterone.The cytoplasm contains receptors which bind progesterone specifically and transport it as a complex into oviduct nuclei. Only a small fraction of the receptors are active in vitro; this fraction is increased to 30% by brief warming of the receptor-hormone complexes before incubation with the nuclei. The binding reaction is slow, and the receptors cannot be released from nuclei by DNase treatment. The reaction occurs preferentially with oviduct nuclei, which contain at least twice as many acceptor sites (8000/nucleus) as other chick tissues (< 2000/nucleus). The binding constants are the same in all tissues (Kd ~ 10−8 M), and are identical to the constant for receptor binding to isolated chromatin.We have isolated the progesterone receptor component which binds to the chromatin. Oviduct cytosol from laying hens was prepared and progesterone receptors were precipitated with ammonium sulfate (30% sat.). The re-dissolved pellet was eluted from a steroid-affinity column (Sepharose 4B-BSA-deoxycorticosterone) with 3M urea. The receptors were reconstituted by dialysis and labeled with [3H]-progesterone. The [3H]-progesterone-receptor complexes were then purified by sequential chromatography and elution with the indicated slats at pH 74 from DEAE-cellulose (0.2 M KCl), phosphocellulose (0.26 M KCl), and hydroxylapatite (0.15 M KxPO4). The peak fraction was finally chromatographed on an agarose A-1.5 M column (Kav = 0.28). Yield was 1 %, and purity approached the theoretical maximum specific activity, 109 d.p.m./mg protein.
    Journal of Investigative Dermatology 01/1977; 68(1):1-4. DOI:10.1111/1523-1747.ep12485116 · 6.37 Impact Factor
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    ABSTRACT: The ovalbumin structural gene was purified and amplified by cloning in the bacterial strain χ1776 using the tetracycline-resistant plasmid pMB9. To prepare this recombinant plasmid, a full-length copy of the mRNA was first synthesized with avian myeloblastosis virus (AMV) reverse transcriptase. A double-stranded copy of the DNA was then synthesized and joined to the plasmid by the polyA-polydT “tailing” procedure. Transformed colonies containing ovalbumin DNA were selected by in situ hybridization with 32P-cRNAov.Digestion of the chimeric clones with HhaI, which does not cleave the ovalbumin DNA, can be used to calculate the size of the inserted DNA. One chimeric plasmid, pOv230, contained a 1950 base pair insert, which is longer than the 1850 bases of the mRNAov. The vast majority of this DNA is complementary to ovalbumin gene sequences, since a complete DNA copy of the mRNAov (cDNAov = 1850 bp) is protected from S1 nuclease digestion (>95%) by pOv230. The ramaining bases are probably comprised of the poly(dA-dT) linkers. Multiple restriction enzyme digests of the inserted ovalbumin DNA demonstrated that sites present in the double-stranded ovalbumin DNA are preserved in the chimeric plasmid and allowed us to determine the orientation of the inserted ovalbumin DNA. Finally, the chimeric plasmid pOv230 is able to transform χ1776 with an efficiency similar to the parent plasmid pMB9.
    Gene 12/1977; 2(s 5–6):217–231. DOI:10.1016/0378-1119(77)90002-6 · 2.08 Impact Factor
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    Critical Reviews in Biochemistry and Molecular Biology 01/1981; 10(1):1-38. DOI:10.3109/10409238109114634 · 5.81 Impact Factor

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