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Cloning and characterization of a 67 kDa polypeptide and a 52 kDa polypeptide of eukaryotic initiation factor 2 from rabbit reticulocytes
Abstract
A 67 kDa polypeptide (p$\sp{67}$) is often associated with eukaryotic initiation factor-2, called eIF-2. Under certain physiological conditions e.g., heme deficiency, the small subunit ($\alpha$) of eIF-2 is phosphorylated thereby inhibiting protein synthesis. The p$\sp{67}$ renders the eIF-2 preparation resistant to phosphorylation so that the protein synthesis is not inhibited. In order to isolate the gene sequence of p$\sp{67}$ polypeptide, a $\lambda$ gt11 rabbit liver cDNA library was screened using monoclonal antibodies against p$\sp{67}$. Several clones containing p$\sp{67}$ gene sequences were obtained and the DNA inserts from these clones were isolated and subcloned into pTZ19, pGEM7 and pIN-II series vectors. Most of the subsequent work was done with clone #1 having a 2.2 kbp insert. The clone was further characterized as follows: (1) The Northern blot hybridization showed a mRNA of size 2.3 kb corresponding to p$\sp{67}$. (2) The vector pGEM7 containing 2.2 kbp insert was linearized and transcribed in vitro using SP6 RNA polymerase. The transcript thus generated was used for in vitro translation in nuclease treated rabbit reticulocyte lysate. The translated product was analyzed by SDS-PAGE, followed by autoradiography. (3) Expression of p$\sp{67}$ gene sequence was carried out using pIN-II series expression vector in E. coli. Cell extract was analyzed by SDS-PAGE followed by Western blot analysis. (4) A nested set of deletions was made using Exo III/S1 nuclease system and deleted DNA inserts were sequenced at the DNA sequencing facility of the University of Nebraska-Lincoln. In another study, I have tried to clone and characterize the gene sequence of the p$\sp{52}$ polypeptide. A $\lambda$ gt11 human liver cDNA library was screened using polyclonal antibodies against p$\sp{52}$. Several clones containing p$\sp{52}$ gene sequences were obtained and were subcloned in pUC18 vector for further characterization. Most of the subsequent work was done with clone #1 having a 1.5 kbp insert as follows: (1) Northern blot hybridization of rabbit liver poly A$\sp+$ RNA showed a mRNA of size 1.6 kb corresponding to p$\sp{52}$. (2) The poly A$\sp+$ RNA specific for p$\sp{52}$ gene sequence was isolated by hybrid selection and used for in vitro translation in rabbit reticulocyte system, which encoded a polypeptide with a mass of 52 kDa. (3) The NH$\sb2$-terminal sequences of intact p$\sp{52}$ was determined by automated Edman degradation. (4) A nested set of deletions was made using Exo III/S1 nuclease system and part of the insert DNA was sequenced by dideoxy sequencing method of Sanger.
Subject Area
Biochemistry|Molecular biology
Recommended Citation
Nag, Mihir Kumar, "Cloning and characterization of a 67 kDa polypeptide and a 52 kDa polypeptide of eukaryotic initiation factor 2 from rabbit reticulocytes" (1989). ETD collection for University of Nebraska-Lincoln. AAI8925252.
https://digitalcommons.unl.edu/dissertations/AAI8925252