Cell-free translation of avian adipose tissue lipoprotein lipase messenger RNA

Poly(A)+ mRNA isolated from chicken adipose tissue directed cell-free translation in a rabbit reticulocyte lysate system. Immunoadsorption with polyclonal antibodies against lipoprotein lipase detected a protein of 56 +/- 2 kDa. Immunodetection of this protein was prevented by inclusion of purified lipoprotein lipase in the assay mixture. Identification of the 56 kDa protein as lipoprotein lipase was confirmed by immunoadsorption to the monoclonal antibody CAL 1-11. Inclusion of dog pancreatic microsomal membranes in the translation system resulted in isolation of an additional protein of 62 kDa. Treatment of the 62 kDa protein with endo-beta-N-acetylglycosaminidase H or endo-beta-N-acetylglucosaminidase F decreased the observed molecular mass to that of the primary translation product, indicating that the increase in molecular mass resulted from the addition of N-linked oligosaccharides. Starving and refeeding chickens prior to poly(A)+ mRNA isolation resulted in a 3-fold increase in the amount of immunodetectable lipoprotein lipase synthesized.     

Cell-free translation of messenger RNA coding for a precursor of human calcitonin

Polyadenylated RNA, extracted from a human medullary thyroid carcinoma, was translated in cell-free systems prepared from wheat germ and reticulocyte lysates. The major product of the translations was a protein of 15,000 M_R which was immunoprecipitated specifically with an antiserum to synthetic human calcitonin. Addition to the translation reactions of microsomal membranes, prepared from canine pancreas, resulted in the partial disappearance of the 15,000 M_R polypeptide and the concomitant appearance of a smaller peptide (11,000 M_R), also immunoprecipitated specifically by antisera to calcitonin. These results indicate that human calcitonin is synthesized in the form of a precursor of 15,000 M_R and suggest that the precursor contains a leader sequence that is cleaved from the polypeptide by enzymes associated with microsomal membranes.     

Cell-free synthesis of a large translation product of prolactin messenger RNA.

RNA prepared from rat anterior pituitaries or from prolactin-secreting pituitary tumors has been shown to direct the synthesis of a large form of prolactin in a cell-free system derived from wheat germ. Immunoprecipitation of cell-free reactions demonstrated the synthesis of a product which was recognized by a specific antiprolactin antisera. Analysis of the immunoprecipitate on sodium dodecyl sulfate containing polyacrylamide gels suggested that the cell-free product has a molecular weight of approximately 28,000 compared to 22,500 for prolactin. RNA prepared by completely different techniques from rat pituitary and a pituitary tumor resulted in identical large translation products. Translation of tumor RNA in a cell-free system from Krebs ascites cells also resulted in a similar large product. The identity of the cell-free product as prolactin was confirmed by comparing peptides derived from the cell-free product and prolactin. The results of these studies suggest that prolactin messenger RNA directs the cell-free synthesis of a product which contains the amino acid sequence of prolactin but which has an addition at one or both ends of the molecule.     

Cell-free translation of messenger RNA from oocytes of Xenopus laevis

The poly(A)⁺ RNA which accumulates during oogenesis in the amphibian Xenopus laevis is shown to be functional mRNA; the RNA was active in the mRNA-dependent “shift assay” for initiation sites in the rabbit reticulocyte lysate, and was an efficient template for protein synthesis in the wheat-germ cell-free system. Analysis of the in vitro protein products showed no differences between the coding properties of poly(A)⁺ RNA extracted from oocytes at all stages of development from previtellogenesis to maturity. In previtellogenic oocytes, the in vitro products of polysomal and of mRNP-associated poly(A)⁺ RNA were also identical. Neither was there any evidence for changes in the coding properties of the poly(A)⁺ mRNA of the oocyte. However, the patterns of oocyte in vivo protein synthesis changed markedly during early vitellogenesis. We conclude that the mRNP-associated poly(A)⁺ RNA present in mature oocytes constitutes the stored maternal mRNA, and that during oogenesis the coding composition of the poly(A)⁺ mRNA synthesised does not change markedly, while some form of translational control operates to direct the changing pattern of protein synthesis.     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Cell-free translation of messenger RNA for chondroitin sulphate proteoglycan core protein in rat cartilage.

Total RNA was extracted from the cartilage tissues rat Swarm chondrosarcoma, neonatal-rat breastplate and embryonic-chicken sterna and translated in wheat-germ cell-free reactions. The core protein of the chondroitin sulphate proteoglycan subunit was identified among translation products of rat mRNA by its apparent Mr of 330 000 and by its immunoprecipitation with specific antisera prepared against rat or chicken proteoglycan antigens. The apparent Mr of the rat proteoglycan core protein is 8000-10000 less than that of the equivalent chicken cartilage core-protein product.     

Cell-free translation of human pheochromocytoma messenger RNA yields protein(s) containing methionine-enkephalin

Cell-free translation of messenger RNA extracted from a human pheochromocytoma yields protein(s) of apparent molecular weight >70,000 daltons which contain the pentapeptide methionine-enkephalin. It is estimated that 0.8–1.0% of the total pheochromocytoma mRNA codes for the methionine-enkephalin-containing protein, based on percent incorporation of [³⁵S]methionine into methionine-enkephalin during cell-free translation. These results demonstrate that human pheochromocytoma mRNA contains the message for a high-molecular weight methionine-enkephalin-containing protein or proteins, presumably the methionine-enkephalin precursor molecule(s).     

Purification of high molecular mass species of calf thymus terminal deoxynucleotidyltransferase

We have developed a simplified column chromatographic procedure for the simultaneous purification of two high molecular mass forms (58 kd and 45 kd) and a standard two subunit 44 kd from of terminal deoxynucleotidyltransferase (TdT) from calf thymus chromatin. The procedure involves high salt extraction of the chromatin fraction followed by successive chromatographies on phosphocellulose, DEAE sephadex, and hydroxylapatite matrices. While all 3 species of TdT comigrate throughout these steps, separation of individual species is achieved on a single stranded DNA agarose column. The combined yield of the 45 kd and 58 kd TdTs is quite high (approximately 8 mg/5000g tissue), 45 kd being the major species (approximately 60%) and the 58 kd constituting about 30%. The 44 kd species containing two subunits usually represents under 10% of the total. All the three forms of TdT showed similar specific activity and preference for purine deoxynucleoside triphosphates (dNTPs). The Km for individual dNTP with all three species of TdT is quite similar and decreases in the order dCTP greater than dTTP greater than dATP greater than dGTP. The Km for both synthetic primer and activated DNA with the 3 TdTs was, in increasing order, two subunit 44 kd less than 45 kd less than 58 kd TdT. Both 58 kd and 45 kd TdT displayed two optima for Mn++ (0.1 mM and 1 mM) and a single sharp optimum for Mg++ (2.5 mM). The two subunit 44 kd TdT exhibited a single but broad optimum for Mn++ (1 mM) and for Mg++ (10 mM).     

Cell-free translation of murine coronavirus RNA.

The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murine hepatitis virion RNA have been investigated by cell-free translation. The six murine hepatitis virus-specific subgenomic RNAs were partially purified by agarose gel electrophoresis and translated in an mRNA-dependent rabbit reticulocyte lysate, and the cell-free translation products were characterized by gel electrophoresis, immunoprecipitation, and tryptic peptide mapping. These studies have shown that RNA 7 codes for the nucleocapsid protein, RNA 6 codes for the E1 protein, RNA 3 codes for the E2 protein, and RNA 2 codes for a 35,000-dalton nonstructural protein. Genomic RNA directs the cell-free synthesis of three structurally related polypeptides of greater than 200,000 in molecular weight.     

Cell-free translation of avian erythroblastosis virus RNA

Avian erythroblastosis virus (AEV) RNA rescued from nonproducer cells by superinfection with a helper virus is translated into three polypeptides in the messenger-dependent rabbit reticulocyte lysate. A 75,000 molecular weight polypeptide (P75AEV) is synthesized from 28S RNA and is encoded by the 5' section of the AEV RNA, including gag-related and AEV-specific sequences. The P75AEV synthesized in infected cells and the P75AEV synthesized in the cell-free system are electrophoretically identical. A 44,000 molecular weight polypeptide (P44AEV) is synthesized from 20-24S RNA, apparently from the 3' section of the AEV-specific RNA sequence. A minor 37,000 molecular weight polypeptide (P37AEV) is synthesized from 20S AEV RNA. A comparison is drawn between the cell-free products of MC29 and AEV RNAs.     

Cell-free translation of bovine viral diarrhea virus RNA.

Bovine viral diarrhea virus RNA was translated in a reticulocyte cell-free protein synthesizing system. The purified, 8.2-kilobase, virus-specific RNA species was unable to serve an an efficient message unless it was denatured immediately before translation. In this case, several polypeptides, ranging in molecular weight from 50,000 to 150,000 and most of which were immunoprecipitated by bovine viral diarrhea virus-specific antiserum, were synthesized in vitro. When polyribosomes were used to program cell-free synthesis, mature viral 80,000- and 115,000-molecular-weight proteins were detected; no precursor to the viral 55,000-molecular-weight glycoprotein was noted. The implications of these results with respect to virus-specific protein synthesis are discussed.     

Isolation and translation of plant messenger RNA

A fraction of the RNA species isolated from Lemna gibba G-3 consists of molecules with attached sequences of polyadenylic acid. This polyadenylic acid-containing fraction, separated from total RNA by adsorption onto oligothymidylic acid-cellulose, was shown to be mRNA by its ability to serve as template in a cell-free translation system derived from wheat germ. The products of translation were characterized by electrophoresis. This method permitted the comparison of mRNA from plants grown under different light conditions. Such plants were shown to possess qualitative and quantitative differences in their mRNA complements.     

Cell-free translation products of basement membrane RNA from the EHS tumor

The biosynthetic products of the Engelbreth-Holm-Swarm (EHS) tumor and the cell-free translation products of EHS tumor cell RNA were characterized. Six distinct gene products (three laminin polypeptides, entactin/nidogen, and two collagen IV chains) comprising the basement membrane matrix were identified by a combination of proteolytic digestion and immunologic techniques. Analysis of the cell-free translation products using EHS tumor RNA precipitated by anti-laminin serum confirms earlier evidence that there are at least two B chains encoded by different genes. The anti-laminin serum also recognized entactin/nidogen, which was further identified by specific immunoprecipitation with anti-entactin serum. Radiolabeled laminin A chains, synthesized by the EHS tumor in organ culture, were also identified by the anti-laminin serum but were not detected among the cell-free translation products of EHS tumor RNA. Pulse-chase studies of EHS tumor in organ culture as well as in vitro translation of EHS tumor RNA suggest that the precursor forms of alpha 1(IV) and alpha 2(IV) collagen chains are nearly identical in size, with apparent molecular weights of 170,000. The mRNAs encoding these two polypeptides migrate differently on sucrose gradients. It is likely that glycosylation and hydroxylation of collagen IV account for the major differences in molecular weight of mature alpha 1(IV) and alpha 2(IV) chains in the EHS tumor matrix.     

Biochemistry of terminal deoxynucleotidyltransferase. Affinity labeling and identification of the deoxynucleoside triphosphate binding domain of terminal deoxynucleotidyltransferase

Using the technique of UV-mediated cross-linking of nucleotides to their acceptor sites (Modak, M. J., and Gillerman-Cox, E. (1982) J. Biol. Chem. 257, 15105-15109), we have labeled calf terminal deoxynucleotidyltransferase (TdT) with [32P]dTTP. The specificity of dTTP cross-linking at the substrate binding site in TdT is demonstrated by the competitive inhibition of the cross-linking reaction by other deoxynucleoside triphosphates, and ATP and its analogues, requiring concentrations consistent with their kinetic constants. Tryptic peptide mapping of the [32P]dTTP-labeled enzyme showed the presence of a single radioactive peptide fraction that contained the site of dTTP cross-linking. The amino acid composition and sequence analysis of the radioactive peptide fraction revealed it to contain two tryptic peptides, spanning residues 221-231 and 234-249. Since these two peptides were covalently linked to dTTP, the region encompassed by them constitutes a substrate binding domain in TdT. Further proteolytic digestion of the tryptic peptide-dTTP complex, using V8 protease, yielded a smaller peptide, and its analysis narrowed the substrate binding domain to 14 amino acids corresponding to residues 224-237 in the primary amino acid sequence of TdT. Furthermore, 2 cysteine residues, Cys-227 and Cys-234, within this domain were found to be involved in the cross-linking of dTTP, suggesting their participation in the process of substrate binding in TdT.     

Specificity of reticulocyte initiation factors for the translation of globin messenger RNA

Initiation factors from rabbit reticulocytes can select globin mRNA for translation in an ascites cell-free system in the presence of either encephalomyocarditis viral RNA or endogenous ascites mRNAs. It appears that the viral RNA cannot compete for either α- or β-globin-specific factors.