Molecular biology of terminal transferase

Terminal transferase is an unusual deoxynucleotide polymerizing enzyme found only in prelymphocytes. The protein was purified to homogeneity from calf thymus glands in 1971 as a 32 kDa protein with a two peptide structure. Subsequent biochemical and immunological analyses of terminal transferase protein in crude extracts from a number of animal species showed a single peptide with a molecular weight of about 58,000. The two peptide structure found earlier was caused by proteolysis. Homogeneous 58 kDa terminal transferase has now been produced from human lymphoblastoid cells and calf thymus glands by immunoaffinity chromatography. In vitro phosphorylation studies showed that the terminal transferase protein contains one phosphorylation site near one end of the polypeptide chain, and the phosphorylation of the enzyme has been confirmed by in vivo labeling experiments. Unambiguous demonstration of the molecular weight of the human terminal transferase was obtained by translation of the cloned human terminal transferase DNA sequence to a 58,308 Da protein. The translated amino acid sequence also provided a possible phosphorylation site near the amino-terminus of the protein. Preliminary analysis of the genomic structure shows a simple intron/exon pattern with the total human terminal transferase gene spanning at least 65 Kb.     

Antigenic relationships in calf thymus and human leukemic cell terminal deoxynucleotidyl transferase.

Antibody to purified terminal deoxynucleotidyl transferase (nucleosidetriphosphate : DNA deoxy-nucleotidylexotransferase, E.C. 2.7.7.31) from calf thymus was prepared in rabbits using terminal deoxynucleotidyl transferase crosslinked to bovine serum albumin. These antibodies, partially purified by 60% ammonium sulfate precipitation and Sephadex G-200 column chromatography, produced one precipitation band with calf thymus terminal deoxynucleotidyl transferase on immunodiffusion. This antibody preparation also inhibited the in vitro activity of terminal deoxynucleotidyl transferase from calf thymus, acute leukemic lymphoblasts and Molt-4 cells but not that of DNA polymerases alpha, beta and psi from these cells     

Purification of a high molecular weight human terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyltransferase has been purified from lymphoblasts of leukemic patients. The enzyme has a molecular weight of approximately 62,000 as determined by gel filtration and nondenaturing gel electrophoresis and is not dissociated into subunits by sodium dodecyl sulfate. In contrast, the terminal transferase enzyme from calf thymus has a molecular weight of 42,000 as determined by gel filtration, and is dissociated into 2 subunits of Mr 30,000 and 8,000 by sodium dodecyl sulfate. The enzyme has an isoelectric point of 8.2 and kinetic characteristics which are similar to those of calf thymus terminal transferase. The apparent Km for purine nucleotide polymerization at saturating initiator concentration with Mg2+ is 0.2 mM and with Mn2+ is 0.05 mM. Like calf terminal transferase, the reaction velocity is higher in the presence of Mg2+ than Mn2+. ATP inhibits the reaction catalyzed by terminal transferase isolated from human lymphoblasts due to mutual recognition of ATP and dATP by a common site on the enzyme. Preliminary experiments indicate that human terminal transferase may contain a small amount of carbohydrate. This report represents the first purification to near homogeneity of terminal transferase from a tissue source other than calf thymus.     

Synthesis of DNA polymerase by in vitro translation of calf RNA

Synthesis of alpha-polymerase in translation mixtures containing calf thymus poly(A+) RNA was examined by activity gel analysis and by immuno-binding with a monoclonal antibody to calf thymus alpha-polymerase. Activity gel analysis indicated that a DNA polymerase catalytic polypeptide of Mr = approximately 120,000 had been synthesized. Immunobinding experiments indicated that an immunoreactive polypeptide of about the same size had been formed in vitro. Sucrose gradient centrifugation of calf thymus total RNA revealed that mRNA encoding the approximately 120,000-Mr DNA polymerase polypeptide sedimented at about 16S. This approximately 120,000-Mr catalytic polypeptide corresponds in size to an alpha-polymerase catalytic polypeptide found earlier in crude extracts of calf cells.     

Expression of human terminal deoxynucleotidyl transferase in Escherichia coli

A cloned DNA fragment related to pT17 containing a partial cDNA sequence of human terminal deoxynucleotidyl transferase was used as a probe to screen for the full length cDNA sequence of the enzyme in a lambda gt11 library constructed from human lymphoblastoid KM-3 cDNA. A recombinant containing a 2068-base pair insert was isolated and recloned into the EcoRI site of the sequencing plasmic pUC-8 as two subclones, pT711 and pT106. DNA sequencing and hybridization studies showed that pT711 contains the pT17 sequence and an additional 172 upstream nucleotides. pT711 represents the coding sequence for the carboxyl half of the terminal transferase protein. pT106, containing a 965-base pair insert, hybridizes to the same mRNA as pT711 on Northern blots and contains an open reading frame that is in phase with the reading frame of the insert in pT711. Amino acid sequencing of the 58-kDa peptide of the calf thymus terminal transferase failed, indicating that the N terminus is blocked. N-Terminal sequencing of a 56-kDa form of the protein produced 24 amino acids corresponding to the translated human cDNA coding sequence starting at residue 398 of the insert in pT106 with 83% homology between bovine and human sequence. The initiation codon is assigned to an ATG sequence at nucleotide 329 of the insert in pT106. Comparison of the translated human terminal transferase sequence with peptides from the calf thymus enzyme showed that the homology between the human and bovine enzyme is better than 90% among 263 amino acids determined. The coding sequences in pT106 and pT711 were recloned into an expression plasmid pUC-19 downstream from the lac promoter and in phase with the coding sequence of the lac Z gene. Lysates of bacteria carrying the reconstructed coding sequence of human terminal transferase contain a fused protein of 60 kDa that reacts with rabbit antibody to terminal transferase on immunoblots and exhibits enzyme activity. Isolation of this fused protein from bacterial lysates with mouse monoclonal antibody to human terminal transferase produces the expected protein of 60 kDa.     

In vitro translation, post-translational processing and secretion of pulmonary surfactant protein B precursors

Surfactant proteolipid SP-B is a hydrophobic protein of Mr = 8000 identified in organic solvent extracts of pulmonary surfactant. Analysis of the human SP-B RNA predicts that the active surfactant peptide is derived by proteolysis of an Mr = 40,000 precursor. In the present work, characteristics of synthesis, secretion and processing of SP-B were demonstrated in a pulmonary adenocarcinoma cell line by immunoprecipitation of radiolabelled precursors. Treatment of cells with tunicamycin resulted in synthesis and secretion of unglycosylated proSP-B of Mr = 39,000. Immunoprecipitation of protein produced by in vitro translation of human lung poly(A)+ RNA detected an Mr = 40,000 protein; the size discrepancy is likely related to cleavage of a leader signal sequence. Endoglycosidase-H-sensitive precursors of Mr = 41,000-43,000, pI = 5.1-5.4 were the first isoforms detected within the cells and were processed to endoglycosidase-H-resistant isoforms and secreted. Neuraminidase and endoglycosidase-F-sensitive forms of proSP-B were first detected in the media at 60 min as Mr = 42-46,000 isoforms with pI = 4.6-5.1. Proteolytically processed isoforms of proSP-B were detected primarily in the media and were generated by cleavage of an amino-terminal Mr = 16,000 peptide resulting in Mr = 27,000-33,000 isoforms (pH = 5.6-6.8). The Mr = 27,000-33,000 isoforms were sensitive to neuraminidase, resulting in isoforms with pH = 6.0-6.8. Digestion of the Mr = 27,000-33,000 peptide with endoglycosidase-F resulted in isoforms of Mr = 23,000, pH = 6.0-6.8. The endoglycosidase-F-resistant peptide of Mr = 16,000, pI = 4.2-4.4 was identified with an antiserum generated against synthetic peptides derived from the amino-terminal domain, as deduced from the SP-B DNA sequence. Further proteolytic processing of the Mr = 27,000-33,000 isoforms to the Mr = 8000 peptide detected in surfactant was not observed in this cell line. Thus, in the H441-4 cells (a cell line with morphologic features of Clara cells), SP-B is synthesized as a preproprotein which undergoes cleavage of a signal sequence and addition of asparagine-linked carbohydrate; proSP-B is secreted by processes which are independent of glycosylation. SP-B peptides of Mr = 27,000-33,000 and Mr = 16,000, representing carboxy and amino-terminal domains, accumulate in the media.     

Large polypeptides of 10S DNA polymerase alpha from calf thymus: rapid isolation using monoclonal antibody and tryptic peptide mapping analysis

The polypeptides recognized by a monoclonal antibody against calf thymus DNA polymerase alpha (secreted from a hybridoma CL22 -2- 42B , Nucleic Acids Res. (1982) 10, 4703-4713) were identified by the immunoblot method as the large polypeptides of the partially-purified 10S DNA polymerase alpha fraction. Using an immunoprecipitation technique with the monoclonal antibody, a rapid immunological isolation of the polypeptides has been achieved. By this method, the large polypeptides with Mr = 140,000, 145,000, and 150,000 were isolated from a partially-purified preparation of 10S DNA polymerase alpha. On the other hand, the polypeptides with Mr = 150,000, 180,000, and 240,000 were obtained from a crude extract of calf thymus. Tryptic peptide maps showed that the large polypeptides with Mr = 150,000, and 180,000 were very similar in primary structure and that the structures of Mr = 180,000 and 240,000 polypeptides contained partially common sequences. Among these polypeptides, the Mr = 150,000 polypeptide was shown to correlate with the enzyme activity. These results suggest that the large polypeptide of 10S DNA polymerase alpha is initially synthesized as Mr = 180,000 or larger polypeptide, then converted to the form with Mr = 150,000. The Mr = 140,000 and 145,000 polypeptides in the purified preparation may be artificial products formed during purification.     

Immunological and sequence interrelationships between multiple human liver and rat glutathione S-transferases

The 13 forms of human liver glutathione S-transferases (GST) (Vander Jagt, D. L., Hunsaker, L. A., Garcia, K. B., and Royer, R. E. (1985) J. Biol. Chem. 260, 11603-11610) are composed of subunits in two electrophoretic mobility groups: Mr = 26,000 (Ha) and Mr = 27,500 (Hb). Preparations purified from the S-hexyl GSH-linked Sepharose 4B affinity column revealed three additional peptides at Mr = 30,800, Mr = 31,200, and Mr = 32,200. Immunoprecipitation of human liver poly(A) RNAs in vitro translation products revealed three classes of GST subunits and related peptides at Mr = 26,000, Mr = 27,500, and Mr = 31,000. The Mr = 26,000 species (Ha) can be precipitated with antisera against a variety of rat liver GSTs containing Ya, Yb, and Yc subunits, whereas the Mr = 27,500 species (Hb) can be immunoprecipitated most efficiently by antiserum against the anionic isozymes as well as a second Yb-containing isozyme (peak V) from the rat liver. The Mr = 31,000 band can be immunoprecipitated by antisera preparations against sheep liver, rat liver, and rat testis isozymes. Human liver GSTs do not have any subunits of the rat liver Yc mobility. Antiserum against the human liver GSTs did not cross-react with the Yc subunits of rat livers or brains in immunoblotting experiments. The human liver GST cDNA clone, pGTH1, selected human liver poly(A) RNAs for the Ha subunit(s) in the hybrid-selected in vitro translation experiments. Southern blot hybridization results revealed cross-hybridization of pGTH1 with the Ya, Yb, and Yc subunit cDNA clones of rat liver GSTs. This sequence homology was substantiated further in that immobilized pGTH1 DNA selected rat liver poly(A) RNAs for the Ya, Yb, and Yc subunits with different efficiency as assayed by in vitro translation and immunoprecipitation. Therefore, we have demonstrated convincingly that sequence homology as well as immunological cross-reactivity exist between GST subunits from several rat tissues and the human liver. Also, the multiple forms of human liver GSTs are most likely encoded by a minimum of three different classes of mRNAs. These results suggest a genetic basis for the subunit heterogeneity of human liver GSTs.     

The two molecular weight forms of rat phenylalanine hydroxylase are encoded by different messenger RNAs

Immunoprecipitation of the phenylalanine hydroxylase formed by translation of rat liver RNA in a rabbit reticulocyte cell-free protein synthesis system was used to examine the origin of the molecular weight heterogeneity of the enzyme. Sodium dodecyl sulfate-polyacrylamide electrophoresis of the immunoprecipitated products showed that in most cases a single specifically immunoprecipitated polypeptide was produced which corresponded to the higher molecular weight (H) form of phenylalanine hydroxylase (Mr = 50,000). The identity of the product was confirmed by immunological competition and peptide mapping. RNA from other rats, however, coded for both the H-form and the lower molecular weight (L) form of phenylalanine hydroxylase or for only the L-form. The evidence suggests that the L-form derives from a different mRNA, rather than by proteolysis of the H-form, an interpretation which is supported by the isolation of the lower form of phenylalanine hydroxylase from livers of some rats.     

Two developmentally regulated messenger RNAs differing in their coding region may exist for the myelin-associated glycoprotein

Mouse and rat brain RNA were translated in vitro in a rabbit reticulocyte lysate translation system, and myelin-associated glycoprotein-related polypeptides were immunoprecipitated. Two products of Mr = 72,000 (p72MAG) and Mr = 67,000 (p67MAG) were specifically immunoprecipitated. These polypeptides were characterized as follows: 1) exogenously added purified myelin-associated glycoprotein (MAG) competitively eliminates their immunoprecipitation; 2) peptide maps obtained from them are very similar; 3) they are both destined to become glycoproteins, as assayed by insertion into dog pancreas microsomes and their subsequent sensitivity to endoglycosidase H. Translatable mRNA for the two polypeptides have different developmental expressions. p72MAG mRNA appears at an earlier age than p67MAG mRNA and remains the dominant MAG mRNA species throughout the period of rapid myelination. As the rate of myelination decreases, p67MAG mRNA becomes the dominant MAG mRNA species. Finally, two endoglycosidase H-sensitive polypeptides were specifically immunoprecipitated from mouse brain microsomes. Therefore, there exist two MAG proteins that differ slightly in their polypeptide structure and that are developmentally regulated. The possibility that the two polypeptides are synthesized de novo from two coordinately regulated mRNAs that differ in their coding region is discussed.     

Biosynthesis of mammalian DNA ligase

A monospecific antibody against calf thymus DNA ligase composed of a single polypeptide with Mr = 130,000 cross-reacts with rodent and calf thymus DNA ligases. The antibody precipitates a single Mr = 200,000 polypeptide from detergent lysates of [3H] leucine-labeled mouse Ehrlich tumor cells and calf thymocytes. Pulse-chase experiments show that the Mr = 200,000 polypeptide in Ehrlich tumor cells has a half-life of about 0.5 h. In addition to the Mr = 200,000 polypeptide, a Mr = 130,000 polypeptide is detected in the partially purified enzyme preparations from radiolabeled Ehrlich tumor cells. These results suggest that DNA ligase is synthesized in mammalian cells as a Mr = 200,000 polypeptide and that the Mr = 200,000 polypeptide is degraded to a Mr = 130,000 polypeptide by a limited proteolysis in vitro.     

Structural homology among calf thymus alpha-polymerase polypeptides.

A sample of highly purified calf thymus alpha-polymerase contained an abundant 118,000 Mr polypeptide as well as five lower molecular weight polypeptides in the range of 54,000- to 64,000-Mr. This 118,000-Mr polypeptide was capable of DNA polymerase activity, as revealed by in situ assay after SDS-polyacrylamide gel electrophoresis. Tryptic peptide mapping indicated that the 118,000-Mr polypeptide shared extensive primary structure homology with 57,000-, 58,000- and 64,000-Mr polypeptides and some limited homology with 54,000- and 56,000-Mr polypeptides. This is the first evidence that lower and higher Mr polypeptides of purified calf thymus alpha-polymerase share sequence homology; these results are interpreted in the context of a model that predicts the existence of a common precursor with molecular weight greater than 140,000.     

Characterization of messenger RNA for chick-embryo DNA polymerase beta and its translation product in vitro

A specific immunoprecipitation method, using rabbit anti-(chick DNA polymerase beta) IgG was applied to detect the polypeptide of DNA polymerase beta among translation products obtained in vitro with mRNA extracted from chick embryos. A polypeptide of Mr = 40 000 was specifically immunoprecipitated from [35S]methionine-labeled translation products and was competitive with the purified DNA polymerase beta for the antibody. Furthermore, the 40 000-Mr translation product obtained in vitro had DNA polymerase activity, which was detected by assay in situ after electrophoresis in a polyacrylamide gel containing DNA. The mRNA for DNA polymerase beta was polyadenylated and its content was estimated as the range of 0.001% of total poly(A)-rich RNA on the basis of [35S]methionine incorporation in the translation in vitro. The size of this mRNA was determined to be about 1800 nucleotides by zone sedimentation and agarose gel electrophoresis under denaturating conditions.     

Structural analysis of the hepatic glucagon receptor. Identification of a guanine nucleotide-sensitive hormone-binding region

[125I-Tyr10]Monoiodoglucagon [( 125I]MIG) was cross-linked to liver membrane glucagon receptors with hydroxysuccinimidyl-p-azidobenzoate, and the products were analyzed by sodium dodecyl sulfate-gel electrophoresis. Autoradiograms of the gel obtained after a 24-h exposure showed one major band at Mr = 63,000 that was sensitive to GTP and excess unlabeled glucagon. Exposure for 7 days showed labeling of an additional Mr = 33,000 species that was also sensitive to excess unlabeled glucagon. The Mr = 33,000 peptide can be obtained by subtilisin, trypsin, elastase, or Staphylococcus aureus V8 protease treatment of the [125I]MIG-occupied receptor in the membrane or in Lubrol-PX solution. In contrast, limited proteolysis of membranes containing vacant receptors results in labeling of a Mr = 24,000 peptide. The Mr = 24,000 peptide specifically binds [125I]MIG in a GTP-sensitive manner. The Mr = 33,000 peptide also retains GTP sensitivity since it releases bound [125I]MIG upon addition of GTP. Elastase treatment of the electroeluted Mr = 33,000 peptide yields the Mr = 24,000 and 15,000 fragments. The Mr = 15,000 peptide is the smallest fragment of the receptor as yet identified. Treatment of the Mr = 63,000 receptor with [125I]MIG cross-linked to it with endo-beta-N-acetylglucosaminidase F results in four distinct fragments with Mr values of 61,000, 56,000, 51,000, and 45,000; prolonged treatment resulted in the accumulation of the last two. Neither the Mr = 33,000 nor the Mr = 24,000 fragment appeared to be substrates for endo-beta-N-acetylglucosaminidase F. These data indicate that glucagon receptor is a glycoprotein of approximately 60,000 daltons which contains at least four N-linked glycans accounting for 18,000 daltons of its mass. Both its glucagon binding function and its capacity to interact with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans. Hormone occupancy of the receptor results in a conformational change so as to expose a region that is susceptible to proteolysis by proteases of varying specificities to yield a peptide of approximately 30,000 daltons that also does not contain N-linked glycans.     

An antibody and anti-idiotypic antibody against the extra signal peptide of pre-aspartate aminotransferase

A peptide (extra signal peptide) comprising amino acids 1-29 of pig liver pre-mitochondrial aspartate aminotransferase (p-mAAT) was synthesized chemically. The peptide was found to block the import of rat liver p-mAAT into rat liver mitochondria. An antibody raised against the peptide immunoprecipitated rat liver p-mAAT synthesized in a rabbit reticulocyte cell-free translation system. These results suggested that the extra signal peptide sequence of p-mAAT is essential for import of p-mAAT into the mitochondria and that there is structural homology between the extra signal peptides of pig and rat liver p-mAAT. An anti-idiotypic antibody against the peptide was also prepared and purified by affinity chromatography on an Affi-Gel 10 anti-peptide IgG column and was then characterized.     

Cloning and expression in Escherichia coli of cDNA for arginase of rat liver

A cDNA expression library constructed in a plasmid pUC8 from poly(A)+ RNA of rat liver was screened immunologically, using an antibody against arginase of rat liver. A cDNA clone was isolated and identified by hybrid-selected translation. The clone contained an insert approximately 1.35 kilobase pairs in length. In the bacterial clone, we detected a specific protein of Mr = about 43,000 that is slightly larger than the purified arginase (Mr = about 40,000) and a high activity of arginase was expressed. The arginase mRNA species of about 1600 bases long was detected in the liver, but not in the small intestine, kidney, spleen and heart of the rats.     

Electrophoretic separation of in vitro translation products on giant two-dimensional gels allows detailed analysis of cellular mRNAs

The in vitro translation products of mRNA pretreated with methylmercuric hydroxide were examined by giant two-dimensional gel electrophoresis. In addition to increasing overall translational efficiency approximately 2.5-fold, methylmercuric hydroxide selectively increases the translation of mRNAs coding for higher molecular mass (greater than 45 kDa) proteins, allowing the routine resolution of 1500 [35S]methionine-labeled proteins. This yields 3 to 4-fold the number of translation products seen with smaller size two-dimensional gels. With this method we compare thymus cell proteins synthesized in vivo with the products of in vitro translation of mRNA recovered from thymus cells. Fifty-eight percent of the translation products are qualitatively the same as proteins synthesized in vivo (similar Mr, pI, and neighboring proteins), with 64% of these also being quantitatively similar (less than 5-fold difference). A comparison of thymus mRNA in vitro translation products with those coded for by mRNA from liver reveals only 32% qualitative similarity, with 63% of these also being quantitatively similar. These results are discussed in relation to predictions of mRNA abundance and complexity based on DNA:RNA hybridization data. Giant two-dimensional gel separations of in vitro translation products appear to be useful for detecting less abundant cellular mRNAs, including those that may be regulated by hormones or other physiological mediators.     

Limited proteolysis of calf thymus terminal deoxynucleotidyl transferase

Controlled, limited proteolysis of homogeneous calf thymus terminal deoxynucleotidyl transferase (EC 2.7.7.31) using immobilized Staphylococcus aureus V-8 protease results in a low molecular weight form of the enzyme which possesses unaltered catalytic activity. Analysis of the products of limited proteolysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that only the large subunit, β, is modified from a molecular weight of 30,500 to 25,500. The small subunit, α, which has a molecular weight of 9500, is unchanged. A shift in the apparent isoelectric pH of the calf enzyme following proteolysis is observed from pI = 8.2 to 7.8. Both forms of the enzyme are homogeneous in the isoelectric focusing gel system, as determined by coincidence of single protein bands with terminal transferase activity on the gel. The specific activities of cleaved and uncleaved terminal transferase proteins, as well as their thermal stabilities, are comparable. These results suggest that the polypeptide domain involved in terminal transferase enzymatic activity can be probed further by novel methods involving limited proteolysis without concomitant loss in enzymatic function.