Processing of herpes simplex virus proteins and evidence that translation of thymidine kinase mRNA is initiated at three separate AUG codons

The role which post-translational modification plays in the genesis of herpes simplex virus-induced polypeptides was investigated. Two-dimensional gel electrophoresis was used to identify those polypeptides (i) synthesized in vitro, (ii) labeled in vivo during a pulse, and (iii) labeled after a chase. Excluding glycoproteins, we detected 36 precursor or short-lived polypeptides, 8 polypeptides which were generated by post-translational modification, 46 polypeptides which were apparently not modified after synthesis, and 19 polypeptides which were either transient intermediates or not modified. Comparison of polypeptides synthesized in vitro and during an in vivo pulse showed that translation in vitro resembles quite closely translation in vivo and that amounts of protein synthesized in vivo are determined largely by the levels of mRNA. This analysis provided the basis for an investigation of the suggestion (C.M. Preston and D.J. McGeoch, J. Virol. 38:593-605, 1981) that the two polypeptides of apparent molecular weights of 43,000 (VI 43) and 39,000 (VI 39) encoded by the herpes simplex virus type 1 thymidine kinase gene are translated from a single mRNA by two in-phase initiation codons. Hybrid arrest was used to identify in vitro translation products encoded by the thymidine kinase gene. Two-dimensional gel electrophoresis showed that VI 39 was more acidic than VI 43, consistent with the predicted amino acid composition of a polypeptide whose synthesis was initiated at the second AUG codon, located 135 bases downstream from the first. Furthermore, two-dimensional gels revealed a third polypeptide whose synthesis was arrested by the same fragment. Its pI and apparent molecular weight (38,000) were compatible with initiation of translation at a third AUG codon an additional 42 bases downstream. Our findings provide strong evidence that downstream initiation codons within the thymidine kinase mRNA are used.     

Translation of Rauscher leukemia virus RNA in heterologous cell-free systems.

Rauscher leukemia virus RNA (RLV RNA) is translated in mammalian cell-free systems into distinct polypeptides which are immunoprecipitable by an antiserum directed against RLV proteins. These polypeptides partially comigrate electrophoretically with native viral proteins synthesized in vivo in JLS-V9 cells. Besides 72000-, 65000- and 50000-dalton polypeptides a 15000-dalton polypeptide is also synthesized in vitro. Analysis of incubations of RLV RNA in different cell-free systems reveals that no virus-specific factors are required in the translation of RLV RNA in vitro.     

Developmentally regulated alternate modes of expression of the Gpdh locus of Drosophila.

Immunoblot analyses have been performed on extracts prepared from Drosophila melanogaster. Those analyses have revealed two subunit forms of enzyme glycerol 3-phosphate dehydrogenase (GPDH) in larval tissues and in adult abdominal tissues. Thoracic tissue, which accounts for the bulk of the adult GPDH, has only one subunit form, the smaller. The two subunit forms differ by approximately 2400 daltons. In agreement with previous genetic and biochemical data indicating that this enzyme is encoded by a single structural gene, analyses of extracts prepared from a strain carrying a GPDH null mutation detect no GPDH polypeptides in larvae or adults. Similarly, analyses of extracts prepared from a strain carrying a mutation which produces a GPDH polypeptide that differs in size from wild-type reveal a change in the adult thoracic GPDH polypeptide as well as a change in both GPDH polypeptides found in larvae. Total Drosophila RNA prepared from larvae or newly eclosed adults has been translated in a mRNA-dependent cell-free system. GDPH was immunoprecipitated from the translation products and analyzed. Two subunit forms of GPDH were immunoprecipitated from translation products whose synthesis was directed by larval RNA and only one was detected in the polypeptides synthesized from adult RNA. The GPDH polypeptides synthesized in vitro are approximately the same size as the corresponding polypeptides found in vivo. The relative proportion of total GPDH represented by each subunit form synthesized in vitro is similar to those found in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of mRNA translationin vitro andin ovo followed by crossed immunoelectrophoretic autoradiography to study the biosynthesis of human cholinesterases

The synthesis of various cholinesterases in different fetal human tissues was studied using in vitro and in ovo translation of poly(A)+ RNA, followed by crossed immunoelectrophoretic autoradiography. When unfractionated poly(A)+ mRNA from fetal brain, muscle, or liver was translated in vitro, in the reticulocyte lysate cell-free system, polypeptides were synthesized which reacted with antibodies against either "true" acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) or "pseudo", butyrylcholinesterase (acylcholine acylhydrolase; EC 3.1.1.8). The two nascent cholinesterases could be separated by crossed immunoelectrophoresis followed by autoradiography, suggesting that acetylcholinesterase and butyrylcholinesterase are produced in all three tissues from nascent polypeptides containing different immunological domains. To examine whether the biosynthesis of cholinesterases includes posttranslational processing events, Xenopus oocytes were microinjected with mRNA from these tissues. Immunoelectrophoretic analysis of oocyte intracellular homogenates and incubation medium revealed various precipitation arcs, reflecting the synthesis and posttranslational processing of multiple forms of tissue-specific exported and intracellular acetylcholinesterase and butyrylcholinesterase. These findings demonstrate that polymorphic cholinesterases are produced from nascent polypeptide products which undergo further posttranslational processing events in a tissue-specific manner before they become mature compartmentalized cholinesterases.     

Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C

The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.     

Cholera toxin is synthesized in precursor form on free polysomes in Vibrio cholerae 569B.

Membrane-bound and free polysomes have been isolated from Vibrio cholerae 569B. Nacent polypeptide chains were completed in a cell-free translation mixture containing Escherichia coli S-300 extracts and [3H]leucine or [35S]methionine. Cholera toxin-related polypeptides synthesized in vitro were immunologically detected after treatment with either anti-subunit A or anti-subunit B serum. Immunoreactive translation products were removed from reaction mixtures with formalinized Cowan's strain of Staphylococcus aureus, electrophoresed on sodium dodecyl sulfate-polyacrylamide gels, and visualized by fluorography. Anti-subunit A serum precipitated two major polypeptide species (molecular weights 52,000 and 45,000) from translation mixtures programed with free polysomes, whereas anti-subunit B serum precipitated only the 45,000-molecular-weight polypeptide. No cholera toxin-related polypeptides were detectable in translation mixtures programed with membrane-bound polysomes. Purified subunit A and cholera toxin competed for anti-subunit A binding sites and blocked the immunoprecipitation of the 35S-labeled 52,000- and 45,000-dalton polypeptides from in vitro translation mixtures. The data presented suggest that cholera toxin is synthesized in the cytoplasm in a precursor form on free polysomes and is secreted post-translationally.     

Orientation of the cleavage map of the 200-kilodalton polypeptide encoded by the bottom-component RNA of cowpea mosaic virus

The genomic organization of the bottom-component RNA of cowpea mosaic virus was studied. In vivo, this RNA encodes at least eight different polypeptides of 170, 110, 87, 84, 60, 58, 32, and 4 kilodaltons (K), the last polypeptide representing the genome-bound protein VPg. In rabbit reticulocyte lysates, bottom-component RNA is translated into a 200K polypeptide which is then processed to give the 32 and 170K polypeptides also found in vivo. By pulse-labeling the 200K primary translation product, we now show that the 32 and 170K polypeptides are derived from the NH₂-terminal and COOH-terminal parts of this polypeptide, respectively. Comparison of the proteolytic peptide patterns of 170K polypeptides synthesized in vitro and pulse-labeled at either the NH₂-terminal or the COOH-terminal end with the patterns of the 170 and 110K polypeptides found in vivo demonstrates that the order within the 200K primary translation product of cowpea mosaic virus bottom-component RNA is as follows: NH₂-32K polypeptide-58K polypeptide-VPg-24K polypeptide-87K polypeptide-COOH.     

Respiratory syncytial virus mRNA coding assignments.

The polypeptide coding assignments for six of the respiratory syncytial virus-specific mRNAs were determined by translation of the individual mRNAs in vitro. The coding assignments of the RNAs are as follows. RNA band 1 is complex and can be separated into at least two components on the basis of electrophoretic mobility (molecular weights [MWs] approximately equal to 0.21 X 10(6) and 0.31 X 10(6), respectively) that code for three polypeptides of 9.5, 11, and 14 kilodaltons (K). RNA 2 (MW, 0.39 X 10(6)) codes for a 34K polypeptide; RNA 3 (MW, 0.40 X 10(6)) codes for a 26K polypeptide; RNA 4 (MW, 0.47 X 10(6)) codes for a 42K polypeptide; and RNA 5 (MW, 0.74 X 10(6)) codes for a 59K polypeptide. By limited-digest peptide mapping, the 34, 26, and 42K polypeptides synthesized in vitro appeared to be unique. Additionally, peptide mapping showed that the 34, 26, and 42K polypeptides synthesized in vitro were indistinguishable from their counterparts synthesized in infected cells. Thus, the 34, 26, and 42K polypeptides coded for by mRNAs 2, 3, and 4, respectively, were identified as the respiratory syncytial virus phosphoprotein (34K), matrix protein (26K), and nucleocapsid protein (42K), respectively. RNA 5 was shown to code for a 59K polypeptide. The 59K polypeptide synthesized in vitro did not comigrate with any polypeptide specific to infected cells, suggesting that it is a candidate for co- or post-translational modification.     

Distribution of yolk polypeptides in the follicle cells during the differentiation of the follicular epithelium in Sarcophaga bullata egg follicles

In S. bullata, the ovaries contribute to the synthesis of yolk polypeptides. A specific antiserum for yolk polypeptides was used to visualize the presence of yolk polypeptides in the follicle cells during their differentiation. After vitellogenesis has started, all follicle cells contain yolk polypeptides. The squamous follicle cells covering the nurse cells and the border cells lose yolk polypeptides before mid-vitellogenesis, whereas the follicle cells over the oocyte contain yolk polypeptides until after late vitellogenesis. All follicle cells are immunonegative afterwards. In vitro translation of poly(A)+ RNA demonstrated that the presence of yolk polypeptide mRNA correlates well with follicle cell immunopositivity for yolk polypeptides. This suggests that the follicle cells synthesize the ovarian yolk polypeptides. Differences in cellular and nuclear morphology, total and poly(A)+ RNA synthesis and the rate of yolk polypeptide synthesis were shown to be correlated with the presence or absence of yolk polypeptides in the differentiating follicular epithelium. The possible relationship between these different aspects of follicle cell differentiation, follicle cell polyploidy and the extracellular current pattern around follicles are discussed.     

Optimization of protein synthesis in isolated higher plant chloroplasts. Identification of paused translation intermediates

Protein synthesis in isolated, intact pea chloroplasts was optimized and compared to translation within chloroplasts in vivo. Many polypeptides labeled with [35S]methionine in isolated intact chloroplasts did not comigrate with polypeptides which were labeled within chloroplasts in vivo. Antibodies to the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) immunoprecipitated [35S]-labeled large subunit plus several lower-molecular-mass translation products of isolated chloroplasts. The lower-molecular-mass soluble translation products synthesized in pulse-labeled chloroplasts were converted into full-length large-subunit polypeptides during a subsequent chase period. This result suggests that many of the polypeptides observed in pulse-labeled chloroplasts are incomplete translation products which are the result of ribosome pausing at discrete points along chloroplast mRNAs. The pulse-chase technique was used to follow synthesis of the 34.5-kDa precursor of the psb A gene product and its processing to the mature 32-kDa polypeptide in isolated chloroplasts. Chloroplast translation profiles obtained using the pulse-chase assay were very similar to translation profiles obtained in vivo thus extending the utility of protein synthesis in isolated chloroplasts.     

In vivo and in vitro synthesis of adenovirus type 2 early proteins.

The synthesis of adenovirus type 2 (Ad2)-induced early polypeptides was examined in vivo and in vitro by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis alone and specific immunoprecipitation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of total [35S]methionine-labeled polypeptides synthesized in vivo at 3 h postinfection allowed us to detect in infected cells at lease 13 distinct polypeptides that are either absent or less conspicuous in extracts from mock-infected cells. These Ad2-induced early polypeptides have molecular weights ranging from 72 x 10(3) to 10.5 x 10(3) and have accordingly been designated as E72K to E10.5K. Nine of the in vivo synthesized early polypeptides can be precipitated specifically from infected cell extracts by antisera with specificity against early adenovirus proteins. In vitro translation of mRNA extracted from mock-infected cells and from Ad2-infected cells was carried out in preincubated Ehrlich ascites cell extracts. All the early Ad2-induced polypeptides identified in the extracts from infected cells labeled in vivo were also detected among the polypeptides immunoprecipitated specifically from the in vitro reaction mixtures programmed by RNA extracted at 4 h postinfection from Ad2-infected cells.     

Isolation and characteristics of poly(A+) RNA in the lactating bovine udder

Total cellular poly(A+)-RNA was isolated from a lactating cow mammary gland. The poly(A+)-RNA molecules exhibit a heterogeneous distribution from 500 to 5000 nucleotides (average size--1600 nucleotides) and are made up of three main fractions (1550, 950 and 600 nucleotides) possessing a high template activity during translation in vitro. Optimal conditions for poly(A+)-RNA translation in a cell-free protein-synthesizing system from wheat embryos were elaborated. Immunochemical analysis of translation products revealed that 30% of the synthesized polypeptides are precipitated by immunoglobulins against cow milk proteins. Using hybridization with homologous cDNA, the kinetic complexity and heterogeneity of total cellular poly(A+)-RNA were investigated. This population was shown to consist of four classes differing in the diversity of their nucleotide sequences and the number of copies per cell. The total amount of the poly(A+)-RNA species in the cells of a lactating cow mammary gland is 9200, i.e., 0.46% of the genome complexity.     

In vitro synthesis of bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases. Characterization and processing of a precursor polypeptide for the chloroplast enzyme

Bean (Phaseolus vulgaris) chloroplastic and cytoplasmic leucyl-tRNA synthetases differ in their structural and catalytic properties and do not share common antigenic determinants. Polyadenylated mRNAs, prepared from young bean leaves, have been translated in vitro in a rabbit reticulocyte lysate cell-free system. The newly synthesized polypeptides have been submitted to immunoadsorption on protein A-Sepharose in the presence of the antibodies raised against the chloroplastic or the cytoplasmic leucyl-tRNA synthetase. The specificity of the immunoadsorption has been checked by competition experiments involving the pure enzymes. Bean chloroplastic leucyl-tRNA synthetase is synthesized in vitro from a polyadenylated mRNA as a precursor polypeptide of 130 kDa, which is somewhat larger than the mature enzyme of 120 kDa. Bean cytoplasmic leucyl-tRNA synthetase is synthesized in vitro as a polypeptide which has the size of the mature monomer (130 kDa). Processing of the precursor polypeptide of the chloroplastic leucyl-tRNA synthetase, yielding the mature enzyme, has been obtained by performing the in vitro translation in the presence of canine pancreatic microsomal membranes. These results suggest that in vivo bean chloroplastic leucyl-tRNA synthetase could be synthesized in the cytoplasm as a precursor which would be transported into the chloroplasts.     

Identification of adenovirus 2 early region 4 polypeptides by in vitro translation and tryptic peptide map analysis.

The mRNA species encoded by early region 4 (E4) (map position [mp] 91.5 to 99.3) of adenovirus 2 were isolated from the polysomes of infected KB cells and were purified by hybridization to the cloned HindIII-F fragment (mp 89.5 to 97.3) or to EcoRI-C fragment (mp 89.7 to 100). The mRNA's were translated in vitro using [35S]methionine as a labeled precursor in rabbit reticulocyte lysates treated with micrococcal nuclease as well as in wheat germ lysates. Five major (35,000-molecular-weight [35K], 23K, 22K, 21K, 18K) polypeptides were observed when the reticulocyte lysate was used. The 23K, 22K, 21K, and 18K polypeptides were also observed with the wheat germ lysate, as well as a very prominent 11K polypeptide; the 35K polypeptide was not observed. Assignment of these polypeptides to E4 was further established by hybrid arrested translation. Two-dimensional gel electrophoresis of a wheat germ translate resolved five polypeptides ranging from 18K to 23K, the major 11K polypeptide, and polypeptides of 10K and 9K. The in vitro 23K to 18K and 11K polypeptides migrated to approximately the same positions on two-dimensional gels as did seven 26K to 21K polypeptides and an 11K polypeptide synthesized in vivo (Brackmann et al., J. Biol. Chem, 255:6772--6779, 1980). Two-dimensional tryptic peptide maps demonstrated that the 35K, 23K, 22K, 21K, and 18K polypeptides are related. The peptide map of 11K is different from those of the above polypeptides, although 11K may share one tryptic methionine polypeptide with them. These results indicate that E4 encodes a major 11K polypeptide, as well as major 35K, 23K, 22K, 21K, and 18K polypeptides.     

Diversity of abundant mRNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings

The diversity of abundant mRNA sequences in various parts of 4-d etiolated pea seedlings (Pisum sativum L. var. Rondo CB) was compared by a cell-free translation of the mRNAs in the presence of [³⁵S]methionine and by an analysis of the products by two-dimensional electrofocussing/ electrophoresis (2D separation). The various parts of the seedlings were also examined for the pattern of protein synthesis in vivo. Proteins were labeled by injection of [³⁵S]methionine into the cotyledons, followed by 2D separation of the products. Over 95% of the abundant mRNA sequences and newly synthesized abundant polypeptides were shared by all parts of etiolated seedlings, including the cotyledons. However, a few distinct differences were observed when comparing mRNAs of roots and shoots; the most prominent among these were a group of six abundant mRNA sequences found exclusively in shoots. Only about 30% of the polypeptides synthesized on isolated RNA could be traced in equivalent positions on the gels as the polypeptides synthesized in vivo. Analysis of total RNA from light-grown pea seedlings showed the appearance of some twenty-five translation products not found with total RNA from etiolated seedlings, while about nine other translation products disappeared. At least ten of the light-induced RNA sequences were also present after growth in low-intensity red light (>600 nm) and are therefore thought to be controlled by the phytochrome system. Comparison of 11-d light-grown pea plants with 4-d light-grown seedlings did not reveal additional translatable RNA sequences, indicating that the major morphogenetic changes that occur after 4 d are not accompanied by significant changes in the pattern of abundant RNA sequences.     

Identification of lactate dehydrogenase-M polypeptide translated in vitro from human and mouse tumor cell poly(A)-containing messenger RNA

Rabbit anti-human lactate dehydrogenase-5(M4) antisera were raised which cross-reacted with mouse lactate dehydrogenase M polypeptide. The antisera were used for identification of human and mouse LDH-M polypeptides synthesized using an in vitro system directed by the mRNAs. The in vitro translation products directed by both mRNAs were similar in size and immunologically identical to the authentic LDH-M polypeptides. The sizes of the mRNAs encoding for both human and mouse LDH-M polypeptides were similar, about 15S (1445 nucleotides) and were shorter than the corresponding rat mRNA which is about 18S (1765 nucleotides).     

Characterization of Measles Virus-Specific Proteins Synthesized In Vivo and In Vitro from Acutely and Persistently Infected Cells

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.