In vitro synthesis of laminin and entactin polypeptides

Total RNA and poly(A+) RNA, isolated from 13.5-day-old mouse embryo parietal endoderm cells and from differentiated F9 teratocarcinoma cells that synthesize laminin and entactin, were translated in the reticulocyte lysate. Antiserum raised against purified and denatured laminin B chains specifically immunoprecipitated from the translation reaction polypeptides of Mr = 205,000, 200,000, and 185,000. Antiserum against the native complex of laminin and entactin also immunoprecipitated these polypeptides, although less efficiently. In addition, this antiserum immunoprecipitated polypeptides of Mr = 300,000, 270,000, and 140,000. Antiserum against purified and denatured entactin immunoprecipitated only the Mr = 140,000 polypeptide. In contrast, no polypeptides were immunoprecipitated from translation reactions programmed with RNA from undifferentiated F9 cells that produce only small amounts of laminin and entactin. The in vitro synthesized polypeptides migrate on NaDodSO4-polyacrylamide gel electrophoresis slower than the respective unglycosylated laminin and entactin chains isolated from cells treated with tunicamycin. Supplementing the reticulocyte lysate with dog pancreas microsomal membranes yields in vitro translation products which co-migrate with the respective glycosylated laminin and entactin chains of control cells. Taken together, these results suggest that the polypeptides described represent in vitro synthesized laminin and entactin chains.     

Abnormal Expression of the Myelin-Associated Glycoprotein in the Central Nervous System of Dysmyelinating Mutant Mice

Total cytoplasmic brain RNA was isolated at two different ages from three neurological mutant mice (qk/qk, jp/Y, and shi/shi) and their apparently normal littermates. This RNA was translated in vitro in a rabbit reticulocyte lysate system. Myelin-associated glycoprotein (MAG)-related polypeptides were immunoprecipitated from equal amounts of total translation products derived from mRNA of mutant animals, normal littermates, or control animals. The developmentally regulated synthesis of MAG polypeptides was compared among the mutants and normal animals. mRNA from qk/qk brains synthesized an overabundance of p67MAG (five- to sevenfold) which may be compensation for a decreased synthesis of p72MAG. mRNA from jp/Y brains synthesized less than 10% of normal amounts of both MAG polypeptides. The quantity of MAG synthesized by 15-day shi/shi brain mRNA was slightly decreased compared with normal brain mRNA but the quantity of MAG synthesized by adult shi/shi brain mRNA was normal. No apparent differences were detected in the sizes of the MAG polypeptides synthesized by any of the mutants studied. The data suggest that the genetic defect in qk/qk mutants directly or indirectly affects the coordinated developmental regulation of MAG polypeptide synthesis leading to an overabundance of the MAG polypeptide that is normally found in older animals. The jp/Y mutation appears to affect general myelin protein synthesis. Finally, shi/shi mutants may have a delayed synthesis of MAG. The data are discussed in the light of recent observations concerning the synthesis of myelin proteins and their proposed role in myelin assembly.     

Storage protein precursor polypeptides in cotyledons of Pisum sativum L. Identification of, and isolation of a cDNA clone for, an 80000-Mr legumin-related polypeptide

An 80 000-Mr polypeptide, which bound to anti-legumin IgG, was detected among labelled polypeptides from cotyledons at late stages of development. When poly(A)-containing RNA from similar cotyledons was translated in a cell-free protein-synthesizing system, an 80 000-Mr polypeptide was also detected. Immunoprecipitation of translation products with anti-legumin IgG showed that, in addition to the major legumin precursor polypeptides of Mr approximately 60 000, the 80 000-Mr polypeptide was specifically immunoprecipitated. A cDNA clone, pCD32, was found to select an RNA coding for an 80 000-Mr polypeptide in hybrid-selection experiments. Additional minor polypeptides of Mr 63 000 and 65 000 were present in translation products of RNA selected by pCD32; all three polypeptides were immunoprecipitated by anti-legumin IgG. Thermal elution of RNAs bound to pCD32 showed that the affinity of pCD32 to the RNA coding for the 80 000-Mr polypeptide was greater than to the RNAs coding for the 63 000-Mr and 65 000-Mr polypeptides. In similar hybrid-selection experiments, another cDNA clone, pCD40, selected RNAs coding predominantly for polypeptides of Mr 63 000 and 65 000. A minor polypeptide of Mr 80 000 was also detected among these products; again all three polypeptides were immunoprecipitated by anti-legumin IgG. Peptide mapping revealed close similarities between the 80000-Mr polypeptide and the 63 000-Mr/65 000-Mr polypeptides obtained by translation of RNAs selected by pCD32. There were similarities also between maps obtained from translation products of RNA selected by pCD32 and those obtained from anti-legumin IgG immunoprecipitates of total translation products of poly(A)-containing RNA.     

Translation products of mRNA from infective larvae of Trichinella spiralis include an antigenic polypeptide

Total RNA was extracted from packed infective larvae of Trichinella spiralis by centrifugation through a 5.7 M caesium chloride cushion. Polyadenylated messenger RNA was separated from total RNA in an oligothymidylic acid-cellulose gel column. The in vitro translation of the mRNA, isolated from infective larvae of T. spiralis, was carried out using the rabbit reticulocyte cell-free translation system. Incorporation of 35S-methionine into the trichloroacetic acid precipitates in the lysate containing mRNA was 5 times greater than that in control. The translation products were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Many polypeptides with molecular weights of less than 100,000 were synthesized in the lysate. A T. spiralis positive mouse serum was mixed with translation products to form antigen-antibody complexes, which were then absorbed by Staphylococcus aureus Cowan 1 strain and analysed by autoradiography of SDS-PAGE. An antigenic polypeptide with a molecular weight of 48,000 was demonstrated to react specifically with IgG antibody in T. spiralis positive mouse serum. T. spiralis larvae were cultured in methionine-free medium containing 35S-methionine, and antigenic polypeptides in somatic extracts and ES products were compared with those in translation products by autoradiography of SDS-PAGE. Several polypeptides in ES products and somatic extracts reacted specifically with IgG antibodies in positive serum. Especially the polypeptide with a molecular weight of 48,000 in ES products strongly reacted with IgG antibody in positive serum.     

Developmental and functional analysis of Jonah gene expression

The Jonah genes are expressed twice in development: Jonah RNA is detected during all larval stages, disappears at the end of the third larval instar, and then reappears shortly after eclosion, in the adult midgut. Construction and analysis of Jonah cDNA clones reveals that multiple Jonah genes are transcribed; cDNA clones deriving from at least four different clusters of Jonah genes have been identified. In at least one case, multiple genes in a cluster are transcribed, and one cluster is found to be transcribed both in larvae and adults. Evidence that different Jonah genes are under different control with respect to both spatial and temporal patterns of expression has been provided. Jonah RNA encodes a 28-kDa translation product or products for which we consider a possible function. Jonah RNA of constant length is found to be conserved in all strains of Drosophila melanogaster examined, Jonah genes are found at a minimum of three common chromosomal sites in all of seven D. melanogaster strains examined, and multiple Jonah genes are found in other Drosophila species.     

Ventralization of the Drosophila embryo by deletion of extracellular leucine-rich repeats in the Toll protein.

Dorsoventral polarity of the Drosophila embryo is established by a signal transduction pathway in which the maternal transmembrane protein Toll appears to function as the receptor for a ventrally localized extracellular ligand. Certain dominant Toll alleles encode proteins that behave as partially ligand-independent receptors, causing embryos containing these proteins to become ventralized. In extracts of embryos derived from mothers carrying these dominant alleles, we detected a polypeptide of approximately 35 kDa in addition to full-length Toll polypeptides with antibodies to Toll. Our biochemical analyses suggest that the smaller polypeptide is a truncated form of Toll lacking extracellular domain sequences. To assay the biological activity of such a shortened form of Toll, we synthesized RNA encoding a mutant polypeptide lacking the leucine-rich repeats that comprise most of Toll's extracellular domain and injected this RNA into embryos. The truncated Toll protein elicited the most ventral cell fate independently of the wild-type Toll protein and its ligand. These results support the view that Toll is a receptor whose extracellular domain regulates the intrinsic signaling activity of its cytoplasmic domain.     

Biosynthesis of intestinal microvillar proteins. Translational evidence in vitro that aminopeptidase N is synthesized as a Mr-115000 polypeptide.

A crude RNA fraction, prepared from pig small intestine, was found to be more efficient than a fraction enriched in polyadenylated RNA in directing translation of polypeptides with Mr greater than 100000 in a rabbit reticulocyte lysate system. Aminopeptidase N (EC 3.4.11.2) synthesized in vitro was immunopurified from the translation mixture and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It was found to have an apparent Mr of 115000 regardless of whether the translation was performed in the absence or presence of proteinase inhibitors. This result contradicts the possibility of aminopeptidase N being synthesized as a large single-chain precursor polypeptide.     

Developmental regulation of glycerol-3-phosphate dehydrogenase synthesis inDrosophila

Methods have been developed to measure the synthesis of glycerol-3-phosphate dehydrogenase (GPDH) during the development of Drosophila melanogaster. In emerged adult flies, GPDH is a principal component of protein synthesis, comprising between 1 and 2% of the protein synthetic effort. This high relative rate of protein synthesis continues throughout adult life during a period of stable enzyme concentration. Therefore, it is evident that GPDH undergoes continual turnover. Analysis of GPDH synthesis in the adult segments reveals that this enzyme is synthesized in head, thorax, and abdomen. In 5-day-old flies, the relative rates of GPDH synthesis in the thorax and abdomen are similar. However, the concentration of GPDH in the thorax greatly exceeds that found in the abdomen. Therefore, it appears that the turnover rate of GPDH in the abdomen must be greater than the turnover rate of GPDH in the GPDH-containing cells (flight muscle) of the thorax. GPDH represents between 0.5 and 0.9% of the protein synthetic effort of larvae. The principle GPDH-containing tissue of larvae is fat body. The turnover of GPDH in larvae is similar to that in adult abdomen. This may be related to the concurrent presence of GPDH isozyme-3 in both tissues. Our studies indicate that the cell type-specific control of GPDH occurs at several levels.     

Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.     

HPLC purification and preparation of antibodies to cholic acid-inducible polypeptides from Eubacterium sp. V.P.I. 12708

The role of bile acid-inducible polypeptides in 7-dehydroxylation was investigated in Eubacterium sp. V.P.I. 12708. Cholic acid-inducible bile acid 7 alpha-, 7 beta-dehydroxylase, and delta 6 reductase activities co-eluted from a gel filtration high performance liquid chromatography (HPLC) column. Antibody (Ab) was prepared to these enzymatically active fractions, immunoadsorbed with uninduced cell extract coupled to Sepharose 4B, and used for immunoprecipitation of [35S]-methionine-labeled polypeptides. Ab immunoprecipitated polypeptides with molecular weights of 45,000, 27,000, and 23,500 from induced but not uninduced cell extracts. Immunoinhibition experiments showed that this Ab preparation inhibited (60%) bile acid 7 alpha-dehydroxylase activity in cell extracts. The 45,000 mol wt polypeptide was purified by (NH4)2SO4 fractionation, HPLC gel filtration, and HPLC-DEAE chromatography. Ab prepared to the 45,000 mol wt polypeptide immunoprecipitated only that polypeptide. This Ab, however, did not inhibit bile acid 7 alpha-dehydroxylase activity. Ab specific for the 27,000 mol wt polypeptide was prepared by partial purification and immunoadsorption with uninduced cell extracts. Immunochemical staining, following SDS-PAGE of crude cell extracts, shows a single immunoreactive protein band at 27,000 daltons. This Ab immunoprecipitated the 27,000 mol wt polypeptide as well as small amounts of the 45,000 and 23,000 mol wt polypeptides. Immunoinhibition studies showed that this Ab preparation inhibited (25%) 7 alpha-dehydroxylase activity. These data suggest that the 27,000 mol wt polypeptide is involved in enzyme catalysis. This does not, however, eliminate some role for the 45,000 and 23,500 mol wt polypeptides in bile acid metabolism in this organism.     

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.     

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.     

CYTOPLASMIC BIOSYNTHESIS OF LIGHT-HARVESTING POLYPEPTIDES IN THE GREEN FLAGELLATE MANTONIELLA SQUAMATA (MICROMONADOPHYCEAE)1

The biosynthesis of the light-harvesting complex (LHC) polypeptides of the green flagellate Mantoniella squamata (Manton et Parke) Desikachary (Micromonadophyceae, Chlorophyta) was examined by in vivo polypeptide labeling and immunoprecipitation of in vitro translation products. Using protein synthesis inhibitors, the LHC polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes and not in the chloroplasts of cells. Poly (A)+ RNA was isolated and proteins were synthesized by a rabbit reticulocyte lysate system, with antisera raised against M. squamata LHC used for immunoprecipitation from the translation products. One polypeptide 3-5 kDa larger than mature LHC polypeptides was immunoprecipitated. These studies indicate that although the LHC of M. squamata is quite different from the LHC of most green plants, the LHC polypeptides are synthesized as precursors in the cytoplasm of the cell and suggest that the genes encoding these polypeptides are located in the nucleus.     

The biogenesis of the cyanellae of Cyanophora paradoxa. III. In vitro synthesis of cyanellar polypeptides using separated cytoplasmic and cyanellar RNA

RNA from Cyanophora paradoxa was separated into cytoplasmic and cyanellar fractions by using a combination of subcellular fractionation and oligo-dT chromatography. In vitro translation of the separated cytoplasmic and cyanellar RNAs in a rabbit reticulocyte lysate system in the presence of [35S]methionine resulted in the incorporation of radiolabel into electrophoretically distinct sets of polypeptides. Monospecific and polyspecific antibodies that react with cyanellar polypeptides were used to probe the in vitro translation products by indirect immunoprecipitation by using Staphylococcus protein A conjugated to Sepharose beads. The results indicate that linker polypeptide L1 of the phycobilisome, the gamma subunit of coupling factor CF1, and subunit II of PS I are synthesized in the cytoplasm as precursor molecules that are 5-8 kDa larger than their mature sizes. Antibodies directed against the psbA gene product (the D1 protein) precipitated a polypeptide found in the translation products of the cyanellar RNA-directed reactions, which is about 1.5 kDa larger than the mature protein.     

RNA polymerase II subunit composition, stoichiometry, and phosphorylation.

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.     

Differential expression and isozymic composition of sn-glycerol-3-phosphate dehydrogenase in tissues from variant lines of Drosophila melanogaster

The tissue-specific expression and isozymic composition of Drosophila sn-glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8) have been determined for a high-activity control line and two variant lines that alter either the temporal or systemic expression of GPDH through a reduction in rates of polypeptide synthesis. The temporal variant exhibits a reduction in enzyme levels in all larval tissues and in the adult abdomen, while levels of activity in the adult thorax are equal to the control line. Isozymic analyses of these tissues demonstrate that it is the GPDH-3 species that is reduced in a temporal and tissue-specific manner. In contrast, the systemic variant demonstrates a uniform reduction of all isozymic species in each tissue and developmental stage. Analyses of the tissues of F1 hybrid offspring of each variant line and appropriately marked electrophoretic variants demonstrate that the tissue-specific effects observed are due to cis-acting elements that are tightly linked to the structural gene.