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.     

Differentiation of dicyclohexylcarbodiimide reactive sites of the ATPase complex bovine heart mitochondria

1. In isolated bovine heart mitochondria, the 14C-labelled dicyclohexylcarbodiimide (DCCD) induced inhibition of the ATPase activity is accompanied by labelling of three polypeptides of Mx 9000, 16 000 and 33 000. Of these, only the 9000 polypeptide reacts with [14C]DCCD proportionally to the inhibitory effect, being saturated when the enzyme is maximally inhibited. 2. The 9000 and 16 000 polypeptides are extracted by neutral chloroform/methanol (2 : 1 v/v) while the 33 000 polypeptide remains in the non-extractable residue. No disaggregation of the polypeptides takes place during the extraction. 3. In the ATPase complex immunoprecipitated with antibody against F1, the 9000 and 16 000 polypeptides are present, but the 33 000 polypeptide is absent. 4. The results obtained indicate that the 33 000 polypeptide is not a component of the ATPase complex. As far as F0 is concerned, two types of the binding sites for DCCD were demonstrated, corresponding to the 9000 and 16 000 polypeptides. Their existence is explained by a non-random arrangement among individual monomers of the DCCD-binding protein.     

The polypeptide structure of Vitellogenin and Vitellin from the cockroach,Leucophaea maderae

Vitellogenin (Vg) synthesized by the fat body of Leucophaea maderaeis made up of four polypeptides with molecular weights of 160,000, 105,000, 98,000, and 57,000. Other polypeptides previously reported as part of Vg are associated with other proteins. Vitellin (Vt), the yolk protein (YP) isolated from mature oocytes and from newly formed oothecae, is a protein with a sedimentation coefficient of 28s and consists of three polypeptides with molecular weights of 105,000, 85,000, and 57,000. During vitellogenesis, the YP of developing oocytes contains both Vt and a 14s component. The 14s component is made up of four polypeptides with molecular weights of 105,000, 90,000, 85,000, and 57,000. The data suggest that 14s may not be a discrete protein but rather a form in transition between Vg and Vt in which the 98,000 dalton polypeptide is converted to the 85,000 dalton polypeptide of Vt through a 90,000 dalton intermediate. The 160,000 dalton peptide of Vg does not appear to be a part of Vt. Under alkaline conditions, both the 14s component and Vt are reduced to a polypeptide with a lower sedimentation rate in sucrose gradients. When acid conditions are restored, a protein resembling 14s is obtained. This suggests that the YP is a loosely held aggregate of similar or identical proteins with a molecular weight of about 250,000.     

Temperature and androgens regulate the biosynthesis of secretory proteins from rabbit cauda epididymidis

We have compared the biosynthesis of secretory proteins in rabbit cauda epididymidis maintained for 15 days at abdominal temperature with that of the scrotal cauda. Explants from both situations were incubated in vitro in the presence of [³⁵S] methionine, and the labelled proteins released into the incubation medium were analyzed by polyacrylamide gel electrophoresis. Body temperature specifically inhibited the synthesis of at least two polypeptides of 43 kDa and 21 kDa (designated EP21), whereas the synthesis of polypeptides of 80, 39, 31, and 24 kDa was increased. These changes resembled those produced by castration, but androgen treatment was not able to reverse the effect of body temperature. To confirm these observations, poly (A)⁺ RNA from the scrotal and the abdominal cauda respectively, was translated in vitro and the synthesized products were immunoprecipi-tated with an antibody against EP21 polypeptide. Both castration and body temperature strongly decreased the concentration of EP21 mRNA. In vivo testosterone administration restored the content of EP21 mRNA in cauda from castrated animals, but not in cauda maintained at body temperature. The changes observed might be related to the adverse effect of body temperature on sperm storage in the cauda epididymidis. © 1993 Wiley-Liss, Inc.     

CAP binding proteins associated with the nucleus.

Cap binding proteins of HeLa cells were identified by photo-affinity labelling using the cap analogue gamma-[32P]-[4-(benzoyl-phenyl)methylamido]-7-methylguanosine-5'- triphosphate. Photoreaction with whole cell homogenates resulted in specific labelling of five major polypeptides. The small molecular weight polypeptide appeared to be identical to the 24 000 to 26 000 dalton cap binding protein previously identified in initiation factors. A cap binding protein of 37 000 dalton was found in initiation factors as well as in preparations of crude nuclei. It was released from nuclei by washing with buffer of moderate salt concentration. Three high molecular weight cap binding proteins (approximately 120 000, approximately 89 000, approximately 80 000 dalton) were found in the nuclear fraction and were only partly released upon nuclease digestion and high salt extraction.     

Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Translation of HTLV (human T-cell leukemia virus) RNA in a nuclease-treated rabbit reticulocyte system

A human type-C retrovirus, designated HTLV (human T-cell leukemia virus), was isolated from the HTLV producer cell line MT-2. Agarose gel electrophoresis analysis 32P-labeled HTLVMT-2 virion RNA revealed that HTLVMT-2 virion RNA consists mainly of 24S and small amounts of 35S and 32S RNAs. The 24S HTLVMT-2 virion RNA and unfractionated HTLVMT-2 virion RNA were translated in a rabbit reticulocyte lysate system in vitro. The predominant polypeptide synthesized from 24S RNA had an apparent mol. wt. of 28 000 (28 K); unfractionated HTLVMT-2 virion RNA directed the synthesis of 53 000 (53 K), 33 000 (33 K) and 28 000 (28 K) polypeptides as main components. Most of the polypeptides synthesised in vitro by translation of HTLVMT-2 virion RNAs possess the same sizes as the proteins formerly designated as ATLA (ATL-associated antigen) in SDS-polyacrylamide gel electrophoresis and immunologically precipitated with sera of ATL patients. Therefore, the antigens termed ATLA, found by the serological study of ATL, are HTLVMT-2 encoded polypeptides.     

Identification of the in vitro translation products of adenovirus mRNA by immunoprecipitation.

Adenovirus type 2 mRNA was translated in S30 extracts from Ehrlich ascites and wheat embryo cells. The in vitro products were identified by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation with specific antisera in the presence of urea. Seven virion polypeptides could be identified by immunoprecipitation. Three of these appear to be precursors to polypeptides of the virion. mRNA isolated late in adenovirus infection was separated into three size classes by zonal sedimentation. Material sedimenting at 26S was translated into polypeptides corresponding to the largest virion polypeptides II to IV, a 22S fraction corresponding to polypeptide V, and smaller polypeptides and a 15S fraction corresponding to polypeptide IX. A significant amount of polypeptide IX was also synthesized by the 26S and 22S RNA.     

The effect of inhibition of RNA synthesis by actinomycin D on the population of basic polypeptides of the 30S unclear ribonucleoprotein particles

In rats injected intraperitoneally with actinomycin D (2 mg/kg body weight) 12 h earlier, the yield of the 30S ribonucleoprotein particles isolated from liver nuclei by extraction with 0.1 M NaCl at pH 8.0 decreased by 60 per cent. The protein-to-RNA ratio of these particles increased to 32:1 from the ratio 4.4:1 found in the same particles isolated from the nuclei of liver of control rats. The particles isolated from the liver nuclei of rats injected with actinomycin D were depleted of all charge isomers of the two most prominent polypeptides (33,000 and 39,000 daltons) present in the particles of liver of control animals. The most abundant protein in these particles was a 43,000 dalton polypeptide. This polypeptide is the least prominent of the 3 major polypeptides present in the control particles. The same charge isomers of the 43,000 dalton polypeptide were present in the nuclear ribonucleoprotein particles isolated from the liver of control animals and from the liver of animals treated with actinomycin D 12 h earlier. In control animals the nuclear ribonucleoprotein monoparticles isolated from kidney contained 3 major polypeptides of the same molecular weight with the same distribution of their charge isomers as were present in the particles isolated from liver nuclei. The injection of actinomycin D 12 h earlier was without any effect on the protein composition of the 30S nuclear ribonucleoprotein particles of rat kidney.     

Comparison of polypeptides from cultured human fibroblasts and sarcoma cells.

The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000--180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [35S]methionine and [3H] mannose incorporation experiments but not using the surface labeling method.     

Photosynthetic functions and thylakoid membrane polypeptide composition in light-sensitive mutants of Chlamydomonas reinhardii

In this paper we compared the pigment composition, photochemical activity, chloroplast ultrastructure, thylakoid membrane polypeptide composition and ribosomal content of wild-type and seven light-sensitive mutants of Chlamydomonas reinhardii.All the mutants had low chlorophyll and carotenoid content compared to wild-type. Mutants lts-30 and lts-135 were also characterized by a complete absence of visible carotenoids, while mutant lts-19 was fully deficient in chlorophylls.In most mutants, the chloroplast fragment could not carry out any DCIP photoreduction and O₂ evolution was also blocked. The PSI/P₇₀₀/activity was decreased in most cases.The mutant strains contained mostly single lamellae in their plastids, that is the stacking capacity of the thylakoid membranes was very decreased or fully absent. In most cases the number of lamellae was also very low.The relative amounts of 70 S ribosomes were decreased in all of the mutants. The thylakoid membranes showed anomalies in the region of 24 000–30 000 dalton polypeptides. The common characteristic for them was the relatively higher amount of the 30 000 dalton polypeptide and considerably decreased level of the 27 000 and 24 000 dalton polypeptides relative to the wild-type. These polypeptides were probably constituents of the chlorophyll-protein complex II which has been suggested to be the light harvesting pigment complex for PSII. The polypeptide of 30 000 daltons is the precursor for the LHCP apoprotein (24 000 dalton protein). It may be that the lighstimulated conversion of this precursor into LHCP apoprotein was blocked in our pigment-deficient mutants.Abbreviations CPI Chlorophyll-protein complex I - PSI Photosystem I - PSII Photosystem II - LHCP Light-harvesting pigment complex - DCIP 2,6-dichlorophenolindophenol - RuDPC-ase Ribulose-1,5-biphosphate-carboxylase - SDS Sodium dodecyl sulfate - LIDS Lithium dodecyl sulfate - PAG Polyacrylamide gel - TKM buffer 25 mM Tris-HCl, pH 7.S; 25 mM KCl; 25 mM Mg acetate     

Human term placenta contains transforming growth factors

Growth factors of apparent molecular weights of 6,000, 10,000, 20,000 and one in excess of 30,000 daltons can be isolated from acid-ethanol extracts of human term placentas. Each size class of growth factor resembles transforming growth factor (TGF) in that it stimulates anchorage independent growth of normal rat kidney cells and competes with EGF for binding to EGF membrane receptors. The 6,000, 10,000 and 20,000 molecular weight polypeptides also resemble TGF in their acid and heat stability, and their requirement for intact disulfide bonds for growth promoting activity. By homologous radioimmunoassay, neither the 6,000 nor 10,000 dalton polypeptide is related to human epidermal growth factor (hEGF). The presence of these TGFs in ample concentrations (approximately 100 ng of EGF equivalents per term placenta for the 10,000 dalton polypeptide) indicates the usefulness of this tissue source for study of human TGFs.     

Dietary regulation of levels of active mRNA coding for amylase and serine protease zymogens in the rat pancreas

Translation products of a reticulocyte lysate reaction, programmed with poly(A)-rich RNAs from the male mouse submaxillary gland, were subjected to affinity chromatography on a tubulin-Sepharose column. Analysis of the bound proteins in sodium dodecylsulfate/polyacrylamide gels revealed two polypeptides of Mr 27 000 and 45 000, that were shown to bind to tubulin in a specific manner. These polypeptides were absent from the translation products coded by poly(A)-rich RNAs from the female mouse. They were eluted from the tubulin-Sepharose resin under conditions similar to those employed for the dissociation of immune complexes. The Mr-27 000 and Mr-45 000 proteins were identified by immunoprecipitation with specific antisera as the precursors of the gamma subunit of the nerve growth factor (NGF) and renin respectively. These two precursors as well as a third, unidentified polypeptide of Mr 38 000, probably unrelated to the beta subunit of NGF, bound also to microtubules. The mature form of renin, purified from the submaximillary gland, also displayed an affinity for the microtubules. In contrast, the mature form of the gamma subunit of NGF did not bind to the microtubules. The possible involvement of the microtubules (tubulin) in the biosynthesis of these two secretory proteins is discussed.     

Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L

Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined.

The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N₂-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides.

     

Translation and characterization of poly(A)-lacking RNA from lupin root nodules

Total polysomal RNA from yellow lupin root nodules was fractionated by double oligo(dT)-cellulose chromatography. Poly(A)-containing and poly(A)-lacking RNA fractions showed considerable messenger activity in wheat germ and rabbit reticulocyte cell-free systems. The sizing of poly(A)-lacking RNA on sucrose-density gradient gives rise to separation of 14S mRNA from 22-24S mRNA species. A single polypeptide with molecular weight of 22,000 was coded for by 14S mRNA, while two polypeptides with an apparent mol. wt. of 90,000 and 87,000 were the main products of 22-24S mRNA fraction. High concentrations of unfractionated poly(A)-lacking RNA as well as the addition of poly(A) led to preferential synthesis of the 22,000 product. Preliminary results suggest the presence of m7GpppX cap structure at 5' terminus of the separated 14S and 22-24S mRNA species. This comes from the competition experiments with m7GMP and m7GTP as well as from the fact that the poly(A)-lacking RNA preparation was susceptible to methylation by methyl-transferase from vaccinia virus (methylated is the 2'-O-nucleotide adjacent to 7-methylguanosine). Digestion by T1 RNAase of methylated poly(A)-lacking RNA produced two short 5'-terminal oligonucleotides 10 and 17 nucleotides in length.     

Androgen induction of beta-glucuronidase translational yield in submaxillary gland of B6.N mice

Androgens induce the synthesis of murine beta-glucuronidase (GUS) 10-fold in the submaxillary gland of B6.PAC-Gusn mice without a concomitant increase in GUS mRNA levels. Since the rate of GUS synthesis per mRNA molecule, or translational yield, is a function of both the efficiency with which the message is translated and the fraction of newly made polypeptides that are incorporated into GUS tetramers, we conclude that androgen induces at least one of these components in the submaxillary gland of B6.N mice. Genetic variation in the submaxillary gland induction response was tested for using six congenic mice strains, each carrying a different haplotype of the Gus gene complex on a C57BL/6J genetic background. The results indicate that the DNA sequences determining androgen responsiveness of the Gus gene in the submaxillary gland are linked to the Gus gene complex and that the DNA sequences determining the submaxillary gland response are distinct from those determining the androgen induction of GUS mRNA in kidney.     

Identification of psbA and psbD gene products,D1 and D2, as reaction centre proteins of photosystem 2

A recent report (Nanba O, Satoh K: Proc. Natl. Acad. Sci. USA 84: 109–112, 1987) described the isolation from spinach of a putative photosystem 2 reaction centre which contained cytochrome b-559 and three other electrophoretically resolvable polypeptide bands, two of which have molecular weights comparable to the D1 and D2 polypeptides. We have used in vivo labelling with radioactive methionine and probed with D1 and D2 monospecific antibodies (raised against synthetically expressed sequences of the psbA and psbD genes) for specific detection of these proteins in a similarly prepared photosystem 2 reaction centre preparation. These techniques identified a 32 000 dalton D1 band, a 30 000 dalton D2 band and a 55 000 dalton D1/D2 aggregate, the latter apparently arising from the detergent treatments employed. Digestions with a lysine-specific protease further confirmed the identification of the lysine-free D1 polypeptide and also confirmed that the D1 molecules in the 55 000 dalton band were in aggregation with other bands and not in self-aggregates. The D1 and D2 polypeptides (including the aggregate) are considerably enriched in the photosystem two reaction centre preparation compared to the other resolved fractions.     

Cell-free translation of adenovirus 2 E1a- and E1b-specific mRNAs and evidence that E1a-related polypeptides are produced from E1a-E1b overlapping mRNA

We have characterized the polypeptides translated in vitro by mRNAs of early region 1 (E1) of human adenovirus (Ad) type 2. Poly (A+) polyribosomal RNA was isolated from early Ad2-infected cells, the viral specific mRNAs were selected by hybridization to Ad2 E1a and E1b DNA, and the mRNAs were translated in vitro using [35S]methionine as a labeled precursor with a rabbit reticulocyte lysate. E1a-selected mRNA was translated to the 45-58-kDa cluster of polypeptides. We show here that E1b-selected mRNA can also be translated to the 45-58-kDa cluster of polypeptides in addition to the major 19-kDa polypeptide. The E1b 58-kDa polypeptide was produced only at a low level unless E1b mRNA is fractionated before translation to enrich for the 58-kDa mRNA. Translation of E1b region-selected mRNAs that have been fractionated by size shows that the 22 S mRNA fraction is translated to at least the 53-58-kDa E1a-related polypeptides as well as to E1b 58- and 19-kDa polypeptides. Our experiments suggest that the 22 S mRNA fraction includes E1a-E1b overlapping mRNA which was translated to E1a-related polypeptides as well as E1b 22 S mRNA. When compared by two-dimensional gel electrophoresis and by tryptic peptide mapping, the cluster of polypeptides translated from E1a-selected mRNA and the cluster translated from E1b-selected mRNA were distinguishable. A possible explanation for this is discussed, based upon splicing sites of the E1a-E1b overlapping mRNA which would result in an amino acid sequence with a COOH-terminal end slightly different from that of E1a polypeptides.     

Isolation of mRNA from membrane-bound polysomes synthesizing Sepia officinalis haemocyanin in Xenopus laevis oocytes

Isolation and translation in Xenopus laevis oocytes of the mRNAs from the nuclear pellet and the cytoplasmic supernatant of the branchial glands from Sepia officinalis, homogenized in the presence of vanadyl-ribonucleoside complexes, revealed that the membrane-bound polysomes for haemocyanin sedimented together with the nuclei. Centrifugation on a 15-50% sucrose gradient of these polysomes, released by treatment with Triton X-100, yielded a major peak of heavy ones. The messenger fraction, isolated from this heavy fraction, directed the synthesis of the large 390 000 Mr haemocyanin polypeptide chain in oocytes. A large mRNA on heavy membrane-bound polysomes thus synthesizes the whole haemocyanin chain of S. officinalis.