Purification and cell-free translation of mRNA coding for a variant specific antigen from Trypanosoma brucei gambiense

Messenger RNA (mRNA) coding for a variant specific surface antigen (VSA) from Trypanosoma brucei gambiense was isolated from total trypanosomal polyribosomes by indirect immunoprecipitation. An IgG fraction of antisera to purified VSA was obtained by ion exchange chromatography and Protein A-agarose affinity chromatography. These antibodies were then subjected to affinity chromatography on a VSA-agarose column to remove non-specific IgG. Polyribosomes from the same antigenic variant of T. b. gambiense were isolated from a cell lysate and those polysomes bearing nascent VSA were bound to the IgG by gentle mixing and the complexes formed were retrieved by precipitation with fixed Staphylococcus aureus cells. The VSA-specific mRNA was separated from these complexes be dissociation of the polysomes, deproteinization, and affinity chromatography on oligo(dT)-cellulose. The mRNA isolated in this way was shown to be undergraded, active in protein synthesis and homogeneous electrophoretically. The products of the cell-free translation of this mRNA were precipitable by specific IgG but not by antiserum to heterologous VSA. The translation product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography. The molecular weight of the mRNA was measured by electron microscopy, agarose and polyacrylamide gel electrophoresis. Enough highly purified mRNA can be isolated in this manner to be used for hybridization analysis of the VSA gene(s).     

Purification of gamma-enolase messenger ribonucleic acid from rat brain by an immunoadsorption method

Messenger ribonucleic acid (mRNA) coding for the brain-specific protein gamma-enolase was isolated by an immunopurification procedure. Rat brain polysomes including nascent polypeptide chains were reacted with specific gamma-enolase antibody. The polysome-antibody complexes were subsequently adsorbed to protein A-Sepharose. After extensive washing, RNA was eluted and applied to an oligo(dT)-cellulose column. Purified mRNA was translated in vitro in a mRNA-dependent rabbit reticulocyte lysate system. The synthesized product was identical to gamma-enolase synthesized by free polysomes from rat brain. Immunoisolated gamma-enolase mRNA was enriched 380-fold compared to total mRNA extracted from free polysomes. This result indicates that low-abundance mRNAs may conveniently be isolated from brain tissue by immunoadsorption of polysomes.     

Control of Type I Collagen Systhesis: Evidence for Pretranslational Coordination of Proα1 (I) and Proα2 (I) Chain Synthesis in Embryonic Chick Bone

The normal type I collagen molecule contains two αl (I) chains and one α2 (I) chain. In embryonic chick calvaria, the two chains are synthesized in a 2: 1 ratio, and total polysomes from this tissue contain twice as much mRNA for proal (I) as for proα2 (I).⁹ To further investigate the mechanism by which synthesis may be coordinated, RNA isolated from various cell fractions of embryonic chick calvaria was translated in a rabbit reticulocyte lysate cell-free system. The procollagen chain products were separated by gel-electrophoresis and densitometrically quantitated from autoradiograms of the gels. Total cellular RNA, total cytoplasmic RNA, and polysomal RNA each directed the synthesis of proal (I) and proa2 (I) in a proportion of 2: 1, whereas no procollagen mRNA activity was found in nonpolysomal cytoplasmic RNA. These results indicate that in the chick bone cells, all compartments contain twice as much proal (I) mRNA as proa2 (I) mRNA, and that virtually all procollagen mRNA in the cytoplasm is poly some-bound. The coordination of procollagen chain synthesis thus presumably occurs at a pretranslational level, through differential rates of formation and/or degradation of the two mRNAs.     

Cell-free translation of carnation latent virus RNA and analysis of virus-specific dsRNA

Carnation latent virus was shown to direct the synthesis of virus-specific polypeptides in both reticulocyte lysate and wheat germ in vitro translation systems. The L-(4,5-³H)-leucine-labeled products ranged in molecular mass from M_r 190 to 33 kD. The 33 kD product, synthesized after only 15 min incubation, was the only major polypeptide that immunoprecipitated with antiserum to CarLV. Coatprotein synthesis does not occur as a result of proteolytic processing, but may arise as a result of translation of a subgenomic RNA species. Subgenomic RNA species were not detected by Northern hybridization of CarLV cDNA to either viral RNA or total nucleic acid from systemically infected plants, although CarLV-specific dsRNA species equivalent to 1.6 and 2.1 kb were detected.     

The importance of tRNA for the in vitro cell-free translation of messenger RNA isolated from the malaria parasite Plasmodium lophurae

Preliminary studies had indicated the inadequacy of the wheat germ and rabbit reticulocyte cell-free translation systems for the in vitro translation of mRNA isolated from Plasmodium lophurae. To identify the factors which are important for the efficient translation of parasite proteins, an homologous system was established using polysomes, the pH 5 fraction, and tRNA prepared from P. lophurae. For comparison, the same components were isolated from the host duck reticulocytes and tested. The effect of each of these factors was evaluated by analysis of the translation products and by comparison with products synthesized in vivo. The results indicated that P. lophurae tRNA had a marked stimulatory effect on the synthesis of parasite proteins while it inhibited the synthesis of host proteins. Duck reticulocyte tRNA could not be used as a substitute for the parasite tRNA. Based on these findings, a commercially available rabbit reticulocyte system was supplemented with P. lophurae tRNA, which markedly increased the efficiency of translation of P. lophurae proteins by this system.     

In vitro translation of adenovirus type 12-specific mRNA complementary to the transforming gene

Specific mRNA complementary to the adenovirus type 12 (AM) transforming gene was selected from Ad12-infected cells by hybridization with EcoRI-C (0–16.5 map units) and AccI-H (0–4.7 map units) DNA restriction fragments, and translated in vitro in [³⁵S]methio-nine-containing reticulocyte lysate. The products were analyzed by two-dimensional gel electrophoresis. The result showed that the EcoRI-C region encoded at least three polypeptides with molecular weights of 40,000 (40K), 38K, and 10K, while the AccI-H region encoded only 40K and 38K polypeptides. According to molecular weights and pI values, it is found that 40K or 38K and 10K polypeptides correspond to T antigen g and f, respectively. The result indicates that T antigen g is coded for by the left end of the transforming gene (0–4.7 map units, region la) and suggests that T antigen f is coded by the rest region (4.7–6.8 map units, region Ib). The size of mRNA for T antigen g was examined by translation with mRNA fractionated by sedimentation through a sucrose gradient and electrophoresis of ³²P-labeled mRNA selected by the Accl-H fragment. The results showed that the size of mRNA for T antigen g was about 13 S. The structure of the 13 S mRNA was examined by nuclease S1 mapping described by Berk and Sharp, and two :kinds of mRNA were detected. These two mRNAs are considered to be translated into two species of T antigen g polypeptides (40K and 38K).     

Isolation and cell-free synthesis of variant surface glycoproteins from Trypanosoma congolense

Two Trypanosoma congolense stocks, 1/148 FLY and TREU 921, were cloned in A/J strain mice immunosuppressed with cyclophosphamide. The cloned populations, AmNat 1.1 and AmNat 3.1, each characterized by a different variant antigen type, were checked for homogeneity by the indirect fluorescent antibody test using 6-day antisera developed in rabbits. The variant surface glycoproteins (VSGs) from both AmNat clones were purified to homogeneity. Electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gradient gels revealed that the apparent Mr values of the two VSGs were 51 700 (AmNat 1.1) and 49 900 (AmNat 3.1). Monospecific antisera prepared in rabbits to each VSG were used to confirm the homogeneity of the clones by the indirect fluorescent antibody test. The VSGs were susceptible to endoglycosidase H digestion, indicating the presence of high-mannose type oligosaccharides in these glycoproteins. The apparent Mr values of the endoglycosidase H-digested VSGs were 48 800 and 46 900 for AmNat 1.1 and 3.1, respectively. Poly(A+)-enriched RNA isolated from each clone was assayed for template activity using a mRNA-dependent rabbit reticulocyte lysate for in vitro protein synthesis. Radioactively labeled polypeptides were initially characterized by SDS-polyacrylamide gradient gel electrophoresis and visualized by fluorography. VSG-specific translation products were immunoprecipitated with IgGs isolated from the homologous monospecific antisera and analyzed on SDS-polyacrylamide gradient gels. The apparent Mr values for the AmNat 1.1 and 3.1 precursor VSGs synthesized in vitro were 39 000 and 43 000, respectively.     

Comparison of the in vitro translation capacity of Taenia crassiceps metacestode mRNA prepared by the phenol and cesium chloride method

Total RNA of T. crassiceps metacestodes harvested from male and female NMRI mice was prepared by both the phenol extraction technique and cesium chloride (CsCl) gradient centrifugation. mRNA was selected by oligo (dT)-cellulose affinity chromatography and used as the template for the in vitro translation of parasite polypeptides in a cell-free rabbit reticulocyte lysate. The template activity of the mRNA obtained after CsCl preparation was clearly higher, as shown by the amount of ³5S-methionine incorporated into the translation products and by fluorographed SDS-PAGE of the synthesized labelled polypeptides. SDS-PAGE fluorographs of antigens encoded by the mRNA prepared by CsCl centrifugation and selected by immunoprecipitation using purified IgG antibodies of T. crassiceps-infected mice (day 80 postinfection) exhibited seven labelled polypeptides of about 65, 46, 45, 42, 34, 29 kDa and a predominant 20-kDa antigen. The latter polypeptide was the only one recognized by the antibodies amongst the in vitro translation products directed by mRNA prepared by the phenol method.     

Messenger RNA from allotype-defined rabbits directing the cell-free synthesis of immunoglobulin heavy and light chains

In vitro synthesis studies were performed utilizing poly A(+)-RNA and lymphocytes from the spleens of rabbits hyperimmunized with Micrococcus luteus or Streptococcus pneumoniae (type III). Poly A(+)-RNA isolated after 4 M guanidinium thiocyanate extraction and oligo(dT)-selection appeared to be undegraded on CH₃HgOH-agarose gel electrophoresis and demonstrated biological activity when translated in a rabbit reticulocyte cell-free system. The electrophoretic patterns of the specifically immunoprecipitated cell-free products were compared with those of Nonidet-P40 extracts (lysates) and secretions (supernatants) from rabbit spleen lymphocyte cultures and serum proteins. Kappa light chains with specific b allotypes, as well as immunoglobulin heavy chains, were identified. The efficient translation of mRNA of defined allotypes was a necessary prerequisite for production of characterized cDNA clones and identification of genomic sequences for rabbit immunoglobulin heavy and light chains.     

Cell-free synthesis of enzymatically active vaccinia virus thymidine kinase

Mouse L cells that lacked thymidine kinase (L_tk^? cells) were infected with wild-type vaccinia virus or with a mutant that was deficient in the viral thymidine kinase. Cytoplasmic messenger RNA preparations were isolated 2 hr after infection and translated in a messenger RNA-dependent reticulocyte lysate. Both messenger RNA preparations directed the efficient synthesis of early vaccinia virus polypeptides. Furthermore, translation of the messenger RNA from cells infected with wild-type virus resulted in a substantial increase in the thymidine kinase activity of the lysate which was prevented by inhibitors of cell-free protein synthesis. Translation of the messenger RNA from cells infected with the viral tk^? mutant, or from uninfected L_tk^? cells, did not increase the thymidine kinase activity of the extract. The enzyme synthesized in vitro was compared with that isolated from vaccinia virus-infected cells. The two enzymes were indistinguishable with respect to their rates of thermal inactivation, their sedimentation coefficients in glycerol gradients, and their relative electrophoretic mobilities in nondenaturing polyacrylamide gels. We conclude that active viral thymidine kinase can be synthesized de novo in a cell-free system directed by early messenger RNA from vaccinia virus-infected cells.     

Polymerase chain reaction-based construction of cDNA libraries from minute amounts of third-stage and fourth- and fifth-stage larvae of Dictyocaulus viviparus

 A strategy is described for the amplification and cloning of cDNA from minute amounts of Dictyocaulus viviparus larvae. Initially, third-stage larvae (L3) were used to establish the procedure. Amplification of cDNA synthesized from approximately 400 ng total RNA from 5,000 L3 generated products that were more than 800 bp in length. The unidirectional cloning of amplified cDNA products led to the construction of a UNI ZAP cDNA library with 1×10⁶ clones. Screening with a homologous oligo(dT)-primed digoxigenin-labeled cDNA probe as well as sequencing of seven randomly picked clones confirmed the successful cloning of lungworm cDNA. Subsequently, approximately 600 ng total RNA was isolated and polymerase chain reaction (PCR) products of up to 2,400 bp were amplitied from 400 fourth- and fifth-stage larvae (L4/L5). Cloning of these products resulted in a L4/L5 cDNA library of D. viviparus consisting of 5×10⁵ recombinant clones. In all, 11 clones were randomly picked and sequenced, all revealing typical mRNA/cDNA characteristics. Comparison of the predicted amino acid sequence of the 5′ end of clone DvL5/7 revealed 100% homology with the actin gene of several other helminths. Received: 3 July 1996 / Accepted: 12 August 1996     

Control of type I collagen synthesis: evidence for pretranslational coordination of pro alpha 1 (I) and pro alpha 2 (I) chain synthesis in embryonic chick bone

The normal type I collagen molecule contains two alpha 1 (I) chains and one alpha 2 (I) chain. In embryonic chick calvaria, the two-chains are synthesized in a 2:1 ratio, and total polysomes from this tissue contain twice as much mRNA for pro alpha 1 (I) as for pro alpha 2 (I). To further investigate the mechanism by which synthesis may be coordinated, RNA isolated from various cell fractions of embryonic chick calvaria was translated in a rabbit reticulocyte lysate cell-free system. The procollagen chain products were separated by gel-electrophoresis and densitometrically quantitated from autoradiograms of the gels. Total cellular RNA, total cytoplasmic RNA, and polysomal RNA each directed the synthesis of pro alpha 1 (I) and pro alpha 2 (I) in a proportion of 2:1, whereas no procollagen mRNA activity was found in nonpolysomal cytoplasmic RNA. These results indicate that in the chick bone cells, all compartments contain twice as much pro alpha 1 (I) mRNA as pro alpha (I) mRNA, and that virtually all procollagen mRNA in the cytoplasm in polysome-bound. The coordination of procollagen chain synthesis thus presumably occurs at a pretranslational level, through differential rates of formation and/or degradation of the two mRNAs.     

Establishment and analysis of a system which allows assembly and disassembly of alphavirus core-like particles under physiological conditions in vitro

Core-like (CL) particles which closely resemble alphavirus cores in size, shape, and relative amount of nucleic acid and protein have been assembled in vitro from Sindbis (SIN) virus core (C) protein and single-stranded nucleic acids in buffer containing 1 M urea [G. Wengler, U. Boege, G. Wengler, H. Bischoff, and K. Wahn (1982)Virology118, 401–410]. We have now analyzed the interaction of SIN virus C protein and nucleic acids in vitro under conditions designed to resemble those present in the cell during core assembly. In buffer containing 100 mM K-acetate, 1.7 mM Mg-acetate, pH 7.4, CL particles are efficiently assembled from all single-stranded nucleic acids analyzed, and even heparin and polyvinylsulfate are incorporated into such particles. A reticulocyte lysate translates SIN virus-specific mRNA into C protein under these ionic conditions. Interactions of C protein with nucleic acids and ribosomes in a reticulocyte lysate have also been analyzed. The following conclusions can be drawn from these analyses: (1) In accordance with earlier findings [N. Glanville and I. Ulmanen (1976) Biochem. Biophys. Res. Commun.71, 393–399] the C protein translated in vitro efficiently binds to ribosomes. (2) Exogenously added C protein binds to the large subunit of the ribosomes in the lysate. (3) CL particles can be assembled in the lysate from exogenous added 42 S genome RNA and exogenous added C protein if both components are present at sufficiently high concentrations. (4) The C protein translated from viral mRNA in the lysate is transferred from the ribosomes into preassembled CL particles containing 42 S RNA in the lysate. (5) If only small amounts of CL particles are added into a lysate these particles disaggregate and core protein molecules are transferred from the particles to the large subunit of the ribosomes. The results on the assembly of CL particles in vitro allow the formulation of some hypotheses concerning the assembly and disassembly of core particles in vivo.     

Biosynthesis of Trypanosoma brucei variant surface glycoproteins — Analysis of carbohydrate heterogeneity and timing of post-translational modifications

The variant surface glycoproteins from two cloned populations of Trypanosoma brucei brucei which were known to migrate as multiple bands on SDS gels have been studied. The heterogeneity present was located in those oligosaccharide side chains the addition of which is tunicamycin-sensitive. The time required for the trypanosome to synthesize and express a variant surface glycoprotein molecule in vitro was found, from pulse-chase and limited trypsinisation experiments, to be approximately 40 min. In the light of these data, pulse-chase experiments on the two antigens known to have heterogeneity in their oligosaccharide side chains demonstrated that the heterogeneity probably arose by two different mechanisms. Pulse-chase experiments on three different clones of trypanosomes have also been used to investigate the timing of cleavage of the carboxyl-terminal extension, known to be encoded on variant surface glycoprotein mRNA. Similar pulse-chase experiments followed by immunoprecipitation using affinity purified antiserum have been used to investigate the addition of the cross-reacting determinant. The timing of both these events has been discussed in relation to the time necessary for the synthesis and expression of the variant surface glycoprotein on the surface of the trypanosome.     

Mycoplasma pulmonis V-1 surface protein variation: occurrence in vivo and association with lung lesions

The V-1 antigen of Mycoplasma pulmonis is exposed on the surface of the mycoplasma and has an immunoblot banding pattern that varies in vitro between and within strains. To determine if V-1 variation occurs in vivo, we infected C3H/HeNCr mice intranasally with 5 × 10⁸ colony-forming units of M. pulmonis strain 5782C. We isolated M. pulmonis clones from the respiratory tracts of mice up to 28 days post-infection, then used anti-V-1 monoclonal antibody P39 to visualize their V-1 immunoblot banding patterns. By the 28th day following infection, 92% of the recovered clones had variant V-1 banding patterns. Additionally, there was a significant correlation between the severity of lung lesions and the percentage of V-1 variant clones recovered from the respiratory tracts of individual mice.These studies prove that V-1 variation does occur in vivo, and suggest that mice with more severe pulmonary lesions tend to have more V-1 variant clones as a percentage of the M. pulmonis population. Thus, variation in the V-1 protein may be a mechanism by which M. pulmonis persists in the in vivo environment, possibly by evasion of host immune surveillance or by alteration of its surface membrane to take better advantage of its environmental niche in the host.     

T cell receptor specificities of Toxoplasma gondii-reactive mouse CD4+ T lymphocytes and Th1 clones

The Toxoplasma gondii-directed CD4⁺ T cell response in chronically infected mice was studied with respect to both T cell receptor diversity and antigen specificities. T cell receptor chains Vβ4, 6, 8, 10, and 14 were predominantly found on toxoplasma-reactive CD4⁺ splenocytes. This repertoire was also detected among T. gondii-specific CD4⁺ T cell clones. Analysis of clonotypic cytokine profiles revealed typical Th1 clones secreting interleukin-2, interferon-γ and tumour necrosis factor activity and Th2 clones producing interleukin-4 and interleukin-10. Five distinct toxoplasma antigens (p26, p40, p55, p58 and p60) were detected in electrophoretically separated toxoplasma lysate by five individual Th1 clones. Parallel testing of CD4⁺ T lymphocytes from infected mice confirmed that these specificities constitute the peak immunogenic fractions of toxoplasma lysate. The expression patterns of two clonotypic, T cell-stimulatory parasite antigens were studied in detail. While p55 was expressed by mouse-virulent and avirulent T. gondii isolates and in both the tachyzoite and bradyzoite stages, p58 was detected only in virulent strains from intraspecies subgroup I. Thus, we describe the heterogeneity of toxoplasmic immunodominant T cell antigens including a 58-kDa group I-restricted molecule which may provide a marker for virulent isolates. Received: 3 February 1997     

Cloning of TPA-inducible early (TIE) genes by differential hybridization using TPA-Nonresponsive variant of mouse 3T3-L1 cells

The tumor promoter 12-O -tetradecanoylphorbol-13-acetate (TPA) induces DNA synthesis in quiescent 3T3-L1 cells but not in its variant VT-1 cells. A gt10 cDNA library was constructed using poly(A)⁺ RNA from 3T3-L1 cells that were stimulated by TPA for 20 min. Radioactive cDNA probes were prepared from mRNAs of TPA-treated 3 T3-L1 and VT-1 cells and used for screening of the 3T3-L1 cDNA library by differential hybridization. Nine of 6000 phage plaques hybridized only to the 3T3-L1 cDNA probe. Analysis of the nucleotide sequence of five of these clones indicated a high degree of homology with human or mouse type I and type III collagen genes. Three other independent clones showed no homology with any known DNA sequences. These isolated clones of TPA-inducible early (TIE) genes may be useful to study the signal transduction pathway of phorbol esters.     

Functional characterization of the early and late mRNAs of simian virus 40.

We have characterized the early SV40 mRNAs by size fractionation on acrylamide gels followed by in vitro translation and immunoprecipitation. The mRNA for the 17K T antigen migrates more slowly than that for the 100K T antigen, with the apparent size difference being 250 nucleotides This finding is consistent with the mRNA for the 17K T antigen corresponding to an almost complete copy of the early region while the mRNA for the 100K T antigen would lack the sequences between 0.59 and 0.54 map unit. By in vitro translation of SV40-specific RNA followed by immunoprecipitation we have been unable to detect the synthesis of any T antigen intermediate in size between the 100K and 17K T antigens. We have demonstrated the synthesis of two proteins from intracellular SV40-specific mRNAs which appear to be identical to the VP2 and VP3 found in the virion on the basis of their mobilities in acrylamide gels and their tryptic peptide maps. Both VP2 and VP3 are labeled with formyl-[³⁵S]methionine when synthesized in the presence of [³⁵S]fMet-tRNA_f^Met. By analyzing partial proteolysis products of such N-terminally labeled proteins we could show that VP2 and VP3 share common C-termini. Therefore, VP3 is initiated and made independently of VP2 and is not derived from VP2 by proteolytic processing. VP2 and VP3 are both made from mRNAs about 19 S in size. The mRNA for VP2 migrates marginally more slowly than the mRNA for VP3 in acrylamide gels.