scholarly journals Heterogeneity of guinea-pig caseins synthesized and sequestered by cell-free protein-synthesizing systems

1981 ◽  
Vol 196 (2) ◽  
pp. 567-574 ◽  
Author(s):  
J C Pascall ◽  
A P Boulton ◽  
D Parker ◽  
L Hall ◽  
R K Craig
Keyword(s):  
Dna Sequences ◽  
Milk Proteins ◽  
Two Dimensions ◽  
Two Dimensional Gel Electrophoresis ◽  
Free System ◽  
Free Protein ◽  
Mrna Species ◽  
Reticulocyte Lysate ◽  
Alpha Lactalbumin

1. Individual mRNA species encoding guinea-pigs caseins A, B and C, and alpha-lactalbumin, were purified by hydridization to recombinant milk-protein plasmid DNA immobilized on diazobenzyloxymethyl-paper or diazobenzyloxymethyl-cellulose. Addition of the purified mRNA species to a reticulocyte-lysate cell-free system, in the presence or absence of a dog pancreas microsomal membrane fraction, established a precursor-product relationship between the primary translation products and those sequestered within microsomal vesicles, as determined by polyacrylamide-gel analysis in one and two dimensions. 2. Three sequestered variants of sequestered casein A were identified, but only single forms of sequestered casein B and alpha-lactalbumin. Sequestered variants of casein C proved to be unexpectedly basic, and did not focus on the pH gradient utilized. 3. Comparative analysis of milk proteins synthesized in the reticulocyte-lysate and wheat-germ cell-free systems by two-dimensional gel electrophoresis demonstrated both quantitative and qualitative differences. In particular, marked but variable heterogeneity was apparent within the primary translation products of casein A and casein B. Pre-casein C did not focus. Limited N-terminal processing of the primary translation products was also evident. These observations are discussed in relation to (i) unscheduled post-translational modifications by cell-free protein-synthesizing systems and (ii) multiplicity of signal sequences. 4. Overall we demonstrate that complex precursor-product relationships between primary translation products and their sequestered variants, programmed in vitro by a mixed mRNA population, may be readily analysed by using individual mRNA sequences purified by hybridization to immobilized cloned complementary-DNA sequences.

scholarly journals Characterization of the translation products of the major mRNA species from rabbit lactating mammary glands and construction of bacterial recombinants containing casein and α-lactalbumin complementary DNA

1982 ◽  
Vol 201 (1) ◽  
pp. 81-90 ◽  
Author(s):  
Y M L Suard ◽  
M Tosi ◽  
J P Kraehenbuhl
Keyword(s):  
Dna Sequences ◽  
Protein A ◽  
Mammary Glands ◽  
Cdna Clones ◽  
Complementary Dna ◽  
Mrna Species ◽  
Polyadenylated Rna ◽  
Alpha Lactalbumin

Total cytoplasmic polyadenylated RNA from lactating rabbit mammary glands was analysed on methylmercury hydroxide-agarose gels. The size of the most abundant mRNA species ranged between 0.5 and 5.0 kb (kilobases), with major bands at 0.55, 0.84, 0.92, 1.18 and 2.4 kb and discrete minor bands of 1.5, 1.7, 3.0 and 3.9 kb. Translation in vitro of total mRNA with [3H]leucine or [35S]methionine as precursor yielded four major bands with apparent Mr values of 16 000, 25 000, 26 000 and 29 000. The four protein bands were identified by immunoprecipitation by using specific antisera as alpha-lactalbumin and x-, kappa- and alpha-caseins, respectively. Labelling with (35S]cysteine followed by immunoprecipitation with anti-transferrin or anti-alpha-lactalbumin sera allowed the identification of two whey proteins. Translated transferrin was resolved as an 80 000-dalton band and alpha-lactalbumin appeared as a 16 000-dalton protein. A library of recombinant plasmids containing cDNA (complementary DNA) sequences representing cytoplasmic polyadenylated RNA was used to isolate clones for the major rabbit caseins and alpha-lactalbumin. A preliminary characterization of these cDNA clones was achieved by colony hybridization with enriched RNA fractions as probes. Positive clones were identified by use of hybrid-promoted translation in vitro and immunoprecipitation of the translation products. The corresponding mRNA species were further identified by hybridizing RNA blots with radioactively labelled cDNA clones. We present the restriction map of alpha-casein and kappa-casein cDNA clones.

scholarly journals Asynchronous globin chain synthesis in rabbit reticulocyte lystates induced by hemin-controlled repressor

Blood ◽  
1978 ◽  
Vol 51 (4) ◽  
pp. 653-658 ◽  
Author(s):  
RS Franco ◽  
JW Hogg ◽  
OJ Martelo
Keyword(s):  
Globin Chain ◽  
Free System ◽  
Globin Chains ◽  
Reticulocyte Lysate ◽  
Chain Imbalance ◽  
Globin Synthesis ◽  
Chain Synthesis ◽  
Globin Chain Synthesis

Abstract To define further the role of hemin-controlled repressor (HCR) in globin synthesis, we studied its effect on the synthesis of individual globin chains in a rabbit reticulocyte lysate cell-free system. In the presence of HCR there was a marked globin chain imbalance, resulting in a lowered alpha/beta ratio. These findings in vitro may have relevance to certain clinical heme deficiency states in which a similar globin chain imbalance has been observed.

scholarly journals Generation of a mutant form of protein synthesis initiation factor eIF-2 lacking the site of phosphorylation by eIF-2 kinases.

1988 ◽  
Vol 8 (2) ◽  
pp. 993-995 ◽  
Author(s):  
V K Pathak ◽  
D Schindler ◽  
J W Hershey
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Covalent Modification ◽  
Site Directed Mutagenesis ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Mutant Form ◽  
Two Dimensional Gel Electrophoresis ◽  
Reticulocyte Lysate

The phosphorylation of the alpha-subunit of initiation factor eIF-2 leads to an inhibition of protein synthesis in mammalian cells. We have performed site-directed mutagenesis on a cDNA encoding the alpha-subunit of human eIF-2 and have replaced the candidate sites of phosphorylation, Ser-48 and Ser-51, with alanines. The cDNAs were expressed in vitro by SP6 polymerase transcription and rabbit reticulocyte lysate translation, and the radiolabeled protein products were analyzed by high-resolution two-dimensional gel electrophoresis. The wild-type and Ser-48 mutant proteins became extensively phosphorylated by eIF-2 kinases present in the reticulocyte lysate, and when additional heme-controlled repressor or double-stranded RNA-activated kinase was present, phosphorylation of the proteins was enhanced. The Ser-51 mutant showed little covalent modification by the endogenous enzymes and showed no increase in the acidic variant with additional eIF-2 kinases, thereby suggesting that Ser-51 is the site of phosphorylation leading to repression of protein synthesis.

In vitroTranslation of Natural mRNAs in a Cell-free System Containing Components from Interferon-treated Chicken Fibroblasts and Factor Preparations from Mouse Ascites Cells or Rabbit Reticulocytes

1975 ◽  
Vol 30 (5-6) ◽  
pp. 398-405 ◽  
Author(s):  
G. Hiller ◽  
G. Viehhauser ◽  
I. Winkler ◽  
D. Pohl ◽  
C. Jungwirth ◽  
...  
Keyword(s):  
Chick Embryo ◽  
In Vitro Translation ◽  
Interferon Treatment ◽  
Mixed Cell ◽  
Free System ◽  
Free Protein ◽  
Cell Free System ◽  
Mouse Ascites ◽  
Rabbit Reticulocytes

Abstract Interferon Mechanism, in vitro Translation, Viral and Cellular mRNA, Chick Embryo Fibroblasts, Vaccinia Infection The effect of interferon has been studied in a mixed cell-free protein synthesizing system. Hemo­ globin (Hb) and Encephalomyocarditis virus (EMC) -RNA can be efficiently translated in vitro in a system containing S-30 lysates or run-off ribosomes from primary chick embryo fibroblasts (CEF) and a postmicrosomal supernatant from mouse ascites cells or a ribosomal-wash preparation from rabbit reticulocytes. Ribosomes prepared from CEF pretreated with high doses of homologous inter­ feron (500 units/ml) were able to translate Hb-RNA in the presence of heterologous factors with the same efficiency as ribosomes prepared from control cells. Translation of EMC-RNA was slightly reduced if ribosomes from interferon-treated cells were used in the mixed cell-free system, confirming previous reports. No inhibitory effect caused by interferon treatment of CEF cells could be detected on in vitro translation of natural mRNAs if the cells had, in addition to interferon treatment, been infected with vaccinia virus. Possible reasons for the different observations made with our cell-free protein synthesizing system from CEF and with cell-free systems prepared from mouse cells are discussed.

Generation of a mutant form of protein synthesis initiation factor eIF-2 lacking the site of phosphorylation by eIF-2 kinases

1988 ◽  
Vol 8 (2) ◽  
pp. 993-995
Author(s):  
V K Pathak ◽  
D Schindler ◽  
J W Hershey
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Covalent Modification ◽  
Site Directed Mutagenesis ◽  
Initiation Factor ◽  
Alpha Subunit ◽  
Mutant Form ◽  
Two Dimensional Gel Electrophoresis ◽  
Reticulocyte Lysate

The phosphorylation of the alpha-subunit of initiation factor eIF-2 leads to an inhibition of protein synthesis in mammalian cells. We have performed site-directed mutagenesis on a cDNA encoding the alpha-subunit of human eIF-2 and have replaced the candidate sites of phosphorylation, Ser-48 and Ser-51, with alanines. The cDNAs were expressed in vitro by SP6 polymerase transcription and rabbit reticulocyte lysate translation, and the radiolabeled protein products were analyzed by high-resolution two-dimensional gel electrophoresis. The wild-type and Ser-48 mutant proteins became extensively phosphorylated by eIF-2 kinases present in the reticulocyte lysate, and when additional heme-controlled repressor or double-stranded RNA-activated kinase was present, phosphorylation of the proteins was enhanced. The Ser-51 mutant showed little covalent modification by the endogenous enzymes and showed no increase in the acidic variant with additional eIF-2 kinases, thereby suggesting that Ser-51 is the site of phosphorylation leading to repression of protein synthesis.

scholarly journals Posttranslational incorporation of contractile proteins into myofibrils in a cell-free system.

1988 ◽  
Vol 107 (2) ◽  
pp. 587-596 ◽  
Author(s):  
M Bouché ◽  
S M Goldfine ◽  
D A Fischman
Keyword(s):  
In Vitro Translation ◽  
Homologous Proteins ◽  
Free System ◽  
Cell Free System ◽  
Reticulocyte Lysate ◽  
Thin Filament Proteins ◽  
A Cell ◽  
The Many ◽  
Kinetic Equilibrium

The incorporation of newly synthesized protein into myofibrils has been examined in a cell-free system. Myofibrils were added to a reticulocyte lysate after the in vitro translation of muscle-specific poly(A)+RNA. Only a small number of the many synthesized proteins were found to associate with the exogenously added myofibrils. These proteins were all identified as sarcomeric components and had subunit mobilities (Mr) of 200, 140, 95, 86, 43, 38, 35, 25, 23, 20, and 18 kD. The association was rapid (t1/2 less than 15 min) and, for most of the proteins, relatively temperature insensitive. Except for a 43-kD polypeptide, tentatively identified as beta-actin, none of the proteins encoded by brain poly(A)+RNA associated with the myofibrils. When filaments made from purified myosin or actin were used as the "capture" substrates, only thick or thin filament proteins, respectively, were incorporated. Incorporation was substantially reduced when cross-linked myosin filaments were used. These results are compatible with a model in which proteins of the sarcomere are in kinetic equilibrium with homologous proteins in a soluble pool.

scholarly journals Translation of preprochymosin in vitro. Evidence for folding of prochymosin to the native conformation

1990 ◽  
Vol 272 (3) ◽  
pp. 659-664 ◽  
Author(s):  
A Sheikh ◽  
R B Freedman
Keyword(s):  
Translation Vector ◽  
Translation Product ◽  
Native Conformation ◽  
T7 Promoter ◽  
Free System ◽  
Cell Free System ◽  
Core Fragment ◽  
Reducing Conditions ◽  
Reticulocyte Lysate

1. The cDNA coding for preprochymosin has been sub-cloned into the transcription/translation vector pGEM-3Z, the T7 promoter used to transcribe the gene and the product expressed in an ‘in vitro’ cell-free system comprising rabbit reticulocyte lysate and dog pancreatic microsomes. 2. Translations in various conditions, and analyses of the translation product in reducing and non-reducing conditions, indicate that oxidizing translation conditions and the cleavage of the N-terminal ‘pre-’ sequence are essential for generation of a disulphide-bonded translation product. 3. The disulphide-bonded translation product was resistant to proteinases, as expected for a translation product segregated within microsomal vesicles; in the presence of detergent to solubilize the membranes, the product was not readily susceptible to proteolysis, and was converted to a proteinase-resistant core fragment. 4. Segregated prochymosin, synthesized in reducing conditions, was completely degraded by proteinases under similar conditions. 5. Proteinase treatment of purified recombinant prochymosin gave rise to a proteinase-resistant fragment of similar Mr, suggesting that the disulphide-bonded product of translation in vitro was correctly folded. 6. The translocated, disulphide-bonded and folded prochymosin could be converted into pseudochymosin at pH 2.0, and addition of chymosin to the activation mixture resulted in increased pseudochymosin production.

Developmental changes in translatable RNA species associated with meiosis and spore formation in Saccharomyces cerevisiae

1984 ◽  
Vol 4 (4) ◽  
pp. 695-702
Author(s):  
E M Weir-Thompson ◽  
I W Dawes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Control ◽  
De Novo ◽  
Sporulation Medium ◽  
Two Dimensional Gel Electrophoresis ◽  
Vegetative Cells ◽  
Control Of Gene Expression ◽  
Mrna Species ◽  
Reticulocyte Lysate ◽  
Translatable Mrna

During Saccharomyces cerevisiae sporulation distinct changes in translatable mRNA species have been detected by two-dimensional gel electrophoresis of the polypeptides produced in a messenger-dependent, cell-free rabbit reticulocyte lysate primed with RNA prepared from a/alpha, a/a, and alpha/alpha isogenic diploids at different stages of sporulation. The availability of functional mRNA increased by about 25% during the first 4 h after transfer of either sporulating (a/alpha), or nonsporulating (a/a and alpha/alpha) diploids to sporulation medium. Thereafter functional mRNA decreased such that in the a/alpha strain after 24 h there was only about 50% of the amount in vegetative cells; a less marked decrease was observed in the a/a and alpha/alpha strains. Of 750 mRNA species detected, 43 underwent alterations only during sporulation in the a/alpha strain, whereas 36 changes were common to all three strains and one mRNA specific to alpha/alpha vegetative cells was detected. Only four of the sporulation-specific changes were due to the de novo appearance of translatable species, and two of these became predominant species of the total population. The majority of the specific changes were due to either permanent or transient increases in the concentration of individual mRNA species; 11 decreases were found. Changes were found at most stages of sporulation, although many occurred in either of two stages, one early (before 2 h) and the other later (between 6 and 8 h) when commitment to meiotic segregation was beginning. The results provide evidence for both quantitative and, to a lesser extent, qualitative transcriptional control of gene expression during sporulation.

scholarly journals Cell type-dependent expression of tubulins in Physarum.

1983 ◽  
Vol 97 (6) ◽  
pp. 1852-1859 ◽  
Author(s):  
T G Burland ◽  
K Gull ◽  
T Schedl ◽  
R S Boston ◽  
W F Dove
Keyword(s):  
Differential Expression ◽  
Dna Sequences ◽  
Self Assembly ◽  
Peptide Mapping ◽  
In Vitro Translation ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Tubulin Genes ◽  
Beta 2

Three alpha-tubulins and two beta-tubulins have been resolved by two-dimensional gel electrophoresis of whole cell lysates of Physarum myxamoebae or plasmodia. Criteria used to identify the tubulins included migration on two-dimensional gels with myxamoebal tubulins purified by self-assembly into microtubules in vitro, peptide mapping with Staphylococcus V8 protease and with chymotrypsin, immunoprecipitation with a monoclonal antibody specific for beta-tubulin, and, finally, hybrid selection of specific mRNA by cloned tubulin DNA sequences, followed by translation in vitro. Differential expression of the Physarum tubulins was observed. The alpha 1- and beta 1-tubulins were detected in both myxamoebae and plasmodia; alpha 2 and beta 2 were detected only in plasmodia, alpha 3 was detected only in the myxamoebal phase, and may be specific to the flagellate. Observation of more tubulin species in plasmodia than in myxamoebae was remarkable; the only microtubules detected in plasmodia are those of the mitotoic spindle, whereas myxamoebae display cytoplasmic, centriolar, flagellar, and mitotic-spindle microtubules. In vitro translation of myxamoebal and plasmodial RNAs indicated that there are distinct mRNAs, and therefore probably separate genes, for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulins. Thus, the different patterns of tubulin expression in myxamoebae and plasmodia reflect differential expression of tubulin genes.

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