Insulin antagonism of glucocorticoid induction of tyrosine aminotransferase in cultured foetal hepatocytes

Polyadenylated RNA from developing Artemia salina cysts was fractionated by centrifugation through a sucrose gradient containing methylmercuric hydroxide (CH3HgOH). Aliquots of each fraction were directly added to a rabbit reticulocyte lysate to program protein synthesis in vitro. The translation products were assayed for eukaryotic elongation factor Tu (eEF-Tu) by immunoprecipitation with an antibody raised in rabbits and purified by affinity chromatography. The immunoprecipitated radioactivity was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Sequences coding for eEF-Tu sediment in the 20-S region of the gradient and form a major component of the poly(A)-containing RNA. The mRNA of the 20-S region, comprising about 10% of the poly(a)-containing RNA fractionated on the gradient, has been translated in vitro and 30% of the translation products represent immunoprecipitable eEF-Tu protein chains with an Mr of 50000.     

Stage- and tissue-specific expression of the genes encoding calliphorin,the major larval serum protein of Calliphora vicina

Summary The stage- and tissue-specific biosynthesis of calliphorin was analysed during the development of the blowfly, Calliphora vicina. Western blot analyses show that the protein is not present in eggs, whereas it can be detected in fat body, brain, imaginai disk, salivary gland and epidermis throughout all postembryonic stages, including the adult one. By Northern analysis a unique 2.6 kb mol.wt. mRNA coding for calliphorin is identified exclusively in the fat body tissue of larvae, pupae and adults. Hybridization experiments of in vivo labelled poly(A)⁺ RNA with filter-bound calliphorin genes indicate that the genes are transcribed until pupariation. However, the translation of the calliphorin mRNA stops at the end of the feeding stage, as shown by [³⁵S]-methionine incorporation.     

Protein metabolism by the salivary glands and other organs of the larva of the blowfly, Calliphora erythrocephala

Protein metabolism in salivary glands, gut, haemolymph, and fat body during the last larval instar of the blowfly, Calliphora erythrocephala, has been investigated. In salivary glands, protein release, protein synthesis, amylase, and pepsin-like protease activity were maximal in 6 day larvae, this being at a time when the larvae had finished feeding. All these functions declined in glands from the rounded-off white puparial stage (R.O.) while acid phosphatase activity rose throughout the third instar to a maximum at the R.O. stage, Glands from 6 and 7 day larvae released protein which on disk gel electrophoresis separated into four minor bands and two major bands one of the latter possessing protease activity.In the gut, pepsin-like protease activity was maximal in 4 day larvae after which it fell rapidly thus following the feeding pattern of the larva in contrast to that in the salivary glands which did not.In vitro experiments showed that protease was released from 6 day glands through the basal membrane of the cells and not via the duct. A pepsin-like protease was also found in the haemolymph and fat body, the activity in the fat body rising rapidly during the latter part of the third instar, a rise which is attributed to the fat body sequestering protease from the haemolymph. Acid phosphatase activity in the fat body was maximal in 5 day larvae indicating that this enzyme was synthesized early in the third instar. It was shown that fat body sequestered ¹⁴C-labelled protein synthesized by and released from the salivary glands, most of the ¹⁴C activity being associated with a 600 g precipitable, acid-phosphatase rich fraction.It is proposed that in late third instar larvae the salivary glands function as glands of internal secretion, releasing protease into the haemolymph, which is then sequestered by the fat body (and perhaps other tissues) and is subsequently used in the lysis of the tissues at the time of metamorphosis.     

北京鸭珠蛋白mRNA的分离纯化及其cDNA的合成

 从人工贫血的北京鸭网织红细胞中直接提取总RNA,经Oligo(dT)-纤维素柱层析分离获得珠蛋白mRNA,并经蔗糖密度梯度离心首次得到了电泳单一条带的北京鸭球蛋白mRNA。从凝胶电泳以及蔗糖密度梯度离心鉴定其沉降系数为9S。在麦胚无细胞体外翻译体系中测定了它们的蛋白翻译活力。鸭珠蛋白mRNA促进了~3H-亮氨酸参入新生蛋白的活力,达到对照组的10倍。所翻译的蛋白产物在SDS-聚丙烯酰胺凝胶上的电泳行为与天然鸭珠蛋白一致。 经Oligo(dT)-纤维素及蔗糖密度梯度离心提纯的珠蛋白mRNA,在AMV反转录酶及DNA聚合酶的作用下,分别合成了单链及双链cDNA。其双链链长,经凝胶电泳分析,约为500碱基对。     

Isolation and characterization of alpha-amylase messenger RNA from bank vole parotid glands. Evidence for two separate messenger RNAs coding for amylase and an amylase-related protein

Bank vole saliva contains two glycogen-precipitable proteins, both of which show affinity for the alpha-amylase inhibitor cycloheptaamylose. One of these proteins, amylase, has a molecular weight of 55,000, judged from dodecylsulphate/acrylamide gel electrophoresis. The other has an apparent molecular weight of 59,000 and has no amylase activity. We report here that tryptic peptide maps as well as amino-acid composition analyses indicate extensive homology between the two proteins. We have also isolated total poly(A)-containing mRNA from amylase-rich bank vole parotid glands. These mRNAs were translated in the presence of [35S]methionine in an mRNA-dependent cell-free translation system from rabbit reticulocyte lysate. The radioactive translation products were examined by dodecylsulphate/polyacrylamide gel electrophoresis. Two major translation products with apparent molecular weights of approximately 56,500 and 60,500, respectively, were further characterized by tryptic peptide analyses. Our data indicate that the 56,500-Mr product is the biosynthetic precursor of amylase, whereas the 60,500-Mr translation product is a precursor of the 59,000-Mr amylase-like protein. Both precursors appear to contain extra peptide material, presumably as amino-terminal 'pre' or 'signal' peptides, in analogy with that found for other precursors of secretory proteins. Thus, amylase and the 59,000-Mr protein, although very similar, are translated from two separate mRNAs. These two messengers sediment in a sucrose gradient at about 17-S, corresponding to lengths of about 1,800 nucleotides.     

Complete cDNA and gene sequence of the developmentally regulated arylphorin of Calliphora vicina and its homology to insect hemolymph proteins and arthropod hemocyanins.

Two cDNA libraries were prepared from poly(A)+ RNA isolated from fat bodies of last instar larvae of the blowfly Calliphora vicina. The libraries were probed with a genomic clone containing the coding sequence for an arylphorin subunit. Two cDNA clones as well as the genomic clone were mapped and their nucleotide sequences were determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 759 amino acid residues. The deduced primary structure of Calliphora arylphorin and hemolymph proteins of other insect species and arthropod hemocyanine show nearly 30% identity. Highly conserved regions could be also identified.     

Interaction of the anterior fat body protein with the hexamerin receptor in the blowfly Calliphora vicina.

In late larvae of the blowfly, Calliphora vicina, arylphorin and LSP-2 proteins, which belong to the class of hexamerins, are selectively taken up by the fat body from the haemolymph. Hexamerin endocytosis is mediated by a specific membrane-bound receptor, the arylphorin-binding protein (ABP). Using the two-hybrid technique, we found that the anterior fat body protein (AFP) interacts with the hexamerin receptor. AFP, a homologue of the mammalian calcium-binding liver protein regucalcin (senescence marker protein-30), exhibits a strong binding affinity for a naturally occurring C-terminal cleavage fragment of the hexamerin receptor precursor (the P30 peptide) and other receptor cleavage products that contain P30. Expression of AFP mRNA and protein is restricted to the anterior part of the fat body tissue and to haemocytes in last-instar larvae. AFP mRNA occurs in all postembryonic developmental stages. Our results suggest that AFP plays a role in the regulation of hexamerin uptake by fat body cells along the anterior-posterior axis.     

家蚕(Bombyx mori)主要血浆蛋白质及其基因表达的研究

本文用SDS-PAGE法观察不同发育阶段蚕血液中主要血浆蛋白质sp、30KP浓度的变化;从不同发育阶段的蚕脂肪体提取RNA和poly(A)~+-RNA,在兔网织红细胞系作体外翻译并检测翻译产物。结果表明,5龄蚕脂肪体mRNA合成蛋白质的速率为初蛹的2倍;5龄及初蛹脂肪体30KP mRNA活性的发育变化与其相应蛋白质在血液中的浓度变化一致;sp-1在5龄幼虫脂肪体内的表达及卵黄原蛋白(Vg)在蚕蛹脂肪体内的表达具有雌特异性,其表达和性特异性大体是在前翻译水平被调节的。     

Purification of messenger RNA coding for glycine methyltransferase from rat liver

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.     

Purification and characterization of the messenger RNA coding for bovine corticotropin/beta-lipotropin precursor.

The mRNA coding for the common precursor of corticotropin and beta-lipotropin has been purified to homogeneity from neurointermediate lobes of bovine pituitaries. The homogeneity of the mRNA preparation is evidenced by analysis of its translation product, electrophoresis on polyacrylamide gel in the presence of formamide and analysis of the kinetics of hybridization with its cDNA. The purification procedure involves the isolation of RNA from membrane-bound polysomes, chromatography on oligo(dT)-cellulose and on poly(U)-Sepharose and sucrose density gradient centrifugation. The mRNA has a molecular weight of approximately 450000, equivalent to approximately 1360 nucleotides in length, and contains a polyadenylate sequence with an average length of 68 nucleotides. The size of the mRNA is sufficiently large to encode the corticotropin/beta-lipotropin precursor.     

Partial purification and characterization of the mRNA for alpha-amylase from barley aleurone layers

The poly(A)-containing mRNA from barley aleurone layers pretreated with gibberellic acid has been purified by phenol-chloroform extraction and repeated oligo[d(pT)]-cellulose chromatography. This RNA has been translated in both the wheat germ and reticulocyte lysate in vitro translation systems with greater than 50% of the synthesized protein being α-amylase. The mRNA for α-amylase has been further purified by dimethylsulfoxide-formamide-sucrose density gradient centrifugation and by gel electrophoresis. By these methods, its molecular weight has been determined to be 580,000.     

Casein and alpha-lactalbumin messenger RNAs during the development of mouse mammary gland. Isolation, partial purification, and translation in a cell-free system

Messenger RNAs for the milk proteins, casein and α-lactalbumin, were isolated and partially purified from lactating mouse mammary glands by oligo(dT)cellulose chromatography followed by sucrose density gradient centrifugation. The translation of poly(A)⁺ mRNA in a wheat germ cell-free system yielded three casein polypeptides and a putative precursor form of α-lactalbumin which were precipitated by specific antibodies and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The casein polypeptides synthesized in vitro had a molecular weight that was no greater than that of the caseins in mouse milk. The presence of individual casein mRNAs coding for these polypeptides was demonstrated by the translation of various fractions of mRNA obtained by sucrose density gradient centrifugation of poly(A)⁺ mRNA. Casein mRNA activity increased about 250-fold between midpregnancy and the 10th–12th days of lactation, amounting to 50–60% of the total mRNA activity in that tissue. A similar study of α-lactalbumin mRNA showed an increase during lactation amounting to 0.2–0.4% of the total mRNA activity, which corresponds to the percentage of α-lactalbumin in total mouse milk protein.     

Neurospora crassa glutamine synthetase. Translation of specific messenger ribonucleic acid in a cell-free system derived from rabbit reticulocytes.

The total reticulocyte lysate cell-free protein-synthesizing system was incubated in the presence of Neurospora crassa RNA. With the aid of an antibody directed against purified N. crassa glutamine synthetase, the synthesis of a specific protein was detected. This protein precipitates with antiglutamine synthetase using both direct and indirect procedures, migrates with the same molecular weight as the monomer of N. crassa glutamine synthetase when subjected to acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and chromatographs as N. crassa glutamine synthetase on anthranilate-bound Sepharose. These data indicate the translation of the mRNA that codes for N. crassa glutamine synthetase. This RNA behaves as poly(A)-containing material when fractionated on oly(U)-Sepha-rose.     

Purification of immunuglobulin light chain messenger RNA by immunoprecipitation from mouse myeloma tumor, MOPC-31C.

Polysomes producing IgGl(kappa) myeloma protein were specifically selected by an immunoprecipitation method, and immunoglobulin light chain mRNA was purified from the precipitated polysomes. The purified mRNA migrated predominantly as a single band and the molecular weight of this mRNA was calculated to be 410.000 by polyacrylamide gel electrophoresis in 98% formamide. A protein possessing a molecular weight of 25,000, which is the size of the light chain precursor, was synthesized as a major product of translation in a wheat germ cell-free system. DNA complementary to the mRNA (cDNA) was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA had an average size of 8.3S as determined by sedimentation through an alkaline sucrose gradient. Using this cDNA, Crt 1/2 values of template RNA and RNA from various preparations were calculated from the results of molecular hybridization. The relative content of the mRNA increased 4,4-fold during the immunoprecipitation of polysomes.     

Membranes des globules lipidiques du laithumain. Preparation,etude morphologique et composition chimiqueHuman milk fat globule membranes. Preparation,morphological studies and chemical composition

Two procedures for the preparation of the human milk fat globule membranes have been used, and morphological and enzymatic comparisons were made. Membranes obtained by the churning procedure were investigated for their protein, lipid, carbohydrate and amino acid contents. Analysis of the proteins by acrylamide gel electrophoresis in sodium dodecyl sulfate were also performed. Several bands are observed: two of these represent glycoproteins. Nucleic acids were analysed on a sucrose density gradient.     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.     

Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial Purification and Characterization of Messenger RNA Coding 14-3-2 Protein from Rat Brain

14-3-2 Protein (neuron-specific enolase) is a neuron-specific protein. Using a reticulocyte lysate cell-free system for translation of 14-3-2 protein mRNA, we have partially purified this mRNA by several procedures, including formamide sucrose density centrifugation, formamide polyacrylamide gel electrophoresis (PAGE) and polyuridylic acid (poly(U))-Sepharose affinity chromatography. Using mRNA obtained by these procedures, we could increase the translation ratio of 14-3-2 protein synthesized/total soluble protein synthesized to 7.31%. The overall purification was 37.8-fold. The size of 14-3-2 protein mRNA appears to be about 19-20S, because translation activity of mRNA obtained by sucrose density gradient centrifugation or formamide PAGE was the most active in this RNA size.     

The purification and polypeptide composition of avian infectious bronchitis virus.

Purification of egg-grown infectious bronchitis virus (IBV) by sucrose density gradient centrifugation alone, or sucrose density gradient centrifugation plus pH 8.0 treatment, concanavalin A precipitation or metrizamide density gradient centrifugation, failed to produce any differences in the virus polypeptide pattern following polyacrylamide gel electrophoresis in the presence of SDS(SDS-PAGE). SDS-PAGE of purified IBV on 7.5% acrylamide gels separated 16 polypeptides which were detectable by staining with Coomassie blue or measurement of radioactivity following electrophoresis of (3H)-leucine labelled IBV. The molecular weights of the polypeptides were within the range 15,000-135,000. The polypeptides of egg and chick kidney (CK) cell-grown IBV were identical in both size and number but quantitative differences were detected. In particular the relative proportion of the major 52,000 molecular weight polypeptide was greatly reduced in IBV grown in CK cells. SDS-PAGE of purified IBV and staining with Schiff's reagent to detect carbohydrate revealed four.bands with molecular weights of 128,000, 86,000, 67,500 and 37,000. The 128,000 band did not correspond to any of the detected polypeptides. Use of 5% acrylamide gels for SDS-PAGE of IBV failed to resolve all the minor polypeptides and only seven bands were detected.