Antje Huth

Abundant embryonic mRNA in field bean (Vicia faba L.) codes for a new class of seed proteins: cDNA cloning and characterization of the primary translation product.

AbstractcDNA clones have been constructed bearing inserts for a specific mRNA class of high abundance in developing seeds of field bean (Vicia faba L.). Three full-length clones representing transcripts of different genes were sequenced and conceptually translated into a M=30 000 primary gene product. The structural analysis of the derived amino acid sequence revealed distinct domains: (i) a cleavable signal peptide; (ii) a hydrophilic N-terminal stretch possessing two serine clusters; (iii) a valine cluster and a hydrophobic domain in the C-terminal part of the polypeptide. The amino acid sequence of the polypeptide does not show homology with other known proteins.The corresponding mRNA could be isolated using cDNA clones and was efficiently translated in various systems. In a cell-free system the presence of a functional signal peptide was shown, which interacts with the signal recognition particle resulting in a cotranslational translocation across the membrane of the endoplasmic reticulum. If synthesized in Xenopus oocytes the translation product of the mRNA was secreted out of the cell. Homologous mRNA was found to be present also in developing cotyledons of pea (Pisum sativum L.) and french bean (Phaseolus vulgaris L.).

Envelope proteins of Semliki Forest virus synthesized in Xenopus oocytes are transported to the cell surface.

The mRNA coding for the structural proteins of Semliki Forest virus, the 26S RNA, was injected into Xenopus oocytes. Synthesis of the capsid protein and the three envelope glycoproteins E1, E2 and E2 was observed. The proteins, which are normally incorporated into the plasma membrane of infected cells, are transported to the surface of the oocytes. The transport of the membrane proteins takes place in the presence of tunicamycin. The results show that the proteins foreign to the oocyte reach their destination in the plasma membrane. Consequently, the mRNA contains the information for the transport and proteolytic cleavage of the polypeptides.

Regulation of the biosynthesis of insulin in isolated brockmann bodies of the carp (Cyprinus carpio)

The biosynthesis of proinsulin and insulin was followed by the incorporation of radiolabeled leucine and phenylalanine into carp islet protein. Even in the absence of glucose or amino acids in the incubation medium the share of proinsulin in the total protein biosynthesis amounted to ∼ 40% of the incorporated [3H]leucine during 2 hr of incubation at 17°. Glucose increased only the rate of total protein synthesis by approximately 20% but did not stimulate the (pro)insulin synthesis. Furthermore, leucine did not stimulate the [3H]-phenylalanine incorporation into proinsulin. Cyproheptadine, a specific inhibitor of proinsulin synthesis in mammals, had no effect on carp islets. The rate of [3H]leucine incorporation into carp islet protein was strongly dependent on the temperature. The share of proinsulin in the total protein synthesis decreased to about 20% at 4°. The effect of temperature on the enzyme system converting proinsulin to insulin was most dramatic. Whereas the half-time of conversion was between 3 and 4 hr between 25° and 17°, it was 58 hr at 9° and undetectably slow at 4°. The results indicate that, in contrast to mammals, the regulation of insulin biosynthesis in islets of the poikilotherm carp is not determined by the supply of metabolic substrates, but by the environmental temperature.

Biosynthesis of proinsulin of carp (Cyprinus carpio) and characterization and cloning of mRNA.

In spite of the long and intensive research that has brought brilliant successes, a number of basic aspects of the biology of insulin are still unexplained. They include among others the regulation of its biosynthesis and the transport of the insulin molecule into membranes. These are the problems that have occupied us. We have chosen to study the islets of Langerhans of fish (Brockmann bodies), which appeared to be a suitable object for this purpose since they are much bigger than those of mammals and are located apart from the exocrine tissue of the pancreas and therefore can be isolated with little contamination. Fish islets are also of considerable interest for comparative purposes. The present paper presents data on the synthesis of carp preproinsulin and its conversion to proinsulin which support the signal hypothesis proposed by Blobel and Dobberstein. We present first direct evidence for specific membrane receptors of signal peptides. Furthermore, evidence is given for the predominance of preproinsulin mRNA in the total mRNA of carp islets and for a regulatory behavior of the biosynthesis of proinsulin in carp which is different from that in mammalian islets. Finally, we report on the successful cloning of preproinsulin mRNA sequences in a bacterial system.

Internally transposed signal sequence of carp preproinsulin retains its functions with the signal recognition particle

It is shown that the signal sequence of carp preproinsulin is functional with the dog pancreatic signal recognition particle (SRP) both when present at its normal location at the amino‐terminus of the protein or when engineered to an internal location. Inhibition of translation by SRP in the absence of microsomal membranes, reconstitution by SRP of the translocation competence of high‐salt inactivated microsomes and signal peptide cleavage all occur with the signal sequence being preceded by a highly charged peptide segment of 39 amino acid residues (the distance from the amino‐terminus to the cleavage site of the signal peptidase is increased to 56 residues).

SYNTHESIS AND EXPRESSION OF CHIRONOMUS THUMMI BALBIANI RING RNA

ABSTRACT The DNA of Balbiani rings BR1 and BR 2 from Chironomus thummi salivary gland chromosomes is transcribed into large RNA molecules of about 10 − 20 × 10 6 D. This giant RNA is transported into the cytoplasm without any significant size reduction and becomes in all likelihood “in toto” translated on membrane-bound polysomes into secretory proteins. The secretory proteins are composed of two serologically different reacting proteins which are separated each into three main components of about 17,500 D, 35,000 D and 175,000 D by SDS gel electrophoresis. All three components are serologically indistinguishable from each other. Several lines of evidenoe suggest the 17,500 D component to be the basic unit of secretory proteins. On the basis of our experimental data, a model of the struoture and translation of BR RNA is proposed.

BIOSYNTHESIS OF PROINSULIN OF CARP

Publisher Summary This chapter discusses the biosynthesis of proinsulin of carp (cyprinus carpio). Islets of Langerhans of fish—Brockmann bodies—are suitable objects for the study of the biosynthesis of insulin. The islets of fish are much bigger than those of mammals and permit the isolation of relatively large amounts of mRNA. Therefore, it is possible to study the biosynthesis of insulin both on the cellular and subcellular level. The chapter discusses the primary sequence of carp insulin and reviews the possible structure-function relationships. Studies on the regulation of the biosynthesis of proinsulin and insulin in carp islets show that glucose has no effect whereas the temperature plays an essential role. Carp insulin may also serve as a model for the study of the biosynthesis of secretory proteins in general. Recently, the amino acid sequence of the carp insulin B-chain has been confirmed by sequencing on the nucleotide level.