Studies on a nonpolysomal ribonucleoprotein coding for myosin heavy chains from chick embryonic muscles. (1/2686)

A messenger ribonucleoprotein (mRNP) particle containing the mRNA coding for the myosin heavy chain (MHC mRNA) has been isolated from the postpolysomal fraction of homogenates of 14-day-old chick embryonic muscles. The mRNP sediments in sucrose gradient as 120 S and has a characteristic buoyant density of 1.415 g/cm3, which corresponds to an RNA:protein ratio of 1:3.8. The RNA isolated from the 120 S particle behaved like authentic MHC mRNA purified from chick embryonic muscles with respect to electrophoretic mobility and ability to program the synthesis of myosin heavy chain in a rabbit reticulocyte lysate system as judged by multi-step co-purification of the in vitro products with chick embryonic leg muscle myosin added as carrier. The RNA obtained from the 120 S particle was as effective as purified MHC mRNA in stimulating the synthesis of the complete myosin heavy chains in rabbit reticulocyte lysate under conditions where non-muscle mRNAs had no such effect. Analysis of the protein moieties of the 120 S particle by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows the presence of seven distinct polypeptides with apparent molecular weights of 44,000, 49,000, 53,000, 81,000, 83,000, and 98,000, whereas typical ribosomal proteins are absent. These results indicate that the 120 S particles are distinct cellular entities unrelated to ribosomes or initiation complexes. The presence of muscle-specific mRNAs as cytoplasmic mRNPs suggests that these particles may be involved in translational control during myogenesis in embryonic muscles.  (+info)

Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes. (2/2686)

Initiation factor IF-E2 was isolated from rabbit reticulocytes and purified 120-fold to near homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and phosphocellulose, and, when suitable, by sucrose density gradient centrifugation. The factor is a complex protein containing three nonidentical polypeptides of molecular weight 57,000, 52,000, and 36,000. It behaves as a complex throughout its purification and during polyacrylamide gel electrophoresis in nondenaturing buffer but its thress components are readily separated by electrophoresis in denaturing buffers. None of its components corresponds to any of the polypeptides of the other initiation factors or to any proteins of ribosomes washed in buffers containing a high salf concentration. A stoichiometric ratio of 1:1:1 was determined for the three polypeptides; based on the assumption of one copy each per complex, the calculated factor molecular weight is 145,000, a value in agreement with the measured value of 160,000. Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase also isolated from rabbit reticulocytes. Neither procedure causes a measurable change in the ability of the factor to form a ternary complex with GTP and the initiator methionyl-tRNA. 5'-Guanylyl-methylenediphosphonate may substitute for GTP, but only at relatively high concentrations. The binding of labeled initiation factor IF-E2 and methionyl-tRNA to the 40 S ribosomal subunit was studied by sucrose density gradient centrifugation. Appreciable binding of the factor is seen only when all three components of the ternary complex are included in the reaction mixture. The binding of either the factor or methionyl-tRNA was not stimulated by the addition of globin messenger RNA and initiation factor IF-E3. It was shown that all three polypeptide components of initiation factor IF-E2 are bound to these nascent initiation complexes.  (+info)

BE-31405, a new antifungal antibiotic produced by Penicillium minioluteum. I. Description of producing organism, fermentation, isolation, physico-chemical and biological properties. (3/2686)

A new antifungal antibiotic, BE-31405, was isolated from the culture broth of a fungal strain, Penicillium minioluteum F31405. BE-31405 was isolated by adsorption on high porous polymer resin (Diaion HP-20), followed by solvent extraction, precipitation and crystallization. BE-31405 showed potent growth inhibitory activity against pathogenic fungal strains such as Candida albicans, Candida glabrata and Cryptococcus neoformans, but did not show cytotoxic activity against mammalian cells such as P388 mouse leukemia. The mechanism studies indicated that BE-31405 inhibited the protein synthesis of C. albicans but not of mammalian cells.  (+info)

unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA. (4/2686)

Initiation of translation of the animal picornavirus RNAs occurs via a mechanism of direct ribosome entry, which requires a segment of the 5' UTR of the RNA, known as the internal ribosome entry site (IRES). In addition, translation of the enterovirus and rhinovirus (HRV) subgroups requires cellular trans-acting factors that are absent from, or limiting in rabbit reticulocytes, but are more abundant in HeLa cell extracts. It has been shown previously that HeLa cells contain two separable activities, each of which independently stimulates HRV IRES-dependent translation when used to supplement reticulocyte lysate; one of these activities was identified as polypyrimidine tract-binding protein (PTB). Here, the purification of the second activity is achieved by use of an RNA-affinity column based on the HRV 5' UTR. It comprises two components: a 38-kD protein (p38), which is a novel member of the GH-WD repeat protein family and has no intrinsic RNA-binding activity; and a 96- to 97-kD protein doublet, which was identified as unr, an RNA-binding protein with five cold-shock domains. Coimmunoprecipitation with antibodies against either protein shows that the two proteins interact with each other, and thus p38 is named unrip (unr-interacting protein). Recombinant unr acts synergistically with recombinant PTB to stimulate translation dependent on the rhinovirus IRES. In contrast, unr did not significantly augment the PTB-dependent stimulation of poliovirus IRES activity.  (+info)

Cell-free immunology: construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire. (5/2686)

A novel cloning-independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single-chain antibody repertoire and express it directly in a cell-free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed.  (+info)

Gbeta5 prevents the RGS7-Galphao interaction through binding to a distinct Ggamma-like domain found in RGS7 and other RGS proteins. (6/2686)

The G protein beta subunit Gbeta5 deviates significantly from the other four members of Gbeta-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gbeta5 in vivo, we have isolated a native Gbeta5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gbeta5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gbeta5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gbeta5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gbeta5 or anti-RGS7 antibodies. The specific Gbeta5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Ggamma subunits. Deletion of this domain prevents the RGS7-Gbeta5 binding, although the interaction with Galpha is retained. Substitution of the Ggamma-like domain of RGS7 with a portion of Ggamma1 changes its binding specificity from Gbeta5 to Gbeta1. The interaction of Gbeta5 with RGS7 blocked the binding of RGS7 to the Galpha subunit Galphao, indicating that Gbeta5 is a specific RGS inhibitor.  (+info)

Human molybdopterin synthase gene: identification of a bicistronic transcript with overlapping reading frames. (7/2686)

A universal molybdenum-containing cofactor (MoCo) is essential for the activity of all human molybdoenzymes, including sulphite oxidase. The free cofactor is highly unstable, and all organisms share a similar biosynthetic pathway. The involved enzymes exhibit homologies, even between bacteria and humans. We have exploited these homologies to isolate a cDNA for the heterodimeric molybdopterin (MPT)-synthase. This enzyme is necessary for the conversion of an unstable precursor into molybdopterin, the organic moiety of MoCo. The corresponding transcript shows a bicistronic structure, encoding the small and large subunits of the MPT-synthase in two different open reading frames (ORFs) that overlap by 77 nucleotides. In various human tissues, only one size of mRNA coinciding with the bicistronic transcript was detected. In vitro translation and mutagenesis experiments demonstrated that each ORF is translated independently, leading to the synthesis of a 10-kDa protein and a 21-kDa protein for the small and large subunits, respectively, and indicated that the 3'-proximal ORF of the bicistronic transcript is translated by leaky scanning.  (+info)

Evidence for and consequences of chronic heme deficiency in Belgrade rat reticulocytes. (8/2686)

The Belgrade rat has a microcytic, hypochromic anemia inherited as an autosomal recessive trait (gene symbol b). Transferrin-dependent iron uptake is defective because of a mutation in Nramp2 (now DMT1, also called DCT1), the protein responsible for endosomal iron efflux. Hence, Belgrade reticulocytes are iron deficient. We show that a chromatographic method is able to measure the amount of 'free' heme in reticulocytes. Most of the 'free' heme is the result of biosynthesis. Succinylacetone, an inhibitor of heme synthesis, decreases the level of 'free' heme and cycloheximide, an inhibitor of globin synthesis, increases the 'free' heme level. In a pulse-chase experiment with 59Fe-transferrin, the 'free' heme pool behaves as an intermediate, with a half-life of just over 2 h. Belgrade reticulocytes contain about 40% as much 'free' heme as do heterozygous or homozygous reticulocytes. This deficiency of 'free' heme slows initiation of translation in Belgrade reticulocytes by increasing the level of an inhibitor of initiation. Thus the Belgrade rat makes a whole animal model available with chronic heme deficiency.  (+info)