Short-lived Precursor Research Articles

Biosynthesis, membrane association, and release of N-CAM-120, a phosphatidylinositol-linked form of the neural cell adhesion molecule.

The neural cell adhesion molecule (N-CAM) of rodents comprises three distinct proteins of Mr 180,000, 140,000, and 120,000 (designated N-CAM-180, -140, and -120). They are expressed in different proportions by different tissues and cell types. but the individual contribution of each form to cell adhesion is presently unknown. Previous studies have shown that the two N-CAM species of higher relative molecular mass span the membrane whereas N-CAM-120 lacks a transmembrane domain and can be released from the cell surface by phosphatidylinositol-specific phospholipase C. In this report, we provided evidence that N-CAM-120 contained covalently bound phosphatidylinositol and studied N-CAM-120 from its biosynthesis to its membrane insertion and finally to its release from the cell surface. Evidence was presented showing that the lipid tail of N-CAM-120 contained ethanolamine as is the case for other lipid-linked molecules. The phospholipid anchor was attached to the protein during the first minutes after completion of the polypeptide chain. This process took place in the endoplasmic reticulum as judged from endoglycosidase H digestion experiments. Immediately after a 2-min pulse with [35S]methionine, we detected also a short-lived precursor that had not yet acquired the lipid tail. Pulse-chase studies established that N-CAM-120 was transported to the cell surface from which it was slowly released into the extracellular milieu. The molecules recovered in the incubation medium appeared to have lost all of their bound fatty acid but only around half of the ethanolamine. Upon fractionation of brain tissue, approximately 75% of N-CAM-120 was recovered with a membrane fraction and approximately 25% in a membrane-free supernatant. A small proportion (approximately 6%) was found to be resistant to extraction by non-ionic detergent. A major posttranslational modification of N-CAM is polysialylation. Our results showed that also N-CAM-120 was polysialylated in the young postnatal brain and released in this form from cultured cerebellar cells. The presence of N-CAM in a form that can be released from the cell surface and accumulates in the extracellular fluid suggests a novel mechanism by which N-CAM-mediated adhesion may be modulated.

RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus.

The reverse transcriptase polymerase of the human T-cell lymphotropic virus/lymphadenopathy-associated virus has been cloned into an expression vector and expressed in Escherichia coli. Two polypeptides of 66 and 51 kDa molecular mass are detectable in polymerase-expressing bacterial lysates with human patient sera. They are processed from a short-lived 120-kDa polyprotein precursor equivalent to a region consisting of polymerase, protease, and endonuclease. The 51 kDa protein appears to originate from the 66-kDa molecule; additional processing products are 32- and 15-kDa proteins. The bacterially expressed polymerase is enzymatically active and exhibits the template specificities, ion requirements, and response to inhibitors of the authentic enzyme. It was purified by DEAE-cellulose-, phosphocellulose-, and poly(rC)-agarose column chromatography followed by glycerol density gradient centrifugation. It copurifies with an RNase H activity, suggesting the existence of a virus-coded DNA polymerase-RNase H complex. The purified bacterial enzyme allows a safe large-scale screening for inhibitors of both activities.

Synthesis of Globulins in Maize Embryos

The two major components of the globulin fraction in Zea mays embryos are specified by the Prot gene. Pulse-chase analysis of protein synthesis in cultured, immature embryos indicates that the smaller Prot-specific polypeptide, PROT, is derived from the larger polypeptide, PROT'. These experiments also demonstrate that PROT' is derived from a short-lived precursor polypeptide, prePROT'. The primary Prot-specific translation product, as detected by in vitro translation of immature embryo RNA, is of a lower apparent molecular weight than pre-PROT', suggesting the involvement of co- and/or post-translational modification in the production of prePROT'.

Multiple Growth-Associated Nuclear Proteins Immunoprecipitated by Antisera Raised against Human c-myc Peptide Antigens

Different antisera raised against various regions of the human c-myc protein were used to identify four human c-myc proteins with apparent molecular masses in sodium dodecyl sulfate-polyacrylamide gels ranging from 64 to 68 kilodaltons (phosphoproteins pp64 and pp67 and nonphosphorylated proteins p65 and p68). pp64 and p65 were the major detectable c-myc proteins, and pp67 and p68 were minor but specific components of the immunoprecipitates. The c-myc proteins were all localized in the cell nucleus. Accumulation of [35S]methionine-labeled p65 was observed after pulse-labeling and chase, suggesting that the stable p65 c-myc protein is generated posttranslationally from short-lived precursors. pp64, pp67, and p68 possessed short half-lives and may therefore be precursors of the stable p65. Confirmation of the nuclear localization of the human c-myc proteins was obtained by immunofluorescent staining. The human c-myc proteins were revealed as a pattern of punctate nuclear staining with, particularly for p65, nucleolar enhancement that left an unstained annulus surrounding the nucleolus.

Multiple growth-associated nuclear proteins immunoprecipitated by antisera raised against human c-myc peptide antigens.

Different antisera raised against various regions of the human c-myc protein were used to identify four human c-myc proteins with apparent molecular masses in sodium dodecyl sulfate-polyacrylamide gels ranging from 64 to 68 kilodaltons (phosphoproteins pp64 and pp67 and nonphosphorylated proteins p65 and p68). pp64 and p65 were the major detectable c-myc proteins, and pp67 and p68 were minor but specific components of the immunoprecipitates. The c-myc proteins were all localized in the cell nucleus. Accumulation of [35S]methionine-labeled p65 was observed after pulse-labeling and chase, suggesting that the stable p65 c-myc protein is generated posttranslationally from short-lived precursors. pp64, pp67, and p68 possessed short half-lives and may therefore be precursors of the stable p65. Confirmation of the nuclear localization of the human c-myc proteins was obtained by immunofluorescent staining. The human c-myc proteins were revealed as a pattern of punctate nuclear staining with, particularly for p65, nucleolar enhancement that left an unstained annulus surrounding the nucleolus.

Expression of the cytochrome b-URF6-URF5 region of the mouse mitochondrial genome.

The nature of RNA coded by the only light-strand (L-strand) open-reading frame unidentified reading frame 6 (URF6) was studied by using a variety of single- and double-strand DNA subclones derived from the 3.6-kilobase (kb) cytochrome b (cyt b)-URF5 coding region of the mouse mitochondrial genome. Northern blot experiments using single-strand-specific M13 clones indicate that both the heavy (H) and L strands of this genomic region are symmetrically transcribed and processed into poly(adenylic acid) [poly(A)] RNAs of comparable size. The 1.2- and 2.4-kb RNAs coded by the H strand, putative mRNAs for cyt b and URF5 reading frames, respectively, are derived from a common precursor of 3.6-kb RNA. The L-strand-coded 1.15-kb RNA, on the other hand, is derived from a short-lived precursor of 3.6-kb RNA by a multiple-step processing involving a 2.4-kb intermediate RNA. The S1 nuclease protection experiments using both the 3'- or 5'-end-labeled DNA probes and also affinity-purified 32P-labeled RNA probes indicate that the 1.15-kb RNA maps between the start of the URF6 reading frame (3' end) and a region 590-600 nucleotides to the 5' end of this reading frame. The 1.15-kb RNA thus contains the entire URF6 coding sequence and an about 590-nucleotide-long 3' untranslated region. The molar abundance of the three mRNAs in the steady-state mitochondrial RNA varies markedly. The 1.15-kb URF6 mRNA is only one-tenth the level of 1.2-kb cyt b mRNA, although it is nearly as abundant as the 2.4-kb URF5 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Rb—Sr and Sm—Nd geochronology of lower Proterozoic granite—greenstone terrains in French Guiana, South America

Nine samples of metavolcanic rock from the lower parts of greenstone belts in central French Guiana (the Paramaca series) and 14 granitic samples from the intrusive gneisses (the Degrad Roche and Arawa gneisses) were selected for Sm—Nd and Rb—Sr analysis. The Sm—Nd results from the metavolcanic series (including two tholeiites, five peridotitic komatiites and two andesites) yield an isochron age of 2.11±0.09 (2 σ) Ga with an initial 143Nd 144Nd ratio (I Nd) of 0.51002±9 (2 σ), corresponding to ϵ Nd( T) = + 2.1 ± 1.8. This isochron is interpreted as representing the age of initial volcanism of the Paramaca series. Acid intrusives were dated by the Rb—Sr method. A whole rock Rb—Sr isochron, including data points from both the Degrad Roche and Arawa gneisses, yields an age of 2.00±0.07 (2 σ) Ga with initial 87Sr 86Sr ratio (I Sr value) of 0.7019±4 (2 σ). This result is considered to be the time of emplacement of the orthogneiss protoliths. The positive ε Nd value (+ 2.1 ± 1.8) obtained from the metavolcanic rocks of French Guiana suggests that their mantle sources have evolved in reservoirs slightly depleted in Light Rare Earth Elements (LREE). This result confirms the possible existence of ancient LREE-depleted reservoirs within the lower Proterozoic mantle. Moreover, the high ε Nd( T) value for these rocks excludes any significant crustal contamination during magma genesis. The French Guianese orthogneisses yield a low I Sr value (0.7019±4 (2 σ)) which, together with geochemical considerations, suggests that their granitic protoliths could have originated by partial melting of short-lived crustal precursors of basaltic to granodioritic composition. The present geochronological and isotopic study suggests that the Guiana Shield may represent a major continental accretion event during the lower Proterozoic.

Examination of the Immunological Relationships between Flaviviruses Using Yellow Fever Virus Monoclonal Antibodies

Monoclonal antibodies prepared against vaccine strains of yellow fever (YF) virus were initially characterized by fluorescence microscopy of Vero cells infected with YF virus strain 17D. When similarly tested against representatives of all flavivirus subgroups, the antibodies produced a wide spectrum of reactions ranging from the monospecific to the broadly cross-reactive; at least five antigenic domains in the YF virus envelope glycoprotein were identified. Monoclonal antibodies differentiated between YF virus vaccine strains (17D, 17DD, FNV), wild-type viruses and plaque variants selected from a 17D pool. One isolate from a patient with YF was antigenically similar to the Brazilian vaccine strain 17DD. Several of the antibodies reacting with the YF viral envelope glycoprotein in biological tests identified the 54K envelope glycoprotein; 45K and 26K polypeptides in YF 17D virus-infected cells were also identified by radioimmunoprecipitation and polyacrylamide gel electrophoresis. Neither of these polypeptides was found in uninfected cells. They may represent short-lived precursors of the 54K protein, post-translational cleavage or breakdown products. Other antibodies reacted with a 48K polypeptide in virus-infected cell lysates. This may be the non-structural NV3 protein described for YF virus. Its appearance on the surface of unfixed infected cells, but not on released virions, was demonstrated by fluorescence microscopy.

Nature of steady-state and newly synthesized mitochondrial messenger ribonucleic acids in mouse liver and Ehrlich ascites tumor cells.

The steady-state mitochondrial mRNAs in Ehrlich ascites tumor cells and mouse liver were identified by the Northern blot analysis using nick-translated mtDNA and 32P-labeled cDNA probes. The steady-state mRNA species were compared with the poly(A)-containing RNA synthesized in vitro in isolated mitoplasts. The results show that the isolated mitoplast system can efficiently transcribe almost all of the poly(A)-containing RNAs detected in the steady-state RNA population. It is also seen that the mode of transcription and maturation of mitochondrial mRNAs in different mouse tissues are identical. The results of Northern blot analyses suggest that there may be at least two different modes of mRNA maturation depending upon if the reading frames are interrupted by tRNA cistrons or not. mRNAs for reading frames with adjacent tRNA cistrons downstream appear to be processed from very short-lived precursors. In contrast, mRNAs coded by adjacently located reading frames with no interrupting tRNA genes such as URF3 -cyt ox III and URF5 -cyt b are processed from relatively long-lived precursors. The in vitro pulse-labeling studies also show that almost all of the poly(A)-containing mRNAs are transcribed at nearly identical rates, suggesting that the major regulation of mt gene expression may occur at the level of translation or mRNA decay. The present experiments have also identified a 1.85-kb poly(A)-containing RNA as the putative URF5 mRNA.

Uvomorulin: a nonintegral membrane protein of early mouse embryo.

A monoclonal antibody has allowed the characterization of various forms of uvomorulin, a glycoprotein involved in the process of compaction of mouse morula. In addition to various degradation products, uvomorulin exists as a 120-kilodalton exocellular molecule stable at the cell surface. A short-lived 135-kilodalton precursor of uvomorulin has been detected after 10-min pulse labeling. Uvomorulin-like molecules are found on various tissues at various stages of development of the mouse.

Transport and localization of protein S, a spore coat protein, during fruiting body formation by Myxococcus xanthus.

Protein S, the most abundant soluble protein synthesized by Myxococcus xanthus FB during early fruiting body formation, accumulates in the soluble fraction of developing cells, reaching a peak at about 24 h; at late stages of fruiting body formation, protein S is found on the surface of spores (M. Inouye et al. Proc. Natl. Acad. Sci. U.S.A. 76:209-213, 1979). In this study, the transport and localization of protein S were investigated. Cells were fractionated to give osmotic shock, membrane, cytoplasmic, and spore fractions. The various fractions were then analyzed for protein S. Protein S was first detected in the cytoplasmic fraction at about 3 to 6 h of development. However, transport of protein S through the cytoplasmic membrane was not observed until 15 to 18 h of development. Thus, protein S is unusual among translocated proteins in that it accumulates as a soluble cytoplasmic protein before translocation. Biosynthesis of protein S ceased after 48 h; by 72 h, protein S was only found on the surface of spores. Pulse-chase experiments were performed to determine the transport kinetics of protein S. The results showed that in 24-h developing cells, the transport of protein S across the cytoplasmic membrane was rapid, occurring in less than 2 min. However, transport across the outer membrane was slow, requiring 10 to 15 min. Pulses of 15 s with [35S]methionine failed to reveal any short-lived precursor form in immunoprecipitated material separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing also failed to detect any precursor form of protein S. Thus, protein S appears to be translocated in the absence of a cleaved signal peptide.

Intracellular distribution of low molecular weight RNA species in HeLa cells.

Intracellular distributions of the low molecular weight RNA species of HeLa cells were determined by a nonaqueous method of cell fractionation, in which lyophilized cells were homogenized and centrifuged in anhydrous glycerol. The nonaqueous method was used to avoid artifactual extraction of weakly bound nuclear RNA during cell fractionation. We found that the mature small RNA species K, A, C, and D were almost entirely (greater than 95%) nuclear, and that mature 4S tRNA was partially (5-10%) nuclear. Our results gave higher nuclear content of the mature species K, A, C, and 4S than was shown previously with conventional aqueous cell fractionation. The nonaqueous method also gave higher nuclear proportions of some short-lived precursors to mature small RNAs. We found that approximately one-half of recently synthesized pre-4S RNA and more than one-half of recently synthesized 5S RNA were nuclear, whereas these species had been thought to be cytoplasmic from previous work. The species C' and D', precursors to the stable nuclear species C and D respectively, were found to be partially nuclear, also in contrast to earlier work. The stable cytoplasmic species L (oncornavirus 7S RNA) was found to be mostly nuclear shortly after synthesis.

The four cytoplasmically made subunits of yeast mitochondrial cytochrome c oxidase are synthesized individually and not as a polyprotein.

Subunit-specific antisera prepared against each of the four cytoplasmically made subunits (IV, V, VI, and VII) of yeast mitochondrial cytochrome c oxidase (EC 1.9.3.1) were used to precipitate immunoreactive polypeptides that were synthesized either in vitro, in a cell-free protein-synthesizing system programmed with total yeast mRNA, or in vivo, in intact cells and in spheroplasts, under conditions of pulse labeling, pulse-chase labeling, and continuous labeling. Using N-formyl-[35S]Met-rTNA as the only radioactively labeled component in the cell-free system, we demonstrated (i) that each of the four cytoplasmically made subunits is synthesized as a separate entity and not as part of a polyprotein as was claimed by others; (ii) that subunits IV, V, and VI are synthesized as precursors, larger by 1500-3000 daltons than their mature counterparts; in contrast, subunit VII is not synthesized as a larger precursor. Precursor forms of subunits IV, V, and VI identical to those synthesized in vitro were also detected in vivo by pulse-labeling of spheroplasts. The observed disappearance of these larger forms after a chase is compatible with the notion that they represent short-lived precursors that are rapidly converted to their mature counterparts during or shortly after import into mitochondria. Furthermore, using N-formyl-[35S]Met-tRNA, we provide definitive evidence that two of the cytoplasmically made subunits (beta and gamma) of another oligomeric inner mitochondrial membrane protein (F1-ATPase, EC 3.6.1.3) are not synthesized as part of a polyprotein but as individual precursors.

Type I supernovae come from short-lived stars

view Abstract Citations (141) References (17) Co-Reads Similar Papers Volume Content Graphics Metrics Export Citation NASA/ADS Type I supernovae come from short-lived stars. Oemler, A., Jr. ; Tinsley, B. M. Abstract Statistics for a sample of 178 supernovae in external galaxies are presented which show convincingly that at least most Type I supernovae (SN I), viz., those in spiral and irregular galaxies, are associated with a young stellar population. Relative SN rates in galaxies of different types are discussed which establish an association between SN I and newly formed stars, at least in spirals and irregulars, and SN rates in spirals are derived as a function of color, confirming this association. Problems of associating SN I in elliptical galaxies with ongoing star formation are also considered. It is concluded that SN I have fairly short-lived precursors, with lifetimes between a few tens of millions of years and a few hundred million years. Publication: The Astronomical Journal Pub Date: July 1979 DOI: 10.1086/112502 Bibcode: 1979AJ.....84..985O Keywords: Galactic Structure; Stellar Evolution; Supernovae; Distance; Elliptical Galaxies; Protostars; Spiral Galaxies; Stellar Luminosity; Astrophysics; Galaxies:Supernovae; Supernovae:Statistics full text sources ADS |

In vitro synthesis of a 5S RNA precursor by isolated nuclei of rat liver and HeLa cells

Isolated rat liver nuclei were incubated under appropriate conditions in the presence of 0.5 μg/ml α-amanitin and an RNAase inhibitor prepared from cytosol fraction, together with α- 32P-UTP or α- 32P-CTP and three other nucleoside triphosphates. RNA extracted by an SDS-hot phenol procedure was fractionated with sucrose density gradient centrifugation followed by acrylamide gel electrophoresis. Fingerprint analysis of the in vitro synthesized “5S” RNA, which was slightly larger than mature 5S RNA on gel electrophoresis, showed that it contained all the sequences of mature 5S RNA except for the oligonucleotide at the 3′ end. Instead, it contained two additional spots which were not present in mature 5S RNA. Analysis of the extra spots revealed that they were derived from the 3′ end of the in vitro synthesized “5S” RNA, which was sequenced tentatively as -CUU GAUGCUUUoh (extra sequence underlined). The 5′ end of the product was (p)pGU—. Isolated HeLa cell nuclei synthesized similar sized “5S” RNA under the same conditions. We conclude from these results that in isolated nuclei of these mammalian cells, RNA polymerase III starts transcription of 5S RNA gene at the same site as the 5′ end of mature 5S RNA, proceeds toward the 3′ direction and stops at a site probably 8 nucleotides downstream from the 3′ end of mature 5S RNA. Experiments with a short pulse and with various “chases” have demonstrated the presence of a short-lived precursor 5S RNA which is similar in size and sequence to in vitro “5S” RNA, suggesting that 5S RNA is synthesized in vivo as a longer precursor molecule as demonstrated in this in vitro system, and is rapidly processed into mature 5S RNA.

Half-lives andP n values of delayed-neutron precursors in the mass chains 85?87, 92, 135, 136 and 145

Further results are presented on half-lives and neutron emission probabilities (P n ) of short-lived delayed-neutron precursors in the mass chains 85–87, 92, 135, 136 and 145 obtained with the recoil focussing parabola-type mass separator for unslowed fission products LOHENGRIN installed at the Grenoble high-flux reactor. A newP n -value of (44±14)% is given for the (0.73±0.06)s87As; theP n -values of seven precursor nuclides have been redetermined: (1.9±0.1)s85As with (22±8)%, 0.9s86As with (10.5±2.2)%, (0.35±0.04)s92Br with (21±8)%, (1.6±0.15)s135Sb with (14±1)%, (0.9±0.1)s136Sb with (19±9)%, (17.5±0.2)s136Te with (2.0±1.0)% and 0.6s145Cs with (12.5±3.0)%. The results are compared with the existing data, and the neutron emission probabilities are discussed within a simple semi-empirical picture.

Amino-terminal sequence of phenobarbital-inducible cytochrome P-450 from rabbit liver microsomes: Similarity to hydrophobic amino-terminal segments of preproteins

Summary The amino-terminal sequence of two electrophoretically homogeneous forms of rabbit liver microsomal cytochrome P-450, P-450 LM 2 and P-45 LM 4 , has been examined by automated Edman degradation. Methionine is the amino terminus of P-450 LM 2 , and 17 of the first 20 residues are hydrophobic, including two clusters of five consecutive leucines. The composition and sequence of this region are similar to those of the short-lived hydrophobic amino-terminal precursor segments of certain other proteins, especially myeloma immunoglobulin light chains and pancreatic zymogens. Multiple amino-terminal residues, including methionine, were detected for P-450 LM 4 suggesting the presence of several highly similar forms of P-450 or that partial proteolysis had occurred.

Are globin coding sequences expressed in nuclear RNA of chick embryo brain?

The content of globin coding sequences in nuclear RNA from chicken embryo red blood cells and chicken embryo brain was determined by hybridization with globin cDNA to be 0.27% for embryonic red blood cells and 0.001% for embryo brain, i.e. 0.37% of globin coding sequences relative to embryonic red blood cells. By transfusion of [3H]uridine labelled red blood cells it could be shown that the brain nuclear RNA preparation was contaminated by 0.41% RNA originating from blood cells. As this value is in the same range as the hybridization rate there is no evidence for globin transcripts in brain nuclear RNA. It has also not been possible to detect a short-lived globin mRNA precursor in pulse labelled brain nuclear RNA. In further experiments the RNAs were translated in an Ehrlich ascites cell-free system. No globin synthesis could be observed with brain RNA.

Biogenesis of Silk Fibroin mRNA: An Example of Very Rapid Processing?

This chapter reviews that an affinity column containing a bound synthetic polynucleotide has been used as a tool for the isolation of pulse-labeled fibroin mRNA. Two passages through the column are sufficient to purify the pulse-labeled fibroin gene transcripts from the total hnRNA population. The fibroin mRNA molecules isolated after pulse-labeling in vivo are not demonstrably larger than fibroin mRNA. In fact, for short pulse-labeling times the peak of the gel profile of pulse-labeled mRNA is at a position one to two gel slices smaller than mature mRNA. To investigate the possibility that putative RNA processing cuts may be occurring even before the termination of transcription, experiments have been done under conditions in which processing may be expected to be slower. In vivo pulse-labeling of animals at 11.5°C, followed by affinity chromatography and gel electrophoresis, results in a different size-profile of newly synthesized fibroin mRNA. In this case, the peak of the size distribution is a few gel slices larger than the mature mRNA peak. This larger material, which accumulates at low temperature, could represent a very short-lived precursor species, and may in fact be the “primary” transcript of the silk fibroin gene.

The size of pulse-labeled fibroin messenger RNA

A method has been developed for the isolation of fibroin gene transcripts from total RNA of the silk-worm Bombyx mori. It is based on affinity chromatography using Sephadex-bound polynucleotides capable of selectively hybridizing with fibroin mRNA sequences. In vivo pulse labeling of the posterior silk gland for periods of 10–35 min produces labeled heterogeneous nuclear RNA of high molecular weight (>40S). Fibroin gene transcripts can be selected from the total hnRNA population by two consecutive passages through the affinity column. Analysis of the column-bound material in denaturing polyacrylamide-agarose gels reveals that the size of pulse-labeled fibroin mRNA is essentially the same (within 5%) as that of mature cytoplasmic mRNA. This holds true for pulses as short as 6 min, where even nascent mRNA can be observed. However, a small shoulder of material is present on the heavy side of the pulse-labeled mRNA, which could be indicative of an extremely short-lived precursor species. The purified pulse-labeled mRNA (10 min incorporation) has been further analyzed by chromatography in oligo(dT)-cellulose. The data show that the mRNA is polyadenylated within a few minutes after synthesis.