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Biomedical subjects

I Ulmanen

Publications and source records attributed to I Ulmanen.

At least 73 records · Page 4Linked to original sources
PubMed1981

A unique cap(m7GpppXm)-dependent influenza virion endonuclease cleaves capped RNAs to generate the primers that initiate viral RNA transcription.

We propose a mechanism for the priming of influenza viral RNA transcription by capped RNAs in which specific 5'-terminal fragments are cleaved from the capped RNAs by a virion-associated endonuclease. These fragments would serve as the actual primers for the initiation of transcription by the initial incorporation by the initial incorporation of a G residue at their 3' end. We show that virions and purified viral cores contain a unique endonuclease that cleaves RNAs containing a 5' methylated cap structure (m7GpppXm) preferentially at purine residues 10 to 14 nucleotides from the cap, generating fragments with 3'-terminal hydroxyl groups. RNAs containing the 5'-terminal structure GpppG could not be cleaved to produce these specific fragments. Consistent with our proposed mechanism, those capped fragments that function as primers could be linked to a G residue in transcriptase reactions containing alpha-32P-GTP as the only ribonucleoside triphosphate. The pattern of G and C incorporation onto these primer fragments suggests that this incorporation is directed by the second and third bases at the 3' end of the virion RNA template, which has the sequence 3' UCG. Primer fragments with a 3'-terminal A residue were used more efficiently than those with a 3'-terminal G residue, indicating a preference for generating an AGC sequence in the viral mRNA complementary to the 3' end of the virion RNA. Cleavage of the RNA primer and initiation of transcription are not necessarily coupled, because a 5' fragment isolated from one reaction could be used as a primer when added to a second reaction. Uncapped ribopolymer inhibitors of viral RNA transcription inhibited the cleavage of capped RNAs.

Endonucleases↗
PubMed1981

Biosynthesis of the major human red cell sialoglycoprotein, glycophorin A. A review.

The human leukemia cell line K562 is erythroid and expresses the major red cell sialoglycoprotein, glycophorin A. With this cell line we have studied the biosynthesis of glycophorin A after pulse-chase labeling with [35S] methionine. Using lectin-Sepharose affinity chromatography and immune precipitation with specific anti-glycophorin A antiserum followed by polyacrylamide slab gel electrophoresis a precursor of glycophorin A was visualized. This had an apparent molecular weight of 37000 and contained an incompleted N-glycosidic oligosaccharide and unfinished O-glycosidic oligosaccharides. After chase for 10 min, the completed glycophorin A with an apparent molecular weight of 39000 was seen and it appeared at the cell surface in about 30 min. Using tunicamycin N-glycosylation was inhibited but not O-glycosylation. The absence of the N-glycosidic oligosaccharide did not affect the migration of the protein to the cell surface but the yield of glycophorin A was diminished. Translation of glycophorin A messenger-RNA was achieved in a rabbit reticulocyte cell-free system. This yielded a non-glycosylated protein with an apparent molecular weight of 19500, which exceeded that of the glycophorin A apoprotein with about 5000. This indicates the presence of a "signal sequence" in the preprotein. When the translation was performed in the presence of microsomal membranes from dog pancreas the glycophorin A apoprotein aws both N-and O-glycosylated and the apparent molecular weight (37000) of the synthesized protein was identical to that of the precursor obtained from cells.

Amino Acid Sequence↗
PubMed1981

Role of two of the influenza virus core P proteins in recognizing cap 1 structures (m7GpppNm) on RNAs and in initiating viral RNA transcription.

Purified influenza viral cores catalyze the entire process of viral RNA transcription, which includes the endonucleolytic cleavage of heterologous RNAs containing cap 1 (m(7)GpppNm) structures to generate capped primers 10-13 nucleotides long, the initiation of transcription via the incorporation of a guanosine residue onto the primers, and elongation of the viral mRNAs [Plotch, S. J., Bouloy, M., Ulmanen, L & Krug, R. M. (1980) Cell 23, 847-858]. To identify which viral core protein (nucleocapsid protein, P1, P2, or P3) recognizes the cap 1 structure on the RNA primer, we irradiated (UV) endonuclease reactions carried out by viral cores in the absence of ribonucleoside triphosphates, with a primer RNA labeled in its cap 1 structure with (32)P. The labeled cap was crosslinked to a protein that had a mobility similar to that of the P3 protein, the smaller of the two basic P proteins, in both one- and two-dimensional gel electrophoresis. This strongly suggests that this crosslinked protein is the viral P3 protein. Competition experiments with unlabeled RNAs containing or lacking a cap 1 structure established that this protein recognizes the cap 1 structure on RNAs. This protein remained associated with the cap throughout the transcription reaction, even after the viral mRNA molecules were elongated. To identify the viral core protein that catalyzes the initiation of transcription via the incorporation of a guanosine residue onto primer fragments, we irradiated transcription reactions carried out by viral cores in the presence of [alpha-(32)P]GTP as the only ribonucleoside triphosphate with an unlabeled primer RNA. A labeled guanosine residue was crosslinked to a protein that had a mobility similar to that of the P1 protein, the larger of the two basic P proteins, in both one-and two-dimensional gel electrophoresis. The transcription reaction conditions required to bring this protein in close association with a labeled guanosine residue so that crosslinking could occur indicated that this association most likely occurred coincident with the guanosine residue's being incorporated onto the primer. These results suggest that the viral P1 protein catalyzes this incorporation and hence initiates transcription.

Orthomyxoviridae↗
PubMed1981

Cell-free synthesis and glycosylation of the major human-red-cell sialoglycoprotein, glycophorin A.

The human erythroid cell line, K562, synthesizes the major red cell sialoglycoprotein, glycophorin A. We have isolated an mRNA fraction which codes for glycophorin A from K562 cells and studied the synthesis of the sialoglycoprotein in a rabbit reticulocyte cell-free system. In the absence of membranes a precursor form of glycophorin A was synthesized. This was identified using specific anti-(glycophorin A) serum. The apparent molecular weight of the carbohydrate-free precursor of glycophorin A was 19 500. This exceeds the molecular weight of the glycophorin A apoprotein by approximately 5000. In the presence of membranes from dog pancreas, the synthesized glycophorin A precursor was N-glycosylated and probably also O-glycosylated. The oligosaccharide chains remained incomplete and the glycoprotein synthesized in vitro corresponded to the glycosylated precursor of glycophorin A obtained in intact cells.

Animals↗
PubMed1981

In vitro translation of Uukuniemi virus-specific RNAs: identification of a nonstructural protein and a precursor to the membrane glycoproteins.

We isolated the virus-specific RNA species from Uukuniemi virus-infected chicken embryo cells and fractionated them by sucrose gradient centrifugation. In addition to three RNA species cosedimenting with the three viral RNA segments L (29S), M (23S), and S (17S), a fourth major RNA species, sedimenting at about 12S (S2), was found early in the infection. Annealing experiments indicated that the cytoplasmic L and M RNA species consisted of both plus and minus strands, with the plus strands in slight excess. Most of the S1 RNA was of negative polarity, whereas S2 was of positive polarity. The S2 RNA specifically annealed to the virion S RNA segment, indicating that it is transcribed from this segment. In vitro translation of the individual RNA species in micrococcal nuclease-treated cell-free reticulocyte extracts showed that an mRNA cosedimenting with the virion M RNA directed the synthesis of a virus-specific 110,000-dalton polypeptide (p110). This polypeptide could be immunoprecipitated with antiserum prepared against purified virions. When translation was carried out in the presence of dog pancreas microsomes, p110 was absent. Instead, an immunoprecipitable polypeptide band, with a molecular weight of about 70,000 and migrating between the virion surface glycoproteins G1 and G2, was observed. It is thus likely that the glycoproteins are synthesized as a precursor (p110), which during translation is cleaved roughly in the middle to yield G1 and G2. The 12S RNA species directed the synthesis of the nucleocapsid protein and a novel polypeptide with an apparent molecular weight of about 30,000. The latter was not precipitated with antivirion serum and was absent from lysates programmed with the corresponding RNA fraction from a mock-infected extract. Since, in addition, it was not found in purified virions and was present in the cytoplasm of infected cells but not in uninfected cells, it probably represents a nonstructural polypeptide.

Animals↗
PubMed1980

Synthesis and processing of Semliki Forest virus-specific nonstructural proteins in vivo and in vitro.

A large short-lived virus-specific nonstructural protein with an apparent molecular weight of about 250000 (nsp250) has been isolated from cells infected with the temperature-sensitive mutants ts-4 and ts-6 of the Semliki Forest virus. nsp250 contained all peptides characteristic of the two previously identified nonstructural precursor proteins, nsp155 and nsp135, as revealed by limited proteolysis with Staphylococcus aureus V8 protease. Thus nsp250 is probably the translational product of the 5' two-thirds of the 42-S RNA genome which codes for the virus-specific nonstructural proteins. A second viral nonstructural precursor protein, nsp220, was also characterized by peptide mapping. This protein contained all the peptides of nsp155, and several but not all of the peptides of nsp135. Some peptides were demonstrated which possibly are derived from ns60, the only nonstructural protein not yet isolated. Small amounts of proteins with identical mobility to nsp250 and nsp220 were synthesized at 38 degrees C in micrococcal-nuclease-treated rabbit reticulocyte lysate in response to virion 42-S RNA from the ts-6 mutant. The product of the wild-type 42-S RNA in vitro contained, in addition to nsp220 and nsp155, polypeptides which comigrated with ns86, ns72 and ns70, indicating processing of the translational product. The authenticity of nsp220, nsp155 and ns70 synthesized in vitro was confirmed by limited proteolysis with V8 protease.

Animals↗
PubMed1979

Comparison of the structural properties of Sindbis and Semliki forest virus nucleocapsids.

The envelope spikes of Sindbis and Semliki Forest virus are arranged in a T = 4 icosahedral surface lattice and, by deduction, it has been suggested that the nucleocapsid proteins are similarly arranged. After treatment of the virions with a non-ionic detergent the released nucleocapsids sediment in sucrose gradients at about 160S and 150S and have densities in CsCl of 1.42 g/ml and 1.425 g/ml, respectively, for Sindbis and Semliki Forest virus. At pH 6.0 Sindbis nucleocapsids do not contract like those of Semliki Forest virus. Nucleocapsids of both viruses are sensitive to the action of ribonuclease but only those of Semliki Forest virus undergo a drastic structural rearrangement due to the treatment. EDTA treatment in hypotonic conditions results in a decrease in the S-value for both particles. Electron micrographs show that the SFV nucleocapsids are partly 'unfolded' while those of Sindbis appear slightly contracted after exposure to EDTA.

Capsid↗
PubMed1979

[Genetic distance of the malarial mosquitoes, Anopheles beklemishevi and Anopheles messeae (Diptera, Culiccidae), and their intraspecific polymorphism].

Allele frequencies at enzyme loci have been studied in Finnish populations of two species of the Anopheles maculipennis complex: A. beklemishevi and A. messeae. A. beklemishevi is spread over central and northern Finland, whereas A. messeae is found in southwestern and central Finland. The allele frequencies of these two species exhibit both similarities and differences. The results indicate that the two species do not interbreed in the nature. The allele frequencies at two loci--Hydroxybutyrate dehydrogenase (Hbdh) and Superoxide dismutase-2 (Su-2)--are almost totally different and adult individuals of the two species can be reliably diagnosed by these allelic differences. The genetic distance, D, between A. beklemishevi and A. messeae is 0.35. This value is compared with corresponding distances between other dipterans studied.

Alleles↗
PubMed1978

Assembly of Semliki Forest virus nucleocapsid: detection of a precursor in infected cells.

The synthesis of Semliki Forest virus nucleocapsid in infected cells was studied by labelling the virus RNAs with 3H-uridine for different periods at various phases of infection. Short pulses (10 to 20 min) revealed the accumulation of 42S RNA in a ribonucleoprotein which sedimented at about 90S (90S RNP) and contained only small amounts of capsid protein. Only after longer pulses was the labelled 42S RNA found in the virus nucleocapsid, suggesting that the 90S RNP may be its precursor. The life time of the 90S RNP was long in the early phases of infection and short in the late phases, reflecting the increased rate of assembly of the nucleocapsid during infection. The 90S RNP was the only 42S RNA containing RNP found in cells infected with temperature sensitive mutants deficient in nucleocapsid formation or wild type infected cells treated with cycloheximide to inhibit nucleocapsid assembly.

Capsid↗
PubMed1977

Transient association of Semliki Forest virus capsid protein with ribosomes.

HeLa cells infected with Semliki Forest virus were exposed to [35S]methionine for 1 min and chased for various periods. The analysis of labeled ribonucleoproteins showed that the viral capsid protein associated first with the large ribosomal subunit in polysomes, from which it was chased to assembling nucleocapsids and to free monosomes.

Capsid↗
PubMed1976

Semliki Forest virus capsid protein associates with the 60S ribosomal subunit in infected cells.

Semlike forest virus capsid protein cosedimented with the large ribosomal subunit at 60S in sucrose gradients after treatment of cytoplasm from infected cells with Triton X-100 and EDTA. In CsCl gradients the capsid protein banded with the subunit at a density of 1.56 to 1.57 g/cm3. Most of the capsid protein could be detached from the 60S structure by treatment with 0.8 M KCl. The ribonucleoprotein of the 26S RNA had a sedimentation value of 53S and a density of 1.50 g/cm3 and could thus be separated from the 60S structure. The data suggest that the capsid protein binds to the large ribosomal subunit, but not to the viral 26S RNA.

Capsid↗
PubMed1975

The role of pet cats in the seroepidemiology of toxoplasmosis.

The occurrence of toxoplasma antibodies was studied in 3 selected groups of humans by means of complement fixation (CF) and indirect immunofluorescence antibody (IFAT) techniques. High occurrence of antibodies was observed among the owners of pedigree cats (CF: 55%; IFAT: 67%), which differed significantly from the values of the control material (35% and 33%, respectively). Having a pet in the family did not increase the proportion of reactors among controls. No significant correlation was observed between the antibody occurrence and the period for which a pet had been kept, nor the raw meat consumption of the cats or humans themselves, or the hunting habits of the animals.

Adolescent↗
PubMed1975

Replication of Semliki Forest virus.

Replication of Semliki Forest virus, a typical alphavirus, takes place in the cytoplasm of many eukaryotic cells. The virus genome, the 42 S RNA, directs the synthesis of at least two RNA-dependent RNA polymerases. By the aid of these enzymes complementary 45 S RNA is synthesized; it serves as a template for the synthesis of positive RNA strands with sedimentation values of 45 S and 26 S. In BHK cells close to 200,000 molecules of each RNA species are produced per cell. Both 26 S and 42 S RNAs are associated with polysomes synthesizing viral structural proteins. The 26 S RNA is a duplication of the nucleotide sequences coding for the virion proteins. These are translated as a polyprotein with the capsid protein at the N-terminal end followed by the envelope proteins E2 and E1. Usually only small amounts of nonstructural proteins are synthesized at the exponential phase of virus growth, indicating that a translational control operates in Semliki Forest virus-infected cells. One of our temperature-sensitive mutants, ts-1, directs, however, the synthesis of two nonstructural proteins with MWs of 78,000 and 86,000 when grown at the nonpermissive temperature. The assembly of the viral nucleocapsid begins by association of the capsid protein with the 42 S RNA, which is still serving as a messenger. In this process a cytoplasmic structure sedimenting at about 65 S is presumably one of the capsid protein donors. The 140 S nucleocapsid buds through the host cell plasma membrane whereby the capsid protein interacts with the envelope proteins creating a specific viral envelope devoid of host proteins. Altogether 5,000 to 20,000 virus particles are released from each cell by the end of the growth cycle, representing about 10% of the 42 S RNA molecules synthesized during the infection.

Animals↗