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Biomedical subjects

L Philipson

Publications and source records attributed to L Philipson.

At least 109 records · Page 6Linked to original sources
PubMed1982

An adenovirus glycoprotein binds heavy chains of class I transplantation antigens from man and mouse.

The successful killing of virus-infected cells by cytotoxic T lymphocytes (CTL) is dependent on the recognition of both a viral product and class I antigens of the major histocompatibility complex (MHC) on the infected cell surface. Whether these two entities are found independently on the cell surface and therefore recognized by two different CTL receptors, or whether they are associated together and can therefore be recognized by a single receptor is not known. The association between an adenovirus-encoded glycoprotein expressed on the cell surface early after infection and class I antigens has been investigated and it has been found that antisera against class I antigens can co-precipitate the antigen and the viral glycoprotein from an adenovirus-transformed cell line from the Hooded-Lister rat strain. We show here by in vitro affinity chromatography and in vivo immunoprecipitation that the viral glycoprotein specifically binds to the heavy chain of class I antigens in both man and mouse.

Adenoviridae↗
PubMed1982

Detection of a cellular polypeptide associated with adenovirus-coded VA RNA using in vitro labeling of proteins cross-linked to RNA.

Ultraviolet light induced RNA-protein cross-linking for identification of polypeptides interacting with RNA in intact cells (Wagenmakers et al. 1980), is limited by the intensity of the label in the proteins or in residual nucleotides remaining attached to the proteins after RNase treatment of the RNA-protein complexes. Here we report a method, where th cross-linked RNA-protein complexes are treated with RNase T1 and the T1-oligonucleotides covalently linked to the proteins are labeled in the 5' terminus using gamma-32P-ATP and T4 polynucleotide kinase. The cross-linked proteins can then readily be identified owing to the incorporated 32P label. As examples, proteins associated with polyadenylated mRNA, hnRNA and adenoviral VA RNA were identified. A protein with a molecular weight of approximately 50,000 is found associated with adenovirus-coded VA RNA. This was confirmed by binding assays, in which labeled VAI RNA is incubated with proteins from uninfected and adenovirus infected HeLa cells immobilized on nitrocellulose sheets.

Adenoviruses, Human↗
PubMed1982

Non-random localization of ribonucleoprotein (RNP) structures within an adenovirus mRNA precursor.

Heterogeneous nuclear protein complexes (hnRNP) containing the precursor RNA from the adenovirus early region 2 were analysed to determine the specificity of protein-RNA interaction. RNA precursor sequences were present in isolated hnRNP complexes and endogenous 30S particles. At least 20-40 bases long fragments were protected when RNase A was used to remove unprotected RNA sequences in hnRNA complexes. Similarly around 40 bases of RNA were protected in 30S particles. These sequences represent discrete regions of the adenovirus genome. Especially sequences complementary to the EcoRI-F fragment encoding the first leader and the major intron for the DNA binding protein (DBP) RNA precursor, were analysed in detail. Tentatively, sequences resistant to RNase A were located in the middle of the intron and at the splice-donor junction of the first leader of the DBP precursor RNA. The same sequences were identified irrespective whether hnRNP complexes or 30S particles were used suggesting that 30S particles originate from hnRNP complexes. A 38.000 dalton protein appears to be in direct contact with RNA sequences complementary to the EcoRI-F fragment.

Adenoviruses, Human↗
PubMed1982

A candidate gene for human U1 RNA.

Clones containing sequences complementary to the small nuclear RNA U1 were isolated from the human DNA library of Lawn et al. (1978). Three clones were studied by hybridization and restriction enzyme cleavage. The results showed that the inserts in all three clones were different and that each clone contains one single copy of a sequence which hybridizes to U1 RNA. The results revealed moreover that only one of the three clones contains all the cleavage sites which can be predicted from the known sequence of human U1 RNA, suggesting that the three clones comprise one candidate U1 gene and two pseudogenes. A fragment from the recombinant with the candidate U1 gene was subcloned in the pPR322 plasmid and part of its sequence was determined. The results showed that the subclone contains a sequence which matches that of the human U1 RNA perfectly. The sequence "TATAT" which often is found adjacent to RNA polymerase II start sites, was identified 33-37 base pairs upstream from the beginning of the U1 sequence. Two ten base pairs long, nearly perfect, direct repeats were also identified in the vicinity of the U1 sequence and an imperfect inverted repeat follows immediately after the U1 gene.

Base Sequence↗
PubMed1982

Leader arrangement in the adenovirus fiber mRNA.

Four oligonucleotides, complementary to the adenovirus 2 (ad2) fiber mRNA were chemically synthesized and used as primers to study the 5' end of that messenger. The oligonucleotides which were 8, 10, 12, and 14 nucleotides long had a common 3'-terminal sequence, TAC, complementary to the initiating codon of the fiber mRNA. The 5'-32P-labeled oligonucleotides were hybridized to polyadenylated mRNA from ad2-infected cells and the primer was extended, using reverse transcriptase. When the resulting products were analyzed by polyacrylamide gel electrophoresis multiple distinct bands could be identified by autoradiography. The extension products were characterized by hybridization and sequence analysis. The most prominent band contained the well-known tripartite lead which is attached to many different late adenovirus mRNAs. In addition, four differently spliced leaders were characterized which, besides the three segments of the tripartite leader, contain one or two auxilliary segments. A kinetic analysis of the different leaders during lytic adenovirus infection shows no temporal variation.

Adenoviridae↗
PubMed1982

A novel mRNA and a low molecular weight polypeptide encoded in the transforming region of adenovirus DNA.

Immunoprecipitation was used to identify adenovirus type 2 (ad2) tumor antigens synthesized in vivo. The antisera, prepared from tumor-bearing animals, reacted with a wide spectrum of ad2 early proteins including a 11 000-dalton (11 K) polypeptide. The gene for this polypeptide was mapped to the transforming region of the viral genome by hybridization selection followed by in vitro translation and immunoprecipitation. Hybrid arrest translation revealed that the 11 K RNA was transcribed from the leftward reading strand (1-strand) in contrast to other mRNAs from this region. Sucrose gradient analysis of the selected 11 K mRNA revealed that the size of the mRNA was 20S corresponding to approximately 2000 nucleotides. Novel 1-strand transcripts of this length from the transforming region were identified by S1 endonuclease analysis. Taken together, these results suggest that both strands of the transforming region of ad2 DNA are actively transcribed into functional mRNA early after viral infection.

Adenoviruses, Human↗
PubMed1982

Small nuclear RNAs are encoded in the nontranscribed region of ribosomal spacer DNA.

The structure of in vitro synthesized mouse small nuclear RNA transcribed by RNA polymerase I (snPI RNA) was studied by T1 RNase digestion pattern analysis. The patterns of four different snPI RNA species were different from those of the U1 and U2 RNA species. In addition, the four different snPI RNA species, ranging from 130 to 240 nucleotides in length, yielded almost identical patterns. The snPI RNA molecules hybridized to cloned mouse ribosomal DNA containing the nontranscribed spacer DNA and 45S ribosomal precursor RNA molecules did not compete with this hybridization. Southern blot analysis of fragments from the ribosomal DNA confirmed that snPI RNA species exclusively hybridized to sequences corresponding to the so-called nontranscribed ribosomal spacer region.

Animals↗
PubMed1982

Purification of a native membrane-associated adenovirus tumor antigen.

A 15,000-dalton protein was purified from HeLa cells infected with adenovirus type 2. Proteins solubilized from a membrane fraction of lytically infected cells was used as the starting material for purification. Subsequent purification steps involved lentil-lectin, phosphocellulose, hydroxyapatite, DEAE-cellulose, and aminohexyl-Sepharose chromatographies. A monospecific antiserum, raised against the purified protein, immunoprecipitated a 15,000-dalton protein encoded in early-region E1B (E1B/15K protein) of the adenovirus type 2 DNA. Tryptic finger print analysis revealed that the purified protein was identical to the E1B/15K protein encoded in the transforming part of the viral genome. The antiserum immunoprecipitated the E1B/15K protein from a variety of viral transformed cell lines isolated from humans, rats, or hamsters. The E1B/15K protein was associated with the membrane fraction of both lytically and virus-transformed cell lines and could only be released by detergent treatment. Furthermore, a 11,000- to 12,000-dalton protein that could be precipitated with the anti-E1B/15K serum was recovered from membranes treated with trypsin or proteinase K, suggesting that a major part of the E1B/15K protein is protected in membrane vesicles. Translation of early viral mRNA in a cell-free system, supplemented with rough microsomes, showed that this protein was associated with the membrane fraction also in vitro.

Adenoviruses, Human↗
PubMed1982

Regulation of glycosaminoglycan synthesis by thyroid hormone in vitro.

Human skin fibroblasts synthesize and accumulate glycosaminoglycans (GAG). Recently, we reported that fibroblasts incubated in thyroid hormone-deficient media accumulate more GAG than do cultures incubated in the same media enriched with 0.1 muM triiodothyronine (T(3)) (1981. Endocrinology. 108: 2397). The current study characterizes that enhanced accumulation. Confluent cultures were maintained in thyroid hormone-deficient media without or with added T(3), labeled with [(3)H]acetate and analyzed for total [(3)H]GAG and [(3)H]hyaluronic acid content. Addition of T(3) to thyroid hormone-depleted media consistently inhibited the incorporation of [(3)H]acetate into GAG by 28-60% in fibroblast cultures from four different normal human donors. Maximal inhibitory effect was observed within 3 d after hormone addition at concentrations > 1 nM. 73% of the maximal inhibitory effect was observed in the presence of physiologic concentrations of T(3) (0.16 nM total T(3) or 1.4 pM free T(3)). The following observations indicated that T(3) inhibition of [(3)H]GAG accumulation is most likely due to a decrease in GAG synthesis rather than to changes in the acetate pool or GAG degradation: (a) Addition of 0, 100, 500, and 2,500 muM unlabeled acetate progressively decreased [(3)H]acetate incorporation into GAG, up to 80%, without altering the further inhibitory effect of T(3) (35-40%); (b). A similar effect of T(3) on GAG (32% inhibition) was observed using [(3)H]glucosamine as substrate; (c) T(3) decreased hyaluronate synthetase activity by 32%; and (d) There was no effect of T(3) on GAG degradation in a pulse-chase experiment. The effect of T(3) on [(3)H]GAG accumulation appears to be quite specific, since the hormone had no effect on the incorporation of [(3)H]leucine into trichloroacetic acid-precipitable material.Thus, thyroid hormone inhibits GAG accumulation in a dose-, time-dependent, and reversible manner. This inhibition is apparently due to specific effects on the rate of macromolecular synthesis.

Acetates↗
PubMed1981

Human DNA sequences complementary to the small nuclear RNA U2.

Clones containing sequences complementary to the small nuclear RNA U2 were isolated from a human DNA library (1). Three clones, designated U2/4, U2/6 and U2/7 were purified and characterized by restriction enzyme cleavage, hybridization and heteroduplex analysis. Hybridization showed that the three clones each contained one single region which is complementary to U2 RNA. Restriction enzyme cleavage revealed furthermore that the inserted fragments in the three recombinants are different. Heteroduplex analysis identified a 240-380 bp long duplex region in each heteroduplex which includes sequences complementary to U2 RNA. Heteroduplexes between clones U2/4 and U2/7 as well as between U2/4 and U2/6 revealed two additional approximately 200 bp long homologies. The remainder of the inserts were found to lack measurable sequence homology. Two fragments from clone U2/4 were subcloned in the pBR322 vector and the subclones were used to determine the nucleotide sequence of a region in clone U2/4 which is complementary to U2 RNA. A comparison between the established sequence and the sequence for rat U2 RNA (2) reveals several discrepancies.

Base Sequence↗
PubMed1981

The conformation of adenovirus VAI-RNA in solution.

The secondary structure of an adenovirus associated low molecular weight RNA (VAI-RNA) has been studied by partial digestion with T1-RNase and S1-endonuclease followed by T1-fingerprint analysis. The empirical secondary structure has been compared with two computer generated models based on minimal free energy of the structure. The results suggest that VAI-RNA in solution has a compact structure with a free energy of around -60 kcal with two stems and four bulge regions. The implication of this structure for the function of VAI-RNA is discussed.

Adenoviridae↗
PubMed1981

The adenovirus hexon protein. The primary structure of the polypeptide and its correlation with the hexon gene.

The primary structure of the adenovirus hexon polypeptide has been determined by amino acid sequence studies of peptides from all regions of the molecule combined with sequence analysis of selected areas of its gene. The sequence presented contains 966 unique amino acid residues. Overlapping peptides recovered from CNBr cleavage and from digestions with proteolytic enzymes were analyzed, as well as DNA segments around sites for restriction endonucleases in the hexon gene. The primary structure is in good agreement with the total composition of the protein, with the compositions of individual CNBr fragments, and with known locations of restriction enzyme cleavage sites in the gene. Distinct regions of internal homology do not occur in the structure. The entire hexon polypeptide is encoded by a contiguous DNA sequence without intervening sequences.

Adenoviruses, Human↗
PubMed1981

Adenovirus early gene products may control viral mRNA accumulation and translation in vivo.

The mechanisms controlling early adenovirus gene expression in vivo have been studied using inhibitors of protein synthesis. When inhibitors were added shortly before or at the onset of infection, viral mRNA from all early regions was transcribed, spliced and accumulated over a 7 hr period. After longer pretreatment, accumulation of several early mRNAs were suppressed. Addition of inhibitors 1 hr after infection enhanced the accumulation of viral mRNA in the cytoplasm. Translation of early mRNA selected on adenovirus DNA in a cell-free system reflected the amount of viral mRNA present. A viral coded product may therefore control accumulation of viral mRNA. A different pattern emerged when inhibitors of protein synthesis were removed at 5 hr postinfection and cells were removed at 5 hr postinfection and cells were pulse-labeled in vivo. If inhibitors were introduced at or before infection, early viral proteins were synthesized only after a lag of 1-3 hr. However, if treatment was introduced 1 hr postinfection, reversion of the protein synthesis block was instantaneous. It appears that protein synthesis inhibitors reveal an in vivo translational block for viral mRNA. This block could be overcome by preinfection with a related virus. Furthermore, no block was observed in a virus-transformed human embryonic kidney cell line (293) which expresses early region 1 of the viral genome. Viral gene product(s) encoded in early region 1 may control translation of early adenovirus messenger RNA in vivo.

Adenoviruses, Human↗