| 1. |
- Edlund, T, et al.
(author)
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Isolation of cDNA sequences coding for a part of human tissue plasminogen activator.
- 1983
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In: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 80:2, s. 349-52
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Journal article (peer-reviewed)abstract
- We have isolated a cDNA sequence coding for a part of human tissue plasminogen activator. mRNA coding for tissue plasminogen activator was partially purified, copied into double-stranded cDNA, and cloned into Escherichia coli. Two sets of partially overlapping oligodeoxynucleotide mixtures corresponding to all possible coding sequences for a known portion of the tissue plasminogen activator gene were prepared. One set was used as a probe to screen cDNA containing bacterial clones and both were used as probes in hybridization against purified plasmid DNA. Of 4,200 bacterial clones examined, 1 carried a plasmid that hybridized to both sets of oligonucleotides. This plasmid contained a 370-base-pair cDNA insert, which was shown by nucleotide sequence analysis to code for the cleavage site region in the one-chain form of the human tissue plasminogen activator.
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| 2. |
- Gustafsson, A, et al.
(author)
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Rapid induction of seven proteins in human lymphocytes by interferon; correlation to natural killer cell activity.
- 1982
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In: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 129:5, s. 1952-9
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Journal article (peer-reviewed)abstract
- The early effects of interferon (IFN) on the synthesis of protein in human nylon wool-nonadherent lymphocytes have been stimulated by use of two-dimensional electrophoresis. IFN-alpha or -beta as well as Escherichia coli-produced IFN-alpha 2 induced the rapid formation of seven proteins (Mr 80, 75, 62, 53, 38, 36, and 33 kD). At least five proteins were expressed within 2 hr of incubation with IFN. The synthesis of the seven proteins seemed to require rapid transcription of new RNA, because actinomycin D markedly inhibited their formation only when added less than 30 min after IFN. A good correlation was found between the ability of actinomycin D to inhibit both the formation of new proteins and the augmentation of natural killer (NK) cell activity. Screening of a panel of 10 hematopoietic and two anchorage-dependent cell lines revealed that p62 and p38 were induced in most cell lines, whereas p80 and p33 were preferentially induced in lymphoid cell lines. Three proteins could not be induced by IFN in any of the 12 cell lines, and thus could represent molecules mediating differentiated functions, possibly involved in NK cell function.
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| 3. |
- Leanderson, T, et al.
(author)
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Interferon-specific effects on protein synthesis in P3HR-1 cells.
- 1982
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In: EMBO Journal. - 0261-4189 .- 1460-2075. ; 1:12, s. 1505-11
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Journal article (peer-reviewed)abstract
- The effect of interferon (IFN) on protein synthesis was studied in the Burkitt's lymphoma cell line P3HR-1 by [35S]methionine labelling of the cells, followed by two-dimensional gel electrophoresis of cell extracts. De novo synthesis of three proteins (mol. wts. 33 000, 62 000, and 98 000, respectively) and alterations in the rate of synthesis for a small number of additional proteins were observed during the first 12 h of treatment, while the rate of overall protein synthesis was unaffected. Treatment of P3HR-1 cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or hydrocortisone (HC), which induce similar changes in cell cycle distribution as does IFN, did not induce comparable changes in the rates of protein synthesis. Thus, the effects were specific for IFN and not induced by the change in cell cycle distribution per se, i.e., accumulation in G0. Treatment of cells with 2'-5' pA core did not mimic the effect of IFN at the translational level. A substrain of P3HR-1 cells, selected for resistance to the anti-proliferative effect of IFN, lacked six proteins found in the wild-type. The 62 000 mol. wt. protein was induced in this substrain as well as in native P3HR-1 cells on addition of IFN. The resistant substrain still developed an anti-viral effect in response to IFN. Thus, it seems as if the anti-proliferative and anti-viral effects of IFN, at least in some cells are mediated by different intracellular molecular mechanisms.
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| 4. |
- Lee, J Y, et al.
(author)
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Insect immunity. Isolation of cDNA clones corresponding to attacins and immune protein P4 from Hyalophora cecropia.
- 1983
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In: EMBO Journal. - 0261-4189 .- 1460-2075. ; 2:4, s. 577-81
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Journal article (peer-reviewed)abstract
- Diapausing pupae of the Cecropia moth (Hyalophora cecropia) respond to an injection of live bacteria by the selective synthesis of certain types of RNA and immune proteins (designated P1-P9). The in vitro translation products of RNA from both injured and infected pupae showed specific patterns with a defined number of extra bands. Some proteins characteristic of the normal RNA were reduced in the immune RNA translation products. Antibody reaction was used to show the selective synthesis of immune proteins P4 and P5 with mRNA from pupae subjected to injury or infection. The protein synthesized in vitro, which cross-reacted with P5 antibodies, is most likely a precursor of the attacins described in the preceding paper. A cDNA clone bank was prepared and two clones were isolated and shown to contain 750 bp corresponding to P4 and 250 bp of attacin information. These clones were used to estimate the sizes of the mRNAs by Northern blotting and to estimate, by RNA/DNA hybridization, the levels of P4 and P5 mRNA. In vivo incorporation of [35S]methionine into attacins and P4 during different conditions was compared with the levels of the corresponding mRNA.
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| 5. |
- Lund, B, et al.
(author)
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Novel cluster of alpha-interferon gene sequences in a placental cosmid DNA library.
- 1984
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In: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 81:8, s. 2435-9
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Journal article (peer-reviewed)abstract
- A human cosmid library was constructed and probed with a human alpha interferon (IFN-alpha) cDNA clone. One clone giving a strong hybridizing signal was isolated and characterized. The cosmid DNA insert represents a section of the human genome containing three regions of IFN-alpha-like sequences. The DNA was characterized with restriction endonuclease mapping, thereby allowing comparison to similar linkage groups reported recently and determination of homologous regions on the known physical map. The three IFN-alpha-like sequences were analyzed by a partial sequence analysis. Mapping and sequence data establish this section as a not-yet-described cluster of IFN-alpha sequences in the human genome; however, a part of the section matches to some degree to a previously described genomic region. The region described here could represent genetic polymorphism or a duplicated segment.
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| 6. |
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| 7. |
- Ny, Tor, et al.
(author)
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Cloning and restriction mapping of the trmA gene coding for transfer ribonucleic acid (5-methyluridine)-methyltransferase in Escherichia coli K-12.
- 1980
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In: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 142:2, s. 371-9
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Journal article (peer-reviewed)abstract
- A hybrid plasmid from the Clarke and Carbon collection has been isolated. This plasmid carries the trmA gene of E. coli, which is necessary for the formation of 5-methyluridine (m5U,ribothymidine) present in all transfer ribonucleic acid (tRNA) chains of the organism so far sequenced. A restriction map of the argCBH-trmA regions is presented. By using cloning in vitro, the trmA gene was located on a 2.9-kilobase pair deoxyribonucleic acid (DNA) fragment. These results and comparison with lambda dargECBH transducing phages established the gene order: argECBH trmA bfe in the 88-min region of the E. coli chromosomal map. Plasmids carrying this 2.9-kilobase pair DNA fragment overproduce the enzyme tRNA(m5U)methyltransferase (EC 2.1.1.35) 20 to 40 times. When this 2.9-kilobase pair chromosomal DNA fragment was expressed in a minicell system, a polypeptide of a molecular weight of 42,000 was synthesized. This polypeptide was tentatively identified as the tRNA(m5U)methyltransferase. These results support the earlier suggestion that the trmA gene is the structural gene for the tRNA(m5U)methyltransferase.
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| 8. |
- Ny, Tor, et al.
(author)
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Growth rate-dependent regulation of transfer ribonucleic acid (5-methyluridine) methyltransferase in Escherichia coli B/r.
- 1980
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In: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 141:1, s. 67-73
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Journal article (peer-reviewed)abstract
- Enzymes catalyzing the transfer of methyl groups from S-adenosyl-l-methionine to a precursor transfer ribonucleic acid (tRNA) and forming 5-methyluridine (m(5)U), 1-methylguanine (m(1)G), or 5-methylaminomethyl-2-thio-uridine (mam(5)s(2)U) are denoted tRNA(m(5)U)-(EC 2.1.1.35), tRNA(m(1)G)-(EC 2.1.1.31), and tRNA(mam(5)s(2)U)methyltransferase. We have studied the regulation of these tRNA biosynthetic enzymes in Escherichia coli under various physiological conditions and in bacterial mutants known to affect the regulation of components of the translational apparatus. Such studies have revealed that tRNA(m(5)U)-methyltransferase increases with the growth rate in the same fashion as stable RNA, whereas the activity of two other tRNA methyltransferases remains constant in relation to the growth rate. Thus, these tRNA biosynthetic enzymes were not coordinately regulated. Regulation of both tRNA(m(5)U)methyltransferase and stable RNA was similar during shift-up and shift-down experiments. This enzyme showed a stringent regulation in relA(+) strain (T. Ny and G. R. Björk, J. Bacteriol. 130:635-641, 1977) but also in two temperature-sensitive mutants, fusA and fusB, known to influence the accumulation of guanosine 5'-diphosphate 3'-diphosphate and RNA synthesis at nonpermissive temperatures. The tRNA(m(5)U)methyltransferase showed a gene dose effect when its structural gene, trmA, was carried on a plasmid or on lambda transducing phages. Although the regulation of tRNA-(m(5)U)methyltransferase was surprisingly coupled to that of stable RNA, this enzyme was expressed at a much lower level.
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| 9. |
- Ny, Tor, et al.
(author)
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Non-coordinate regulation of enzymes involved in transfer RNA metabolism in Escherichia coli.
- 1980
-
In: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 607:2, s. 277-84
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Journal article (peer-reviewed)abstract
- During different steady state growth conditions in Escherichia coli the level of the three tRNA-modifying enzymes, the tRNA(m5Urd)-, tRNA(m1Guo)- and tRNA(mam5s2Urd)methyltransferase and of five aminoacyl-tRNA synthetases, the leucyl-, valyl-, isoleucyl-, arginyl- and threonyl-tRNA-synthetase, has been determined. It is shown that those two classes of tRNA affecting enzymes are not coordinately regulated and that even within these two groups of enzymes the constituents are regulated independently of each other. Furthermore it is demonstrated that none of the aminoacyl-tRNA synthetases and only one of the three tRNA-methyltransferases, the tRNA(m5Urd)methyltransferase, is under control of the relA+-gene.
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| 10. |
- Ny, Tor, et al.
(author)
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The structure of the human tissue-type plasminogen activator gene : correlation of intron and exon structures to functional and structural domains.
- 1984
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In: Proceedings of the National Academy of Sciences of the United States of America. - 0027-8424 .- 1091-6490. ; 81:17, s. 5355-9
-
Journal article (peer-reviewed)abstract
- A genomic clone carrying the human tissue-type plasminogen activator (t-PA) gene was isolated from a cosmid library, and the gene structure was elucidated by restriction mapping, Southern blotting, and DNA sequencing. The cosmid contained all the coding parts of the mRNA, except for the first 58 bases in the 5' end of the mRNA, and had a total length of greater than 20 kilobases. It was separated into at least 14 exons by at least 13 introns, and the exons seemed to code for structural or functional domains. Thus, the signal peptide, the propeptide, and the domains of the heavy chain, including the regions homologous to growth factors, and to the "finger" structure of fibronectin, are all encoded by separate exons. In addition, the two kringle regions of t-PA were both coded for by two exons and were cleaved by introns at identical positions. The region coding for the light chain, comprising the serine protease part of the molecule was split by four introns, revealing a gene organization similar to other serine proteases.
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