Five hundredfold overproduction of DNA ligase after induction of a hybrid lambda lysogen constructed in vitro.

A lambda vector that contains the gene for Escherichia coli DNA ligase (lambdagt4-lop-11 lig+) has been modified to achieve overproduction of this enzyme. The third Eco RI site in the lambda chromosome has been altered by mutation, and the left-hand Eco RI fragment has been shortened. The new vector, lambdagt4-lop-11 lig+, forms a stable lysogen which, upon induction, produces a 100-fold increase in DNA ligase activity. Introduction of a phage mutation (S7) that prevents cell lysis results in an even greater increase (500-fold).

Induction of DNA ligase during stimulation of DNA synthesis in intact rat liver by a dietary manipulation.

After a nutritional shift from a protein-free to a diet containing 50% casein, the activity of DNA ligase increases in intact rat liver in correlation with the induction of hepatic DNA replication. The treated rat liver as well as control rat liver contains a single species of DNA ligase having a sedimentation coefficient of about 5.5 S. The administration of cycloheximide in vivo completely inhibits the increase in DNA ligase activity and in DNA synthesis, indicating that DNA ligase is induced in the hepatic cells replicating DNA. In contrast to DNA ligase, DNA kinase is unchanged in the activity level by the dietary manipulation.

Computer-assisted analysis demonstrates that polypeptides induced by natural and recombinant human interferon-alpha are the same and that some have related primary structures.

The biological effects on diploid and trisomy 21 human fibroblasts of pure human interferon IFLrA, a single IFN-alpha species produced from cloned DNA, were compared with those of partially purified natural IFN-alpha. Twelve interferon-induced polypeptides were visualized by two-dimensional gel electrophoresis and autoradiography. Seven of these were shown to have related primary structures and are therefore products of related genes or are related through post-translational modification. Qualitative visual comparisons and computer-aided quantitation of autoradiograms revealed no differences in the patterns of polypeptide induction following treatment with the two types of IFN-alpha, and the two interferons also induced (2'-5') oligoisoadenylate synthetase equally. By these criteria, the activities of the two interferons are qualitatively and quantitatively indistinguishable. In addition, the effects of trisomy 21 on IFLrA-induced polypeptide synthesis and on antiviral response were similar to those previously demonstrated with natural IFN-alpha.

Induction of poly(I):poly(C)-binding 50 K protein and 2',5'-oligoadenylate synthetase in interferon-treated mouse L929 cells.

A new protein retained by poly(I):poly(C)-Sepharose was induced together with dsRNA-dependent enzymatic activities, a protein kinase and 2',5'-oligoadenylate synthetase (2,5A synthetase), in interferon-treated mouse L929 cells; it had an apparent molecular weight of 50,000 (50 K) and was not phosphorylated by the protein kinase. The kinetics of the induction of the poly(I):poly(C)-binding 50 K protein were similar to those of dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase, and their inductions were all dependent on the interferon dose added, though a relatively higher dose was required for the 50 K protein. When the interferon preparation was heated to 100 degrees C in the presence of sodium dodecyl sulfate, its effect on cells of inducing the activity of 2',5'-oligoadenylate synthetase was preserved completely, indicating that the interferon molecule itself is responsible for the induction of the synthetase. Since the induction of the enzymatic activity was inhibited by addition of either actinomycin D or cycloheximide, it may not be an activation of a latent enzyme but a de novo synthesis of the enzyme.

Induction, purification, and properties of 2'5' oligoadenylate synthetase.

The results presented above relate to several aspects of the 2-5A system. A simple but insensitive radiochemical assay fro 2-5A and its synthetase is described. Progress towards the molecular characterization of the synthetase suggested that it is composed of a single 56,000 dalton polypeptide chain that is synthesized in response to IF treatment. Degradation of 2-5A occurs at the same rapid rate in extracts of IF-treated and untreated chick cells. However, this breakdown can be inhibited by its end-product, 5'AMP, or by the activated synthetase which can further elongate 2-5A and thereby protect it from degradation. The direction of elongation is from the 5' to the 2' terminus. Molecules other than 2-5A can act as substrates fro 2'-adenylation by the activated synthetase. These include some dinucleoside monophosphates, ADP-ribose and NAD+, and methylene-bridged analogues of ATP. The methylene-bridged analogues of 2-5A that are synthesized in the latter case retain some of the biological activity of authentic 2-5A, indicating that cleavage of the 5'-terminal phosphate group(s) is not involved in the mechanism of nuclease activation by 2-5A.

Induction of 3-methyladenine DNA glycosylase II is recA+-independent.

The recA1 mutation was transduced into the tag-2 mutant of E. coli, thus making a strain deficient in the induction of SOS repair as well as in the constitutive repair of 3-alkylated adenines in DNA. The double mutant recA tag is more sensitive to methyl methanesulfonate exposure than either single mutant, indicating that recA and tag mutations block different pathways in repair of alkylation damage. The double mutant is more deficient in host cell reactivation of alkylated phages than the tag single mutant. However, alkylation induction of the double mutant with N-methyl-N'-nitro-N-nitrosoguanidine resulted in killing adaptation of the cells to methyl methanesulfonate and restored the host cell reactivation capacity for alkylated lambda phage to wild-type levels. These adaptive responses can be ascribed to the induction of 3-methyladenine DNA glycosylase II which is shown by enzyme analysis to proceed normally in the recA mutant background. The results imply that the induction of the alkA gene encoding 3-methyladenine DNA glycosylase II is independent of SOS induction.

Regulation of expression of the cloned ada gene in Escherichia coli.

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned in multicopy plasmids. O6-Methylguanine-DNA methyltransferase and 3-methyladenine-DNA glycosylase II, which are known to be inducible as part of the adaptive response, were produced in ada- cells bearing ada+ plasmids, even without treatment with alkylating agents. When such cells had been treated with methyl methanesulfonate, even higher levels of the enzyme activities were produced. Maxicell experiments revealed that the ada gene codes for a polypeptide with a molecular weight of 38 000. We constructed a hybrid plasmid carrying an ada'-lacZ' fused gene, with the proper control region for ada expression. beta-Galactosidase synthesis from the fused gene was strongly induced only when cells were treated with low doses of methylating agents, but was weakly induced with relatively high doses of ethylating agents. The induction was autogenously regulated by the ada gene product, in a positive manner.

Ability of various alkylating agents to induce adaptive and SOS responses: a study with lacZ fusion.

We used alkA'-lacZ' and umuC'-lacZ' fused genes and determined the ability of various alkylating agents to induce adaptive and SOS responses. The degree of induction of expression of these genes was quantitatively measured by a simple colorimetric assay of beta-galactosidase activity. SN1 type methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea, were more effective inducers for the alkA than for the umuC system, while SN1 type ethylating agents, such as N-ethyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea, were more potent inducers for the umuC than for the alkA system. Similar but less striking effects on the two systems were obtained with SN2 type alkylating agents.

[Identification of the isoadenylate synthetase in the messenger RNA translation products of antiviral proteins]. / Identifikatsiia izoadenilatsintetazy v produktakh transliatsii informatsionnykh RNK antivirusnykh belkov.

A further study of conditions for induction, determinations of activity and products of mRNAs translation of AVP having nonspecific antiviral activity from L-929 cells treated with poly(I) . poly(C) was carried out. Under conditions of superinduction of interferon the yield of AVP mRNA is reduced unlike that of interferon mRNA. Manifestations of the antiviral effect of AVP mRNA translation products do not seem to require the stages of transcription as dihydrorifampicin exerts no influence on this process. The inhibiting effect of actinomycin D is more likely to be associated with its effect on the energy of cells and synthesis of macroergic compounds of ATP type. Active AVP mRNAs from L-929 cells like interferon mRNA consist mainly of poly-A-deficient mRNAs. The sedimentation rate of AVP mRNAs differs from that of interferon mRNA and is about 10S. AVP mRNA translation products in ovocytes of Xenopus laevis, in contrast to control mRNAs, contain isoadenylate synthetase responsible for synthesis of 2'5'-oligoisoadenylate, a nonspecific translation inhibitor the mechanism of action of which consists in activation of cellular endogenous nuclease. The results suggest that the mode of development of the antiviral condition by activation of isoadenylate synthetase is similar in the cells treated with interferon and poly(I) . poly(C).

Assay of an interferon-induced enzyme in white blood cells as a diagnostic aid in viral diseases.

Interferon is part of the system of defence against viral infections and has important cell-regulatory and immunoregulatory functions. It is, however, not always possible to quantify circulating interferon in patients. An assay has been developed to measure an interferon-induced enzyme in white blood cells. The activity of this enzyme is constant in healthy subjects but increases by 2-10 times in 85% of patients with acute viral infections. It is also enhanced in autoimmune diseases and in virus-related malignancies and neurological disorders. The enzyme level was not raised in bacterial infections or non-infectious diseases studied. This simple and rapid biochemical assay of the interferon system could be used for diagnosis and evaluation of many diseases.

Genetic analyses of an amber mutation in Escherichia coli K-12, affecting deoxyribonucleic acid ligase and viability.

Genetic analyses of an Escherichia coli K-12 mutant possessing the amber mutation lig-321 were carried out. This mutant is defective in deoxyribonucleic acid (DNA) ligase and conditionally lethal. We constructed strains harboring an F'lig+ or F'lig-321 plasmid. Genetic complementation analyses were done by using these plasmids and by constructing a lig-4/F'lig-321 merodiploid. It was shown that lig-321 does not complement lig-4, unless the former is suppressed by an amber suppressor. The same was found to be the case between lig-321 and lig-ts7. Transductional mapping of lig-321, by a four-factor cross, revealed that lig-321 is very closely linked to lig-4. The frequency of recombinants between the two alleles was not unreasonable for assuming that they arose by intragenic recombination. The lig-4 and lig-ts7 alleles are known to reside in the structural gene for DNA ligase, in which lig-321 may also be located.

Relationship between thymus ontogeny and DNA ligase expression in the chicken.

DNA ligase expression was studied in the embryonic chicken thymus. As previously reported, during thymus ontogeny two separate and successive (8S and 6S) forms of enzyme are found. The switch from the heavy to the light form takes place at around 18 days of incubation in situ. The demonstration that two successive generations of thymic lymphoid precursor cells develop in the embryonic bird thymus and that the replacement of the first by the second thymocyte generation takes place at around 18 days suggested the possibility that each form of enzyme could be restricted to a given thymocyte generation. By either culturing or transplanting embryonic thymuses of different ages it appears that the switch from the heavy to the light form of DNA ligase is not related to a change over of the first by the second generation of embryonic thymocytes but rather to some maturation process of the thymic lymphocytes starting from 17 days of embryonic life. The influence of extrathymic factors on the turning on of the expression of the 6 S DNA ligase in the post-natal environment is also suggested.

Lysogenization of Escherichia coli by bacteriophage Lambda: complementary activity of the host's DNA polymerase I and ligase and bacteriophage replication proteins Q and P.

When bacteriophage lambda DNA replication is blocked by mutation in phage genes O or P, the efficiency of lysogenization drops to a very low value unless high multiplicities of infecting phage are used. Our results show that even at high multiplicity, lambda O or P mutants cannot efficiently lysogenize some hosts that are defective in either DNA polymerase I or DNA ligase. Covalent closure of infecting DNA molecules, a preliminary step for insertion according to Campbell's model and an obvious candidate for this lysogenization defect, appears to occur normally under our conditions. In addition, prophage excision as measured by the frequency of curing O- and P- lysogens seemed normal when tested in the poll- strain. These results suggest that the Escherichia coli enzymes DNA polymerase I and ligase, and phage proteins O and P, are able to provide some complementary activity whose function is required specifically for prophage integration.

Biosynthesis of mammalian DNA ligase.

A monospecific antibody against calf thymus DNA ligase composed of a single polypeptide with Mr = 130,000 cross-reacts with rodent and calf thymus DNA ligases. The antibody precipitates a single Mr = 200,000 polypeptide from detergent lysates of [3H] leucine-labeled mouse Ehrlich tumor cells and calf thymocytes. Pulse-chase experiments show that the Mr = 200,000 polypeptide in Ehrlich tumor cells has a half-life of about 0.5 h. In addition to the Mr = 200,000 polypeptide, a Mr = 130,000 polypeptide is detected in the partially purified enzyme preparations from radiolabeled Ehrlich tumor cells. These results suggest that DNA ligase is synthesized in mammalian cells as a Mr = 200,000 polypeptide and that the Mr = 200,000 polypeptide is degraded to a Mr = 130,000 polypeptide by a limited proteolysis in vitro.

Interferon induction of (2'-5') oligoisoadenylate synthetase in diploid and trisomy 21 human fibroblasts: relation to dosage of the interferon receptor gene (IRFC).

Trisomy 21 human fibroblasts are more sensitive to human interferon-alpha (IFN-alpha) than are diploid controls, consistent with the location of the gene (IFRC) which codes for the IFN-alpha receptor on chromosome 21. When compared in the antiviral assay, the difference in sensitivity is five- to tenfold, much greater than the 50% difference in IFRC gene dosage. An understanding of the mechanism by which this amplification of gene dosage occurs is relevant to the specific pathology of Down's syndrome and as a model system for studying the pathogenic effects of chromosomal aneuploidy. The enzyme (2'-5') oligoisoadenylate synthetase (2-5A synthetase), which is believed to be central to the interferon-induced antiviral response, is induced 50% more in trisomy 21 fibroblasts than in diploid controls. Thus the amplification in response occurs subsequent to the binding of IFN-alpha to its receptor and the triggering of the first set of intracellular events, the latter exemplified by the induction of 2-5A synthetase. Similar results were obtained with IFN-gamma, consistent with other evidence which indicates that a gene coding for a separate IFN-gamma receptor is also located on chromosome 21.

Analysis of expression of the rII gene function of bacteriophage T4.

Temperature-sensitive (ts) mutants of the T4 phage rII gene were islated and used in temperature shift experiments that revelaed two different expressions for the normal rII (rII+) gene function in vivo: (i) an early expression (0 to 12 min postinfection at 30 C) that prevents restriction of T4 growth in Escherichia coli hosts lysogenic for gamma phage, and (ii) a later expression (12 to 18 min postinfection at 30 C) that results in restriction of T4 growth when the phage DNA ligase (gene 30) is missing. The earlier expression appeared to coincide with the period of synthesis of the protein product of the T4 rIIA cistron, whereas the later expression occurred after rIIA protein synthesis had stopped. The synthesis of the protein product of the rIIB cistron continues for several minutes after rIIA protein synthesis ceases (O'Farrell and Gold, 1973). The two rII+ gene expressions might require different molar ratios of the rIIA and rIIB proteins. It is possible that the separate expressions of rII+ gene function are manifestations of different associations between the two rII proteins and other T4-induced proteins that are synthesized or activated at different times after phage infection.

In vitro construction of bacteriophage lambda carrying segments of the Escherichia coli chromosome: selection of hybrids containing the gene for DNA ligase.

DNA from lambdagt-lambdaB bacteriophage was cleaved with EcoRI endonuclease and fragments from EcoRI-digested E. coli DNA were inserted. This DNA was used to infect E. coli, and phages containing the gene for DNA ligase were isolated by genetic selection. Two different hybrids were found with the same E. coli segment inserted in opposite orientations. Both hybrids produced similar levels of ligase as measured in crude extracts of infected cells.

[Biosynthesis of early enzymes induced by bacteriophage T4]. / Issledovanie biosinteza rannikh fermentov, indutsiruemykh bakteriofagom T4.

The biosynthesis of early enzymes of DNA-ligase, RNA-ligase, DNA-polymerase, polynucleotide kinase, exonuclease A induced by bacteriophage T4amN82 was studied. The maximal activity of DNA-ligase was observed at the 60th min after the infection, while that of the other enzymes was revealed at the 90th min and reached 4, 45, 529, 120 and 78 units per mg of protein for DNA-ligase, RNA-ligase, polynucleotide kinase, DNA-polymerase and exonuclease A, respectively. Bacteriophage T4amN82 induced the maximal biosynthesis of the tested enzymes, when E. coli B-23 cells were grown in medium I containing trypton bacto ("Merck", West Germany) and a yeast extract ("Difco", USA). Similar events were observed when E. coli B-23 cells infected with phage T4amN82 were grown in a medium (II) with casein hydrolysate and yeast extract. Ultrasonication used for the disruption of E. coli B-23 cells infected with bacteriophage T4 had no effect on the enzyme activities.

Suppression of a mutation in gene 3 of bacteriophage T7 (T7 endonuclease I) by mutations in phage and host polynucleotide ligase.

Bacteriophage T7 bearing amber mutations in both gene 1.3 (T7 DNA ligase) and gene 3 (T7 endonuclease I) are viable when grown in suppressor-negative, ligase-negative hosts. This is evidenced by a high plating efficiency and a large burst size compared to the single mutants. These findings may be explained by a limited destruction of cellular DNA by the double mutant.