Long-range fragmentation of the eukaryotic genome by exogenous and endogenous nucleases proceeds in a specific fashion via preferential DNA cleavage at matrix attachment sites.

Small cell lung cancer cells (OC-NYH-VM) were permeabilized and treated with different nucleases. The long-range distribution of DNA cleavage sites in the amplified c-myc gene locus was then analyzed by pulsed field gel electrophoretic separation of the released 50-kilobase to 1-megabase DNA fragments followed by indirect end labeling. Exogenous DNase I and nucleases specific for the single-stranded DNA were found to generate similar nonrandom patterns of large DNA fragments. The cleavage sites were located close to or even colocalized with matrix attachment regions, which were mapped independently using a recently developed procedure for DNA loop excision by DNA topoisomerase II-mediated DNA cleavage. Endogenous acidic nuclease with the properties of DNase II also digested DNA preferentially in proximity to the matrix attachment regions, generating characteristic patterns of excised DNA loops and their oligomers. A similar, although less specific, pattern of DNA fragmentation was observed after incubation of permeabilized cells under conditions favoring the activity of endogenous neutral Ca(2+)- and Mg(2+)-dependent nucleases. These findings are discussed in the context of the current model of the spatial domain organization of eukaryotic genome.

The channels model of nuclear matrix structure.

The specificity of eukaryotic DNA organization into loops fixed to the nuclear matrix/chromosomal scaffold has been studied for more than fifteen years. The results and conclusions of different authors remain, however, controversial. Recently, we have elaborated a new approach to the study of chromosomal DNA loops. Instead of characterizing loop basements (nuclear matrix DNA), we have concentrated our efforts on the characterization of individual loops after their excision by DNA topoisomerase II-mediated DNA cleavage at matrix attachment sites. In this review the results of applying this mapping approach are compared with the results and conclusions from studies of nuclear matrix DNA. An attempt is also made to reconsider all data about the specificity of DNA interactions with the nuclear matrix and to suggest a model of spatial organization of the eukaryotic genome which resolves apparent contradictions between these data.

Different topoisomerase II antitumor drugs direct similar specific long-range fragmentation of an amplified c-MYC gene locus in living cells and in high-salt-extracted nuclei.

We have analyzed the long-range distribution of topoisomerase II-mediated cleavages induced in an amplified human c-MYC gene locus in the presence of several antitumor agents. The long-range cleavage patterns were found to be nonrandom and similar for all antitumor drugs tested. Cleavages occurred within several kilobase-long areas (approximately 5 kb) highly accessible to topoisomerase II and separated by extended regions (approximately 70-100 kb) of less accessibility, possibly reflecting the mode of DNA organization into loops along the chromosome. Within the cleavage areas, the patterns of cleavage sites showed a certain dependence on the type of drug used for entrapment of topoisomerase II-DNA complexes. Importantly, distribution of cleavage areas in native chromatin and histone-depleted nuclei was very similar, if not identical, suggesting that the primary target of antitumor agents in vivo is topoisomerase II associated with the high-salt-insoluble nuclear matrix. These data show that matrix-attached DNA is preferentially damaged by topoisomerase II-targeting agents, which may be an important cellular event contributing to drug-induced cell death.

Specificity and functional significance of DNA interaction with the nuclear matrix: new approaches to clarify the old questions.

In this chapter the specificity of chromosomal DNA partitioning into topological loops is discussed. Different experimental approaches used for the analysis of the above problem are critically reviewed. This discussion is followed by presentation of a novel approach for mapping the DNA loop anchorage sites that we have developed. This approach, based on the excision of the whole DNA loops by topoisomerase II-mediated DNA cleavage at matrix attachment sites, seems to constitute a unique tool for the analysis of topological organization of chromosomal DNA in living cells. We also discuss experimental results indicating that the DNA-loop anchorage sites form "weak points" in chromosomes that are preferentially sensitive to cleavage with both endogenous and exogenous nucleases. In connection with this discussion, rationales for the supposition that DNA loops constitute basic units of eukaryotic genome organization and evolution are considered. The chapter concludes by suggesting a new model of spatial organization of eukaryotic genome within the cell nucleus that resolves apparent contradictions between different data on the specificity of DNA interaction with the nuclear matrix.

[Evaluation of immunoreactivity of class 2 HLA-determinant using mixed lymphocyte culture]. / Otsenka immunoreaktivnosti HLA-determinant klassa 2 metodom smeshannoi kul'tury limfotsitov.

Immunoreactivity (proliferative capacity) of normal donor peripheral blood mononuclears expressing HLA-DR antigens in MLC test was studied. HLA-DR cellular phenotype was found to influence the intensity of proliferative response. HLA-DR highly reactive (HLA-DR5, HLA-DR7, HLA-DR4) and low-reactive (HLA-DR1, HLA-DR2, HLA-DR6) determinants were distinguished.

Characterization of an altered DNA catalysis of a camptothecin-resistant eukaryotic topoisomerase I.

We investigated topoisomerase I activity at a specific camptothecin-enhanced cleavage site by use of a partly double-stranded DNA substrate. The cleavage site belongs to a group of DNA topoisomerase I sites which is only efficiently cleaved by wild-type topoisomerase I (topo I-wt) in the presence of camptothecin. With a mutated camptothecin-resistant form of topoisomerase I (topo I-K5) previous attempts to reveal cleavage activity at this site have failed. On this basis it was questioned whether the mutant enzyme has an altered DNA sequence recognition or a changed rate of catalysis at the site. Utilizing a newly developed assay system we demonstrate that topo I-K5 not only recognizes and binds to the strongly camptothecin-enhanced cleavage site but also has considerable cleavage/religation activity at this particular DNA site. Thus, topo I-K5 has a 10-fold higher rate of catalysis and a 10-fold higher affinity for DNA relative to topo I-wt. Our data indicate that the higher cleavage/religation activity of topo I-K5 is a result of improved DNA binding and a concomitant shift in the equilibrium between cleavage and religation towards the religation step. Thus, a recently identified point mutation which characterizes the camptothecin-resistant topo I-K5 has altered the enzymatic catalysis without disturbing the DNA sequence specificity of the enzyme.

DNA-specific antiidiotypic antibodies in the sera of patients with autoimmune diseases.

Blood sera of patients with autoimmune diseases scleroderma (Scl), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) have been shown to yield a specific immune response to topoisomerase I, the product of expression of a cDNA fragment cloned into lambda gt11 and monoclonal antibodies (MAB) to the enzyme. The 'topoisomerase test' is not absolutely specific for Scl. The stable positive response of autoimmune sera to anti-topoisomerase monoclonal antibodies has a specific character and is associated with the interaction of the Fab fragment of MAB with the IgG fraction of autoimmune serum. The response observed indicates the induction of anti-idiotypic antibodies against topoisomerase. The anti-idiotype, isolated by HPLC and affinity chromatography demonstrated the following functional activities: (i) the immunological reaction against DNA; (ii) high-affinity DNA-binding with topoisomerase-specific consensus; (iii) ability to compete with the native enzyme for binding with DNA and MAB to topoisomerase; (iv) immunological reaction against MAB to topoisomerase.

Camptothecin inhibits both the cleavage and religation reactions of eukaryotic DNA topoisomerase I.

We investigated the mode of action of the antitumor drug, camptothecin, by use of a partly double-stranded suicide DNA substrate which enables uncoupling of the cleavage and religation half-reactions of topoisomerase I. The suicide DNA substrate contains a single topoisomerase I site at which SDS cleavage is strongly enhanced by camptothecin on normal double-stranded DNA. The results show that the religation reaction of topoisomerase I per se is strongly inhibited at this site compared to site that is only marginally affected by camptothecin on double-stranded DNA. This study hereby directly demonstrates that camptothecin-mediated stability of a topoisomerase I-DNA complex is sequence-dependent. The influence of camptothecin on the suicide cleavage reaction of topoisomerase I was also investigated. Surprisingly, the cleavage reaction per se is strongly inhibited by the drug. However, reformation of a cleavable suicide DNA substrate, which is fully double-stranded downstream from the cleavage position except for a nick, completely reverses the inhibitory effect of the drug on the cleavage reaction. The results suggest that the inhibitory effect of camptothecin on cleavage is due to a general decrease in the noncovalent interaction of topoisomerase I with partly double-stranded suicide DNA substrates. Based on the findings, a plausible model for camptothecin action is discussed.

New technique for uncoupling the cleavage and religation reactions of eukaryotic topoisomerase I. The mode of action of camptothecin at a specific recognition site.

A new technique for uncoupling the cleavage and religation half-reactions of topoisomerase I at a specific site has been developed. The technique takes advantage of a suicidal DNA substrate to attain enzyme-mediated cleavage without concomitant religation. Efficient religation can be achieved, subsequently, by addition of an oligonucleotide capable of hybridising to the non-cleaved strand of the suicide DNA substrate. The technique was used to study the effect of different compounds on the half-reactions of topoisomerase I. It was shown that topoisomerase I-mediated cleavage was inhibited by NaCl concentrations higher than 200 mM, while the religation reaction seemed unaffected by concentrations as high as 3 M-NaCl. The divalent cations Mg2+, Ca2+ and Mn2+ were found to enhance the cleavage but not the religation reaction of topoisomerase I, whereas Cu2+ and Zn2+ inhibited both reactions. Furthermore, the effect of the anti-neoplastic agent, camptothecin, on the half-reactions of topoisomerase I was investigated. It was found that the drug did not affect the cleavage reaction of topoisomerase I at the studied site, while the religation reaction of the enzyme was inhibited. Camptothecin was found to stabilise the enzyme-DNA cleavage complex even when the drug was added after complex formation.

[Dynamics of the immune status of patients with postoperative peritonitis during treatment by the open method]. / Dinamika immunnogo statusa bol'nykh s posleoperatsionnym peritonitom pri lechenii otkrytym sposobom.

The values of cellular and humoral immunity were studied in 53 patients with postoperative peritonitis during open treatment and after closure of the abdominal cavity. Analysis of the results showed that postoperative peritonitis is attended by marked depression of cellular and humoral immunity, and by suppression of functional activity of mononuclear phagocytes. When the open method is applied in conjunction with specific combined and nonspecific immunotherapy restoration of cellular immunity begins on the 5th-7th day after relaparotomy and restoration of humoral immunity on the 3rd-4th day. In successful treatment the immune status is normalized on the 8th-10th day. In unfavourable outcomes depression of cellular and humoral immunity increases continuously and death occurs.

Specific cleavage of chicken alpha A-globin and human c-Ha-ras genes by two molecular forms of calf thymus topoisomerase I.

Two molecular forms of topoisomerase I differing in size and sensitivity to camptothecin were isolated from calf thymus. Mapping of topo I cleavage sites of the cloned chicken alpha A-globin and human c-Ha-ras genes was carried out. Camptothecin was shown to affect site specificity of the topoisomerases.

[Interactions of sera from patients with autoimmune diseases with cDNA expressed fragment of topoisomerase I and monoclonal antibodies against enzyme]. / Vzaimodeistvie syvorotok krovi bol'nykh autoimmunnymi zabolevaniiami s ékspressirovannym fragmentom kDNK topoizomerazy I i monoklonal'nymi antitelami k fermentu.

The interaction of sera from 34 patients with different autoimmune diseases with the expressed fusion protein cloned in lambda gt11 vector (topoisomerase I--beta galactosidase) and monoclonal antibodies against enzyme was studied. It was demonstrated that 100% of Scl cases possessed positive activity against fusion protein. It was shown that this test is not absolutely specific for Scl, i. e. 57.1% of Sle and 84.6% of RA demonstrated positive activity against "topoisomerase test". Autoimmune sera had the positive activity against monoclonal antibodies for topoisomerase I. This activity was shown to be due to the presence of antiidiotypic antibodies against topoisomerase in the autoimmune sera.

Sequence dependent modulating effect of camptothecin on the DNA-cleaving activity of the calf thymus type I topoisomerase.

High-resolution mapping of topol cleavages in the regions of human DNA including the oncogene c-Ha-ras and p53, has revealed three kinds of topol cleavage sites: cleavage sites not affected by camptothecin; cleavage sites reinforced only in the presence of camptothecin, and cleavage sites which weaken in the presence of camptothecin. Statistical analysis of sequences revealed certain nucleotide or dinucleotide preferences for three groups studied. The preferences in camptothecin-reduced sites predominate upstream from the cleavage point, whereas in camptothecin-induced sites the situation is reversed. The influence of camptothecin on cleavage sites induced by two molecular forms of topol has been also studied.

[Topoisomerase I from human placenta. Functional activity of products of expression of cloned cDNA fragments]. / Topoizomeraza I iz platsenty cheloveka. Funktsional'naia aktivnost' produktov ékspressii klinirovannykh fragmentov kDNK.

Immunoscreening of the human placenta cDNA-library in the expression vector lambda gt11 using non-isotope detection based on the avidin-biotin system allowed to identify a number of clones encoding human topoisomerase I. The fusion protein from an extract of Escherichia coli cells infected with the recombinant phage lambda gt11 interacts with the monoclonal antibody raised against topoisomerase I from calf thymus; the dissociation constant being 5.7.10(-8) M. The restricted DNA fragments coding for the topoisomerase polypeptide in the composition of the fusion protein were recloned, and expression in the pEX vector was obtained. The functional analysis of the expression products has enabled localization of the epitope of binding the monoclonal antibody. It was demonstrated that the identified fusion protein can be applied for diagnosis of autoimmune diseases.

[Various indicators of humoral immunity in the population of Moldavian SSR]. / Nekotorye pokazateli gumora'lnogo immuniteta u zhitelei Moldavii.

The first grade tests were used to study the state of humoral immunity in the population of the Moldavian SSR. Shifts in the content of circulating immune complexes and hemolytic activity of the blood serum complement have been recorded.

[Specific splitting of the alpha-A-globin gene by molecular forms of DNA-topoisomerase type I from calf thymus]. / Spetsificheskoe rasshcheplenie alpha-A-globinovogo gena molekuliarnymi formami DNK-topoizomeraz I tipa iz timusa telenka.

The specificity of splitting of the cloned alpha A-globin gene from chicken erythrocytes induced by three topoisomerases I differing in molecular masses was demonstrated. The localization and relative number of topoisomerase breaks in the alpha A-globin gene vary in different topoisomerase I forms.

[Effect of camptothecin on the DNA-relaxing and DNA-cleavage activity of calf thymus topoisomerase I]. / Vliianie kamptotetsina na DNK-relaksiruiushchuiu i DNK-rasshchepliaiushchuiu aktivnosti topoizomerazy I iz timusa telenka.

Nucleotide sequences that are cleaved by calf thymus type I topoisomerase have been determined using cloned human Ha-ras and p53 genes. Localization and relative frequency of single-strand cleavages within these sequences were observed to change in the presence of the cytotoxic alkaloid camptothecin.