[The legacy of Andrey Mirzabekov and his laboratory in the studies of chromatin structure using DNA-protein cross-linking].

The work of A.D. Mirzabekov and his collaborators on chromatin structure is reviewed. The methods of DNA-protein covalent binding with subsequent analysis of cross-linked products developed in the laboratory were used to localize the contacts in both DNA and individual proteins. This original approach played an important role in the deciphering of the molecular structure of nucleosome. In addition, it made possible the investigation of structural transitions in chromatin upon gene activation.

[Microchips based on three dimensional gel cells: history and perspective]. / Mikrochipy na osnove trekhmernykh iacheek gelia: istoriia i perspektivy.

The review describes the history of creation and development of the microchip technology and its role in the human genome project in Russia. The emphasis is placed on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous versions. The microchips are manufactured by photo-initiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical strength and stability, which allowed one to work with the DNA fragments of up to 500 nt in length, as well as with rather large protein molecules. At present, the gel-based microchips are widely applied to address different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA-protein interactions. The oligonucleotide microchips are a cheap and reliable tool of diagnostics designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; for diagnostics of orthopoxviruses, including the smallpox virus; for diagnostics of the anthrax pathogen; and for identification of chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteomics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which open a wide potential for creation biosensors on the basis of microchips.

[Biological microchips with hydrogel-immobilized nucleic acids, proteins, and other compounds: properties and applications in genomics]. / Biologicheskie mikrochipy, soderzhashchie immobilizovannye v gidrogele nukleinovye kisloty, belki i drugie soedineniia: svoistva i prilozheniia v genomike.

The MAGIChip (MicroArrays of Gel-Immobilized Compounds on a chip) consists of an array of hydrophilic gel pads fixed on a hydrophobic glass surface. These pads of several picoliters to several nanoliters in volume contain the gel-immobilized nucleic acids, proteins, and other compounds, as well as live cells. They are used to conduct chemical and enzymatic reactions with the immobilized compounds or samples bound to them. In the latter case, nucleic acid fragments can be hybridized, modified, and fractionated within the gel pads. The main procedures required to analyze nucleic acid sequences (PCR, detachment of primers and PCR-amplified products from a substrate, hybridization, ligation, and others) can be also performed within the microchip pads. A flexible, multipurpose, and inexpensive system has been developed to register the processes proceeding on a microchip. The system provides unique possibilities for research and biomedical applications, allowing one to register both equilibrium states and the course of reaction in real time. The system is applied to analyze both kinetic and thermodynamic characteristics of molecular interaction in the duplexes formed between nucleic acids and the probes immobilized within the microchip gel pads. Owing to the effect of stacking interaction of nucleic acids, the use of short oligonucleotides extends the possibilities of microchips for analysis of nucleic acid sequences, allowing one to employ the MALDI-TOF mass spectrometry to analyze the hybridization data. The specialized MAGIChips has been successfully applied to reveal single nucleotide polymorphism of many biologically significant genes, to identify bacteria and viruses, to detect toxins and characterize the genes of pathogenic bacteria responsible for drug resistance, and to study translocations in the human genome. On the basis of the MAGIChip, the protein microchips have been created, containing the immobilized antibodies, antigens, enzymes, and many other substances, as well as the microchips with the gel-immobilized live cells.

[The genomic organization and localization on the chromosomes of the HvRT family of DNA repetitive sequences in barley]. / Genomnaia organizatsiia i lokalizatsiia na khromosomakh HvRT-semeistva povtoriaiushchikhsia posledovatel'nostei DNK iachmenia.

HvRT family of repetitive DNA sequences from barley genome appears to have complex hierarchical organization. Tandem repetition of 118-bp monomers constitutes lower level of HvRT-family organization. Amplification units of the higher level consist of several contiguous 118-bp monomers. RFLP between different species and cultivars of barley resulted from the differences in the higher-order repeat structure. Individual chromosomes of barley contain specific HvRT subfamilies. This family also possesses separate domains differing in the restriction enzyme sites density. HvRT family is presented in the genomes of H. vulgare, H. leporinum, H. murinum, H. jubatum, but is absent in the genomes of H. marinum, H. geniculatum and wheat.

[Organization of simple sequences in the Drosophilia melanoga ter genome]. / Organizatsiia prostykh posledovatel'nostei v genome Drosophilia melanogaster.

Fragments of Drosophila melanogaster DNA that intensively hybridize with simple sequences poly[(dG-dT).(dC-dA)], poly[(dA).(dT)] and poly[(dG-dA).(dC-dT)] were cloned. The first two types of simple sequences are organized in these clones as separated stretches of moderate length, repeated many times within 12-15 kb. Each cluster contains only one type of the simple sequences and originates from a unique in the genome. In contrast, poly[(dG-dA).(dC-dT)] occurs in the genome as several isolated motifs.

[A new class of mobile genetic elements of Drosophila melanogaster]. / Novyi klass mobil'nykh geneticheskikh élementov Drosophila melanogaster.

Clone Dm A89 was obtained upon cloning of DNA fragments coding abundant poly(A+)RNA's of D. melanogaster. Dm A89 was identified as a new transposable element using in situ hybridization with polytene chromosomes of two independent highly isogenic lines of D. melanogaster oregon RC and gt wa Dm A89 hybridizes with approximately 20 sites in each line. A portion of Dm A89 is homologous to the distal part of type I ribosomal gene insertion sequence and is highly repetitive. Two other sections of the clone have much less redundancy. The unity of the three fragments is not casual, as revealed by cloning of some other genomic sequences homologous to Dm A89. Dm A89 is actively transcribed throughout the development of D. melanogaster and produces polyadenylated RNA 1.1 kb long.

[Gradient condensation of chromatin in ribosomal genes of Drosophila melanogaster]. / Gradientnaia kondensatsiia khromatina v ribosomnykh genakh Drosophila melanogaster.

The organization of chromatin in D. melanogaster ribosomal repeats with and without insertions was studied. We have shown earlier that upon digestion with micrococcal nuclease a "non-transcribed" intergenic spacer produces unusual chromatin particles containing DNA fragments 200-280 b.p. in length. These particles sediment like H1-containing nucleosomes, are stable only in the presence of polyamines, and are probably bound to some non-histone protein. The content of core histones and H1 in different regions of ribosomal genes has been studied by two-dimensional electrophoresis of chromatin particles and by "protein-image" hybridization. The content of histones and respectively the degree of chromatin condensation increase in the following order: the 1kb-long region surrounding the initiation site is practically free of histones less than the region of 240 b.p. repeats from the intergenic spacer, containing homologies with the ribosomal promotor less than coding region preceding the usual site of insertions less than coding region lying behind this site less than inactive type II ribosomal insertion. Therefore, the region of the beginning of transcription of most ribosomal genes is in an active conformation, even though at least 75% of the genes are repressed. Ribosomal insertions are in a compact, repressed form. We suggest that their inhibitory action on the transcription of corresponding genes at the molecular level is similar to the position effect of heterochromatic regions at the chromosomal level.

[Chromatin structure of ribosomal genes of Drosophila melanogaster. Random location of nucleosomes in DNA and characteristics of organization of non-transcribed spacer]. / Struktura khromatina ribosomnykh genov Drosophila melanogaster. Sluchainoe raspolozhenie nukleosom na DNK i osobennosti v organizatsii netranskribiruemogo speisera.

Chromatin structure of ribosomal genes from nuclei of Drosophila melanogaster embryos was studied by using micrococcal nuclease cleavage. End-directed labelling with short cloned fragments of the ribosomal repeat was carried out. It shows, that the micrococcal nuclease prefers specific sites in naked DNA, and the pattern of DNA cleavage is essentially conserved when the nuclei are digested. Only minor differences are visible. Hence, there are no specific positions of nucleosomes on the ribosomal repeat. The DNA fragments from the nuclei treated with micrococcal nuclease were electrophoresed, transferred to DBM-paper and hybridized with different probes subcloned from the ribosomal repeat. The non-transcribed spacer and the region of the beginning of transcription are hydrolysed significantly faster than the coding region or inactive ribosomal insertion. The region of NTS and the beginning of transcription do not give normal nucleosomal fragment in the range of 145-185 bp; instead they produce a heterogeneous band 200-280 bp in length even after prolonged digestion. Dinucleosomal fragments are also slightly longer and more heterogeneous than in other parts of the ribosomal repeat. Higher oligomers are similar throughout the ribosomal repeat. We suggest that a hypothetical transcription factor interacts in a way with histones and protects unusual fragments of DNA from digestion.

[Cloning and study of inserted sequences of gene 28S in Drosophila melanogaster ribosomal RNA]. / Klonirovanie i izuchenie vstavochnykh posledovatel'nostei v gene 28S ribosomnoi RNA Drosophila melanogaster.

Cloning of fragments of ribosomal genes containing insertions in the 28S RNA gene has been reported earlier. Subcloning of DNA fragments corresponding to insertion sequences and their hybridization with DNA, RNA and polytene chromosomes from different flies is described. Type 1 insertions (containing BamI sites) are highly heterogeneous in length and sequence even in homozygotes. Type 2 insertions (with EcoRI sites) are rather homogeneous. Two types of insertions are represented in the D. melanogaster genome by 50 and 30 copies, respectively. Restriction fragments with insertions significantly differ in DNA from embryos and larvae. D. simulans and D. virilis also contain the sequences of both types of insertions, though in fewer number of copies. Type 1 insertions seem to be poorly transcribed, and type 2 insertions are not transcribed at all. Among 2000 recombinant clones screened a number of DI plasmids hybridizing to isolated insertions were obtained. Six of them were mapped with restriction endonucleases and hybridized with insertion fragments. rRNA and polytene chromosomes. All of these DI plasmids hybridize with the nucleoli, one with the chromocenter and one with the 79F 3L site. In LI9, not coding for rRNA, the sequences, corresponding to two types on insertions are located only a few kilobases apart. D17a does not encode for rRNA, but hybridizes in situ only with the nucleoli.

[Simple method of cloning eukaryote DNA: production of certain new ribosomal genes of Drosophila]. / Prostoi metod klonirovaniia DNK éukariot: poluchenie nekotorykh novykh ribosomnykh genov drozofily.

We propose a simple method which allows to receive a collection of clones containing recombinant plasmids. It is based on the ligation of the longer fragment of pBR332 formed by EcoRI and BamH1 with eukaryotic DNA (from Drosophila melanogaster embryo in this case) partially cleaved with EcoRI and BamHI. This approach gave us 10(4) colonies from 1 microgram of Drosophila DNA and 0.1 microgram of the BamHI--EcoRI "vector". About 0.5% of all clones carried the fragments of ribosomal genes with insertions in the 26S gene. Ribosomal genes lacking insertions did not enter the collection due to some peculiarities in their restriction map. The sites of cleavage are mapped in eight recombinant plasmide for HindIII, BamHI and EcoRI. These maps show that some insertions within 26S gene have not been cloned earlier. The mean length of cloned fragments is 11.8 kilobases, the mean number of EcoRI and BamHI restriction sites are 1.2 and 1.0, respectively. The electrophoretical screening of plasmids using cetyl trimethyl ammonium bromide was developed.

[Length of nucleosomal DNA repeat in whole cells, fixed by freezing in the presence of formaldehyde]. / Razmer nukleosomnoi DNK v tselykh kletkakh, fiksirovannykh zamorazhivaniem v prisutstvii formal'degida.

It is shown that whole cells can be effectively fixed in the presence of formaldehyde at -12 degrees C. This reaction is used for the study of the native structure of chromatin. In the nuclei isolated from fixed cells the chromatin has the nucleosomal structure. The size of nucleosomal DNA in these nuclei estimated by hydrolysis with staphylococcal nuclease does not differ significantly from repeat length in the nuclei fixed after isolation or in non-fixed nuclei. However it is shown that mono- and oligonucleosomes in the nuclei from fixed whole cells are significantly more stable to the exonucleolytic degradation than in either nuclei fixed after isolation or non-fixed nuclei. The results suggest that the nuclei isolation does not appreciably affect the chromatin structure. The fixation of whole cells by formaldehyde in frozen suspension can be used also to study the structure of other cellular components and macromolecular complexes directly in the whole cell.

[State of DNA slots in mono- and oligonucleosomes and in DNA complexes with individual histones]. / Sostoianie borozdok DNK v mono- i oligonukleosomakh i v kompleksakh DNK s individual'nymi gistonami.

The state of the major and the minor DNA grooves in purified mono- and oligonucleosomes and in the complexes of DNA with different histones have been studied by means of methylation of DNA with dimethyl sulphate. In nucleosomes histones shielded major groove by 18--20%. This result agrees well with our previous data obtained with chromatin, nuclei and whole cells. Each of the purified histones H2a, H2b, H3 and H4 as well as N-terminal peptides of H4 histone cause relative shielding of the DNA major groove by 15--18% like whole histone does. H1 histone protects neither DNA grooves from methylation. Our results suggest that histones are buried partly in the major groove of DNA in chromatin and purified nucleosomes. The arrangement of histones in the major groove does not depend probably on their specific organization in nucleosomes.

[Structure of the complexes of distamycin type antibiotics and actinomycin D with DNA: new data on the localization of these antibiotics within the DNA narrow groove]. / O strukture kompleksov antibiotikov distamitsinovogo tipa i atkinomitsina D s DNK: novye eksperimental'nye dannye o lokalizatsii antibiotnkov v uzkoi borozdke DNK

It is shown that antibiotics actinomycin D (AM), netropsin (Nt), distamycin A (DM) and the propyl analogue of distamycin A (pDM) being complexed with DNA are located within the narrow groove of DNA. A comparative investigation of the 3H-dimethyl sulphate methylation extent of free calf thymus DNA and its complexes with AM, Nt, DM and pDM reveals that upon DNA saturation these antibiotics decrease the methylation level of the narrow groove (AM by 30%, pDM by 50%, DM by 65% and Nt by 70%). In the triple complex of DNA+AM+DM the methylation level of the narrow groove drops by 80%. The large groove is not shielded by these antibiotics at all. However, the methylation level of the large groove decreases by 50% for T6 phage DNA due to the presence of glucosyl residues linked to 5-hydroxymethylcytosine within the large groove. The binding of AM to DNA saturated with Nt or with the analogue of distamycin A (DM2) containing the 2 N-methylpyrrole residues has been investigated by spectrophotometry. The apparent number of binding sites for AM in these 2 complexes is about half as much as observed for free DNA while the saturation level of the binding decreased only by about 20%. This proves simultaneous presence of AM and Nt (DM2) within the narrow groove of DNA.