Simple repetitive sequences in the genomes of archaebacteria.

Stretches of simple sequences poly(dG-dT).poly(dC-dA), poly(dG-dA).poly(dC-dT), poly(dG).poly(dC) and poly(dA).poly(dT), the occurrence of which is a characteristic feature of eukaryotic genomes, are found in the genomes of archaebacteria Halobacterium halobium and Sulfolobus acidocaldarius. In S. acidocaldarius these sequences constitute a considerable portion of the genome; they belong to a class of repetitive sequences dispersed throughout the genome, being transcribed and found in RNAs of different lengths.

A restriction endonuclease SuaI from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A type II restriction endonuclease (SuaI) has been isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. The enzyme is an isoschizomer of BspRI. It does not cut S. acidocaldarius DNA, as the recognition sequence GGCC in this DNA contains modified nucleotide(s). The enzyme is most active at 60-70 degrees C and is highly thermostable.

Strand-specific transcription of the simple sequences poly[dG-dT):(dC-dA)] and poly[(dG-dA):(dC-dT)] in D. melanogaster embryos revealed by S1 nuclease treatment.

The transcription of different strands of the simple sequences poly[(dG-dT):(dC-dA)] and poly[(dG-dA):(dT-dC)] in Drosophila melanogaster embryos was studied. After selective labelling of one strand, the polymers were hybridized with immobilized RNA and hybrids were treated on filters with S1 nuclease. Appropriate control has shown that this treatment destroys sandwich-like complexes and leaves only true hybrids. Transcription of poly[(dG-dA):(dT-dC)] proved to be nearly symmetric, while poly(dG-dT) is transcribed 3-times more intensively than poly(dA-dC). Our approach allowed us to estimate the relative content of simple sequences in RNA.

[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.

[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.

[Nuclear proteins from Drosophila melanogaster specifically binding to simple homopolymeric sequences of poly[d(T-G)].poly[d(C-A)] type]. / Iadernye belki Drosophila melanogaster, spetsificheski sviazyvaiushchie prostye gomopolimernye posledovatel'nosti tipa poli[d(T-G)].poli[d(C-A)]

Binding of simple homopolymeric sequences to Drosophila melanogaster nuclear proteins has been studied. Proteins with Mr 65-72 kDa have been found, which specifically bind to synthetic poly[d(T-G)].poly[d(C-A)], as well as to D. melanogaster DNA containing a block of poly[d(T-G)].poly[d(C-A) 40 b.p. in length. It has been shown, that these proteins bind only to poly[d(T-G).poly[d(C-A)] and not to other types of simple sequences, for example poly[d(G-A)].poly[d(T-C)] and poly[d(A-T)].

[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.

[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.

[Complexes of histone H1 with supercoiled DNA]. / Issledovanie kompleksov gistona H1 s superspiral'noi DNK.

The action of DNA topoisomerase I on the complexes of supercoiled DNA (DNA I) with histone H1 and other histones was studied at different ionic conditions and histone/DNA ratios. In the presence of 0.15 M NaCl at histone H1/DNA ratios lower than 0.25 (w/w) the relaxation of DNA I is not inhibited. Raising of H1/DNA I ratio up to 0.7 is followed by significant inhibition of DNA I relaxation. At fixed H1/DNA I ratio maximal inhibition is detected at 0.25 M NaCl. At NaCl concentrations lower than 0.1 M and higher than 0.3 M increasing of DNA I relaxation in the presence of H1 was observed. Electron microscopic studies show that increase of ionic strength or H1/DNA I ratio causes more dense packing of DNA molecules in the H1.DNA complexes. Changes in the structure of complexes agree with the increase of DNA I relaxation inhibition in these conditions. DNA I relaxation inhibition by H1 is drastically decreased by iodination of tyrosine 72 residue in the globular part of H1 molecule. Individual core histones inhibit DNA I relaxation at much higher histone/DNA ratios and show different dependence of inhibition on ionic strength. The results are discussed in terms of the possible role of H1 in chromatin condensation-decondensation.

[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.

[Distribution and transcription of determinants homologous to the alpha-hemolysin (hlY) gene in the genomes of HLY- and HLY+ staphylococci]. / Raspredelenie i transkriptsiia determinant, gomologichnykh genu alpha-gemolizina (hlY), v genomakh HLY- i HLY+ stafilokokkov.

The molecular organization of the alpha-hemolysin gene from Staphylococcus aureus strain O15 has been studied. Hybridization of the DNA from this strain with the [32P]-labeled cloned fragment containing alpha-hemolysin gene has shown the studied gene to be unique in the genome of the strain. Sequences homologous to the gene were not found to be dispersed along the genome of Staphylococcus aureus O15. The presence of alpha-hemolysin gene in some Staphylococcus epidermidis strains has been monitored. The strains 1413, 303 have been demonstrated to contain no gene for alpha-hemolysin. The hemolysin genes were found in the genomes of the strains 159, 169, 180 by DNA hybridization technique. In the genome of the strain 180 the long region including the right end of alpha-hemolysin gene is deleted. Hybridization with the net RNA of these strains shows the absence of transcription of intact as well as deleted alpha-hemolysin genes.

[Cloning of alpha-hemolysin gene from Staphylococcus aureus strain 015]. / Klonirovanie gena alpha-gemolizina shtamma Staphylococcus aureus 015.

The genomic library of Staphylococcus aureus O15 has been constructed on the EMBL-3 vector. The synthetic oligonucleotide probes to N- and C-end regions of alpha-hemolysin permitted identification of the recombinant bacteriophage clone RS-1 containing a gene for this protein. The restriction map of the cloned fragment has been constructed for restriction endonucleases SalGI, EcoRV, PstI, PvuII. Expression of the alpha-hemolysin gene in phagolysate of the recombinant clone RS-1 (1000 units per ml) has been demonstrated.

Uneven distribution of cloned transcribed DNA sequences in polytene chromosomes of Drosophila melanogaster.

DNA fragments actively transcribed at different developmental stages of Drosophila melanogaster were cloned. In situ hybridization was used to study their distribution in the polytene chromosomes. The clones were dot-hybridized to poly(A)+ RNA isolated at different developmental stages. Fifty-four of 67 clones were unique, 37 of them were localized. An irregular distribution of the cloned sequences was revealed. Twenty-three clones are localized in chromosome 3, six clones in the X-chromosome, and only eight in chromosome 2. There are transcriptionally active regions where, on a relatively small area of the chromosomes, a number of clones are localized (sections 18-19, 69-71, 82-85, 95-97). RNAs transcribed from the cloned sequences code for about 0.005 to 0.2% of the total poly(A)+ RNA.

The genome of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius does not contain repetitive sequences.

Characteristics of genome organization in the sulfur-dependent thermoacidophilic archaebacterium Sulfolobus acidocaldarius have been studied. By means of hybridization analysis it is shown that the genome of S. acidocaldarius, unlike the genome of the extremely halophilic archaebacterium Halobacterium halobium, does not contain repetitive sequences.

DNA supercoiling by core histone fractions and protamine.

The nuclear extract isolated from late Drosophila melanogaster embryos has Mg2+-dependent DNA topoisomerase 1 and nuclease activities. The extract facilitates the closed circular duplex DNA supercoiling in the presence of calf histone fractions H2A, H2B, H3 and H4, and fish protamine but not HI histone.

Nuclear proteins from Drosophila melanogaster embryos which specifically bind to simple homopolymeric sequences poly [(dT-dG).(dC-dA)].

The binding of nuclear proteins from Drosophila melanogaster embryos to simple homopolymeric DNA sequences was studied. Nuclear proteins were electrophoresed, transferred onto nitrocellulose and incubated with labelled synthetic homopolymers or natural fragment containing simple sequences. Several protein bands were found in the 65-72 KDa region, which specifically bind both poly [(dG-dT).(dA-dC)] and a natural fragment containing 40 bp of this sequence. These proteins do not bind to homopolymers poly [(dA).(dT)] and poly [(dG-dA).(dC-dT)], or other foreign DNAs.

[Drosophila melanogaster embryo factor capable of supercoiling circular covalently closed DNA in the presence of core histones H2a, H2b, H3, H4 or protamine]. / Superspiralizatsiia kol'tsevoi kovalentno zamknytoi DNK v prisutstvii gistonov H2, H2b, H3, H4 ili protamina i ekstrakta iz embrionov Drosophila melanogaster.

The Mg2+-dependent factor capable of introducing the superhelical turns into circular closed DNA in the presence of core histones H2a, H2b, H3, H4 or protamine in solution at physiological conditions has been isolated from Drosophila melanogaster embryos. The factor does not work with histone H1, cytochrome c, poly-L-lysine or tetralysine. It is assumed that the factor is responsible for folding of core histones into nucleosomes.

[Isolation and properties of the SuaI restriction endonuclease from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius]. / Vydelenie i svoistva éndonukleazy restriktsii SuaI iz termoatsidofil'noi arkhebakterii Sulfolobus acidocaldarius.

A new restriction endonuclease SuaI was isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. The enzyme is an isoschizomer of BspR1; it recognizes tetranucleotide GGCC and cleaves DNA in the center of this sequence. SuaI requires Mg2+, the optimal concentration being 6 mM. KCl at concentrations above 25 mM significantly inhibits the enzyme activity. The pH optimum lies within the range of 6--7 at 70 degrees C, the temperature optimum is at 70--75 degrees C. The enzyme is highly stable at temperatures up to 80 degrees C. DNA of S. acidocaldarius is not cleaved by the enzyme.