[Diagnostic prospects for using multilocus DNA markers in systematizing wild hooved animals (Artiodactyla)]. / Diagnostichestkie vozmozhnosti mul'tilokusnykh markersov DNA v sistematike dikikh kopytnykh zhivotnykh (Artiodactyla).

The possibility to use three types of molecular genetic DNA markers for studying genomic variations and differentiation in wild ungulates (exemplified by Cervidae) was estimated. DNA markers were revealed via DNA fingerprinting, taxonomic typing, and random amplification of polymorphic DNA by polymerase chain reaction (RAPD PCR). The highest polymorphism of the markers was detected in roes (Capreolus spp.) RAPD PCR appeared to be the most efficient method for diagnosis of families, genera, and species. The degree of genetic similarity between species estimated by RAPD agreed with the generally accepted taxonomy of Cervidae. DNA polymorphism and interspecies differentiation of European and Siberian roe deer (C. capreolus and C. pygargus, respectively) are discussed.

[The craniometric characteristics of the elk (Alces alces L.) of Eurasia]. / Kraniometricheskaia kharakteristika losia (Alces alces L.) Evrazii.

Comparative craniometric investigation was done on the elk in Eurasia. Four morphological forms are distinguished, thus favoring the subdivision of Alces alces L. into four subspecies: A. a. alces, A. a. cameloides, A. a. pfizenmayeri, and A. a. buturlini.

Vertebrate liver cytokeratins: a comparative study.

The structure and composition of intermediate filaments isolated from liver of representatives of different vertebrate classes have been studied by electron microscopy and biochemical and immunochemical methods. It has been shown that the methodological approach for isolation of rat liver intermediate filaments can be efficiently applied to all other classes of vertebrates. The intermediate filaments studied have the same electron microscopic morphology and are species undistinguishable. The molecular weight of intermediate filament proteins varies from 40,000 to 60,000 and their isoelectric point varies from 5.0 to 6.45. Immunological investigations show that in all animals studied the intermediate filaments are built up of cytokeratins belonging to both types of keratins: type I and type II. Only one protein of the type II cytokeratins is present in all vertebrate classes, whereas in lower vertebrates two or even three type I cytokeratins contribute to the structure of liver intermediate filaments. The biochemical and immunochemical results are discussed with regard to the evolution of liver cytokeratins.

Sequence heterogeneity of the human alphoid satellite DNA and thermal stability of mismatched alphoid DNA duplexes.

1. 340 bp (dimer) and 680 bp (tetramer) fractions of the human alphoid satellite DNA (h alpha RI DNA) were isolated after complete cleavage of total human DNA with EcoR I and cloned in pBR 32.5. 2. Ten clones containing 340 bp inserts and one clone containing 680 bp insert were sequenced in order to investigate the sequence heterogeneity of this satellite DNA and the sequence data were compared with the consensus h alpha RI DNA sequence of Wu and Manuelidis (1980). 3. It was shown that in all clones studied the mutations are nonrandomly distributed along the human alphoid monomers forming distinct conservative and variable regions. 4. This mutation distribution pattern was compared with the nucleotide variations between the consensus sequences of different primate alphoid DNAs and it was found that the interspecies nucleotide divergency of this satellite DNA is quite similar to the intragenomic one. 5. The sequenced h alpha RI DNA clones were used for preparation of DNA-DNA hybrids with a known percentage of base pair mismatching. 6. These hybrids were melted on hydroxyapatite (HAP) and the results obtained were used to determine the relationship between the thermal stability (Tm) and the extent of base pair mismatching for naturally diverged DNA sequences. 7. A value of 0.7 degrees C decrease in Tm per 1% base pair mismatching was found.

Actin participates in the structure of liver intermediate filaments.

A dominating protein fraction (p45) having molecular weight of 45000 and pI 5.45 was found in the intermediate filaments pellet obtained from rat liver besides the present cytokeratins. Peptide mapping and radioimmunological assays with antibodies against this protein and muscle actin proved that the p45 protein belongs to the actin group. Immunoelectron microscopy revealed that this protein is located on the liver intermediate filaments. By melting of the cytokeratin complexes in urea it was established that p45 protein is complexed with the low molecular weight cytokeratin.

Does the concentration of DNA (Co) and the time of incubation (t) as parameters of Cot influence the thermal stability of the DNA duplexes?

It has been shown in a previous paper (8) that the prime product of reassociation of related DNA sequences under open experimental conditions are mismatched duplexes which undergo 'maturation' upon further incubation. Due to this feature, the Tm value of the duplexes of a large number of DNAs is strongly dependent on the Cot value. Here we present data showing that the Tm of the duplexes of such type of DNAs depends also on the concentration of DNA in the range of one and the same Cot value. The significance of this finding in studying the taxonomic relationship by DNA-DNA hybridisation is discussed.

Complex organization of a cryptic satellite DNA in the genome of the marine invertebrate Rapana thomasiana Grosse (Gastropoda).

Isopicnic centrifugation in Cs2SO4-Ag+ gradients at pH 7.0 reveals that the genome of the marine snail Rapana thomasiana Grosse (Gastropoda) contains an AT-rich satellite fraction comprising 5% of the DNA. Restriction enzyme analysis shows that the satellite DNA is composed of a number of related subsets arranged in tandem arrays. They have evolved from the segmental amplification of an 1460 bp long monomer unit with a complex inner organization. Most probably, the present basic repeat originates from an ancestral 400-500 bp long sequence in which some insertions and/or deletions have occurred.

Histone acetylation in chromatin containing mouse satellite DNA.

Histone acetylation in transcriptionally inactive chromatin has been studied with chromatin containing mouse satellite DNA. The latter was obtained by digestion of nuclei from Ehrlich ascites tumor cells with the restriction nuclease Bsp, which degrades main-band DNA but leaves satellite DNA intact. The enzyme-resistant material was separated by gel filtration. Satellite DNA amounted to 65% of the total DNA in this fraction. When the cells were grown in the presence of sodium n-butyrate to inhibit histone deacetylation, a few, if any, hyperacetylated forms of core histones were found in satellite chromatin. Conversely, the highest quantity of tetraacetylated H4 molecules was found in the fractions containing the most extensively degraded chromatin.

Interaction of homo-aza-steroidal ester of [p-[bis-(2-chloroethyl) amino]phenyl]acetic acid (ASE) with DNA of Ehrlich ascites tumor cells.

A cytostatic, homo-aza-steroidal ester of [p-[bis-(2-chloroethyl) amino]phenyl]acetic acid (ASE) was reduced with NaB3H4 and [3H]ASE-treated DNA prepared in vitro. We found that: (1) ASE reacts preferentially with purines; (2) ASE decreases the thermal stability of the double helix upon binding to DNA; (3) [3H]ASE binding sites are clustered along the DNA molecules; (4) ASE binding sites probably represent oligo- or polypurine sequences.

Studies on the thermal denaturation of histone-H1-depleted chromatin.

The multiphasic thermal denaturation profile of histone-H1-depleted chromatin was studied by using a nucleoprotein preparation which lacked the first high temperature transition at about 72 degrees C. Such a preparation was obtained by heating at 72 degrees C H1-depleted chromatin, the DNA of which was cross-linked with psoralen to ensure a complete renaturation of DNA upon cooling. When this nucleoprotein was redenatured, its melting profile was found to be significantly altered: only one high temperature peak centered at about 82 degrees C was observed in addition to the low temperature transition at about 53 degrees C. The kinetics of digestion of this material with micrococcal nuclease showed a limit digest equal to that found for the 'native' H1-depleted chromatin but the rate of hydrolysis was higher. The monomer particles prepared from this nucleoprotein were found similar to the 'native' monosomes in respect to protein:DNA ratio and size of DNA but showed an altered melting profile: the premelt area was broader, bigger, and centered at lower temperature; the main peak was reduced in size with no change in its melting temperature. On the basis of these data, the following conclusions were drawn: (a) the last two thermal transitions in H1-depleted chromatin most likely reflect the presence within each nucleosome of two regions with different stability of DNA; (b) DNA involved in the first high thermal transition of H1-depleted chromatin belongs to nuclease-accessible DNA, and (c) the main peak in the biphasic melting profile of the monomer particles reflects the denaturation of DNA regions which in the polymer nucleoprotein are involved in the two high temperature transitions.

Structural rearrangement of histone-H1-depleted chromatin during thermal denaturation.

The influence of thermal denaturation on the nucleosomal structure of histone-H1-depleted chromatin was studied using psoralen-treated nucleoprotein preparations subjected to partial thermal denaturation. DNA was cross-linked with psoralen to ensure its complete renaturation upon cooling. The structure of the preheated nucleoprotein was investigated by thermal denaturation, kinetics of hydrolysis and DNA fragment pattern obtained upon digestion with micrococcal nuclease. The electron micrographs of the partially denatured nucleohistone showed gross changes in the nucleosomal structure which were consistent with a sliding of histone cores along DNA as recently reported by Tsaneva et al. (Tsaneva, I., Dimitrov, S., Pashev, I. and Tsanev, R., FEBS Lett., (1980) 112, 143-146). This interpretation is strongly supported by the following features of the partially denatured material: a, increased rate of degradation of DNA by micrococcal nuclease; b, melting of a part of DNA as a protein-free DNA; and c, shortening of the DNA repeat length upon digestion with micrococcal nuclease. The sliding of the core histones is parallelled by the denaturation of histones, which accounts for the very intensive background in the DNA digestion pattern, the loss of nucleosome morphology at higher temperatures, and the disappearance in the melting profile of the transition at 72 degrees C.

Fractionation of native and reconstituted chromatin by digesting with deoxyribonuclease ii.

Native and reconstituted chromatin from Ehrlich ascites tumor cells were fractionated into template-active and inactive fractions by the DNAase II/Mg2+-solubility method of Gottesfeld et al. (Gottesfeld, J.M., Garrard, W.T., Bagi, G., Wilson, R.F. and Bonner, J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2193-2197). The Mg2+-soluble (template-active) fractions were compared in respect to sedimentation behavior in sucrose gradients and the relative content of specific transcribed (ribosomal) and non-transcribed (satellite) DNA sequences. It was found that the Mg2+-soluble fraction of the native chromatin was enriched in ribosomal DNA while almost completely devoid of satellite DNA; the nucleoprotein monomer of this fraction sedimented in sucrose gradient at 14 S. Similar-results were obtained if chromatin was fractionated in the presence of 3 M urea. With reconstituted chromatin, however, neither the sedimentation profile, nor the relative content of ribosomal and satellite DNA sequences were recovered, thus indicating that reconstitution did not yield nucleoprotein structurally similar to native chromatin.

Denaturation of mouse satellite DNA upon melting of chromatin in solution.

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.

On the heterogeneity of the slow reassociating ("unique") DNA.

The slow reassociating fraction of mouse DNA ("unique DNA"), when allowed to reassociate in 0.14 M sodoum phosphate buffer at 50 degrees C showed a biphasic melting curve with a transition at 78--80 degrees C. On the basis of this feature, the slow reassociating DNA was separated preparatively into two fractions: "unique DNA" I and II. Their duplexes showed differences with respect to thermal stability, S1 nuclease resistance and rate of reassociation. About one third of the sequences in each fraction were fraction-specific. The conclusion was drawn that for "unique DNA" I these should be the low repetitive or single copy related sequences (multigene families) and for "unique DNA" II--the unrelated single copy sequences or recent families of low repetitive not yet diverged sequences.

Comparative investigation of DNA from mouse and Ehrlich ascites tumor cells.

Mouse DNA and DNA of Ehrlich ascites tumor have been comparatively studied in the search for changes in DNA during tumor progression. No differences were found in kinetics of homologous (mouse X mouse) and heterologous (mouse X tumor) DNA reassociation; in thermal stability of homologous and heterologous duplexes of repeated, unique and satellite DNA; in percentage of hybridization with mouse liver heterogenous nuclear RNA; in thermal stability of the RNA.DNA hybrids. The negative results suggest that the considerable evolution of transplantable tumors (both in biological properties and in karyotype) has not been accompanied by alterations of the genome which could be detected by the now available methods of molecular hybridization for studying DNA divergence. In the light of the results obtained the functions ascribed to the mouse satellite DNA are discussed.