A. E. Braunstein Plenary Lecture. Nuclear skeleton, DNA domains and control of replication and transcription.

Chromosomal DNA is organized in loops or domains of about 100 kb. Their ends seem to be attached to special protein skeletal structures. The DNA-attachment sites can be subdivided into permanent and transient types. The permanent or constitutive attachment sites, which are retained in all types of cells (including those inactive in replication and transcription), either coincide with or are located close to replication origins. This observation provides a simple way for isolation of DNA fragments containing replication origins. Such fragments from the chicken alpha-globin gene domain and other regions of the chicken genome contain DNA sequences which interact with nuclear proteins present in dividing cells, but absent from non-dividing cells. Several new consensus sequences interacting with nuclear proteins were detected. The 5' end region of the alpha-globin gene domain containing a replication origin was found to possess enhancer activity lacking tissue specificity. Hence, the domain organization of DNA is related to the organization of replication process. Other sets of data indicate that the integrity of DNA domains is important for maintaining transcription within the domain. According to these data, even a single nick at an distance of about 100 kbp seems to be sufficient for blocking transcription within the whole domain at the stage of RNA elongation. Thus, topological integrity of DNA may be an important factor involved in formation of active chromatin.

Complexes of nuclear matrix DNA with proteins tightly bound to DNA contain a specific small-size RNA of a novel type.

Analysis of DNA-protein structures composed of nuclear matrix attached DNA and the most tightly bound proteins was performed. Although the previously described non-histone proteins (1) were present the buoyant density of the complex was the same as that of pure DNA. RNA inaccessible to RNase in 0.4 M NaCl but digestible in low ionic strength buffer was detected. This RNA is not a nascent one. It turned out to be homogeneous and represent a novel type of small nuclear RNA. Partial sequence of this RNA is presented.

DNA-protein complexes of the nuclear matrix: visualization and partial characterization of the protein component.

Complexes composed of DNA attached to the nuclear matrix and of proteins most tightly bound to DNA are visualized as globular particles 25-35 nm in diameter. Their morphology depends greatly on the isolation conditions: a Cs salts/urea combination permits the isolation while CsCl/sarcosyl destroys the particles. The preparation is shown to have the same protein content regardless of the treatment employed. The proteins of the complex are resistant to SDS and pronase treatment and to phenol/chloroform extraction while being associated with DNA.

The distribution of tightly bound proteins along the DNA chain reflects the type of cell differentiation.

Distribution of proteins tightly bound to DNA (TBP) along the DNA chain was found to depend on the cell lineage. DNA sequences coding for alpha globin genes are preferentially represented in the TBP-associated DNA fractions isolated from erythroid cells (erythroblasts, mature erythrocytes, differentiated and non-differentiated Friend erythroleukemia cells), but not from cultured fibroblast cells. In transcriptionally active nuclei, complexes of TBP interact with the nuclear matrix, while in mature erythrocytes this interaction disappears.

[DNA-bound proteins mediate attachment to the nuclear skeleton of transcriptionally active DNA fraction]. / Prochno sviazannye s DNK belki oposreduiut prikreplenie k iadernomu skeletu transkriptsionno aktivnoi fraktsii DNK.

The proteins tightly bound to DNA are non-randomly distributed along the DNA chain and are concentrated within the transcriptionally active areas. The distribution of tightly bound proteins along the DNA reflects the type of cell differentiation and does not depend directly on transcription. In functionally active nuclei, the DNA-tightly bound proteins complexes are fixed at the nuclear matrix which is therefore associated with the whole transcriptionally active DNA fraction.

[Composition, structure and various properties of DNA-protein complexes located at the sites of DNA loop attachment to the nuclear skeleton]. / Sostav, stroenie i nekotorye svoistva kompleksov DNK s belkom, raspolozhennykh v uchastkakh prikrepleniia petel' DNK k iadernomu skeletu.

The intimate structure of the complexes located at the sites of DNA loops attachment to the nuclear skeleton was analysed. It is shown that: there are at least three components of the attachment site complex: DNA, protein, RNA; protein moiety consists of 7-8 species with Mr 70-17 kDa. Their association with DNA is resistant to ionic detergents, high salt and urea treatments. The DNA-protein complex is also resistant to the SDS-pronase-phenol deproteinisation procedure; the buoyant density of the complex is the same as DNA density. RNase digestion at low ionic strength reduces density of the complex while the same treatment at 0,4 M NaCl has no effect; DNA-protein complexes isolated with urea-high salt treatment are visualised as globular particles 25-35 nm in diameter with DNA loops attached. These particles were not observed after detergent treatment although protein composition of the complex remained the same.

[Isolation and fractionation of the nuclear matrix from cultured cells]. / Vydelenie i fraktsionirovanie iadernogo matriksa iz kul'tiviruemykh kletok.

A new method for the isolation of tissue culture cell nuclei is presented which involves incubation of the nuclei in the presence of Cu2+- or Zn2+-ions. This method eliminates the danger of nuclear aggregation and permits nuclear matrix isolation and subsequent fractionation. Stabilization of the inner matrix by Cu2-ions permits analysis of the role of nucleic acids in the maintenance of the matrix structure. It is shown that solubilization of more than 95% of matrix-bound DNA and more than 90% of matrix-bound RNA did not cause any significant changes in the nuclear matrix structure.

[Sodium permeability of cardiac cells exposed to nembutal]. / Natrievaia pronitsaemost' serdechnykh kletok pri deistvii nembutala.

A study was made of the effect of anesthetic concentrations of pentobarbital on sodium permeability of the myocardial cell membranes by recording action potentials of the frog myocardial cells, tension fixation by the double sucrose bridge method on frog atrial trabecula and by the voltage clamp method during tension fixation on a rat isolated myocardial cell membrane. It is concluded that pentobarbital has no effect on rapid sodium current of the myocardial cell membranes.

Calmodulin-dependent regulation of calcium-activated outward current in frog atrial membrane.

The steady-state outward current underlying the inward-going rectification in frog atrial fibers has been studied by the double sucrose gap technique. Similar to the case in sheep Purkinje fibers (18,19), the inward rectifying potassium channels in frog atrium are blocked by cesium (5 mM) and activated by increasing cytosolic calcium concentration (replacing 30% of the Na by sucrose). It was shown that trifluoperazine (5 x 10(-6) M), a widely used blocker of the Ca-calmodulin complex, inhibits the inward rectifying potassium channels. Diphenylhydantoin (5 x 10(-6) M), a putative inhibitor of Ca-calmodulin-mediated membrane phosphorylation produces a similar action. The inhibitory action of both blockers is not manifested after preliminary Cs+ (5 mM) exposure. The data obtained suggest that calcium activates the inward rectifying potassium channels in frog atrial fibers via Ca-calmodulin regulatory phosphorylation of the membrane proteins.

Proteins tightly bound to DNA in the regions of DNA attachment to the skeletal structures of interphase nuclei and metaphase chromosomes.

Proteins tightly bound to the DNA of mouse L cells were examined both in the regions of DNA attachment to the protein skeleton and in the DNA loops. The interphase nuclear matrix and the metaphase chromosomal scaffold, containing from 2 to 15% of the original DNA, as well as the material released from the skeleton were isolated by mild nuclease treatment. At least six proteins resistant to sarcosyl-Cs2SO4 treatment are associated exclusively with skeleton-attached DNA both in interphase nuclei and metaphase chromosomes. Two (p52 and p60) can be dissociated from the complex by mercapto-ethanol-sarcosyl treatment. Both skeleton-attached and released DNA fractions contain a group of tightly bound low molecular weight polypeptides consisting of a major p18 band and one or two minor bands. They seem to be randomly distributed throughout the whole DNA loop. Some of the specific proteins that are tightly associated with DNA at the site of its interaction with the skeleton may directly be responsible for DNA attachment to the skeleton.