[Dynamic binding of the telomeric human protein TRF1 to intrachromosomal blocks (TTAGGG)n in living Chinese hamster cells depends on transcription].

In some vertebrates tandem repeats (TTAGGG)n are located not only in telomeres, but also in intrachromosomal sites. In Chinese hamster cells such interstitial repeats which may be called "telomeric" heterochromatin (THC), representing up to 5% of genome. Earlier we have shown, that blocks of THC dynamically bind telomeric protein TRF1 in Chinese hamster cells. In this work question has been studied whether this interaction depends on a transcription. In cells with the normal transcription around 85% of initial fluorescence intensity of GFP-TRF1 is restored in 60 sec after the photobleaching. Treatment of the cells with transcription inhibitor actinomycin D (ActD) in the concentration completely inhibiting activity of DNA-dependent RNA polymerases I and II (RPI and RPII) leads to fast and practically full suppression of exchange GFP-TRF1 (10% of initial fluorescence is restored only) whereas an inhibitor of protein synthesis cycloheximide (CHD) has not effect. At the low ActD concentration, suppressing only RPI, efficiency of recovery of fluorescence was not changed. Since some fractions of heterochromatin in mammalian cells are actively transcribed, exchange of GFP-TRF 1 can be connected to transcription of THC which may be necessary for synthesis of small interfering RNA and self-maintenance of the heterochromatin, or with inhibition of expression of other genes effecting TRF1 stability.

Dephosphorylation of histone gamma-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A.

The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called gamma-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that gamma-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase. Here we studied the dynamics of dephosphorylation of gamma-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of gamma-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the gamma-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, gamma-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites. We also found that calyculin A inhibited early elimination of gamma-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from gamma-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and gamma-H2AX and the coupling of its in situ dephosphorylation to DSB repair.

[Recognition of internal (TTAGGG)n repeats by telomeric protein TRF1 and its role in maintenance of chromosomal stability in Chinese hamster cells]. / Uznavanie vnutrennikh povtorov (TTAGGG)n telomernym belkom TRF1 i ego rol' v podderzhanii stabil'nosti khromosom v kletkakh kitaiskogo khomiachka.

Mammalian telomeres contain long tandem (TTAGGG)n repeats, which are protected by a complex of different proteins. Telomeric repeat-binding factors TRF1 and TRF2 play the key role in protection of telomeres through the formation of terminal loops (called T-loop). A T-loop isolates the 3' strand telomeric end and with this mechanism protects telomeres from the influence of enzymes of DNA reparation and telomere fusions and also interferes with the interaction of telomerase with telomeres. Many vertebrate species also contain large blocks of (TTAGGG)n sequences in pericentric and interstitial chromosome bands. The Chinese hamster genome contains a total of 18 arrays of these non-telomeric internal (TTAGGG)n sequences (ITs). Chromosome bands containing these arrays are unstable and should be protected with the help of another mechanism, rather than that using telomeres. In this study we analysed association of Green Fluorescent Protein (GFP)-tagged TRF1 in Chinese hamster V79 cells with ITs. We found that in these cells GFP-TRF1 associates with ITs in the interphase nucleus. We detected a little overlap between IT-associated GFP-TRF1 and random DSB sites visualized after the treatment of V79 cells with ionizing radiation. We found that the treatment of V79 cells with WM significantly increases the frequency of spontaneous chromosome aberrations. These WM effects are possible due to inhibiting phosphorylation of TRF1 by ATM. TRF1 is known to be eliminated from telomeres by overexpression of TANK1, which induces TRF1 poly(ADP-ribosyl)ation. We transfected V79 cells by plasmid encoding TANK1 and found that the frequency of chromosome rearrangements increased in these cells independently of their treatment by IR. Taken together, our results suggest that TRF1 may be involved in the sequence-specific protection of internal non-telomeric (TTAGGG)n repeats.

[The BCL-xL and ACR-1 genes promote differentiation and reduce apoptosis in muscle fibers of mdx mice]. / Geny BCL-xL i ACR-1 sposobstvuiut differentsirovke i umen'shaiut gibel' myshechnykh volokon myshei mdx.

The effects of the human BCL-xL and ACR-1 genes on dystrophin expression in cross-striated muscle fibers (CSMF) and on CSMF viability were studied in mdx mice after ballistic cotransfection with the human dystrophin minigene. In control mice, the proportion of dystrophin-positive (D(+)) and dying CSMF were 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. Introduction of the dystrophin minigene (20 micrograms of the pSG5dys plasmid) increased the proportions of D(+) and dying CSMF to 5.6 +/- 1.4% and 4.5 +/- 0.9%, respectively. When pSG5dys was introduced along with the pSFFV-Neo plasmid carrying the BCL-xL gene (10 micrograms of each plasmid per shot), the death of CSMF decreased to 3.7 +/- 1% and the proportion of D(+) CSMF significantly (P < 0.05) increased to 12.2 +/- 2.2%. Contransfection with the dystrophin minigene and the BCL-xL gene at 20 micrograms of each plasmid per shot did not stimulate generation of D(+) CSMF, but did reduce the CSMF death to 1.5 +/- 0.3%. Introduction of pSG5dys along with the pRc-CMV-10.1 plasmid containing the ACR-1 gene (10 micrograms of each plasmid per shot) reduced the proportion of D(+) CSMF to 1.1 +/- 0.5% and significantly reduced the proportion of dying CSMF to 0.9 +/- 0.3% as compared with the proportions observed in intact mice or in mice subjected to transfection with pSG5dys. Introduction of the pSG5dys plasmid substantially reduced the proportion of CSMF with peripheral nuclei, suggesting disturbed CSMF differentiation. After cotransfection with the human-dystrophin minigene, the BCL-xL and ACR-1 genes did not affect the extent of CSMF differentiation as compared with that observed in the case of the dystrophin minigene alone. Thus, ballistic transfection of mdx mice with the human dystrophin gene used along with the BCL-xL or ACR-1 gene was shown to suppress the death of muscle fibers and to expedite dystrophin synthesis and cell differentiation.

[Gene hACR-1 suppresses apoptosis of striated muscle fibres of m. quadriceps femoris after ballistic transfection of mdx mice]. / Gen hACR-1 podavliaet apoptoz poperechnopolosatykh myshechnykh volokon m.quadriceps femoris myshei mdx pri ballisticheskoi transfektsii.

Human minidystrophin gene (pSG5dys plasmid) and hACR-1 gene (pRc-CMV-10.1 plasmid) were cotransfected by means of "gene-gun" to M. quadriceps femoris of mdx mice. Effects of transfection on dystrophin expression and survival of striated muscle fibres (SMF) were studied on the 21st day after shots. In the control mdx dystrophin-positive muscular fibers [D(+)] SMF and destroyed SMF made 2.1 +/- 0.1 and 2.1 +/- 0.3%, respectively. In mice transfected with pSG5dys plasmid (20 mkg of DNA per mouse), the shares of D(+) SMF and dead SMF raised, respectively, up to 5.6 +/- 1.4 and 4.5 +/- 0.9%. Transfection of mice with pRc-CMV-10.1 (DNA dose is 20 mkg per mouse) reduced the levels of apoptosis in SMF and D(+) SMF level to 1.6 +/- 0.6 and 1.1 +/- 0.4%, respectively. Cotransfection by pSG5dys and pRc-CMV-10.1 plasmids (10 and 10 mkg of each plasmids DNA per mouse) reduced the share of D(+) SMF to 1.1 +/- 0.5% and SMF destruction to 0.9 +/- 0.3%. pSG5dys transfection considerably reduced the share of SMF having peripherally located nuclei, thus indicating a decrease in SMF differentiation level after transfection. Cotransfection of ACR-1 gene and a dystrophin minigene did not suppress further cytodifferentiation of mdx muscle fibers. A conclusion is made that ballistic transfection by hACR-1 gene reduces the level of apoptosis in mdx mice SMF without changing the level of SMF differentiation. The cotransfection of mdx mice muscle by hACR-1 and human minidystrophin gene reduces SMF destruction and supports SMF differentiation, too.

A negative regulator of telomere-length protein trf1 is associated with interstitial (TTAGGG)n blocks in immortal Chinese hamster ovary cells.

Telomeres of mammalian chromosomes are composed of long tandem repeats (TTAGGG)n which bind in a sequence-specific manner two proteins-TRF1 and TRF2. In human somatic cells both proteins are mostly associated with telomeres and TRF1 overexpression resulting in telomere shortening. However, chromosomes of some mammalian species, e.g., Chinese hamster, have large interstitial blocks of (TTAGGG)n sequence (IBTs) and the blocks are involved in radiation-induced chromosome instability. In normal somatic cells of these species chromosomes are stable, indicating that the IBTs are protected from unequal homologous recombination. In this study we expressed V5-epitope or green fluorescent protein (GFP)-tagged human TRF1 in different lines of mammalian cells and analyzed distribution of the fusion proteins in interphase nucleus. As expected, transient transfection of human (A549) or African green monkey cells with GFP-N-TRF1 or TRF1-C-V5 plasmids resulted in the appearance in interphase nuclei of multiple faint nuclear dots containing GFP or V5 epitope which we believe to represent telomeres. Transfection of immortalized Chinese hamster ovary (CHO) cell line K1 which have extremely short telomeres with GFP-N-TRF1 plasmid leads to the appearance in interphase nuclei of large GFP bodies corresponding in number to the number of IBTs in these cells. Simultaneous visualization of GFP and IBTs in interphase nuclei of transfected CHO-K1 cells showed colocalization of both signals indicating that expressed TRF1 actually associates with IBTs. These results suggest that TRF1 may serve as general sensor of (TTAGGG)n repeats controlling not only telomeres but also interstitial (TTAGGG)n sequences.

[Units of DNA replication in S-phase human cells]. / Edinitsy replikatsii DNK v S-faznykh kletkakh cheloveka.

Current methods of physical mapping allow the estimation of genomic distances (i.e. DNA contents) from linear distances between DNA markers in interphase nuclei, and in this study we estimated the size of focal centers of DNA replication in cultured S-phase human cells. Our results indicate that the conformation of S-phase chromosome fibres in the range of contour lengths 0.1-3.0 microns fits the random walk model and, therefore, the quantitative methods of interphase mapping can be applied to the estimation of sizes of replication units. The obtained data show the existence of multiple non-clustered small units less than 150 kb in size, equivalent to small replicons detected by fiber DNA radioautography, and also a significant fraction of big units more than 500 kb in size, representing groups of small replicons and/or big replicons. These big units are detected as chains of small replication foci, probably reflecting the structural chromatin organization in well-known loop domains, since the experimentally induced decrease of replicons to the average size 12 kb does not lead to any change in the pattern of indicated chains.

[The physical mapping of human chromosomes. II. The production of unique chromosome-specific DNA fragments using the polymerase chain reaction with oligonucleotide primers to conservative regions of Alu repeats]. / Fizicheskoe kartirovanie khromosom cheloveka. II. Poluchenie unikal'nykh khromosom-spetsificheskikh fragmentov DNK s pomoshch'iu polimerzanoi tsepnoi reaktsii s oligonukleotidnymi zatravkami na konservativnye uchastki Alu-povtorov.

Unique DNA fragments localised between Alu-repeats have been produced by PCR. The reaction was carried out with oligonucleotide primers to conservative regions of Alu-repeats. The DNA fragments from different pulls, individual clones, chromosome-specific clonotecs derived from phage lambda, cosmids and individual human chromosomes served as matrixes. The possibilities are discussed of Alu-primer applying in production of exceptional physical features of DNA molecules, suitable for constructing clone couple groups and for direct physical mapping on the DNA of isolated chromosomes, missing the stage of cloning.

[The DNA-binding activity of the membrane protein annexin II and its interaction with antibodies to the chromatin nuclear ribonucleoprotein (alpha-RNP)]. / DNK-sviazyvaiushchaia aktivnost' primembrannogo belka anneksina II i ego vzaimodeistvie s antitelami k iadernomu ribonukleoteinu khromatina (al'fa-RNP).

By screening with labeled Alu DNA, a clone was isolated from cDNA expression library, which appeared identical in sequence to the well-known Ca-phospholipid-binding protein annexin II. To evidence the DNA-binding activity of recombinant annexin II and its presence in the cell nucleus, we have expressed full-length mouse annexin II cDNA in bacteria with pGEMEX vector. The expressed protein was studied with electrophoretic mobility shift assay and for its reaction with polyclonal antibody to chromatin-associated ribonucleoprotein (alpha-RNP), which is one of the major acid-dissolvent components of the nucleus. The obtained results confirm the DNA-binding activity of recombinant annexin II. Annexin II reacts with polyclonal antibody to rat alpha-RNP. So, annexin II is a major nuclear DNA-binding protein in mammalian cells.