Long interstitial (TTAGGG)n arrays do not colocalize with repressive chromatin modifications in Chinese hamster cells.

Interstitial tandem (TTAGGG)n repeats (ITRs) in Chinese hamster (CH) cells are mostly arranged into the long (>100kb) continuous pericentric arrays which do not contain HindIII sites (Faravelli M, Azzalin CM, Bertoni L, Chernova O, Attolini C, Mondello C, et al. Molecular organization of internal telomeric sequences in Chinese hamster chromosomes, Gene 2002;283,11-16), are free of protein-coding genes, and can formally be classified as heterochromatin. ITRs dynamically interact with (TTAGGG)n-binding protein TRF1 and can be visualized using Green Fluorescent Protein (GFP)-tagged TRF1. Here we examined whether mobility of GFP-TRF1 associated with ITRs in CH cells is affected by inhibitors of transcription and whether ITRs colocalize with known repressive chromatin modifications hallmarked by histone H3 trimethylated at lysine-9 (H3K9m3) or lysine-27 (H3K27m3). We found that GFP-TRF1 bodies do not colocalize in the nuclei of V79 cells with H3K9m3 or H3K27m3 indicating that ITRs do not represent typical constitutive or facultative heterochromatin. Mobility of ITR-bound GFP-TRF1 is suppressed by inhibitors of transcription consistent with the view that TRF1 is exchanged during transcription of ITRs. However, GFP-TRF1 bodies do not colocalize with nuclear hubs of intensive transcription detected through in vivo incorporation of 5-bromouridine triphosphate. Using real-time PCR, we also examined transcription of unique sequences adjacent to (TTAGGG)n arrays in CH genome and found that they are transcribed, indicating that these arrays do not generally inhibit transcription in cis. Together, our results suggest that ITRs represent a special kind of moderately transcribed heterochromatin which possible function remains to be established.

[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.

[Evaluation of the heritability of basic parameters of aging in Drosophila melanogaster]. / Otsenka nasleduemosti osnovnykh parametrov stareniia u Drosophila melanogaster.

Based on 28 annuity curves of Drosophila melanogaster, the heritability of Gompertz mortality parameters was estimated; its value was 0.7-0.8. Mortality parameter R0 was shown to be inversely proportional to the square of alpha. Some data indicate the monogenic control of interlinear differences in these parameters. A hypothesis on the localization of these genes on chromosome 2 is discussed.

[A comparative genetic analysis of interlinear differences in intensity of lipid peroxidation and life span in Drosophila melanogaster. Inbred lines and hybrids]. / Sravnitel'no-geneticheskii analiz mezhlineinykh razlichii v intensivnosti perekisnogo okisleniia lipidov i prodolzhitel'nosti zhizni u Drosophila melanogasters. Inbrednye linii i gibridy.

The interrelationship between the level of peroxidation lipid and longevity was studied as based on highly inbred lines of Drosophila melanogaster, bred for adaptively important characters. The concepts of the free-radical theory proved valid for the lines and failed to be supported for the crosses. The dominant role of heterosis in the protecting against the "aging action" of free radicals is postulated.

[The dynamics of mortality in inbred selected strains and their hybrids in Drosophila melanogaster]. / Dinamika smertnosti v inbrednykh selektiruemykh liniiakh i ikh gibridakh u Drosophila melanogaster.

The average life span (ALS) was analyzed in flies of the Drosophila melanogaster inbred stock selected for high embryonic mortality (HEM). In is shown that selection resulted in the reduced ALS of HEM stock, as compared with the initial outbred Leric stock. Two equal peaks of mortality were observed in the hybrid females. The peaks mean values usually corresponded to ALS of the parents. A major peak of mortality was detected in hybrid males, and its mean was higher than ALS values characteristic of both parents. The existence of genes controlling "early" and "late" death in D. melanogaster ontogenesis is proposed.

[The relationship between lipid peroxidation and life span in Drosophila melanogaster]. / K voprosu o sviazi perekisnogo okisleniia lipidov i prodolzhitel'nosti zhizni u Drosophila melanogaster.

Using Drosophila lines pre-selected for adaptive characters, studies have been made on the relationship between the intensity of lipid peroxidation and life span in flies of different genotype, age and physiological condition. It was shown that the intensity of lipid peroxidation depends mainly on different factors (sex, age, virginity) in different lines. No expected negative correlation was found between the level of lipid peroxidation and life span in hybrids between two inbred lines. The intensity of lipid peroxidation was inherited like a dominant character, the dynamics of aging--like a codominant one in females and superdominant in males. Both the level of lipid peroxidation and life span appeared to be sex-dependent characters.

[Biochemical indicators of vitamin provision to Orenburg's schoolchildren and effectiveness of prophylactic vitamin administration]. / Biokhimicheskie pokazateli vitaminnoi obespechennosti shkol'nikov Orenburga i éffektivnost' profilakticheskoi vitaminizatsii.

The study of providing with vitamins of schoolchildren in Orenburg conducted in the spring of 1985-1986 has revealed decreased content of ascorbic acid in 92%, vitamin E in 62%, vitamin A in 39%, folic acid in 38%, vitamin B12 in 14% of the children, the incidence rate of vitamin deficiency among the children in the senior forms was higher as compared to that in the junior forms and in boys it was more frequent than in girls. Additional intake of multivitamin "Undevitum" by schoolchildren during 2-4 months led to a significant improvement of their providing with vitamins that was expressed in normal vitamin levels and elimination of cases of deep vitamin deficiency.

[Providing with vitamins of river transport workers of the Western and Eastern Siberia and oil men of the Tyumen Region]. / Obespechennost' vitaminami rechnikov Zapadnoi i Vostochnoi Sibiri i neftianikov Tiumenskoi oblasti.

The investigation of providing with vitamins of river transport workers in Western and Eastern Siberia and of oil men in the Tyumen Region conducted in the summer period of 1984-1986 has revealed significant deficiencies of vitamins C, E, B1, B2, B6, A and carotene in them. Vitamin administration to the workers with "Undevitum" resulted in a significant improvement of their vitamin status that was expressed in elevation of their biochemical parameters and in diminution of the percentage of subjects with vitamin deficiencies.

[Provision of workers and employees of Kuzbass industrial enterprises with vitamins C, E, A and beta-carotene]. / Obespechennost' rabochikh i sluzhashchikh promyshlennykh predpriiatii Kuzbassa vitaminami C, E, A i beta-karotinom.

The content of vitamins C, E, A and beta-carotene was studied in the blood serum of 404 workers and employees of the Kuzbass industrial enterprises. Insufficient providing of the body with ascorbic acid, tocopherol and beta-carotene has been revealed. The number of workers and employees with vitamin C deficiency comprises at an average 95%, with vitamin E deficiency--9%, and with beta-carotene deficiency--34%. Industrial enterprises characterized by hard physical labor and occupational health hazard have been found to be most unfavourable with respect to providing with vitamins. The results of the investigations conducted have evidenced the necessity of correcting the vitamin nutrition of the subjects studied.

[Vitamin allowance of cosmonauts during preflight training and after completing short space flights]. / Obespechennost' vitaminami kosmonavtov v period predpoletnoi podgotovki i posle zaversheniia kratkovremennykh kosmicheskikh poletov.

Supply of the main and doubling crews of cosmonauts with vitamins C, A, D, E, B1, B2, B6, PP, and B12, folic acid and carotinoids was studied during training and after the completion of short-term space flights (from 4 to 13 days). As the result of the investigation conducted during the intensive training, 1 month before the space flight some cases of certain vitamins deficiency were revealed. Administration of additional vitamins during 2 weeks before the flight led to a rise in the cosmonauts' supply with vitamins. The findings obtained after the short-term space flights evidence an intensive metabolization of vitamins and increased vitamin requirements during this period.

[Vitamin supply of premature infants with intracranial birth injuries fed in different ways]. / Obespechennost' vitaminami nedonoshennykh detei s vnutricherepnoi rodovoi travmoi, nakhodivshikhsia na raznykh vidakh vskarmlivaniia.

A total of 136 premature infants with intracranial birth trauma were studied during the first 2 months of life for vitamin B, C, A and carotinoid supply with regard to their body mass at birth and type of feeding. Premature children with different patterns of birth trauma showed low supply with thiamine, riboflavin, niacin and vitamin A in the presence of breast feeding. Combined feeding raised the supply of the children under observation with thiamine and riboflavin. The data obtained attest to the necessity of further study in this direction with the purpose of developing pathogenetically validated schemes for vitamin therapy of premature children.

[Vitamin status of premature infants in the 1st 2 months of life on natural feeding]. / Vitaminnyi status nedonoshennykh detei pervykh 2 mes zhizni na estestvennom vskarmlivanii.

Fifty premature neonates were followed during the first 2 months of life to study the supply of the body with vitamins A, B6, ascorbic acid, riboflavin, thiamine, niacin and carotenoids with regard to the body weight upon birth and prematurity grade. The children were kept on natural feeding (donor's breast milk). Premature neonates were found to be short of individual vitamins during certain periods of life. The data obtained indicate that it is desirable to prevent vitamin deficiency by additional vitaminization with regard to the body weight upon birth and prematurity grade.