[Radiation biology of structurally different Drosophila melanogaster genes. Report I. The vestigial gene: molecular characteristic of "point" mutations].

The screening of PCR-detected DNA alterations in 9 spontaneous and 59 gamma-ray-, neutron - or neutron + gamma-ray-induced Drosophila vestigial (vg) gene/"point" mutations was carried out. The detected patterns of existence or absence of either of 16 overlapping fragments into which vg gene (15.1 kb, 8 exons, 7 introns) was divided enable us to subdivide all mutants into 4 classes: (i) PCR+ (40.7%) without the detected changes; (ii) "single-site" (33.9%) with the loss of a single fragment; (iii) partial detections (15.2%) as a loss of 2-9 adjacent fragments and (iv) "cluster" mutants (10.2%) having 2-3 independent changes of(ii) and/or (iii) classes. All spontaneous mutants except one were found to be classified as (ii) whereas radiation-induced mutants are represented by all 4 classes whose interrelation is determined by the dose and radiation quality. In particular, the efficacy of neutrons was found to be nine times as large as that of gamma-rays under the "cluster" mutant induction. Essentially, the distribution of DNA changes along the gene is uneven. CSGE-assay of PCR+-exon 3 revealed DNA heteroduplexes in 5 out of 17 PCR+-mutants studied, 2 of which had small deletions (5 and 11 b) and 3 others made transitions (A --> G) as shown by the sequencing. Therefore, gamma-rays and neutrons seem to be significant environmental agents increasing the SNP risk for the population through their action on the germ cells. The results obtained are also discussed within the framework of the track structure theory and the notion of quite different chromatin organization in somatic and germ cells.

Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation.

Cryopreservation as a process can be divided into two methods: conventional freezing and vitrification. The high effectiveness of vitrification in comparison with conventional freezing for human oocytes and embryos is shown, whereas data on human ovarian tissue are limited. The aim of this study was to compare the safety and effectiveness of conventional freezing and vitrification of human ovarian tissue. Ovarian tissue fragments from 15 patients were transported to the laboratory within 22-25 h in a special, isolated transport box that can maintain a stable temperature of between 5 and 8 degrees C for 36 h. Small pieces of ovarian tissue (0.3-1 x 1-1.5 x 0.7-1 mm) were randomly distributed into three groups: group 1, fresh pieces immediately after receiving transport box (control); group 2, pieces after vitrification; and group 3, pieces after conventional freezing. After thawing, all the pieces were cultured in vitro. The viability and proliferative capacity of the tissue by in vitro production of hormones, development of follicles, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression after culture were evaluated. A difference between freezing and vitrification was not found in respect to hormonal activity and follicle quality. The supernatants showed 17-beta estradiol concentrations of 365, 285, and 300 pg/ml respectively, and progesterone concentrations of 3.82, 1.99, and 1.95 ng/ml respectively. It was detected that 95, 80, and 83% follicles respectively were morphologically normal. The molecular biological analysis, however, demonstrated that the GAPDH gene expression in ovarian tissue after vitrification was dramatically decreased in contrast to conventional freezing. For cryopreservation of human ovarian tissue, conventional freezing is more promising than vitrification, because of higher developmental potential.

Pausing, switching and speed fluctuation of the bacterial flagellar motor and their relation to motility and chemotaxis.

Wild-type Escherichia coli and Salmonella typhimurium cells, tethered to glass by their flagella, rotate with brief intermittent pauses, the prevalence of which is decreased by attractants and increased by repellents. By attaching latex beads to filaments of a S. typhimurium mutant having straight rather than helical flagella, it was established that the flagella on free cells also pause intermittently. Pausing is therefore an intrinsic feature of the motor and not an artifact associated with tethering. In tethered cells of wild-type strains and non-chemotactic mutants defective in transducers, chemotaxis proteins, or the flagellar switch, both the classical response to chemotactic stimuli (change in direction of rotation from counterclockwise to clockwise or vice versa), and the pausing response to such stimuli, were linked together. No separate signal for pausing was found. In comparing different strains under different stimulation conditions, it was found that cells that never reversed seldom if ever paused, while cells that reversed frequently paused frequently. It is suggested that pausing is the result of futile switching events. A modified description of tumbling and chemotaxis is provided in which pausing, as well as reversal, has a role. Suppression of reversals and pauses by attractant stimuli commonly resulted in an increase in the speed of counterclockwise rotation; this may be because of suppression of pauses or reversals that are too brief to be detected. The clockwise rotation rate of unstimulated cells, which commonly was faster than their counterclockwise rate, was not further increased by repellent stimuli. The rotation rate of any given cell under any given condition was found to fluctuate on all time-scales measured. The study also revealed that some of the common repellents of E. coli and S. typhimurium slow down or stop the motor; these effects are not mediated by the chemotaxis machinery or intracellular pH.

Relationship between sedimentation behaviour of DNA-protein complexes and DNA single- and double-strand breaks after irradiation with gamma-rays, pulsed neutrons and 12C ions.

The sedimentation behaviour of DNA-protein complexes was studied following irradiation of Chinese hamster cells (V79-4) and human lymphocytes over a wide dose range of 137Cs gamma-rays, pulsed neutrons and accelerated 12C ions. We have shown that the decrease of relative sedimentation velocity of the complexes at low doses is related to the occurrence of single-strand breaks in DNA. Rejoining of the breaks during a repair period increases the sedimentation velocity. At higher doses of radiation, double-strand breaks lead to an increase of sedimentation velocity of DNA-protein complexes. A new method can be devised on the basis of these results enabling estimation of the yields of single- and double-strand breaks in DNA.

[The "2 + 1" mechanism as the basis for synergism in the neutron-photon irradiation of the spermatozoal genome in Drosophila melanogaster]. / Mekhanizm "2 + 1" kak osnova singergizma pri neitron-fotonnom obluchenii genoma spermiev Drosophila melanogaster.

Cytogenetic analysis of polytene chromosomes of Drosophila melanogaster locus-specific mutants induced by consecutive neutron-photon irradiation has shown that their genome contains multiple intra- and inter-chromosome exchanges, including triradials, evidencing the synergistic action of such combined exposure. The appearance of the triradials may be only possible on the base of an interaction between a double and a single DNA strand breaks. The important significance of such interaction as the general mechanism for production of chromosome aberrations in irradiated cells of higher eucaryotes had been postulated by N. V. Luchnik as early as 10 years ago, but only nowadays it has been confirmed experimentally.

[The radiation cytogenetics of multilocus deletions and the principles of the superchromomeric organization of eukaryotic euchromatin]. / Radiatsionnaia tsitogenetika mul'tilokusnykh deletsii i printsipy nadkhromomernoi organizatsii éukhromatina éukariot.

Fine cytological analysis of 72 gamma ray- and neutron-induced multilocus deletions (MLD) at the b, cn and vg regions of Drosophila melanogaster polytene chromosomes combined with the complementation assay at the b region is used to detect precisely the size and location of the MLD ends relatively to chromomeres and to each other. The basic principles of the MLD induction are found to be the same for three studied genome regions, showing that the folding of the interphase chromatin at the superchromomeric level is non-random and follows the megasolenoid-rosette model which is presented and discussed.

[Relative genetic effectiveness of fission neutrons in inducing various types of recessive mutation in Drosophila melanogaster]. / OGE neitronov deleniia pri induktsii retsessivnykh mutatsii raznogo tipa u Drosophila melanogaster.

The RCR-analysis of 53 gamma-Ray- and neutron-induced vg recessive mutations of Drosophila melanogaster combined with complementation assay with the vg[nw83b27] deletion mutation is used to detect precisely the RGE values of neutrons (0.85 MeV) under the chromosome and point (at the DNA level) mutation induction. Simultaneously, the induction-kinetics of gamma-ray- and neutron-induced macrodeletion as well as recessive lethal mutations in the X-chromosome were studied. The results obtained have shown that all genetic end-points increase linearly with gamma-ray or neutron dose. Thereby, the efficacy of neutrons is found to be twice (and more) as large as that of gamma-rays under the all macro- and micro-aberration mutation induction (macrodeletions and recessive lethals in the X-chromosome, multilocus deletions and intragenic deletions as well). Unlike that, the RGE of neutrons are more than twice as low as that of gamma-rays under the gene/point mutation induction. This feature of neutrons have been predicted as far back as in the early days of the radiation genetics (N.W. Timofeeff-Ressovsky, K.G. Zimmer, 1938), but experimentally supported at the DNA level just now.

Analysis of a model for multiseptation in bacteria.

The predictions of a model for multiseptation in bacteria recently proposed by Paulton are derived mathematically. It is shown that independent of the growth rate, the time between septum formation and cell division is given by t(s) = S . t, where S is the average number of successive division sites and t is the generation time. This result is in good agreement with the experimental data.

[Yield of sister chromatid exchanges and survival of V79-4 Chinese hamster cells irradiated with 0.7 MeV neutrons]. / Vykhod sestrinskikh khromatidnykh obmenov i vyzhivaemost' kletok kitaiskogo khomiachka U79-4 pri obluchenii neitronami s énergiei 0.7 MéV.

The dependence of the survival rate and the number of sister chromatid exchanges (SCEs) in Chinese hamster V79-4 cells on the dose of gamma-rays and neutrons with average energy of 0.7 MeV has been investigated. The value of RBE for neutrons is 5.5. The number of SCEs increased with the dose of gamma-radiation while no induction of SCEs could be detected after neutron irradiation.

Model for the chemotactic response of a bacterial population.

We present a mathematical model for the motion of a bacterial population in prescribed attractant or repellent gradients. The model is suggested by the observations of Mesibov et al. (1973, J. Gen. Physiol. 62:203) and Brown and Berg (1974, Proc. Natl. Acad. Sci. U.S.A. 71:1388) who found that the sensitivity of the chemotactic response depends on the concentration of attractant. Predictions of the theory are in general agreement with the experiments of Dahlquist et al. (1972, Nat. New Biol. 236:120) and of Mesibov et al. on populations of motile bacteria in fixed attractant gradients. Additional tests of the model are proposed.

A mathematical model for bacterial chemotaxis.

A differential equation describing the chemotactic migration of a bacterial population in a fixed exponential gradient of attractant has been integrated using the appropriate boundary conditions. The solution predicts an initial bacterial accumulation at the concentration "knee" with the final distribution of bacteria approaching a time-independent state. Specific additional experiments to obtain further data for a rigorous test of the theory are suggested.

A model for traveling bands of chemotactic bacteria.

A theoretical model is used to study band formation by chemotactic populations of Escherichia coli. The model includes the bacterial response to attractant gradients, the chemotactic sensitivity of the bacteria to the concentration of the attractant, and population growth. For certain values of the parameters in the model, traveling bands of bacteria form and propagate with or without growth. Under specific growth conditions the band profile is maintained and the band propagates at constant speed. These predictions are in general agreement with the experiment results of J. Adler and earlier theoretical work by L. Segel and his collaborators. However, our theory differs in several important respects from the latter efforts. Suggestions are made for further experiments to test the proposed model and to clarify the nature of the processes which lead to band formation.

Gliding motility of Cytophaga sp. strain U67.

Video techniques were used to analyze the motion of the gliding bacterium Cytophaga sp. strain U67. Cells moved singly on glass along the long axis at a speed of about 2 micrometers/s, advancing, retreating, stopping, pivoting about a pole, or flipping over. They did not flex or roll. Cells of different lengths moved at about the same speed. Cells sometimes spun continuously about a pole at a frequency of about 2 HZ, the body moving in a plane parallel to that of the glass or on the surface of a cone having either a large or a small solid angle. Polystyrene latex spheres moved to and fro on the surfaces of cells, also at a speed of about 2 micrometers/s. They moved in the same fashion whether a cell was in suspension, gliding, or at rest on the glass. Two spheres on the same cell often moved in opposite directions, passing by one another in close proximity. Small and large spheres and aggregates of spheres all moved at about the same speed. An aggregate moved down the side of a cell with a fixed orientation, even when only one sphere was in contact with the cell. Spheres occasionally left one cell and were picked up by another. Cell pretreated with small spheres did not adhere to glass. When the cells were deprived of oxygen, they stopped gliding, and the spheres stopped moving on their surfaces. The spheres became completely immobilized; they no longer moved from cell to cell or exhibited Brownian movement. Cytophaga spp. are known to have a typical gram-negative cell envelope: an inner (cytoplasmic) membrane, a thin peptidoglycan layer, and an outer (lipopolysaccharide) membrane. Our data are consistent with a model for gliding in which sites to which glass and polystyrene strongly adsorb move within the fluid outer membrane along tracks fixed to the rigid peptidoglycan framework.

[Effect of gamma-irradiation and neutrons on DNA-membrane complexes in mammalian cells]. / Vozdeistvie gamma-izlucheniia i neitronov na DNK-membrannye kompleksy kletok mlekopitaiushchikh.

The first results of the radiobiological studies obtained in a biophysical channel of the IBR-2 reactor at the Joint Institute for Nuclear Research are presented in this report. A study was made of the DNA-membrane complex sedimentation after exposure of Chinese hamster cells V79-4 to gamma-rays within a wide range of doses. The assumption, that had earlier been forwarded by the authors, of the role of double-strand DNA breaks in the alteration of a relative rate of the complex sedimentation upon irradiation of cells with doses above 50 Gy was confirmed.

Pausing of flagellar rotation is a component of bacterial motility and chemotaxis.

When bacterial cells are tethered to glass by their flagella, many of them spin. On the basis of experiments with tethered cells it has generally been thought that the motor which drives the flagellum is a two-state device, existing in either a counterclockwise or a clockwise state. Here we show that a third state of the motor is that of pausing, the duration and frequency of which are affected by chemotactic stimuli. We have recorded on video tape the rotation of tethered Escherichia coli and Salmonella typhimurium cells and analyzed the recordings frame by frame and in slow motion. Most wild-type cells paused intermittently. The addition of repellents caused an increase in the frequency and duration of the pauses. The addition of attractants sharply reduced the number of pauses. A chemotaxis mutant which lacks a large part of the chemotaxis machinery owing to a deletion of the genes from cheA to cheZ did not pause at all and did not respond to repellents by pausing. A tumbly mutant of S. typhimurium responded to repellents by smooth swimming and to attractants by tumbling. When tethered, these cells exhibited a normal rotational response but an inverse pausing response to chemotactic stimuli: the frequency of pauses decreased in response to repellents and increased in response to attractants. It is suggested that (i) pausing is an integral part of bacterial motility and chemotaxis, (ii) pausing is independent of the direction of flagellar rotation, and (iii) pausing may be one of the causes of tumbling.

[Effect of the structure of the cell nucleus chromatin on absorption of low-energy beta-radiation emitted from an incorporated source]. / Vliianie struktury khromatina iadra kletki na pogloshchenie beta-izlucheniia nizkikh énergii ot inkorporirovannogo istochnika.

To study the energy absorption in cell nuclei the analysis was made of the level of registration of beta-particles, emitted by the incorporated tritium, using a liquid scintillation counter. It was shown that the treatment, modifying the chromatin structure, changed the number of beta-particles registered. The pattern of the spectral changes indicated that the effect observed was not the result of the quenching processes. The effect was absent when the source of a higher energy (14C) was used. The authors submit the conceptions concerning the mechanisms of the phenomenon observed which are confirmed by the theoretical studies.

[The effect of intracellular pH on radiation damage to mammalian cells]. / Vliianie vnutrikletochnogo pH na radiatsionnoe porazhenie kletok mlekopitaiushchikh.

In experiments with Chinese hamster cells and Ehrlich ascites tumor cells it has been shown that a decrease in intracellular pH from 7.0 to 6.0, produced by 0.1 M phosphate buffer, ameliorates the radiation injury to cells (DMF = 1.35) with respect to their clonogenic capacity and induction of chromosome aberrations. The rate of repair of DNA single-strand breaks, within the pH range under study, is invariable.