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Horses, asses and zebras belong to the genus Equus and are the only extant species of the family Equidae in the order Perissodactyla. In a previous work we demonstrated that a key factor in the rapid karyotypic evolution of this genus was evolutionary centromere repositioning, that is, the shift of the centromeric function to a new position without alteration of the order of markers along the chromosome. In search of previously undiscovered evolutionarily new centromeres, we traced the phylogeny of horse chromosome 5, analyzing the order of BAC markers, derived from a horse genomic library, in 7 Equus species (E. caballus, E. hemionus onager, E. kiang, E. asinus, E. grevyi, E. burchelli and E. zebra hartmannae). This analysis showed that repositioned centromeres are present in E. asinus (domestic donkey, EAS) chromosome 16 and in E. burchelli (Burchell’s zebra, EBU) chromosome 17, confirming that centromere repositioning is a strikingly frequent phenomenon in this genus. The observation that the neocentromeres in EAS16 and EBU17 are in the same chromosomal position suggests that they may derive from the same event and therefore, E. asinus and E. burchelli may be more closely related than previously proposed; alternatively, 2 centromere repositioning events, involving the same chromosomal region, may have occurred independently in different lineages, pointing to the possible existence of hot spots for neocentromere formation. Our comparative analysis also showed that, while E. caballus chromosome 5 seems to represent the ancestral configuration, centric fission followed by independent fusion events gave rise to 3 different submetacentric chromosomes in other Equus lineages. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

In addition to their location at terminal positions, telomeric-like repeats are also present at internal sites of the chromosomes (intrachromosomal or interstitial telomeric sequences, ITSs). According to their sequence organization and genomic location, two different kinds of ITSs can be identified: (1) heterochromatic ITSs (het-ITSs), large (up to hundreds of kb) stretches of telomeric-like DNA localized mainly at centromeres, and (2) short ITSs (s-ITSs), short stretches of telomeric hexamers distributed at internal sites of the chromosomes. Het-ITSs have been only described in some vertebrate species and they probably represent the remnants of evolutionary chromosomal rearrangements. On the contrary, s-ITSs are probably present in all mammalian genomes although they have been described in detail only in some completely sequenced genomes. Sequence database analysis revealed the presence of 83, 79, 244 and 250 such s-ITSs in the human, chimpanzee, mouse and rat genomes, respectively. Analysis of the flanking sequences suggested that s-ITSs were inserted during the repair of DNA double-strand breaks that occurred in the course of evolution. An extensive comparative analysis of the s-ITS loci and their orthologous ‘empty’ loci confirmed this hypothesis and suggested that the repair event involved the direct action of telomerase. Whereas het-ITSs seem to be intrinsically prone to breakage, the instability of s-ITSs is more controversial. This observation is consistent with the hypothesis that s-ITSs are probably not themselves prone to breakage but represent ‘scars’ of ancient breakage that may have occurred within fragile regions. This study will review the current knowledge on these two types of ITS, their molecular organization, how they arose during evolution, their implications for chromosomal instability and their potential applications as phylogenetic/forensic markers. Copyright © 2008 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The article examines the modulation of gene expression profiles in racing horses. It takes into account the chromosomal location of interleukin 8 (IL8) in Equus caballus. A genomic DNA fragment has been amplified using primers from the horse IL8 mRNA sequence. Overlapping clones 63-D22 and 163-L3 have been isolated. Through the use of bacterial artificial chromosome clone as probe in a fluorescence in situ hybridization, the location of IL8 is determined.

The intrachromosomal location of (T2AG3)n telomeric sequences has been reported in several species. It was proposed that interstitial telomeres (ITs) originated through telomeric fusion of ancestral chromosomes. However, the data so far obtained derive mainly from cytogenetic observations. Cloning and database searching of human IT sequences allowed us to identify three classes: (i) short ITs, composed of few, essentially exact T2AG3 units; (ii) subtelomeric ITs, composed of larger arrays (several hundred base pairs) including many degenerate units within subtelomeric domains; (iii) fusion ITs, in which two extended stretches of telomeric repeats are oriented head-to-head. The number of short ITs is over 50 and subtelomeric ITs are probably present at all chromosomal ends. Surprisingly, the telomeric sequence in 2q13 remains the only fusion IT so far characterized, and evidence presented here suggests that another member of this class may be present in 1q41. Different molecular mechanisms generated the three classes. In particular, several short ITs interrupt precisely repetitive elements or are flanked by direct repeats of 10-41 bp, and are conserved in gorilla and chimpanzee. These features strongly suggest that telomeric repeats were inserted at intrachromosomal sites through the repair of double-strand breaks that occurred in the germline during evolution.

Human blood lymphocytes from two normal and seven Down syndrome (DS) subjects were examined to determine rates of synthesis of individual replicon and adjacent clusters of replicons, using DNA fiber autoradiography. Lymphocytes in 6 of 7 DS patients were shown to have significantly slower synthesis of simultaneously active adjacent replicon clusters compared to normal controls. Rates of synthesis of individual replicons were the same in lymphocytes from all the subjects investigated. These results demonstrate differences in respect to the structural organization of clusters of replicons between DS and normal lymphocytes. A possible relation of the above phenomenon to the chromosomal radiosensitivity in DS cells is discussed.

Several parameters of DNA replicons and replicon clusters have been examined using DNA fiber autoradiography in normal vs. mutant human cell lines showing increased chromosomal sensitivity to ionizing radiation as well as radioresistant DNA synthesis. Rates of synthesis of individual replicons for unirradiated ataxia telangiectasia (AT) AT5MO, basal cell naevus syndrome (BCNS) BCN1SP and Down's Syndrome LCH944 appeared to be in the range seen with two normal fibroblast lines. With the longer labelling times (60-165 min), the average track lengths were longer in normal fibroblasts than mutant cell; after 5 Gy of radiation, normal and mutant cells had similar track lengths for all labelling times (10-165 min), as well as unchanged rates of replicon synthesis. These observations led to the determination of 'chain length', which measures simultaneously active adjacent replicon clusters. The main finding is that 'chain length' in mutant lines was significantly lower than that in the normal fibroblasts; upon 5 Gy irradiation, the values in normal cells were reduced about two-fold while the values for mutant cells remained about the same as controls. Thus, the experiments suggest that in unirradiated mutant cells DNA replication is delayed in a comparable manner as that induced by ionizing radiation in normal cells. A possible relation of the data to the chromosomal radiosensitivity and radioresistant DNA synthesis in the mutant lines is discussed.

Using DNA fiber autoradiography, DNA replication in cells of healthy donors and in those of patients with ataxia telangiectasia (AT) was estimated. A new fact has been demonstrated showing a decreased number of simultaneously operating in tandem groups of replicon clusters in AT cells compared to that in normal cells. The data obtained suggest a reduced frequency of activation in the adjacent replicon clusters in AT cells. It should be noted that the rate of fork movement remained unchanged in AT cells. Besides, the frequency of replication in adjacent replicon clusters remained unaltered after 5 Gy irradiation in AT cells, while the normal cells were radiosensitive to reduction in this replication parameter to the low level seen in both non-irradiated and 5 Gy irradiated AT cells. The relation of the above data to radioresistant DNA synthesis is discussed.

In peripheral blood lymphocytes taken from 7 patients with Down syndrome (DS) and 2 normal donors, using DNA fiber autoradiography, we estimated the rate of DNA-chain growth, which depends oil the number of simultaneously replicating adjacent replicon clusters, but not on the rate of fork movement. No difference was found in the rate of fork movement in these cells. 6 of 7 DS patients showed a significant reduction in the rate of DNA-chain growth as compared to that in normal control. Thus, a new molecular defect in DS lymphocytes has ben first revealed. Besides, new arguments have been provided in favour of genetic heterogeneity of this genetic disorder belonging to premature ageing diseases. Relation of molecular DNA replication defects with ageing is discussed.

Using DNA fiber autoradiography, DNA replication in cultured fibroblasts, derived from normal donors, and from XPII patient with increased sensitivity to ionizing radiation was estimated. Here evidence is provided on the fact that the fork movement, significantly decreased in XPII cells before irradiation, remains the same after exposure to X-rays. The density of replicon clusters simultaneously operating in tandem groups, which is initially much less in XPII cells compared to normal cells, also remains unchanged after exposure to X-rays (5 Gy), since the inhibition of DNA replication occurs to individual replicons only. Our data suggest that the inhibition of DNA replication in normal cells throughout the whole cluster, that drastically reduces the rate of DNA-chain growth, may provide an additional time to restore damaged chromosomes, i. e. it is part of the cellular defence mechanism. It seems likely that XPII cells are deficient in such a defence mechanism.

Using DNA fiber autoradiography, an estimation was made of DNA replication in normal fibroblasts and in those derived from a patient with Cockayne syndrome. The rate of replication fork movement as well as the rate of DNA chain growth, dependent on the frequency of initiation sites in the adjacent clusters of replicons, did not differ in Cockayne syndrome cells, compared to cells of normal donors, either before or after exposure to ionizing radiation.

A cytogenetic observation, that the sister chromatid exchanges (SCE) occur 3 times more frequently in a special form of xeroderma pigmentosum--XPII than in the norm, prompted a study of DNA replication in this rare disease. Using DNA fiber autoradiography, the rate of fork movement and the frequency of initiation in the adjacent clusters of replicons were estimated. The rate of fork movement was significantly slower than that in classical XP and in normal cells. Here evidence was provided on another defect in DNA replication in XPII that involves a significantly decreased number of simultaneously operating adjacent clusters of replicons, which results in a decreased rate of DNA chain-growth. According to the Painter replication model for SCE, the exchanges arise due to double-strand DNA breaks occurring on the border between two adjacent clusters, respectively, completely and partially replicated. A retarded fork-displacement rate together with a decreased rate of DNA-chain growth may cause this situation to persist longer than in the norm. Thus, our data provide a further support of the replication model for SCE. A similar combination of cytogenetic and molecular defects has been obtained earlier in the Bloom syndrome cells.

The influence of Staphylococcus alpha-toxin has been investigated on the duration of S-phase of lymphocyte mitotic cycle and on DNA replication in human fibroblasts in vitro. The duration of the S-phase of lymphocytes was measured by counting labeled metaphases and by making replication curves. Alpha-toxin in a dose of 3 micrograms/ml enhances the onset of S-phase, which is inhibited at a dose of 33 micrograms/ml of alpha-toxin. The action of alpha-toxin resulted in a decreased rate of replication fork and in a progressive activation of replicon groups. This effect was most prominent at 33 micrograms/ml of alpha-toxin. The data obtained allow to suggest that immunodeficiency of the second order, so characteristic of the staphylococcal sepsis, may be due, in many respects, to suppression of DNA replication.

The influence of traumatic shock on some cytogenetic indices in bone marrow cells was investigated in white mongrel rats. The traumatic shock was caused by the Noble-Collip method. Comparative analyses of cytogenetic indices during trauma of different intensity show that the most acute changes involve the percentage of aberrant metaphases, and of average number of chromosome breaks per cell. Dynamics of changes of cytogenetic indices after a heavy trauma show that the traumatic shock may exert a cytogenetic effect maintaining for 18 hours. The results obtained and the analysis of literary data enables us to suggest that the speed of development of changes, and restoration of cytogenetic indices under various forms of trauma is quite different and may serve another confirmation of the hypothesis above the possibility of increasing the process of mutagenesis at the expense of the violation of homeostasis.

Analysis of DNA fiber autoradiograms from basal cell nevus syndrome (BCNS) skin fibroblasts has revealed for the first time a new defect in DNA replication earlier unknown in other chromosomal instability syndromes, that involves a significantly decreased rate of DNA-chain growth in unirradiated cells. Here we present evidence that the defect may be due to a marked reduction in number of simultaneously operating groups of replicons compared to that in normal cells, the rate of fork movement and the fusion of neighbouring units in the group remaining unchanged. Radioresistant DNA synthesis was observed in the BCNS cells. The exposure of cells derived from normal donor to gamma-rays at a dose of 5 Gy reduces the number of simultaneously operating groups of replicons to the level occurring in unirradiated BCNS cells, the rate of folk movement being unchanged in both cell types. However, the incidence of fusion between neighbouring units within the group is lower in the cells exposed to gamma-rays, due perhaps to a radiation-induced lesion in the group. Thus, ionizing radiation reduces the rate of DNA synthesis to the same level, however from different initial levels. Our data suggest that the phenomenon of radioresistant DNA synthesis may be explained by the presence of the initial defect in DNA replication in BCNS or any other chromosomal instability disorders.

Using DNA fiber autoradiography we have revealed a new defect earlier unknown in Down's syndrome but analogous to that earlier shown by the authors in AT and basal cell nevus. This syndrome involves a significantly decreased number of simultaneously operating groups of replicons compared to that in normal cells., the rate of fork movement and the fusion of neighbouring units in the group being unchanged. Ionizing radiation (5 Gy) does not change the rate of DNA chain growth in the cells derived from the affected individuals, however, it significantly reduces this parameter in normal cells due to inhibition of replicon initiation in the whole clusters. Thus, radioresistant DNA synthesis in chromosomal instability syndromes may be explained by some defect in DNA replication in unirradiated cells analogous to that in irradiated normal cells.