Molecular characterization of a novel amplicon at 1q21-q22 frequently observed in human sarcomas

Suppressors of position-effect variegation (Su(var)s) in Drosophila melanogaster are usually studied in the presence of chromosomal rearrangements, which exhibit variegated expression of euchromatic genes moved near to, or heterochromatic genes moved away from, centromeric heterochromatin. However, the effects of Su(var) mutations on heterochromatic gene expression in the absence of a variegating re-arrangement have not yet been defined. Here we present a number of results which suggest that Su(var) gene products can interact to affect the expression of the light gene in its normal heterochromatic location. We initially observed that eye pigment was reduced in several Su(var) double mutants; the phenotype resembled that of light mutations and was more severe when only one copy of the light gene was present. This reduced pigmentation could be alleviated by a duplication for the light gene or by a reduction in the amount of cellular heterochromatin. In addition, the viability of most Su(var) double mutant combinations tested was greatly reduced in a genetic background of reduced light gene dosage, when extra heterochromatin is present. We conclude that Su(var) gene products can affect expression of the heterochromatic light gene in the absence of any chromosomal rearrangements. However, it is noteworthy that mutations in any single Su(var) gene have little effect on light expression; we observe instead that different pairings of Su(var) mutations are required to show an effect on light expression. Interestingly, we have obtained evidence that at least two of the second chromosome Su(var) mutations are gain-of-function lesions, which also suggests that there may be different modes of interaction among these genes. It may therefore be possible to use this more sensitive assay of Su(var) effects on heterochromatic genes to infer functional relationships among the products of the 50 or more known Su(var) loci.     

On the effect of mebendazole on metacestodes of Mesocestoides corti and Echinococcus multilocularis (author''s transl)

Human chromosome 9 shows a high susceptibility for structural rearrangements, particularly pericentric inversions, which often are transmitted. Three types of pericentric inversions can be observed on No. 9: 1) Type I, showing the total constitutive heterochromatin in the short arm. 2) Type II with part of the C heterochromatin on the short arm, the rest located on the long arm proximal to the centromere. 3) Type III: a subtelocentric chromosome with part of the C heterochromatin in the very short arm and the rest located interstitially on the long arm. With these inversions as well as with other structural rearrangements, e.g. translocations, the break-points are located preferentially within the C heterochromatin or close to the heterochromatic-euchromatic junctions. These findings are in contrast to the findings in lymphocytes from 5 patients with fancomi's and after irradiation in vitro, reported in the literature. In lymphocytes break-points seem to be distributed more or less by chance. These observations together led us to speculate that human chromosome 9 primarily was an acrocentric chrosome; in morphology and at least in some functions similar to D- and G-group chromosomes. During evolution this acrocentric chromsome changed to a submetacentric one due to a pericentric inversion.     

Su(UR)ES: a gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes

A genetic locus suppressing DNA underreplication in intercalary heterochromatin (IH) and pericentric heterochromatin (PH) of the polytene chromosomes of Drosophila melanogaster salivary glands, has been described. Found in the In(1)scV2 strain, the mutation, designated as Su(UR)ES, was located on chromosome 3L at position 34. 8 and cytologically mapped to region 68A3-B4. A cytological phenotype was observed in the salivary gland chromosomes of larvae homozygous and hemizygous for Su(UR)ES: (i) in the IH regions, that normally are incompletely polytenized and so they often break to form "weak points," underreplication is suppressed, breaks and ectopic contacts disappear; (ii) the degree of polytenization in PH grows higher. That is why the regions in chromosome arm basements, normally beta-heterochromatic, acquire a distinct banding pattern, i. e., become euchromatic by morphological criteria; (iii) an additional bulk of polytenized material arises between the arms of chromosome 3 to form a fragment with a typical banding pattern. Chromosome 2 PH reveals additional alpha-heterochromatin. Su(UR)ES does not affect the viability, fertility, or morphological characters of the imago, and has semidominant expression in the heterozygote and distinct maternal effect. The results obtained provide evidence that the processes leading to DNA underreplication in IH and PH are affected by the same genetic mechanism.     

The influence of 5-azacytidine on the condensation of the short arm of rye chromosome 1R in Triticum aestivum L. root tip meristematic nuclei

This paper describes the effects of 5-azacytidine on the condensation state of rye (Secale cereale L.) chromatin introduced into the wheat genome (Triticum aestivum L. cv. Beaver). The wheat cultivar Beaver carries a translocation between the short arm of rye chromosome 1R (1RS) and the long arm of wheat chromosome 1B (1BL/1RS). 1RS can be detected using genomic in situ hybridisation and carries a ribosomal DNA (rDNA) locus that can be simultaneously detected using multiple labelling strategies. The rDNA locus divides 1RS into a distal region that is gene rich and a proximal region that is gene poor and highly methylated. 1RS also carries a large block of subtelomeric heterochromatin. The drug, which acts to inhibit DNA methylation in plants, has three pronounced effects on interphase nuclei. (1) It induces aberrant condensation of the rye subtelomeric heterochromatin and in many cases induces sister chromatid separation in the subtelomeric heterochromatin of G2 nuclei. (2) Nuclei trisomic for 1RS are observed at low frequency in treated material and are probably a consequence of aberrant sister chromatid separation or condensation. (3) The drug alters normal condensation of 1RS euchromatin. However, contrary to expectation the effect is not simply to induce decondensation. The proximal region of the arm actually condenses at low levels of drug administration while the distal region remains unaltered or increases its decondensation state. Increasing the concentration of 5-azacytidine induces a biphasic response and at the highest concentration used all regions of the arm show signs of decondensation. Thus the influence of the drug on chromatin condensation depends on the genomic structure.     

Dynamic organization of the beta-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25 degrees C and beta-heterochromatic in X0 males at 14 degrees C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(ILR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin.     

The regulatory properties of autonomous subtelomeric P elements are sensitive to a Suppressor of variegation in Drosophila melanogaster

Genetic recombination was used in Drosophila melanogaster to isolate P elements, inserted at the telomeres of X chromosomes (cytological site IA) from natural populations, in a genetic background devoid of other P elements. We show that complete maternally inherited P repression in the germline (P cytotype) can be elicited by only two autonomous P elements at 1A and that a single element at this site has partial regulatory properties. The analysis of the surrounding chromosomal regions of the P elements at 1A shows that in all cases these elements are flanked by Telomeric Associated Sequences, tandemly repetitive noncoding sequences that have properties of heterochromatin. In addition, we show that the regulatory properties of P elements at 1A can be inhibited by some of the mutant alleles of the Su(var)205 gene and by a deficiency of this gene. However, the regulatory properties of reference P strains (Harwich and Texas 007) are not impaired by Su(var)205 mutations. Su(var)205 encodes Heterochromatin Protein 1 (HP1). These results suggest that the HP1 dosage effect on the P element properties is site-dependent and could involve the structure of the chromatin.     

Treatment of temporomandibular disorders: arthroscopy, orthodontics, kinesitherapy]

In(1LR)pn2a is a pericentric inversion with a euchromatic breakpoint in the 2E polytene region and a heterochromatic breakpoint in the right arm of the X chromosome. It is associated with position-effect variegation (PEV) of the pn, wapl, Pgd and other vital loci of the 2E region, which are relocated near the bulk of the X heterochromatin. Cytological analysis showed that the rearrangement brings the 1A-2E euchromatic segment directly into contact with a major portion of the h34 block, a heterochromatic region that is positively stained by the N-banding technique and contains the AAGAG satellite sequences. Molecular cloning revealed the presence of a new junction between euchromatin and AAGAG satellite sequences and demonstrated that the euchromatic breakpoint of In(1LR)pn2a lies in the vinculin gene. In the X ray-induced secondary rearrangement In(1LR)r30, consisting of a pericentric inversion superimposed on In(1LR)pn2a, the h34 material remains associated with the 2E region but is separated from the rest of the X heterochromatin. In this case, the pn, wapl and Pgd loci no longer variegate, suggesting that the satellite-containing h34 region is not able per se to induce detectable PEV on the adjacent euchromatic genes.     

Association of a redefined proximal mouse chromosome 11 imprinting region and U2afbp-rs/U2af1-rs1 expression

Mice with maternal and paternal disomy for chromosome 11 (Chr 11) show growth retarded and overgrowth phenotypes, respectively, which can be attributed to genomic imprinting. Previous studies have defined the region of Chr 11 responsible (the Chr 11 imprinting region) as lying proximal to the T30H translocation breakpoint at the borders of G-bands 11B1.2 and 11B1.3. Evidence is presented here with two new translocations, T57H and T41Ad, which sequentially reduce the size of the imprinting region and locate it proximal to the T41Ad breakpoint in G-band 11A3.2. It therefore lies close to the centromere. The imprinted gene, U2af1-rs1, is known to be located within the original region and has been regarded as a candidate for the imprinting effects. Meiotic and mitotic chromosome FISH analysis, together with U2af1-rs1 expression studies are now described which show that the gene lies within the newly defined imprinting region and that its expression levels relate to the presence/absence and number of functional paternal alleles. U2af1-rs1 therefore remains a candidate gene for the Chr 11 imprinting effects. However, another recently reported imprinted gene, Meg1/Grb10, that lies within the region is also a good candidate, as it encodes a growth factor receptor. Meg1/Grb10 maps about 15 cM from U2af1-rs1 and is separated by conserved regions showing homology with two different human chromosomes. For these reasons, and because the two human homologues of U2af1-rs1 and Meg1/Grb10 also lie on different chromosomes, it would seem likely that the two genes identify two distinct imprinting domains within the small proximal region of mouse Chr 11.     

Commentary: tensions between the theory and practice of palliative care

Structural variations between great ape and human chromosomes due to pericentric inversions and translocations have created at apparent controversy during the reconstruction of hominoid phylogeny. One such variation involves human chromosome 5, which is equivalent to chromosome 4 in chimpanzee and orangutan but equivalent to segments of chromosomes 4 and 19 in gorilla. Obviously, neither banding patterns nor centromeric indecies in these chromosomes match. The pathological condition of cri du chat syndrome is due to the cytogenetic deletion of band p15.2 of chromosome 5. Is this region involved during pericentric inversion of apes chromosome 4? We used a human cosmid probe for cri du chat syndrome as a phylogenetic marker in search of the aforementioned question. The genomic sequences for cri du chat syndrome region were conserved in chimpanzee (PTR4) and orangutan (PPY4) but displayed a positional divergence in gorilla on chromosome 19(GG019). In addition, we used a human cosmid DNA probe for DiGeorge syndrome which is located on chromosome 22 band q11.2 and was conserved within band 23q11.2 in apes. The loci specific human genomic probes may help to describe the inversions and translocations for other chromosomes.     

Genetic modification of heterochromatic association and nuclear organization in Drosophila

Heterochromatin is the highly compact, usually pericentromeric, region of eukaryotic chromosomes. Unlike the more gene-rich euchromatin, heterochromatin remains condensed during interphase, when it is sequestered to the periphery of the nucleus. Here we show, by using fluorescent in situ hybridization to interphase diploid nuclei of Drosophila, that the insertion of heterochromatin into a euchromatic gene, which results in position-effect variegation (PEV), also causes the aberrant association of the gene and its homologous copy with heterochromatin. In correlation with the gene's mutant variegating phenotype, the cytological association of the heterochromatic region is affected by chromosomal distance from heterochromatin and by genic modifiers of PEV. Proteins that are thought to be involved in the formation of heterochromatin can therefore influence the interphase nuclear position of a chromosomal region. This suggests that heterochromatin and proteins involved in its formation provide a structural framework for the interphase nucleus.     

Gender distinctions and lateral asymmetry in the low-level auditory brainstem response of the human neonate

Given the reported long-range cis-inactivating effect of the XIST gene in early embryonic development and the lack of requirement of X-chromosome-specific elements for propagating the inactive state, there exists the possibility of cis inactivation of autosomal material after de novo translocation to an inactive X chromosome (Xi) in differentiated cells. We have analyzed de novo radiation-induced translocations between the Xi and autosomes to study the maintenance and spreading of X-chromosome inactivation (X inactivation) in relation to the position of the X-inactivation center (XIC)/XIST in differentiated cells. Autosome/Xi translocations were detected by fluorescence in situ hybridization (FISH). The activation status of the chromosomes involved in the translocation was determined by simultaneous immunocytogenetic studies using antibodies against either BrdU incorporated at late S phase or acetylated histone H4. The position of XIC/XIST in the reciprocal products of the translocation was determined by XIST-specific FISH and computer enhancement. In other experiments, the Xq13 region carrying XIC/XIST was localized by computer enhancement of the DAPI banding pattern. Our study in differentiated cells provides a visual demonstration that physical separation from XIC/XIST does not result in reactivation of inactive X-chromosome material and that X inactivation is not spread to the translocated autosomes irrespective of the position of XIC/XIST. This observation suggests that physical linkage to XIC/XIST does not lead to de novo inactivation of autosomal material.     

Structure and DNA methylation pattern of partially heterochromatinised endosperm nuclei in Gagea lutea (Liliaceae)

Pentaploid endosperm nuclei in certain Gagea species exhibit large masses of sticky and dense chromatin, not observed in somatic nuclei. These heterochromatin masses most probably stem from the triploid chalasal polar nucleus of the embryo sac, thus representing an example of facultative heterochromatinisation in plants. In the present investigation, we studied the nuclei in Gagea lutea (L.) Ker-Gawl, endosperm tissue. The position of the heterochromatin in interphase nuclei was observed by confocal laser scanning microscopy (CLSM) and the DNA methylation status of the euchromatin and heterochromatin was analysed by immunolabelling with an antibody raised against 5-methylcytosine (anti-5-mC). In young endosperms, heterochromatin was relatively dispersed, occupying some peripheral and inner parts of the nuclei. In a later endosperm development, the nuclei became smaller and more pycnotic, and the heterochromatin masses were placed predominantly near the nuclear periphery. The distribution of anti-5-mC labelling on the heterochromatic regions was unequal: some parts appeared hypermethylated while other parts were, like the euchromatin, not labelled. During mitosis, the labelling intensity of all the chromosomes was approximately the same, thus indicating that there are no cytologically detectable methylation differences among the individual sets of chromosomes. However, differences in the anti-5-mC signal intensity along individual chromosomes were observed, resulting in banding patterns with highly positive bands apparently representing constitutive heterochromatic regions. From these results it is obvious that facultative heterochromatinisation, in contrast to constitutive heterochromatinisation, need not be strictly accompanied by a prominent DNA hypermethylation.     

Inversion (X)(p11.4q22) associated with Norrie disease in a four generation family

We report on a 4-generation family in which Norrie disease occurs together with a pericentric inversion of the X chromosome in all affected males and carrier females. The breakpoint in the short arm of the X chromosome appears to be at the purported location of the Norrie disease gene. This is the second report of an association between Norrie disease and a chromosome aberration involving Xp11, and the first report of a specific gene disruption, thus physical gene location, due to a pericentric chromosome inversion.     

Epidemiology and scientific methodology applied to Pediatrics II: designs in epidemiologic experimentation

HP1 (Heterochromatin protein 1) is a conserved, non-histone chromosomal protein that is best known for its preferential binding to pericentric heterochromatin and its role in position effect variegation in Drosophila. Using immunolocalization, we show that HP1 is a constant feature of the telomeres of interphase polytene and mitotic chromosomes. This localization does not require the presence of telomeric retrotransposons, since HP1 is also detected at the ends of terminally deleted chromosomes that lack these elements. Importantly, larvae expressing reduced or mutant versions of HP1 exhibit aberrant chromosome associations and multiple telomeric fusions in neuroblast cells, imaginal disks, and male meiotic cells. Taken together, these results provide evidence that HP1 plays a functional role in mediating normal telomere behavior in Drosophila.     

Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocytes, using PCC and FISH

In order to study the initial frequencies and define kinetics of the formation of chromosomal exchanges in X-irradiated human lymphocytes, the premature chromosome condensation (PCC) technique was employed in combination with fluorescence in situ hybridization (FISH) with a composite probe for human chromosome 8 and a pan-centromeric probe for the whole genome. Human lymphocytes were X-irradiated (0.5, 1, 2, 3, 4 and 6 Gy), fused with mitotic Chinese hamster ovary (CHO) cells immediately or 1, 3, 6, 12 and 18 h after irradiation. Immediately after irradiation chromosomal breaks, dicentrics and translocations showed a linear dose-response. Unrejoined chromosome breaks were the most frequent types of aberrations (about 85%) observed. About 15% of total aberrations were chromosome exchanges of 65% of these were translocations and 35% were dicentrics. The chromosomal exchanges initially observed were mostly incomplete, with no complex exchanges at doses of 1 and 2 Gy, at higher doses (3-6 Gy) complex exchanges were observed and their frequencies increased with increasing post incubation time. Following different recovery times, repair kinetics of breaks for different doses of irradiation was studied. The shapes of the curves obtained for breaks as well as chromosome exchanges were linear-quadratic. The linear yield component, alpha, is formed entirely in the fast process that can be manifested in the early plateau, while component beta developed slowly in the subsequent hours. The kinetics of breaks rejoining was exponential, almost 50% of breaks rejoined after 1 h and at 18 h about 20% of breaks remained. At low doses of 1 and 2 Gy most of the exchanges were formed immediately and at higher doses, the frequency of exchanges increased with kinetics similar to that observed for the rejoining of breaks. However, the kinetics was different for different doses of irradiation. The frequency of dicentrics increased at doses above 2 Gy following 3 h recovery time, but for the translocations effect was pronounced even at 1 h recovery time. The frequency of incomplete exchanges (i.e., terminal translocations) decreased with post irradiation time and at 18 h was 30-40% less than the frequency obtained immediately after irradiation. The increase in the total translocations as a function of time between irradiation and fusion was due to a rapid increase in complete exchanges (i.e., reciprocal translocations). The frequency of ring chromosomes immediately after irradiation, also increased linearly, however, it was 3-5 times lower than dicentrics and remained almost constant in number for different doses and at different post-irradiation times.     

Persistence of radiation-induced translocations in human peripheral blood determined by chromosome painting

We have investigated the persistence of translocations and other types of chromosome damage with time using human peripheral blood acutely exposed in vitro to 137Cs gamma rays at doses ranging from 0.5 to 4 Gy. Freshly drawn blood from one donor was irradiated and metaphase chromosomes were prepared 2 to 7 days after exposure. Chromosomes 1, 2 and 4 were painted red-orange and chromosomes 3, 5 and 6 were painted green by fluorescence in situ hybridization (FISH) using "semi-directly" labeled whole-chromosome painting probes. This type of labeling combines direct and indirect labeling and showed significant advantages over both these other methods. All types of structural chromosome aberrations were classified by the Protocol for Aberration Identification and Nomenclature Terminology (PAINT) system. The yields of dicentric chromosomes, acentric fragments and ring chromosomes diminished with time as expected. Translocations exhibited greater persistence but showed a clear and statistically significant reduction in frequency at all doses. The mathematical model suggested that the translocation frequencies would reach a plateau of approximately 4, 15, 51, 106 and 179 translocations per 100 cell equivalents after irradiation with 0.5, 1, 2, 3 and 4 Gy, respectively. When translocations were classified by the conventional system, an analysis of the distribution of translocations and dicentrics per cell indicated that both types of exchanges were Poisson-distributed 48 h postirradiation. However, cells bearing translocations have a higher possibility of having dicentrics than cells without translocations. These findings suggest that dicentrics may contribute to a decline of translocation frequencies with time, and that some translocations are not completely persistent. The results obtained here using human blood exposed in vitro may influence the use of translocations as a retrospective biodosimeter of exposure to ionizing radiation in humans.