Application of chromosome painting to clastogenicity testing in vitro

To maximise sensitivity, protocols for testing chemicals in chromosomal aberration assays in vitro are designed so that cells are sampled when the peak frequency of aberrations might be expected to occur. They are not designed to measure the frequency of aberrations in cells which survive. Only chromosomal aberrations which are heritable, however, can have any relevance to human health, but the detection of those aberrations most likely to be tolerated (inversions, reciprocal translocations) is notoriously difficult with conventional light microscopy. Current protocol design is justified by arguing that the presence of structural aberrations of any type at early times after treatment indicates a risk that a proportion of aberrations will persist and be maintained in the population. Chromosome painting allows reciprocal exchanges to be relatively easily measured and permits the validity of these assumptions to be tested. To date, the kinetics of induction and dose-response relationships of reciprocal translocations induced by chemicals have been little investigated. We compared the frequency of chromosome-type aberrations in human lymphocytes following treatment with two powerful clastogens, streptonigrin and Trenimon, using conventional staining techniques and chromosome painting. The results show that although reciprocal translocations can be shown to arise and persist in treated populations of human lymphocytes for several days following treatment, their frequency is very low, even at concentrations where large amounts of chromosomal damage are induced, indicating that, at present, the value of using chromosome painting as an adjunct to traditional clastogenicity testing is limited.     

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.     

Identification of the chromosome 14 origin of a C-negative marker associated with a 14q32 deletion by chromosome painting

A constitutional chromosome 14 rearrangement was observed in a female with a psychodevelopmental disorder. Karyotype analysis using a variety of chromosome techniques, QFQ, GTG, CBG, Ag-NOR and DA-DAPI, showed a deletion of chromosome 14q32.1-qter region in association with a supernumerary marker chromosome. The marker, resembling a submetacentric, approximately half the size of a G group chromosome is C band and Ag-NOR negative. The heteromorphism of the satellites showed that the deleted chromosome 14 is paternal in origin. Chromosome painting using an Alu-PCR probe specific for the human chromosome 14 and fluorescent in situ hybridization (FISH) showed that the marker contains chromosome 14q32 sequences. It is likely that the marker was generated from the deleted chromosome 14 region through a complex rearrangement.     

Cytogenetic localization of genetic markers on porcine chromosome 7q

We report two cases of port site metastasis as the presenting feature of colonic and ovarian carcinoma after laparoscopic cholecystectomy. Cholecystectomy was performed for upper abdominal pain and gallstones. Six and 4 months after the operation the patients presented with nodules at port sites, other than the site of extraction. Biopsy proved both to be adenocarcinoma and further management found these to be from advanced caecal and ovarian carcinomas.     

Reciprocal chromosome painting between human and prosimians (Eulemur macaco macaco and E. fulvus mayottensis)

We used fluorescence in situ hybridisation to delineate the homology between the human karyotype and those of two lemur species (Eulemur macaco macaco and E. fulvus mayottensis). Human and lemur chromosome-specific probes were established by bivariate fluorescence-activated flow sorting (FACS) and subsequent degenerate oligonucleotide-primed PCR (DOP-PCR). Reciprocal painting of human probes to lemur chromosomes and vice versa allowed a detailed analysis of the interchromosomal rearrangements that had occurred during the evolution of these species. The results indicate that the genomes of both species have undergone only a few translocations during more that 45 million years of lemur and human evolution. The synteny of homologs to human chromosomes 3, 9, 11, 13, 14, 17, 18, 20, 21, X, and Y was found to be conserved in the two lemur species. Taking non-primate mammals as the outgroup for primates, ancestral conditions for various primate chromosomes were identified and distinguished from derived forms. Lemur chromosome painting probes were also used for cross-species hybridization between the two lemur species. The results support an earlier assumption, made on the basis of chromosome banding, that the karyotypes of the two species have evolved exclusively by Robertsonian transformations. All probes derived from E. f. mayottensis chromosomes specific for homologs involved in rearrangements in E. m. macaco exclusively painted entire chromosome arms. The results further indicate that E. f. mayottensis most probably has a more ancestral karyotype than E. m. macaco. Probes derived from prosimians will be useful in comparing the karyotypes of other lower primates, which will improve our understanding of early primate genome evolution.     

Human NK cell and ADCC reactivity against xenogeneic porcine target cells including fetal porcine islet cells

In vitro studies of human NK cell-mediated cytotoxicity and ADCC against porcine target cells were performed. Stimulation of human PBMC responder cells with either allogeneic or xenogeneic porcine cells led to a marked increase in NK cell reactivity. Maximum reactivity was reached following 3-6 days of in vitro culture. The sensitivity of target cells ranked as follows: K562 > porcine PHA-induced lymphoblasts > resting porcine PBMC. Limiting dilution analysis showed that allo- and xeno-stimulation in vitro led to differentiation of similar frequencies of effector NK cells. Split culture experiments showed that single NK effector cells were cytotoxic against both K562 and porcine lymphoblasts, demonstrating that individual NK cells lack species specificity. NK effector cell generation stimulated by xenogeneic cells was cyclosporin A (CsA) sensitive and dependent on the presence of autologous responder T lymphocytes, a dependence that was completely reconstituted by the sole addition of human IL-2. Xenostimulation of enriched CD3+ cells also led to a preferential appearance of CD16+ or CD56+ lymphoblasts. Natural xenoreactive human anti-porcine antibodies are mainly of IgM and IgG2 subclasses, but antibodies in xenoimmunised patients reactive against porcine lymphocytes and fetal porcine islet cells were also of IgG1 and IgG3 subclasses. The same subclass distribution was found among antibodies specific for gal(alpha)1,3 gal epitopes as shown by tests performed with alpha1,3 galactosyltransferase-transfected Raji cells (human Burkitt lymphoma cells). Natural antibodies did not mediate ADCC, whereas gal(alpha)1,3 gal-specific antibodies in sera from xenoimmunised patients did. Fetal porcine islet cells were sensitive to human NK cell-mediated cytotoxicity and to ADCC mediated by xenoimmune sera.     

Identification of complex chromosome rearrangements in the gibbon by fluorescent in situ hybridization (FISH) of a human chromosome 2q specific microlibrary, yeast artificial chromosomes, and reciprocal chromosome painting

Chromosome painting has revealed that the human chromosome homologs in lesser apes are often fragmented and translocated to a number of different hylobatid chromosomes. We investigated the fragmented human chromosome 2 homologs in gibbons to illustrate a new strategy in mapping regional and band-specific chromosomal homologies between species. Previous research showed that the DNA library specific to human chromosome 2 paints parts of four gibbon (lar species group) chromosomes (viz., 1, 10, 12, and 16) and yields five distinct hybridization signals (including two on gibbon chromosome 16). However, the exact segments of human chromosome 2 that were translocated to the various gibbon chromosomes could not be distinguished. To determine the origin of the human chromosome 2 signals, we hybridized a microlibrary for the long arm of human chromosome 2, as well as YACs specific for most of the major bands on this chromosome, to metaphases of the gibbon. For reciprocal chromosome painting, we hybridized flow-sorted gibbon chromosome probes to human chromosome 2. Each method added additional insights that helped clarify the shuffling of human chromosome 2 material in the highly reorganized gibbon genome. There was an excellent correspondence between these complementary techniques. YAC 958d2 identified the breakpoint between human chromosome 2 material present on gibbon chromosomes 10 and 16. The reciprocal chromosome painting permitted a more complete and regional assignment of homology between segments on various gibbon chromosomes to human chromosome 2. The results show that a combination of reciprocal chromosome painting, subregional microlibraries, and band-specific probes (such as YACs) can be used to identify homologies between species and to rapidly construct detailed comparative chromosome maps, especially when the karyotypes are highly rearranged.     

The effect of the wheat Ph1 locus on chromatin organisation and meiotic chromosome pairing analysed by genome painting

The Ph1 locus in wheat influences homo(eo)logous chromosome pairing. We have analysed its effect on the behaviour and morphology of two 5RL rye telosomes in a wheat background, by genomic in situ hybridisation (GISH), using rye genomic DNA as a probe. Our main objective was to study the effect of different alleles of the Ph1 locus on the morphology and behaviour of the rye telosomes in interphase nuclei of tapetal cells and in pollen mother cells at early stages of meiosis. The telosomes, easily detectable at all stages, showed a brightly fluorescing chromomere in the distal region and a constriction in the proximal part. These diagnostic markers enabled us to define the centromere and telomere regions of the rye telosomes. In the presence of functional copies of Ph1, the rye telosomes associated at pre-leptotene, disjoined and reorganised their shape at leptotene, and became fully homologously paired at zygotene - pachytene. In plants without functional alleles (ph1bph1b), the rye telosomes displayed an aberrant morphology, their premeiotic associations were clearly disturbed and their pairing during zygotene and pachytene was reduced and irregular. The Ph1 locus also influenced the behaviour of rye telosomes in the interphase nuclei of tapetal cells: in Ph1Ph1 plants, the rye telosomes occupied distinct, parallel-oriented domains, whereas in tapetal nuclei of ph1bph1b plants they were intermingled with wheat chromosomes and showed a heavily distorted morphology. The results shed new light on the effect of Ph1, and suggest that this locus is involved in chromosome condensation and/or scaffold organisation. Our explanation might account for various apparently contradictory and pleiotropic effects of this locus on both premeiotic associations of homologues, the regulation of meiotic homo(eo)logous chromosome pairing and synapsis, the resolution of bivalent interlockings and centromere behaviour.     

Human aggression and the extra Y chromosome: Fact or fantasy?

Reviews world literature dealing with the association of the extra Y chromosome with aggressive behavior, to shed light on the mounting controversy about this association. The published population surveys were divided into 4 groups: newborn males, normal adult males, adult males in institutions for the mentally ill, and criminal males. The frequency of XYY males was .13% for newborn and normal adult males, .70% for mentally ill males, and 1.93% for criminal males. Although XYY males are only a small proportion of perpetrators of violent crimes, their significantly higher frequency in the criminal population provides strong presumptive evidence for the association of an extra Y chromosome with aggressive behavior. Since Y chromosome is the male determining chromosome, the XYY genotype may be seen as highlighting the association between maleness and aggressive tendencies. (53 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Estrus detection using radiotelemetry or visual observation and tail painting for dairy cows on pasture

The efficiency and accuracy of estrus detection using HeatWatch (DDx Inc., Denver, CO) or visual observation were compared in an autumn-calving Friesian herd (n = 48 per group) and a spring-calving Jersey herd (n = 50 per group) grazing on pasture. Cows in the group monitored by the HeatWatch system were fitted with a pressure-sensitive transmitter that signaled mounting activities associated with estrus. Visual observation was carried out for about 20 min before the morning and afternoon milkings and was aided by a strip of paint applied over the tailhead. Ovarian cyclicity was monitored with progesterone concentrations in milk samples collected twice a week. The efficiency and accuracy of estrus detection were, respectively, 98.4 and 97.6% for visual observation and 91.7 and 100% for HeatWatch detection. Autumn-calving herds differed from spring-calving herds in duration of estrus (9.7 vs. 7.3 h), number of mounts (13.6 vs. 8.5), total duration of mounts (36.8 vs. 19.9 s), and mean duration of a mount (2.6 vs. 2.3 s). There was no significant variation in the distribution of the time of onset of estrus or mounting activities at different hours of the day. Conception rate was similar for AI after estrus detection with HeatWatch (65.8%) or after visual observation (65.0%). The highest conception rate was obtained when AI was carried out between 12 and 18 h after the first mount. Both the HeatWatch system and visual observation plus tail painting can be used for estrus detection of dairy cows on pasture.     

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.     

Characterization of electric-pulse-induced permeabilization of porcine skin using surface electrodes

We measured the transient and long-term changes of permeability of full-thickness porcine skin after the application of a single or a train of electric pulses, as the basis for optimization of the electrical parameters for enhancing transdermal drug or gene delivery by electroporation. Two electrodes were attached to the stratum corneum of excised skin for transdermal electric pulse delivery and impedance measurement. Both transient and long-term permeabilization were found to be dependent on the electrical exposure dose, i.e., the product of pulse voltage and cumulative pulsing (exposure) time. Skin resistance dropped to about 20% of its prepulsing value when pulsed beyond a critical dosage of 0.4 V-s (with 20-40 V across each skin path), but recovered rapidly within seconds after the pulse. Long-term permeabilization of the skin required repeated pulsing with a minimum potential of 160 V (80 V across each skin path). The maximum long-term resistance drop, to 35% of the initial value, required a dose greater than 200 V-s, recovering slowly and seldom completely in tens of minutes to hours. The decrease and recovery of the resistance were dependent on the frequency and pulse length only for low-dose electrical exposure.     

Assignment of ARAF1 to porcine chromosome Xp11.2-p13 by fluorescence in situ hybridization

Combined heteroduplex single-strand conformation polymorphism (HEX-SSCP) analysis of the promoter and coding region of the low density lipoprotein receptor (LDLR) gene revealed a novel C to T mutation at nucleotide position 2056 in a Costa Rican patient with heterozygous familial hypercholesterolemia (FH). This nonsense mutation, Q665X, results in a termination codon in the epidermal growth factor (EGF) precursor homology domain of the mature LDLR.     

Assay of centromere function using a human artificial chromosome

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1-5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes.     

Human chromosome 5 sequence primer amplifies Alu polymorphisms on chromosomes 2 and 17

Members of the Alu family of repetitive elements occur frequently in the human genome and are often polymorphic. Techniques involving Alu element mediated polymerase chain reactions (Alu PCR) allow the isolation of region-specific human DNA fragments from mixed DNA sources. Such fragments are a source of region-specific Alu elements useful for the detection of Alu-related polymorphisms. A clone from human chromosome 5, corresponding to locus D5F40S1, was isolated using Alu PCR differential hybridization. Alu elements within this clone were investigated for the presence of potentially polymorphic 3' polyA tails. Primers were devised to amplify the 3' polyA tail of an Alu element present within the clone. One primer, D5F40S1-T, was specific to the DNA flanking the 3' end of the Alu element, and the other primer was homologous to sequences within the element. When these primers were used in PCR reactions, products from chromosomes 2 and 17 (loci D2F40S2 and D17F40S3) were amplified in addition to the expected product from chromosome 5. The most likely explanation for this nonspecific amplification is that the D5F40S1-T primer is located within a low-copy repetitive element that is 3' of the Alu element. This phenomenon presents a potential problem for the identification of region-specific Alu polymorphisms.