Using three-color chromosome painting to test chromosome aberration models

Ionizing radiation induces DNA double-strand breaks (DSB), which interact pairwise to produce chromosome aberrations. There have long been two main competing theories of such pairwise DSB-DSB interactions. The "classical" theory asserts that an unrepaired DSB makes two ends that separate within the cell nucleus, with each end subsequently able to join any similar (nontelomeric) end. The "exchange" theory asserts that at a DSB the chromatin does not separate completely; rather the DSB ends remain associated until repair, or an illegitimate recombination involving another DSB, occurs. The DSB-DSB interaction mechanism was tested by using three-color fluorescence in situ hybridization to paint chromosomes and observe "three-color triplets": three broken and misrejoined chromosomes having cyclically permuted colors. We observed 18 "three-color triplets" in 2000 cells after 2.25 Gy of gamma-irradiation. On the exchange model in its standard form such three-color triplets cannot occur, so this model is inconsistent with the observations. On the classical model, formalized as a discrete time Markov chain embedded at the transitions of a continuous time Markov chain, the frequency of occurrence of three-color triplets can be computed by Monte Carlo simulations. The number of three-color triplets predicted mathematically by the classical model was found to be slightly larger than the observed number. Thus our data, together with our computer simulations, exclude the standard form of the exchange model but are compatible with the classical model. The results are also compatible with other, more complicated models.     

Hybridization of chromosome 18 alpha-satellite DNA probe to chromosome 22

Fluorescence in situ hybridization (FISH) of uncultured chorionic villus diploid cells with a chromosome 18 alpha-satellite DNA probe (D18Z1) revealed a third small signal in addition to two large signals. FISH analysis of diploid metaphase cells from cultured chorionic villus cells and from maternal lymphocytes revealed that the third signal resulted from hybridization to the centromere of chromosome 22. This is the first report of a variant involving D18Z1 detected by FISH and of hybridization of alpha-satellite from a sub-metacentric chromosome to the centromere of an acrocentric chromosome. We propose that this inherited variant resulted from insertion of chromosome 18 specific alpha-satellite DNA sequences into the centromeric region of chromosome 22.     

Human and porcine correspondence of chromosome segments using bidirectional chromosome painting

The aim of this study was to determine the correspondence between human and porcine chromosome fragments using whole chromosome painting probes from both species in heterologous hybridization experiments (bidirectional heterologous chromosome painting). Bidirectional experiments allow the determination of segment-to-segment homologies between the chromosomes of these two species. Chromosome-specific painting probes from both species were, except one, obtained by DOP-PCR or PARM-PCR amplification of flow-sorted chromosomes. The probes labeled 95% of the total length of the porcine chromosomes with human painting probes and 60% of the human chromosomes in the reverse experiments. Syntenic relationships of chromosomal segments on the karyotype of both species were determined. There was close agreement between com- parative gene mapping data and the identified homologous segments; this comparison enabled orientation of the segments. We demonstrate that bidirectional heterologous chromosome painting is a highly efficient way of generating comparative cytogenetic maps.     

Integrated radiation hybrid and yeast artificial chromosome map of chromosome 9p

OBJECTIVES: To determine concentrations of chondroitin sulphate (CS) and keratan sulphate (KS) epitopes, glycosaminoglycans (GAGs) and hyaluronan (HA) in knee synovial fluid (SF) from normal subjects and patients with osteoarthritis (OA) or rheumatoid arthritis (RA), to test whether these variables may be used as markers of the OA process. METHODS: OA was subdivided into large joint OA (LJOA), nodal generalised OA (NGOA), and OA with calcium pyrophosphate crystal deposition (CPA). Clinical assessment of inflammation (0-6) was undertaken on OA and RA knees. Knee SF was examined by enzyme linked immunosorbent assay for: CS epitopes, using monoclonal antibodies 3-B-3 and 7-D-4; KS epitope using monoclonal antibody 5-D-4; and HA, using biotinylated HA binding region of cartilage proteoglycan. Total sulphated GAGs were measured by dye binding with 1:9 dimethylmethylene blue. RESULTS: Increased SF 3-B-3 concentrations and 3-B-3/GAG ratio were found in OA, compared with RA or normal knees, with higher 3-B-3 and 3-B-3/GAG in LJOA and NGOA than in CPA. SF 7-D-4 and 7-D-4/GAG were reduced in RA, compared with normal and OA; SF 5-D-4 was reduced in OA compared with normal. GAG and HA concentrations were decreased in both OA and RA. No correlations with radiographic scores were observed, but SF 7-D-4 was lower in 'inflamed' compared with 'non-inflamed' RA and OA knees. In patients with bilateral samples there were strong correlations between right and left knees for all SF variables. CONCLUSIONS: Changed concentrations of SF CS and KS can be detected in OA with a profile that differs from that seen in RA. Clinical subgrouping and local joint inflammation may influence these measures, supporting different pathogenesis within OA subgroups and requirement for careful patient characterisation in SF studies.     

Y chromosome and spermatogenesis

The authors present two patients affected by scars resulting from burning of over 60 per cent of the total body area, in which the pre-expansion of a free flap has been used to increase the tissue surface useful for transfer from the only area of residual healthy skin (left forearm, left parascapular region). In both cases it was possible to transfer abundant healthy tissue into the desired areas, obtaining a rapid release of the region, which made possible an early physical rehabilitation of the patient starting after the second postoperative week. One of the main problems encountered, when facing surgical rehabilitation for the seriously burned patient, is the poor availability of skin donor areas suitable for reconstructive flaps. The pre-expansion of free flaps provides an advantage in that it allows the few integral residual areas to be used, improving vascularization and therefore increasing the available surface. Furthermore, as pre-expansion reduces tension on the margins, it allows for the easier closing of the donor area, with a minor risk of complications and a better scar outcome.     

Dynamics of chromosome spreading

Consistency of optimum chromosome spreading during harvest of cytogenetic specimens remains a major concern. We have tested the idea that a precise control of the drying rate (the time with which metaphase cells dry), as fixed cell suspension is placed on a slide or an in situ culture in last fixation, may be the answer. Amniocyte and lymphocyte cultures were allowed to dry at defined combinations of relative humidity (RH) and temperature (T) in a modified Thermotron environmental control unit. We were able to demonstrate, based on 2,250 amniocytes and 1,650 lymphocytes, that the metaphase area after drying was a function of RH and T for both in situ and non-in situ culture systems. As the RH and T increase, the metaphase area increases until a threshold is reached. Also, as RH increases, the slide drying time increases. Data obtained using a response surface regression, proportional hazards regression analysis and slide drying time studies are consistent with our model of chromosome spreading. Optimum metaphase areas can be achieved at various combinations of RH and T. We propose that the use of an environmental control unit is a practical way of achieving optimum chromosome spreading routinely and in a highly consistent manner.     

Seven genes from human chromosome 18 map to chromosome 24 in the bovine

Bovine sequence tagged sites (STSs) were developed for seven genes and used for synteny mapping with a hybrid bovine x rodent cell line panel. The genes were thymidylate synthase (TYMS), pituitary adenylate cyclase activating peptide (ADCYAP1), and melanocortin-2 receptor (MC2R) from the short arm of human chromosome (HSA) 18 and N-cadherin (CDH2), transthyretin (TTR), gastrin-releasing peptide (GRP), and plasminogen activator inhibitor 2 (PAI2) from the long arm of HSA 18. Primers for these genes were designed with human, ovine, or bovine sequences aligned with a sequence from a second species. The bovine PCR product was cloned, and the fragment was sequenced to verify that the homologous gene was indeed amplified. A second set of bovine-specific PCR primers were developed for each gene from these sequences. These STSs were used for synteny mapping, and all seven genes were syntenic with markers of bovine chromosome (BTA) 24. The concordance with BTA 24 was at least 96.5% for all genes.     

X chromosome inactivation in mammals

The most important results of the last 30 years of studies on mammalian X-chromosome inactivation are reviewed. The data on X-chromosome inactivation in cells of embryonic and extraembryonic tissues and in male and female germ cell lines are discussed. Special attention is paid to data on mapping and functioning of the X-inactivation center and of recently discovered gene XIST. The main hypotheses concerning the mechanisms responsible for X-chromosome inactivation are considered. A new model of X-inactivation is proposed, which regards heterochromatin as a nonspecific activator of nucleation of the X-chromosome on which it is located.     

SMC proteins and chromosome structure

The structure of chromosomes is largely determined by chromosome-associated proteins. Members of the SMC (structural maintenance of chromosomes) family play an important role in both prokaryotic and eukaryotic chromosome structure and dynamics. SMC proteins are involved in chromosome condensation, sister-chromatid cohesion, sex-chromosome dosage compensation, genetic recombination and DNA repair. There have been major advances recently in understanding the function of SMC proteins--including the identification of biochemical activities of SMC-containing protein complexes and the realization that individual SMC proteins might link seemingly unrelated aspects of chromosomal metabolism.     

Neurologic diseases and chromosome 17

On the short arm of the 17th chromosome is a peripheral myelin protein (PMP22) the duplication or point mutation of which causes the development of some congenital autosomal dominant hereditary demyelinization neuropathies: the most frequent variants of Charcot-Marie-Tooth disease (CMT1A), some cases of Déjérine-Sottas disease and microdeletion of PMP22 and hereditary pressure neuropathies. The pericentric section of the long arm of chromosome 17 comprises a locus conditioning the development of the most frequent phacomatosis-neurofibromatosis 1. As to rarer neuromuscular diseases, genome mutations of chromosome 17 condition the development of some cases of autosomal recessive forms of severe muscular dystrophy (SCARMD), a clinical analogue of Duchenne's form of muscular dystrophy, metabolic storage myopathy of Pompe's type and some muscle diseases associated with impaired function of the ion channels (hyperkalaemic periodic paralysis, congenital paramyotonia, some cases of malignant hyperthermia). Aspartoacylase deficiency, conditioning Canavan's leucodystrophy was also located in the area of the short arm of chromosome 17.     

Ring Y chromosome without mosaicism

A 53-year-old man with a short stocky build, mild mental retardation, gynecomastia and hypogonadism was found to have a small ring Y chromosome unassociated with mosaicism. The ring Y was represented by a minute portion of chromatin or sometimes paired dots which were no larger than the short arm of the normally expected Y. No brightly fluorescent segment of the ring Y was present nor was it observed elsewhere in the karyotype. A primary medical problem was severe osteoarthritis, necessitating bilateral hip arthroplasties. Plasma testosterone was markedly decreased and plasma gonadotropins were increased. Potency was improved following testosterone injections. We conclude that genes responsible for testicular differentiation, maleness and possibly height are located close to the centromere of the Y chromosome, possibly on both the short and long arm. We also conclude that multiple genes are required for a fully developed male phenotype and apparently some of these genes were deleted or not expressed in this patient.     

Opiates and human chromosome alterations

Methods of monitoring occupational exposure to methanol were investigated in volunteer subjects who had ingested small amounts of methanol. It was confirmed that urinary methanol concentrations accurately reflected those in the blood. This relationship was maintained over a considerable range of concentrations in spite of large variations of urine flow. Concomitant ingestion of ethanolic beverages increased the urinary methanol concentration slightly. Urinary formic acid concentration was too variable to be of value but rate of urinary excretion of formic acid did reflect methanol uptake. The ratio of urinary formic acid to creatinine concentrations (F/C ratio) is a practical monitoring method. However, formic acid elimination rate is reduced by ingestion of ethanolic beverages. Urinary methanol concentration is favoured as a method of monitoring and a concentration of 10 microgram/ml measured at the end of the work shift is suggested as the level above which occupational exposure should be suspected and the appropriate action taken.     

Mechanisms of radiation-induced chromosome aberrations

We have investigated the role of the low-affinity nerve growth factor (NGF) receptor p75NGFR in determining the death of neuronally differentiated PC12 cells after withdrawal of NGF. A range of high and low p75NGFR-expressing cells were obtained by a combination of fluorescence activated cell sorting (FACS) and stable transfection with a p75NGFR expression vector. Cells were readily differentiated to a neuronal phenotype irrespective of the level of p75NGFR expression. However, the rate and extent of neuronal death following NGF deprivation were extremely sensitive to the level of p75NGFR expression. The highest expressing cells died most rapidly. Cells selected for very low levels of p75NGFR expression exhibited resistance to NGF withdrawal, and remained as viable, differentiated neurons, with minimal cell death, for at least 5 days in the absence of NGF. Antisense oligonucleotides against p75NGFR were shown to down-regulate p75NGFR in PC12 cells and, further, to significantly enhance survival in the absence of NGF. These results consolidate and generalize our previous findings that p75NGFR induces cell death in postnatal sensory neurons in the absence of NGF. The ability to induce cell death in the absence of NGF appears to be a more general role of p75NGFR in differentiated neurons, and an important new paradigm for the mechanism of NGF-dependent survival.     

Metaphase analysis of metanephric adenoma reveals chromosome Y loss with chromosome 7 and 17 gain

A 61-year-old man underwent wedge excision of a 3-cm right renal metanephric adenoma. This recently recognized tumor has been considered benign, although no genetic studies have been reported. Metaphase analysis demonstrated a 47,X,-Y,+7,+17 karyotype. These results are consistent with a clonal neoplastic disorder.     

Small extra ring chromosome derived from chromosome 10p: clinical report and characterisation by FISH

We report our experience with a modified Le Fort I osteotomy developed to avoid nasal tip upturning, alar base widening, and upper lip flattening in anterosuperior repositioning of the maxilla. We compare the aesthetic results obtained with this variation of the surgical technique to those obtained using the more traditional Le Fort I osteotomy combined with the alar cinch suture and the anterior nasal spine reduction procedures on a sample of 20 patients.     

A chromosome 14q11/TCR alpha/delta specific yeast artificial chromosome improves the detection rate and characterization of chromosome abnormalities in T-lymphoproliferative disorders

The rate of detection of chromosome abnormalities in T-cell proliferations is lower than that observed in B-cell malignancies. The former frequently involve the TCR alpha/delta locus at chromosome band 14q11. We have identified a YAC encompassing 70% of the TCR alpha/delta locus, which has been used as a fluorescence in situ hybridization probe to detect chromosome rearrangements involving 14q11, both at metaphase and within interphase nuclei, in patients with a variety of T-lymphoproliferative disorders. Its use allowed detection of previously unsuspected TCR alpha/delta rearrangements in 4/13 (30%) immature T-lineage acute leukemias, including two t(10;14) and 2 minor inversion 14s. It also clarified interpretation of complex chromosome 14 abnormalities in mature T-cell proliferations (T-prolymphocytic leukemia and ataxia telangiectasia). Use of this probe will aid the detection and characterization of abnormalities involving the TCR alpha/delta locus, particularly in cases with normal or complex karyotypes and in those proliferations for which mitoses are difficult to obtain.