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Genomic rearrangements can result in losses, amplifications, translocations and inversions of DNA fragments thereby modifying genome architecture, and potentially having clinical consequences. Many genomic disorders caused by structural variation have initially been uncovered by early cytogenetic methods. The last decade has seen significant progression in molecular cytogenetic techniques, allowing rapid and precise detection of structural rearrangements on a whole-genome scale. The high resolution attainable with these recently developed techniques has also uncovered the role of structural variants in normal genetic variation alongside single-nucleotide polymorphisms (SNPs). We describe how array-based comparative genomic hybridisation, SNP arrays, array painting and next-generation sequencing analytical methods (read depth, read pair and split read) allow the extensive characterisation of chromosome rearrangements in human genomes.

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OBJECTIVE: To describe a considerably advanced method of array painting, which allows the rapid, ultra-high resolution mapping of translocation breakpoints such that rearrangement junction fragments can be amplified directly and sequenced. METHOD: Ultra-high resolution array painting involves the hybridisation of probes generated by the amplification of small numbers of flow-sorted derivative chromosomes to oligonucleotide arrays designed to tile breakpoint regions at extremely high resolution. RESULTS AND DISCUSSION: How ultra-high resolution array painting of four balanced translocation cases rapidly and efficiently maps breakpoints to a point where junction fragments can be amplified easily and sequenced is demonstrated. With this new development, breakpoints can be mapped using just two array experiments: the first using whole-genome array painting to tiling resolution large insert clone arrays, the second using ultra-high-resolution oligonucleotide arrays targeted to the breakpoint regions. In this way, breakpoints can be mapped and then sequenced in a few weeks.

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OBJECTIVE: To describe the systematic analysis of constitutional de novo apparently balanced translocations in patients presenting with abnormal phenotypes, characterise the structural chromosome rearrangements, map the translocation breakpoints, and report detectable genomic imbalances. METHODS: DNA microarrays were used with a resolution of 1 Mb for the detailed genome-wide analysis of the patients. Array CGH was used to screen for genomic imbalance and array painting to map chromosome breakpoints rapidly. These two methods facilitate rapid analysis of translocation breakpoints and screening for cryptic chromosome imbalance. Breakpoints of rearrangements were further refined (to the level of spanning clones) using fluorescence in situ hybridisation where appropriate. RESULTS: Unexpected additional complexity or genome imbalance was found in six of 10 patients studied. The patients could be grouped according to the general nature of the karyotype rearrangement as follows: (A) three cases with complex multiple rearrangements including deletions, inversions, and insertions at or near one or both breakpoints; (B) three cases in which, while the translocations appeared to be balanced, microarray analysis identified previously unrecognised imbalance on chromosomes unrelated to the translocation; (C) four cases in which the translocation breakpoints appeared simple and balanced at the resolution used. CONCLUSIONS: This high level of unexpected rearrangement complexity, if generally confirmed in the study of further patients, will have an impact on current diagnostic investigations of this type and provides an argument for the more widespread adoption of microarray analysis or other high resolution genome-wide screens for chromosome imbalance and rearrangement.

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The sequencing of the human genome has led to the availability of an extensive mapped clone resource that is ideal for the construction of DNA microarrays. These genomic clone microarrays have largely been used for comparative genomic hybridisation studies of tumours to enable accurate measurement of copy number changes (array-CGH) at increased resolution. We have utilised these microarrays as the target for chromosome painting and reverse chromosome painting to provide a similar improvement in analysis resolution for these studies in a process we have termed array painting. In array painting, chromosomes are flow sorted, fluorescently labelled and hybridised to the microarray. The complete composition and the breakpoints of aberrant chromosomes can be analysed at high resolution in this way with a considerable reduction in time, effort and cytogenetic expertise required for conventional analysis using fluorescence in situ hybridisation. In a similar way, the resolution of cross-species chromosome painting can be improved and we present preliminary observations of the organisation of homologous DNA blocks between the white cheeked gibbon chromosome 14 and human chromosomes 2 and 17.

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OBJECTIVE: The authors describe a method, termed array painting, which allows the rapid, high resolution analysis of the content and breakpoints of aberrant chromosomes. METHODS: Array painting is similar in concept to reverse chromosome painting and involves the hybridisation of probes generated by PCR of small numbers of flow sorted chromosomes on large insert genomic clone DNA microarrays. RESULTS: and CONCLUSIONS: By analysing patients with cytogenetically balanced chromosome rearrangements, the authors show the effectiveness of array painting as a method to map breakpoints prior to cloning and sequencing chromosome rearrangements.

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A range of fluorescent in situ hybridization techniques have been used to reveal hidden variant Philadelphia translocations in two cases of Ph-positive chronic-phase chronic myeloid leukaemia. In one patient, a highly complex variant Ph translocation affecting four chromosomes had resulted in the formation of structures with the appearance of i(17q) and +8. Misinterpretation of these karyotypes has direct clinical relevance. Our findings illustrate that even established cytogenetic abnormalities may contain cryptic abnormalities beyond the resolution of conventional cytogenetic methods.

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The accumulation of amyloid plaques and amyloid congophilic angiopathy (ACA) in the brains of affected individuals is one of the main pathological features of Alzheimer's disease. Within these deposits, the beta A4 (Ass) polypeptide represents a major component with the C-terminal 39-43 amino acid variants being most abundant. Using a mouse IgG1 MoAb produced by hybridoma beta A4[35-43]-95.2 3B9, which reacts with the epitope is defined by the amino acid residues beta A438[GVV]40, this study has identified a unique conformation within the carboxyl terminus of human beta A4[1-42]. Although the beta A438[GVV]40 sequence is present within the C-termini of human beta A4[1-40] and beta A4[1-43] and the beta A4-containing region of human APP, the beta A4[35-43]-95.2 3B9 MoAb (designated MoAb 3B9) does not bind these polypeptides, demonstrating a high degree of specificity for the beta A438[GVV]40 epitope as presented within the beta A4[1-42] sequence. The beta A4[1-42] epitope bound by MoAb 3B9 is sensitive to heating (100 degrees C for 5 min) and is denatured by SDS but not by oxidative radio-iodination of beta A4 or by adsorption to plastic surfaces or nitrocellulose. The recognition of beta A4 plaque deposits and ACA by MoAb 3B9 within formalin-fixed sections of human AD brain demonstrates the potential of these antibodies for investigating the role of the unique beta A4[1-42] conformation in the development of Alzheimer's disease.

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We report the application of multi-color fluorescence in situ hydribidization (FISH) for bone marrow metaphase cell analysis of hematological malignancies using a sub-set of the human karyotype for chromosome painting. A combination of chromosome probes labeled with three haptens enabled the construction of a "painting probe" which detects seven different chromosomes. The probe was used to screen three chronic myeloid leukemia (CML) derived cell lines and ten CML patient bone marrow samples for aberrations, additional to the Ph rearrangement, that are associated with the onset of blast crisis of CML. This approach was shown to identify karyotype changes commonly seen by conventional karyotyping, and in addition revealed chromosome changes unresolved or undetected by conventional cytogenetic analysis. The seven-color painting probe provides a useful, fast, and reliable complementary tool for chromosome analysis, especially in cases with poor chromosome morphology. This is a simple approach, since the probes can be displayed in a standard red/green/blue format accessible to standard fluorescence microscopes and image-processing software. The proposed approach using panels of locus-specific probes as well as chromosome paints will be useful in all diagnostic routine environments where analysis is directed towards screening for genetic rearrangements and/or specific patterns of chromosome involvement with diagnostic/prognostic value.

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We report a case of severe thrombocytopenia with an abnormal bone marrow karyotype described by G-banding analysis as t(16;21)(p?13;q11). Using fluorescence in situ hybridization (FISH) analysis with whole chromosome paints, the chromosome rearrangement was shown to be more complex, with the additional cryptic involvement of the long arm of chromosome 3. The chromosome rearrangement involved the breakpoints 3q26, 16p13.3, and 21q11; this rearrangement has not been previously described. The size of genomic material translocated from the chromosome 16 homologue was too small to be detected by chromosome paint. A 16p-specific telomeric probe was hybridized to locate the translocated 16p material. The 16p telomeric unique sequence DNA was retained on the der(16) chromosome, indicating a more distal breakpoint. This study demonstrates that telomeric translocations can occur that would be undetected by telomeric-specific FISH probes.

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The transformation of chronic myeloid leukemia (CML) from a chronic phase to an acute phase is frequently accompanied by additional chromosome changes. Extensive chromosome G-banded studies have revealed the secondary changes are nonrandom and frequently include trisomy 8, isochromosome 17q, trisomy 19, or an extra copy of the Philadelphia chromosome. In addition to these secondary chromosome changes, complex structural rearrangements often occur to form marker structures that remain unidentified by conventional G-banded analysis. The CML-derived cell line, K562, has been widely used in research since it was originally established in 1975. The K562 karyotype however, has remained incomplete, and marker structures have never been fully described. Recent advances in fluorescence in situ hybridization (FISH) technology have introduced the possibility of chromosome classification based on 24-color chromosome painting (M-FISH). In this study, we report a clarified karyotype for K562 obtained by a combination of the following molecular cytogenetic techniques: comparative genomic hybridization (CGH), FISH mapping using locus-specific probes, and M-FISH. Multicolor FISH has identified the marker structures in this cell line. The characteristic marker chromosome in K562 has been confirmed by this study to be a der(18)t(1;18). Multicolor FISH confirmed the identity of marker structures partially identified by G-banding as der(6)t(6;6),der(17)t(9;17),der(21)t(1;21),der(5)t(5;6). In addition M-FISH has revealed a deleted 20q and a complex small metacentric marker comprised of material from chromosomes 1, 6, and 20. A cryptic rearrangement was revealed between chromosomes 12 and 21 that produced a structure that looks like a normal chromosome 12 homologue by G-banding analysis. Finally, M-FISH detected regions from chromosome 13 intercalated into two acrocentric markers.

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Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.

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The molecular basis for blast transformation of chronic myeloid leukemia (CML) remains poorly understood. Cytogenetic alterations associated with CML blast crisis have previously been extensively studied by conventional G-banding analysis. However the complexity of some chromosome abnormalities or poor chromosome morphology or both has exceeded the resolution of G-banding analysis in a significant proportion of CML cases, and complex chromosome rearrangements have remained unidentified. In this study, comparative genomic hybridization (CGH) was used to elucidate genome imbalances in chronic phase or blast crisis samples or both from 12 CML patients. CGH and G-banding results were compared, and discrepancies were further clarified by using multipaint chromosome analysis and locus-specific DNA probes. No imbalances were detected in the 4 early disease phase samples studied. Eleven blast crisis samples were analyzed by G-banding and CGH, and the commonest genomic abnormality detected was overrepresentation of the long arm of chromosome 8, which was detected in 5 patients. This overrepresentation was attributable to trisomy 8 in 4 patients, whereas amplification of the entire long arm of chromosome 8 was detected in 1 patient. The formation of isochromosomes of the long arm of chromosome 8 was observed as a mechanism for gene amplification in this patient. Additional material originating from chromosome 8 was also observed intercalated into three marker chromosomes in peripheral blood metaphase spreads from this patient. These markers may further define areas on chromosome 8 that harbor oncogenes implicated in transformation of chronic myeloid leukemia.

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We previously reported that mammalian small intestinal and colonic brush borders (BBs) contained both epithelial Na+/H+ exchangers NHE2 and NHE3. We now show that, in the avian (chicken) colon, NHE2 is the major functional isoform under basal conditions and when stimulated by a low-NaCl diet. Hubbard chickens were maintained for 2 wk on a high- or low-NaCl diet. After the chickens were killed, the ileum and colon were removed, and BBs were prepared by Mg2+ precipitation and 22Na and D-[14C]glucose uptake determined in the BB vesicles. NHE2 and NHE3 were separated by differential sensitivity to HOE-694 (NHE2 defined as Na+/H+ exchange inhibited by 50 microM HOE-694). Chickens on a low-Na+ diet have increased plasma aldosterone (10 vs. 207 pg/ml). On the high-NaCl diet, both NHE2 and NHE3 contributed to ileal and colonic apical Na+/H+ exchange, contributing equally in ileum, but NHE2 being the major component in colon (86%). Low-NaCl diet significantly increased ileal and colonic BB Na+/H+ exchange; the increase in BB Na+/H+ exchange in both ileum and colon was entirely due to an increase in NHE2 with no change in NHE3 activity. In contrast, low-NaCl diet decreased ileal and colonic Na+-dependent D-glucose uptake. Western analysis showed that low-Na+ diet increased the amount of NHE2 in the ileal and colonic BB and decreased the amount of ileal Na+-dependent glucose transporter SGLT1. Both NHE2 and NHE3 were present in the apical but not basolateral membranes (BLM) of ileal and colonic epithelial cells. In summary, 1) NHE2 and NHE3 are both present in the BB and not BLM of chicken ileum and colon; 2) NHE2 is the major physiological colonic BB Na+/H+ exchanger under basal conditions; 3) low-NaCl diet, which increases plasma aldosterone, increases ileal and colonic BB Na+/H+ exchange and decreases Na+-dependent D-glucose uptake; 4) the stimulation of colonic BB Na+/H+ exchange is due to increased activity and amount of NHE2; and 5) the inhibition of ileal D-glucose uptake is associated with a decrease in SGLT1 amount. NHE2 is the major chicken colonic BB Na+/H+ exchanger.

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Membrane vesicles were isolated from the basolateral domains of pig and normal human colonocytes. The activity of the ouabain-sensitive K(+)-activated phosphatase, the basolateral membrane marker, was enriched 13-fold in these membrane vesicles over the original homogenate. The membranes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatibility antigen, both known indicators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-enzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na(+)-independent manner, with a Km of 28.1 +/- 0.8 mM and Vmax. of 3.1 +/- 0.4 nmol/s per mg of protein. The transport was inhibited by 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not by L-glucose or methyl-alpha-D-glucose. Probing the colonocyte basolateral membranes with an antibody against the C-terminus of the human liver GLUT 2 produced a cross-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.

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Monkeys with bilateral transection of the fornix were severely but selectively impaired on learning and retention of visuospatial conditional discriminations, visual conditional discriminations and non-conditional spatial-response tasks. Bilateral transplantation of cholinergic-rich fetal basal forebrain tissue into the hippocampus abolished significant learning impairments on all those tasks impaired by fornix lesions when tested three to nine months after transplantation whereas bilateral transplants of non-cholinergic fetal hippocampal tissue into hippocampus showed no such beneficial effect. Acetylcholinesterase staining was severely depleted throughout the dentate gyrus and hippocampus in fornix-transected monkeys compared with animals with control corpus callosum ablations. Staining was largely restored to normal in the host hippocampus and dentate gyrus in monkeys with cholinergic transplants, whereas acetylcholinesterase staining was abnormal in those with non-cholinergic grafts. These experiments suggest that where a "higher order" cognitive function, in this case the acquisition of specific types of information into long-term memory, is disturbed by a neuropharmacologically simple lesion, cognitive function can be restored by transplantation of neurons containing appropriate neurotransmitters.

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