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.     

Direct cloning of human 10q25 neocentromere DNA using transformation-associated recombination (TAR) in yeast

The transformation-associated recombination (TAR) procedure allows rapid, site-directed cloning of specific human chromosomal regions as yeast artificial chromosomes (YACs). The procedure requires knowledge of only a single, relatively small genomic sequence that resides adjacent to the chromosomal region of interest. We applied this approach to the cloning of the neocentromere DNA of a marker chromosome that we have previously shown to have originated through the activation of a latent centromere at human chromosome 10q25. Using a unique 1.4-kb DNA fragment as a "hook" in TAR experiments, we achieved single-step isolation of the critical neocentromere DNA region as two stable, 110- and 80-kb circular YACs. For obtaining large quantities of highly purified DNA, these YACs were retrofitted with the yeast-bacteria-mammalian-cells shuttle vector BRV1, electroporated into Escherichia coli DH10B, and isolated as bacterial artificial chromosomes (BACs). Extensive characterization of these YACs and BACs by PCR and restriction analyses revealed that they are identical to the corresponding regions of the normal chromosome 10 and provided further support for the formation of the neocentromere within the marker chromosome through epigenetic activation.     

Cytochalasin J affects chromosome congression and spindle microtubule organization in PtK1 cells

PtK1 cells were treated with 10 micrograms/ml cytochalasin J (CJ) for 15 min at various stages of mitosis. When applied at nuclear envelope breakdown (NEB) chromosome congression was blocked or substantially slowed, and chromosomes failed to show organization patterns typical of prometaphase. Spindle microtubule (MT) numbers appeared unaffected as judged by the pattern of birefringent retardation. However, ultrastructural analysis showed MTs to be reorganized within the spindle domain with some exhibiting fragmentation and others failing to interact with poorly defined kinetochore laminae. The spindle domain took on a curved, almost banana-like shape, as related to the position of the centrosomes and lack of orientation of chromosomes. Serial section analysis of kinetochore regions showed reduced contour length and maturation of the kinetochore plate with few MTs associated with this structure. Cells similarly treated with 10 micrograms/ml CJ at NEB for 15 min and then released into conditioned medium for 15 min showed the most chromosomes resumed congression to the metaphase plate. Ultrastructural analysis revealed a more normal organization of spindle MTs, but kinetochore structure remained affected. CJ treatment of cells in prometaphase slightly affected chromosomes congression with most chromosomes aligning at the metaphase plate after 10-15 min of treatment. Ultrastructural analysis showed that astral MTs were disrupted and spindle MTs were fragmented; few MTs coursed from kinetochore to pole. Kinetochore structure was also affected with only small numbers of short MTs seen associated with kinetochores. Application of CJ at anaphase onset had little effect on anaphase A and B, but cytokinesis failed to occur. Anti-tubulin staining of a monolayer of cells treated with 10 micrograms/ml CJ for 15 min showed that over 60% of mitotic figures exhibited changes in MT organization. Cells showing the greatest effect of treatment had several foci of bundles of MTs, as if the spindle were multipolar. Chromosomes were arranged near the periphery of the spindle which could be a result of abnormalities of kinetochore structure. Improper association of spindle MTs with kinetochores and, thus, changes in kinetochore position could account for these changes in spindle architecture.     

Activation of the MKK/ERK pathway during somatic cell mitosis: direct interactions of active ERK with kinetochores and regulation of the mitotic 3F3/2 phosphoantigen

The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal-regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. We have examined the regulation of ERK and MKK in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK1 cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. J. Cell Biol. 122:1311-1321). The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment.     

Localization of Mad2 to kinetochores depends on microtubule attachment, not tension

A single unattached kinetochore can delay anaphase onset in mitotic tissue culture cells (Rieder, C.L., A. Schultz, R. Cole, G. Sluder. 1994. J. Cell Biol. 127:1301-1310). Kinetochores in vertebrate cells contain multiple binding sites, and tension is generated at kinetochores after attachment to the plus ends of spindle microtubules. Checkpoint component Mad2 localizes selectively to unattached kinetochores (Chen, R.-H., J.C. Waters, E.D. Salmon, and A.W. Murray. 1996. Science. 274:242-246; Li, Y., and R. Benezra. Science. 274: 246-248) and disappears from kinetochores by late metaphase, when chromosomes are properly attached to the spindle. Here we show that Mad2 is lost from PtK1 cell kinetochores as they accumulate microtubules and re-binds previously attached kinetochores after microtubules are depolymerized with nocodazole. We also show that when kinetochore microtubules in metaphase cells are stabilized with taxol, tension at kinetochores is lost. The phosphoepitope 3f3/2, which has been shown to become dephosphorylated in response to tension at the kinetochore (Nicklas, R.B., S.C. Ward, and G.J. Gorbsky. 1995. J. Cell Biol. 130:929-939), is phosphorylated on all 22 kinetochores after tension is reduced with taxol. In contrast, Mad2 only localized to an average of 2.6 out of the 22 kinetochores in taxol-treated PtK1 cells. Therefore, loss of tension at kinetochores occupied by microtubules is insufficient to induce Mad2 to accumulate on kinetochores, whereas unattached kinetochores consistently bind Mad2. We also found that microinjecting antibodies against Mad2 caused cells arrested with taxol to exit mitosis after approximately 12 min, while uninjected cells remained in mitosis for at least 6 h, demonstrating that Mad2 is necessary for maintenance of the taxol-induced mitotic arrest. We conclude that kinetochore microtubule attachment stops the Mad2 interactions at kinetochores which are important for inhibiting anaphase onset.     

A novel tumor suppressor locus on chromosome 18q involved in the development of human lung cancer

The high incidence of loss of heterozygosity (LOH) on chromosome 18q in advanced non-small cell lung carcinomas indicates the presence of tumor suppressor gene(s) on this chromosome arm, which plays an important role in the acquisition of malignant phenotypes in lung cancers. In the present study, we examined 62 lung cancer specimens and 54 lung cancer cell lines for allelic imbalance at 11 microsatellite loci to define common regions of 18q deletions. Allelic imbalance of 18q was detected in 24 (55.8%) non-small cell lung carcinoma specimens and in 6 (31.6%) small cell lung carcinoma specimens, whereas a similar frequency of LOH was statistically inferred to occur in cell lines by analyzing marker homozygosity as an indirect measure of LOH. Five specimens and 11 cell lines showed partial or interstitial deletions of chromosome 18q, and 2 of them had homozygous deletions at the 18q21.1 region. A commonly deleted region was assigned between the D18S46 and y953G12R loci. The size of this region is less than 1 Mb, and the coding exons of three candidate tumor suppressor genes, Smad2, Smad4, and DCC, were mapped outside the region. This result suggests that the common region harbors a novel tumor suppressor gene involved in the progression of lung cancer.     

Somatic deletion mapping on chromosome 10 and sequence analysis of PTEN/MMAC1 point to the 10q25-26 region as the primary target in low-grade and high-grade gliomas

The 10q25-26 region between the dinucleotide markers D10S587 and D10S216 is deleted in glioblastomas and, as we have recently shown, in low-grade oligodendrogliomas. We further refined somatic mapping on 10q23-tel and simultaneously assessed the role of the candidate tumor suppressor gene PTEN/MMAC1 in glial neoplasms by sequence analysis of eight low-grade and 24 high-grade gliomas. These tumors were selected for partial or complete loss of chromosome 10 based on deletion mapping with increased microsatellite marker density at 10q23-tel. Three out of eight (38%) low-grade and 3/24 (13%) high-grade gliomas exclusively target 10q25-26. We did not find a tumor only targeting 10q23.3, and most tumors (23/32, 72%) showed large deletions on 10q including both regions. The sequence analysis of PTEN/MMAC1 revealed nucleotide alterations in 1/8 (12.5%) low-grade gliomas in a tumor with LOH at l0q21-qtel and in 5/21 (24%) high-grade gliomas displaying LOH that always included 10q23-26. Our refined mapping data point to the 10q25-26 region as the primary target on 10q, an area that also harbors the DMBT1 candidate tumor suppressor gene. The fact that we find hemizygous deletions at 10q25-qtel in low-grade astrocytomas and oligodendrogliomas - two histologically distinct entities of gliomas - suggests the existence of a putative suppressor gene involved early in glial tumorigenesis.     

Cytogenetic and molecular delineation of a region of chromosome 7 commonly deleted in malignant myeloid diseases

Loss of a whole chromosome 7 or a deletion of the long arm, del(7q), are recurring abnormalities in malignant myeloid diseases. To determine the location of genes on 7q that are likely to play a role in leukemogenesis, we examined the deleted chromosome 7 homologs in a series of 81 patients with therapy-related or de novo myelodysplastic syndrome or acute myeloid leukemia. Our analysis showed that the deletions were interstitial and that there were two distinct deleted segments of 7q. The majority of patients (65 of 81 [80%]) had proximal breakpoints in bands q11-22 and distal breakpoints in q31-36; the smallest overlapping deleted segment was within q22. The remaining 16 patients had deletions involving the distal q arm with a commonly deleted segment of q32-33. To define the proximal deleted segment at 7q22 at a molecular level, we used fluorescence in situ hybridization with a panel of mapped yeast artificial chromosome (YAC) clones from 7q to examine 15 patients with deletion breakpoints in 7q22. We determined that the smallest overlapping deleted segment is contained in a well-defined YAC contig that spans 2 to 3 Mb. These studies delineate the region of 7q that must be searched to isolate a putative myeloid leukemia suppressor gene, and provide the necessary cloned DNA for more detailed physical mapping and gene isolation.     

Chromatin containing CENP-A and alpha-satellite DNA is a major component of the inner kinetochore plate

The pathway of molecular interactions leading to kinetochore assembly on mammalian chromosomes is unknown. Kinetochores could be specified by structural features of centromeric satellite DNA [1-3] or by specific DNA sequences, analogous to budding yeast centromeres, interspersed in centromeric satellite DNA arrays [4,5]. Alternatively, kinetochores could be epigenetic structures that replicate without strict dependence on DNA sequence [6-8]. We purified kinetochore-associated chromatin from human chromosomes by immunoprecipitation of CENP-A, a centromere-specific histone H3 homologue located in the inner plate of the kinetochore [6,9,10]. Hybridization and DNA sequence analyses of cloned kinetochore DNA fragments revealed alpha-satellite as the predominant sequence associated with CENP-A. A major site of micrococcal nuclease digestion was identified by mapping the termini of alpha-satellite clones, suggesting that the inner kinetochore plate contains phased arrays of CENP-A-alpha-satellite nucleosomes. These experiments demonstrate for the first time that complex satellite DNA is a structural component of the kinetochore. Further, because complex satellite DNA is evolutionarily unconserved, these results suggest that molecular recognition events necessary for kinetochore formation take place at the level of DNA conformation or epigenetic mechanisms rather than DNA sequence per se.     

Loss of heterozygosity at 7q31 is a frequent and early event in prostate cancer

It is widely accepted that an accumulation of genetic alterations plays an important role in the genesis of human cancers, but little is known about prostate cancer in this respect. Recent studies have identified regions on chromosome arms 8p, 10q, 16q, and 18q that are frequently deleted in human prostate cancer. We have previously described a loss of heterozygosity (LOH) at the Met locus on chromosome band 7q31 in a study of 20 localized prostate tumors. To determine whether a region on the 7q arm is important in the initiation and/or progression of prostate cancer, prostate tissue from 13 patients with confined prostate tumors, 17 with local extracapsular extension, and 13 with metastatic forms were analyzed for LOH, using a DNA probe for RFLP (pMetH) and 8 CA microsatellite repeats (7 on 7q21-q33 and 1 on 7p). Twenty (47%) of the 43 cases studied showed LOH at one or more 7q loci. The most frequently deleted region was chromosome 7q31.1-7q31.2, whereas the centromeric locus on 7q21 was generally conserved. The percentage of LOH was normally distributed around the D7S480 locus. Moreover, the rate of LOH in the 7q31 region was lower in metastatic tumors than in localized tumors. These results strongly suggest the presence of a tumor suppressor gene on the chromosome band 7q31 with an important role in the early stages of prostate cancer.     

Veterinary medicine comment on camel medicine in Fan-mu tsuan yen-fang]

A small supernumerary chromosome was observed in two Prader-Willi syndrome (PWS) patients. The clinical diagnosis of PWS was confirmed by the ascertainment of the deletion of region 15q11-13 in one case and uniparental disomy (UPD) of the same region in the other. The markers were negative for dystamycinA/DAPI banding, did not contain NOR-positive satellites, and had an appearance consistent with a very small ring chromosome. Fluorescent in situ hybridization (FISH) analysis with the "all human centromere" probe indicated the presence of centromeric sequences in both markers. Chromosomal in situ suppression hybridization with chromosome specific libraries demonstrated that the small markers in the deleted and UPD patient originated from chromosome 15 and X, respectively. To the best of our knowledge these are the only PWS patients reported with a supernumerary marker chromosome other than inv dup(15) characterized by FISH.     

Physical mapping of unique nucleotide sequences on identified rice chromosomes

A physical mapping method for unique nucleotide sequences on specific chromosomal regions was developed combining objective chromosome identification and highly sensitive fluorescence in situ hybridisation (FISH). Four unique nucleotide sequences cloned from rice genomic DNAs, varying in size from 1.3 to 400 kb, were mapped on a rice chromosome map. A yeast artificial chromosome (YAC) clone with a 399 kb insert of rice genomic DNA was localised at the distal end of the long arm of rice chromosome (1q2.1) and a bacterial artificial chromosome (BAC) clone (180 kb) containing the rice leaf blast-resistant gene (Pi-b) was shown to occur at the distal end of the long arm of chromosome 2 (2q2.1). A cosmid (35 kb) with the resistance gene (Xa-21) against bacterial leaf blight was mapped on the interstitial region of the long arm on chromosome 11 (11q1.3). Furthermore a single RFLP marker, 1.29 kb in size, was mapped successfully to the distal region of the long arm of rice chromosome 4 (4q2.1). For precise localisation of the nucleotide sequences within the chromosome region, image analyses were effective. The BAC clone was localised to the specific region, 2q2.1:96.16, by image analysis. The result was compared with the known location of the BAC clone on the genetic map and the consistency was confirmed. The effectiveness and reliability in physically mapping nucleotide sequences on small plant chromosomes achieved by the FISH method using a variety of probes was unequivocally demonstrated.     

Structural aberrations of the long arm of chromosome no. 22. Report fo a family with translocation t(11;22) (q25;q11)

A chromosomal translocation t(11;22) (q25q11) is described in a family. Four members, in two generations, had the same translocation but showed phenotypic variation. Case reports of chromosome aberrations involving the long arm of chromosome 22 associated with and without chronic myeloid leukemia (CML) are reviewed. It appears that the distal segment of the long arm or chromosome 22 is either translocated or deleted, resulting in congenital anomalies, presumably due to chromosome imbalance. In other instances, a specific breakpoint on 22q results in the origin of Philadelphia chromosome (Ph1) associated with CML.     

What explains the negative consequences of adverse childhood experiences on adult health? Insights from cognitive and neuroscience research

Hepatocellular carcinoma (HCC) frequently shows a loss of heterozygosity (LOH) on chromosome 4q. In order to define the commonly affected region on chromosome 4q for further positional cloning of the putative tumor suppressor gene, we carried out allelic imbalance (AI) studies in 41 HCCs using a panel of 43 microsatellite markers. Thirty-four cases (82.9%) showed AI at one or more loci. Detailed deletion mapping identified 7 independent, frequently deleted regions on this chromosome arm. These were the (1) D4S1615 locus, (2) D4S1598 locus, (3) D4S620 locus, (4) D4S1566 and D4S2979 loci, (5) D4S1617 and D4S1545 loci, (6)D4S1537 locus; and (7) from the D4S2920 to D4S2954 locus. Among these 7 frequently deleted regions, 5 were associated with tumor differentiation. Our results suggest that several putative tumor suppressor genes may be present on chromosome 4q and that the AI of chromosome 4q may play a role in the aggressive progression of HCC.     

Amplification of the genes BCHE and SLC2A2 in 40% of squamous cell carcinoma of the lung

Gene amplification is a common genetic change in human cancer cells. Previously, we provided the first evidence for gene amplification at chromosome band 3q26 in squamous cell lung carcinoma. In this study, the following analyses were performed: (a) we evaluated biopsies and paraffin-embedded tissues of 16 additional squamous cell lung carcinomas for gene amplification using reverse chromosome painting. Of the 16 tumors, 3 tumors showed an amplification of the entire long arm of chromosome 3, and 3 tumors showed various amplifications on 3q, all of which involved chromosome band 3q26; (b) we tested eight genes encompassing region 3q25-qter in two different tumors to identify amplified genes on chromosome 3q. The genes SI, BCHE, and SLC2A2 were amplified in both tumors; and (c) we analyzed 15 additional paraffin-embedded tissues to determine the amplification frequency of these genes. Of the 15 squamous cell lung carcinomas, 6 showed amplification for at least 1 of the genes, with BCHE and SLC2A2 as the genes most frequently amplified. Together, our reverse chromosome painting data and our PCR analysis indicate gene amplification at 3q26 in 40% of all squamous cell lung carcinomas with BCHE and SLC2A2 as possible target genes of the amplification unit in squamous cell lung carcinoma.     

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.     

Changes in pain perception and pain-related somatosensory evoked potentials in humans produced by exposure to oscillating magnetic fields

cDNA selection was used to isolate coding sequences from cosmids mapping to the gene-rich telomeric region of human chromosome 21q. A novel cDNA, termed SMT3A, was isolated and mapped between the loci PFKL and D21S171, about 2.2 Mb proximal to the telomere. The predicted protein of 103 amino acids appears to be a homologue of the Saccharomyces cerevisiae SMT3 protein, whose gene was previously isolated as a suppressor of mutations in the MIF2 gene. The yeast MIF2 gene encodes an essential centromeric protein and shows homology to mammalian CENP-C, an integral component of active kinetochores. SMT3A was found to be highly homologous to two other recently isolated human genes, suggesting the presence of a new gene family. Homologous sequences were also found in protozoa, metazoa, and plants. Moreover, all predicted proteins show significant homology to ubiquitin. The proposed role of yeast SMT3 as centromeric protein and the strong evolutionary conservation of the SMT3A gene suggest an involvement of the encoded protein in the function and/or structure of the eukaryotic kinetochore.     

Model of plasmid-bearing, plasmid-free competition in the chemostat with nutrient recycling and an inhibitor

Glutaraldehyde and formaldehyde were used to fix crane-fly spermatocytes for observation with differential interference contrast (DIC) microscopy. In aldehyde-fixed cells, kinetochores exhibit contrast not normally observed in living cells. Although the mechanism underlying this result is not understood, the visualization of kinetochores as distinct refractile objects opens the way for analysis of unstained kinetochores with the light microscope. The analysis of kinetochore refractility reported in this paper is made possible by the finding that the refractility of chromosomes in formaldehyde-fixed cells decreases as the concentration of formaldehyde is increased. In 4% formaldehyde, the refractility of chromosomes is matched with that of its surround, chromosomes appear invisible, and kinetochores may be analyzed as if chromosomes were not present. Kinetochores were imaged with DIC optics, and then digital image analysis was performed. Gray-level scans through the highlight and shadow of an individual kinetochore parallel to the axis of shear resulted in a curve having a slope proportional to the DIC optical path gradient. Curves from autosomal kinetochores imaged in anaphase had slopes approximately one-half those recorded at metaphase under identical optical conditions. By contrast, kinetochore thicknesses (defined as the distance between the peak and the valley of a gray-level scan) at those two stages were not significantly different. These data suggest a loss of dry mass from autosomal kinetochores during anaphase. Neither the refractility nor thickness of lagging sex kinetochores varied as autosomes went through anaphase. The conclusion drawn from these findings is that the decreased refractility of autosomal kinetochores in anaphase is movement-related.     

Allelic imbalance study of 16q in human primary breast carcinomas using microsatellite markers

The high incidence of allelic imbalance on the long arm of chromosome 16 in breast cancer suggests its involvement in the development and progression of the tumor. Several loss of heterozygosity (LOH) studies have led to the assignment of commonly deleted regions on 16q where tumor suppressor genes may be located. The most recurrent LOH regions have been 16q22.1 and 16q22.4-qter. The aim of this study was to gain further insight into the occurrence of one or multiple "smallest regions of overlap" on 16q in a new series of breast carcinomas. Hence, a detailed allelic imbalance map was constructed for 46 sporadic breast carcinomas, using 11 polymorphic microsatellite markers located on chromosome 16. Allelic imbalance of one or more markers on 16q was shown by 30 of the 46 tumors (65%). Among these 30 carcinomas, LOH on the long arm of chromosome 16 was detected at all informative loci in 19 (41%); 13 of them showed allelic imbalance on the long but not on the short arm, with the occurrence of variable "breakpoints" in the pericentromeric region. The partial allelic imbalance in 11 tumors involved either the 16q22.1-qter LOH region or interstitial LOH regions. A commonly deleted region was found between D16S421 and D16S289 on 16q22.1 in 29 of the 30 tumors. The present data argue in favor of an important involvement of a tumor suppressor gene mapping to 16q22.1 in the genesis or progression of breast cancer.