YAC Clones Research Articles

Clinicopathological and Chromosomal Analysis of Small Intestinal Malignant Lymphoma using Fluorescence in situ Hybridization on Paraffin-Embedded Tissue Sections.

<Background> Specific chromosomal translocations are closely associated with distinctive subtypes of non-Hodgkin's lymphoma (NHL). However, in small intestinal malignant lymphoma, the frequency of specific chromosomal abnormalities and their relationship to clinicopathological features remain unclear. The aim of this study was to demonstrate the frequencies of the chromosomal translocations involving BCL1, BCL2, c-MYC, MALT1 and BCL6 genes by using fluorescence in situ hybridization directly on paraffin-embedded tissue sections (tissue-FISH) and to evaluate the relationship between clinical outcome and these chromosomal translocations in small intestinal malignant lymphoma.<Materials and Methods> Twenty-five consecutive patients (22 men and 3 women; age ranging from 10 to 78 years old) diagnosed as having small intestinal malignant lymphoma between 1996 and 2003 were investigated in this study. Tissue-FISH was performed with LSI IGH/BCL1 (Vysis, CA), LSI IGH/BCL2 (Vysis, CA), LSI IGH/MYC (Vysis, CA), and LSI BCL6 (Vysis, CA) probes. API2/MALT1 rearrangement was detected using two YAC clones, y966e4 for 11q21 and y943b8 for 18q21, on paraffin-embedded tissue sections as described previously.<Result> The primary tumor was located at the duodenum (12 cases, 40%), jejunum (4 cases, 16%), ileum (9 cases, 32%) duodenum to ileum (2 cases, 8%) and duodenum to jejunum (1 case, 4%). Histopathologically, 14 patients were diagnosed as having diffuse large B-cell lymphoma (DLBCL) (56%), 6 as marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT) (24%), 3 as follicular lymphoma (FL) (12%) and 2 as mantle cell lymphoma (MCL) (8%). IGH/BCL2 translocation was detected in 3 out of 3 cases of FL (100%), and 3 out of 14 cases of DLBCL (21%), but none of MALT lymphoma. IGH/c-MYC translocation was detected in 1 out of 14 cases of DLBCL (7%). API2/MALT1 translocation was detected in 1 out of 6 cases of MALT lymphoma (17%), and 1 out of 14 cases of DLBCL (7%). IGH/BCL1 translocation was detected only in MCL (2 cases out of 2: 100%). BCL6 translocation was detected in 5 out of 14 cases of DLBCL (36%). As for survival time, DLBCL with either BCL6 or IGH/BCL2 translocation showed poorer prognosis than DLBCL without these translocations (5-year survival rate; 20% vs 65%).<Conclusion> The current study disclosed that the duodenum was preferentially involved in small intestinal malignant lymphoma (40%). Cytogenetically, 13 out of 25 cases (52%) showed specific chromosomal abnormalities. In DLBCL, BCL6 translocation was detected in five cases, t(14;18) in two, t(8;14) and t(11;18) in one each, indicating that DLBCL is cytogenetically heterogeneous. DLBCL with either BCL6 or IGH/BCL2 translocation appeared to be progressive as shown by shorter survival time. The current study validated that tissue-FISH is a routine diagnostic tool for detecting specific chromosomal translocations in NHL.

Abstract 1549: Ectopic expression of a glutamate receptor gene, glur6, induces growth arrest and senescence in breast and ovarian tumor cells

Abstract Applying a functional strategy we determined that Glur6 gene plays a role in the cell senescence. Using chromosome transfer method, we previously identified a YAC clone that carries a cell senescence gene, SEN6A, mapping at 6q21. Human data base search identified GluR6, a kainate type ionotropic glutamate receptor gene, to represent SEN6A. Glutamate receptors are primarily located on the membrane of neuronal cells and are important mediators of rapid synaptic transmission in central nervous system. We cloned GluR6 gene by RT-PCR and defined the structure, splicing pattern and its role in the cell growth and cancer. Five GluR6 splice variants (A-E), transcribed from two different promotes, are differentially expressed in brain and other tissues and cultured cells. GluR6A, specifically expressed in brain, is transcribed from promoter 2, while GluR6 (B-E), transcribed from promoter 1, are expressed in other tissues. GluR6 is either turned off or expressed at reduced level in breast and ovarian tumor cells, whereas its expression is elevated in aged (senescent) cells. Full length cDNAs (open reading frames) for each of the five transcript isoforms of GluR6 were cloned, in frame with EGFP, in a retroviral vector, PQCXIP, and introduced into breast and ovarian tumor cell lines T47D, MCF.7 and SKOV3. Analysis of gene transfer clones revealed that GluR6 expression inhibit the proliferation of breast and ovarian tumor cells leading to senescence. Cells expressing transferred GluR6 gene displayed the doubling time of 60-96 hours that gradually extended to 3-4 days, ending in complete growth arrest and senescence. The gene transfer senescent cells lacked DNA synthesis and were positive for SA-βgal staining. In controls, the transfer of empty vector or vector carrying a truncated GluR6 gene did not affect the growth of tumor cells. In addition control cultures were negative for SA-βgal staining and positive for DNA replication. Our preliminary data shows that GluR6 may function through AKT and ROS pathways. In conclusion, in this study we show that GluR6, which normally functions in neurotransmission and is implicated in a number of neurological disorders, may also be involved in the development and progression of cancer. Thus, pathways related to GluR6 expression may prove to be new novel targets for cancer therapy. Modulation of GluR6 function through small molecule and/or glutamate antagonists has the potential to modify the behavior of tumors in terms of their migration, invasion and metastasis. Drugs already developed for the treatment of neurological disorders can be tested for treatment of cancer. Citation Format: Raghbir S. Athwal, Vikramjit K. Zhawar, Gurpreet Kaur. Ectopic expression of a glutamate receptor gene, glur6, induces growth arrest and senescence in breast and ovarian tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1549. doi:10.1158/1538-7445.AM2014-1549

The abnormal genes of learning and memory in C. elegans and their functions

Objective To research into the function of abnormal gene of learning and memory hab-1 (cn308). Methods 1. There were three YAC clones in the hab-1 area, Y63D3A.6, Y63D3A.7 and Y63D3A.8. The cDNA sequence of Y63D3A.6, Y63D3A.7 and Y63D3A.8 were retrieved in the gene bank, processed by DNASIS-Mac v3.6 software and designed primers, PCR was processed. The PCR product was inserted into pMD ~(18)TVector, culturing and recovering target DNA of Y63D3A.6, Y63D3A.7 and Y63D3A.8, purifying DNA .2. Carrying out rescue experiments; DNA of three YAC clones Y63D3A.6, Y63D3A.7 and Y63D3A.8 were linked with the GFP Protein respectively for preparing DNA:GFP fusion protein. The three DNA:GFP fusion protein solution was microinjected into L4 size C. elegans gonad respectively by OLYMPUS Axiovert S100 confocal inverted microscope operating system, growing and subcultring to F1 generation, observing gene expression site at the selected C. elegans with GFP fluorescence in F1 generation, carrying out Tap test, observing which YAC clone rescued the hab-1 (cn308) phenotype to wild-type phenotype to identify the hab-1 allele. 3. DNA sequencing and Homolog search; DNA sequencing for cn308 after hab-1 allele was ascertained. According to the results of cn308 sequencing and known allele DNA sequences to Homolog search, the genetic structure of cn308 was analyzed and BLST homologic search was carried out, comparing target gene function with other species to clarified gene function of hab-1. Results The result of χ~2 test for the rate of gene expression of Y63D3A.6, Y63D3A.7 and Y63D3A.8 χ~2 showed χ~2 >0.05, P<0.05, there was a significant difference. It showed that although Y63D3A.6, Y63D3A.7 and Y63D3A.8 have linkage with the adjacent location of the first autosomal, expressing rate on the head of C. elegans had no relatedness and had significant differences. It revealed that Y63D3A.7 was expressed strongly on the head of C. elegans, and Tap test proved that Y63D3A.7 rescued hab-1 to wild-type phenotype. The result of DNA sequencing was that hab-1 (cn308) was a missense mutation (GAG→GGG, namely, Glu→Gly ). The result of BLST homologic search showed that Y63D3A.7 was the activity which adjusts mitochondrion complex Ⅰ, it illustrated that hab-1 gene had relations with the function of mitochondrion complex Ⅰ. Conclusion Y63D3A.7 is the allele of hab-1, mainly expressed in the central nervous system. Hypofunction of learning and memory in C. elegans has relations with the function of mitochondrion complex Ⅰ which influences hab-1 mutations. Key words: Tap stimulation; Hab-1; Mitochondrion complex Ⅰ; Learning and memory

Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

The Protein Kinase DYRK1A Regulates Caspase-9-Mediated Apoptosis during Retina Development

The precise regulation of programmed cell death is critical for the normal development of the nervous system. We show here that DYRK1A (minibrain), a protein kinase essential for normal growth, is a negative regulator of the intrinsic apoptotic pathway in the developing retina. We provide evidence that changes in Dyrk1A gene dosage in the mouse strongly alter the cellularity of inner retina layers and result in severe functional alterations. We show that DYRK1A does not affect the proliferation or specification of retina progenitor cells, but rather regulates the number of cells that die by apoptosis. We demonstrate that DYRK1A phosphorylates caspase-9 on threonine residue 125, and that this phosphorylation event is crucial to protect retina cells from apoptotic cell death. Our data suggest a model in which dysregulation of the apoptotic response in differentiating neurons participates in the neuropathology of diseases that display DYRK1A gene-dosage imbalance effects, such as Down's syndrome.

Physical delimitation of the pepper Bs3 resistance gene specifying recognition of the AvrBs3 protein from Xanthomonas campestris pv. vesicatoria

The pepper (Capsicum annuum) Bs3 gene confers resistance to avrBs3-expressing strains of the bacterial spot pathogen Xanthomonas campestris pv. vesicatoria. To physically delimit Bs3, a pepper YAC library was screened with two flanking DNA markers that are separated from Bs3 by 1.0 and 1.2 cM, respectively resulting in the identification of three YAC clones. Genetic mapping of the corresponding YACends revealed however, that these YACs do not cover Bs3 and subsequent screens with newly developed YACend markers failed to identify new YAC clones. Marker saturation at the Bs3 locus was carried out by amplified fragment length polymorphism (AFLP). The analysis of 1,024 primer combinations resulted in the identification of 47 new Bs3-linked AFLPs. High-resolution linkage mapping of Bs3 was accomplished by inspecting more than 4,000 F(2) segregants resulting in a genetic resolution of 0.01 cM. Using tightly Bs3-linked YACend- and AFLP-derived markers we established a Bs3-spanning BAC contig and physically delimited the target gene within one BAC clone. The analysis of the Bs3-containing genomic region revealed substantial local variation in the correlation of genetic and physical distances.

Molecular cytogenetic analysis of a de novo interstitial chromosome 10q22 deletion

Interstitial deletions of 10q are rare, and only one patient with a deletion confined to chromosome band 10q22 has been reported so far. We report on a 2 6/12-year-old girl with a constitutional interstitial deletion of one homologue of 10q [karyotype: 46,XX,del(10)(q22.2q22.3)de novo]. Our patient had muscular hypotonia, developmental delay, growth retardation, mild facial dysmorphism, and hypoplastic labia minora. The precise location and extent (3.6 Mb) of the deletion was determined by fluorescence in situ hybridization (FISH) using 16 YAC and BAC clones. The clinical features in our patient are remarkably similar to the previously reported patient with a 10q22.2 deletion.

Prenatal diagnosis and molecular cytogenetic characterisation of a small de novo interstitial duplication 16q11.2‐q13

We describe the first prenatally detected case of a small de novo interstitial duplication of chromosome 16q. This chromosomal aberration is extremely rare. Amniocentesis was indicated by advanced maternal age only. Ultrasound examinations of the foetus showed no abnormalities. Conventional and molecular cytogenetic analyses on cultured amniocytes by comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH) using partial chromosome paints and a locus-specific YAC clone revealed a de novo direct duplication of the chromosomal region 16q11.2-q13 leading to a partial trisomy 16q (46,XX,dup(16)(q11.2q13)). There are only five postnatal reports of comparable duplications involving this chromosomal region. These patients presented with little or no associated dysmorphic features but with significant neurodevelopmental delay and severe behavioural problems. After genetic counselling, the parents opted for termination of pregnancy. Post-mortem examination showed slight facial dysmorphic signs, minor dysgenesis of the ovaries and an atypical outflow of the arteria thyroidea ima.

Molecular cytogenetic analysis of a de novo interstitial deletion of 5q23.3q31.2 and its phenotypic consequences

We report a 2(3/12)-year-old boy with a constitutional interstitial deletion of 5q,46,XY,del(5)(q23.3q31.2) de novo. Clinical manifestations in this patient included failure to thrive, psychomotor retardation, mild facial dysmorphic features, and long and slender fingers and toes. The precise location and extent (9.5 Mb) of the deletion was determined by fluorescence in situ hybridization (FISH) using 19 YAC and BAC clones. Comparison of the present patient with six other patients with deletions of chromosomal bands 5q22-5q31 allowed further delineation of a constitutional del5q22q31 syndrome. The main features of this syndrome are psychomotor retardation, failure to thrive, hypotonia, hypoplastic muscles, cleft or high arched palate, low-set and dysplastic ears, flat nasal bridge, downslanting palpebral fissures, hypertelorism, anteverted nostrils, and micro- and/or retrognathia.

Phenotypic and molecular characterisation of a de novo 5q deletion that includes the APC gene

We report on a 12-year-old female patient with mild dysmorphic signs, including bilateral epicanthal folds, low-set dysplastic ears, a short nose with anteverted nostrils, conically shaped fingers, generalised increase of subcutaneous fat, multiple fine venous teleangiectasia on her back, mild pectus carinatum, and a general muscular hypotonia. Cytogenetic analysis and fluorescence in situ hybridisation (FISH) studies using region-specific BAC and YAC clones indicated a de novo interstitial deletion of the long arm of chromosome 5, resulting in monosomy 5q21.1-q23.1. Molecular analysis of polymorphic markers helped to narrow down the breakpoints and demonstrated that the derivative chromosome 5 is of paternal origin. By using the same panel of polymorphic markers, a reinvestigation of a similar, already published, 5q deletion case [Raedle et al. (2001) Am J Gastroenterol 96:3016-3020] was performed, allowing a more detailed genotype-phenotype correlation. Phenotypic classification was also carried out. Several known genes, including APC and MCC, were found to map to the common deleted genomic segment. Genetic counselling based on the molecular analysis data was performed for the index family.

Identification of Novel Type of ETV6-TTL Fusion Gene in Pediatric Myeloid/NK Cell Precursor Acute Leukemia Having t(12;13)(p13;q14).

The t(12;13)(p11-13:q11-14) is a recurring chromosomal abnormality found in different types of hematological malignancies. Three genes on 13q12-14, CDX2, FLT3, and TTL have been identified as fusion partners of ETV6(TEL) on 12p13. These genes create different forms of fusion transcripts with ETV6. In the present study, we identified novel types of ETV6-TTL fusion transcripts in a case of a 9 year-old female who developed myeloid/NK cell precursor acute leukemia with t(12;13)(p13;q14). Cytogenetic analyses of the leukemic cells of the patient using a regular G-banding revealed 47, XX, del(2)(q?), -9, der(12)t(12;13)(p13;q14), add(13)(q1?2), +mar1, +mar2 [13/20]. Fluorescence in situ hybridization using YAC clone revealed that ETV6 gene was split in metaphase chromosomes of patient's leukemic cells. We performed reverse-transcriptase-polymerase chain reaction (RT-PCR) analysis to detect the fusion transcripts of ETV6-CDX2, ETV6-FLT3 or TTL-ETV6, however, no fusion transcripts of previously described types were detected. We next performed RT-PCR analysis using various sets of primers to detect unknown types of fusion transcripts involving these genes, and detected novel types of fusion transcripts of ETV6-TTL. These fusion transcripts consisted of exons 1 to 5 of ETV6 and exons 5 to 8 of TTL, and exons 1 to 6 of ETV6 and exon 9 or exons 8 to 9 of TTL. No reciprocal fusion transcripts were detected. Predicted fusion proteins consisted of N-terminal ETV6 lacking whole or part of ETS-binding domain and C-terminal TTL. Previous report showed major type of TTL-ETV6 fusion transcript consisted of exons 1 to 5 of TTL and exons 2 to 8 of ETV6 which contained both helix-loop-helix and ETS-binding domains. These results suggested that novel types of ETV6-TTL act as different fusion proteins from previously reported TTL-ETV6 in leukemogenesis. At the cytogenetic level, it may be difficult to distinguish ETV6-CDX2, ETV6-FLT3, and ETV6-TTL. Further accumulation of the patients with t(12;13) and further analysis of these novel types of fusion transcripts may clarify the pathogenesis of t(12;13)-leukemia.

Unexpected identification of two interstitial deletions in a patient with a pericentric inversion of a chromosome 4 and an abnormal phenotype

Interstitial deletions and pericentric inversions of chromosome 4 appear to be unusual phenomena. Here, we report the case of a 14-year-old boy with severe psychomotor retardation with a de novo 46,XY,der(4)del(p15.2p15.31)inv(4)(p15.2q13.3)del(4)(q13.2q13.2) karyotype. We used FISH analysis with YAC and BAC clones to characterise the inversion's breakpoints. A complex event with six breakpoints was found, characterised by a pericentric inversion and two deletions, the first on the short arm of chromosome 4 (4p) and the second on the long arm of chromosome 4 (4q). The deletion events had removed two segments, one of approximately 5 Mb, from 4p, outside the inversion, and the other 2 Mb from 4q, inside the inversion. These rearrangements were not found in the parents. Microsatellite marker analysis showed that the inversion carrying chromosome 4 was derived from the father. Bioinformatic analysis of the human genome sequence allowed us to identify several hemizygotic genes in the patient, which might be involved in the pathogenesis of this clinical phenotype.

Phosphotyrosine Proteomic Study of Interferon α Signaling Pathway Using a Combination of Immunoprecipitation and Immobilized Metal Affinity Chromatography

Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.

FGF7-like gene is associated with pericentric inversion of chromosome 9, and FGF7 is involved in the development of ovarian cancer

Pericentric inversion of chromosome 9 is a common chromosomal aberration that may be involved in the pathogenesis of several types of cancer. Screening for structural balanced chromosomal aberrations in 58 patients with ovarian carcinoma found a patient with inv(9)(p11;q13) who had repeated spontaneous abortions. To define the breakpoint at the 9p11 region, the human 9p11-specific CEPH-YAC library was first screened with fluorescence in situ hybridization (FISH) analysis, resulting in isolation of the YAC clones 961d9 and 954b9 spanning the breakpoint. Next, screening with FISH analysis of the cosmid library constructed from 961D9 isolated 3 cosmid clones that included the breakpoint. DNA sequence analysis showed that the cosmid clones spanned the 57.7-kb sequence, which contained the FGF7 [keratinocyte growth factor (KGF)]-like gene including exons 2 and 3. FISH analysis showed that the 57.7-kb sequence was duplicated from the 9p11 region to the 9q13 region on the aberrant chromosome 9. Southern blot analysis showed multiple FGF7-like genes in the 9p11 region which were also duplicated to the 9q13 region on the aberrant chromosome 9. Because the FGF7-like genes are located at 9p11 and 9q13, our results are consistent with the hypothesis that the FGF7-like genes at 9p11 and 9p13 initiate the pericentric inversion of chromosome 9. Analysis of surgical specimens of ovarian cancer with the reverse transcription-polymerase chain reaction and immunohistochemical staining showed overexpression of the FGF7 gene in 4 of 9 stage I or II cases and in 10 of 11 stage III or IV cases. These findings suggest that FGF7 plays a significant role in the development of ovarian cancer.

Structure and evolution of the mouse pregnancy-specific glycoprotein (Psg) gene locus.

BackgroundThe pregnancy-specific glycoprotein (Psg) genes encode proteins of unknown function, and are members of the carcinoembryonic antigen (Cea) gene family, which is a member of the immunoglobulin gene (Ig) superfamily. In rodents and primates, but not in artiodactyls (even-toed ungulates / hoofed mammals), there have been independent expansions of the Psg gene family, with all members expressed exclusively in placental trophoblast cells. For the mouse Psg genes, we sought to determine the genomic organisation of the locus, the expression profiles of the various family members, and the evolution of exon structure, to attempt to reconstruct the evolutionary history of this locus, and to determine whether expansion of the gene family has been driven by selection for increased gene dosage, or diversification of function.ResultsWe collated the mouse Psg gene sequences currently in the public genome and expressed-sequence tag (EST) databases and used systematic BLAST searches to generate complete sequences for all known mouse Psg genes. We identified a novel family member, Psg31, which is similar to Psg30 but, uniquely amongst mouse Psg genes, has a duplicated N1 domain. We also identified a novel splice variant of Psg16 (bCEA). We show that Psg24 and Psg30 / Psg31 have independently undergone expansion of N-domain number. By mapping BAC, YAC and cosmid clones we described two clusters of Psg genes, which we linked and oriented using fluorescent in situ hybridisation (FISH). Comparison of our Psg locus map with the public mouse genome database indicates good agreement in overall structure and further elucidates gene order. Expression levels of Psg genes in placentas of different developmental stages revealed dramatic differences in the developmental expression profile of individual family members.ConclusionWe have combined existing information, and provide new information concerning the evolution of mouse Psg exon organization, the mouse Psg genomic locus structure, and the expression patterns of individual Psg genes. This information will facilitate functional studies of this complex gene family.

Supernumerary der(1) marker chromosome derived from a ring chromosome 1 which has retained the original centromere and euchromatin from 1q21.1 ? q21.3 with substantial loss of 1q12 heterochromatin in a female with dysmorphic features and psychomotoric developmental delay

We report on a 5.5-year-old girl with dysmorphic features and psychomotoric developmental delay with a mitotically stable supernumerary marker chromosome. The origin of the marker was identified by microdissection and reverse painting of marker DNA as the pericentromeric region of chromosome 1. Fine mapping by FISH with selected YAC or BAC clones identified no p-arm material on the marker. The marker has retained its original centromere and euchromatin from 1q21.1-q21.3 but only small remnants of the 1q12 heterochromatin. Furthermore, some FISH clones presented single signals on the marker and others presented double signals indicating a partial duplication within the marker. These observations suggest a multi-step origin of the marker most probably with ring formation as the first step.

Trisomy 3 may predict a poor response of gastric MALT lymphoma toHelicobacter pylorieradication therapy

To investigate the relation of the response to Helicobacter pylori eradication therapy to the depth of tumor invasion and chromosome abnormalities in patients with mucosa-associated lymphoid tissue (MALT) lymphoma and to determine the clinical value of aneuploidy. We studied 13 patients with localized gastric MALT lymphoma of stage E1. Before eradication therapy, the depth of tumor invasion was assessed by endoscopic ultrasonography in 8 patients and by endoscopic examination and gastrointestinal series in the remaining patients. To detect chromosomal abnormalities, paraffin-embedded tissue sections of diagnostic biopsy specimens underwent tissue-fluorescence in situ hybridization (FISH), using chromosome-specific alpha-satellite DNA probes for chromosomes 3,7,12, and 18 and YAC clones for t(11;18)(q21;q21). Seven of the 13 patients had complete regression (CR) in response to H pylori eradication therapy. No patient with CR had submucosal tumor invasion. Trisomy 18 was seen in 1 patient with CR, and both trisomies 12 and 18 were present in another patient with CR. All patients with no response or progressive disease had deep submucosal tumor invasion and showed t(11;18)(q21;q21) or trisomy 3. Trisomy 7 was not detected in this series of patients. The depth of tumor invasion is an accurate predictor of the response of stage E1 MALT lymphoma to H pylori eradication therapy and is closely associated with the presence of chromosomal abnormalities. Trisomy 3 may predict the aggressive development of MALT lymphoma.

Specificity of interphase fluorescence in situ hybridization for detection of chromosome aberrations in tumor pathology

Interphase fluorescence in situ hybridization (IFISH) is an interesting and intriguing cytogenetic approach in the study of tumor chromosomal abnormalities when metaphases are not available. This technique can be applied to different types of tumor nuclei, including imprinted nuclei (IM), nuclei obtained from conventional cytogenetic procedures (PB), frozen nuclei, paraffin-embedded nuclei (PE), and nuclei extracted from paraffin-embedded sections (EX). IFISH is a high-sensitivity approach in tumor studies that can give evidence of genetic aberrations present in a small percentage of cells that are likely to escape detection if only molecular techniques are applied. Despite its high sensitivity and versatility, IFISH is an indirect cytogenetic method and needs controls to have adequate specificity. This study includes present data obtained in IFISH experiments using different types of probes (α-satellite and YAC clones) hybridized on different types of normal control nuclei, such as PB, IM, EX, and PE nuclei, to define the threshold level for monosomy and trisomy of different chromosomal regions. My findings demonstrate that the cut-off values depend both on the types of probes and on the types of target nuclei. Therefore, even if IFISH is a versatile, high-sensitivity technique for detecting chromosomal abnormalities, the lack of accurate controls may result in the misdiagnosis of some abnormalities.

Autosomal dominant Brody disease cosegregates with a chromosomal (2;7)(p11.2;p12.1) translocation in an Italian family.

Brody disease is a rare muscle disorder characterized by exercise-induced impairment in muscle relaxation, due to a markedly reduced influx of calcium ions in the sarcoplasmic reticulum. A subset of autosomal recessive families harbour mutations in the ATP2A1 gene, encoding the fast-twitch skeletal muscle sarcoplasmic reticulum Ca(2+) ATPase (SERCA1). Rare autosomal dominant families have been described, in which ATP2A1 was excluded as the causative gene, further supporting genetic heterogeneity. We report four individuals from a three-generation Italian family with a clinical phenotype of Brody disease, in which linkage analysis excluded ATP2A1 as the responsible gene. The disease cosegregates in an autosomal dominant fashion with an apparently balanced constitutional chromosome translocation (2;7)(p11.2;p12.1), suggesting a causal relationship between the rearrangement and the phenotype. FISH analysis using YAC and PAC clones as probes refined the breakpoint regions to genomic segments of about 164 and 120 kb, respectively, providing a possible clue to pinpoint the location of a novel gene responsible for this rare muscle disorder.

Identification of chaperonin CCT gamma subunit as a determinant of retinotectal development by whole-genome subtraction cloning from zebrafish no tectal neuron mutant.

Zebrafish no tectal neuron (ntn) mutant obtained by trimethylpsoralen (TMP) mutagenesis showed defects in tectal neuropil formation and small eyes. We carried out whole-genome subtraction between wild-type and mutant zebrafish embryos using the representational difference analysis (RDA) method. Nineteen subtraction products enabled us to construct genetic and physical maps of the ntn region. Direct selection of cDNAs using a YAC clone encompassing the ntn locus and RT-PCR analysis of transcripts identified a 143 bp deletion in the cct3 gene encoding the gamma subunit of chaperonin containing TCP-1 (CCT). Injection of antisense cct3 morpholino oligonucleotides into zebrafish embryos induced characteristic ntn phenotypes including defects in retinal ganglion cell (RGC) differentiation and tectal neuropil formation. Moreover, injection of cct3 mRNA successfully rescued ntn mutant embryos. Our results suggest that RDA is an efficient and widely applicable cloning strategy in zebrafish genetics. The strong expression of the cct3 mRNA started in the entire embryos by 12 hpf and was sustained thereafter, but there were no detectable abnormalities in body patterning and neurogenesis in ntn mutant embryos at 30 hpf. The expression patterns of transcription factor genes ath5 and brn3b that are essential for the development and maintenance of RGCs were indistinguishable between wild-type and ntn mutant embryos, but those of early and late differentiation markers of RGCs, nicotinic acetylcholine receptor beta 3 and zn5, were diminished in mutant embryos. Immunostaining of acetylated tubulin also revealed the impairment of RGC neurite extension. Thus, the ntn mutation of the cct3 gene impaired the differentiation of retinal neuroepithelial cells to RGCs. Similarly, the expression of brn3b was normal in the tectum of ntn mutants, but tectal neuropil formation was abolished. These results suggest that the gamma subunit of chaperonin CCT plays an essential role in retinotectal development.