RESUMO
Despite an improved understanding of the molecular aberrations that occur in glioblastoma, the use of molecularly targeted therapies have so far been disappointing. We present a patient with three different brain tumours: astrocytoma, glioblastoma and gliosarcoma. Genetic analysis showed that the three different brain tumours were derived from a common origin but had each developed unique genetic aberrations. Included in these, the glioblastoma had PDGFRA amplification, whereas the gliosarcoma had MYC amplification. We propose that genetic heterogeneity contributes to treatment failure and requires comprehensive assessment in the era of personalised medicine.
RESUMO
The beta-amyloid peptide (Abeta) that accumulates in senile plaques in Alzheimer's disease is formed by cleavage of the amyloid precursor protein (APP). The APP gene has several intronic Alu elements inserted in either the sense or antisense orientation. In this study, we demonstrate that binding of SC35 and hnRNPA1 to Alu elements on either side of exon 7 in the transcribed pre-mRNA is involved in alternative splicing of APP exons 7 and 8. Neuronal cells transfected with the full-length form of APP secrete higher levels of Abeta than cells transfected with the APP695 isoform lacking exons 7 and 8. Finally, we show that treatment of neuronal cells with estradiol results in increased expression of APP695, SC35 and hnRNPA1, and lowers the level of secreted Abeta. An understanding of the regulation of splicing of APP may lead to the identification of new targets for treating Alzheimer's disease.
Assuntos
Processamento Alternativo/fisiologia , Elementos Alu/fisiologia , Precursor de Proteína beta-Amiloide/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Processamento Alternativo/genética , Precursor de Proteína beta-Amiloide/genética , Linhagem Celular , Éxons/genética , Ribonucleoproteína Nuclear Heterogênea A1 , Humanos , Neurônios/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fatores de Processamento de Serina-ArgininaRESUMO
Promyelocytic leukemia (PML) bodies are nuclear multi-protein domains. The observations that viruses transcribe their genomes adjacent to PML bodies and that nascent RNA accumulates at their periphery suggest that PML bodies function in transcription. We have used immuno-FISH in primary human fibroblasts to determine the 3D spatial organisation of gene-rich and gene-poor chromosomal regions relative to PML bodies. We find a highly non-random association of the gene-rich major histocompatibilty complex (MHC) on chromosome 6 with PML bodies. This association is specific for the centromeric end of the MHC and extends over a genomic region of at least 1.6 megabases. We also show that PML association is maintained when a subsection of this region is integrated into another chromosomal location. This is the first demonstration that PML bodies have specific chromosomal associations and supports a model for PML bodies as part of a functional nuclear compartment.
Assuntos
Núcleo Celular/metabolismo , Interfase/fisiologia , Complexo Principal de Histocompatibilidade/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Células Cultivadas , Cromossomos Humanos Par 6/genética , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Hibridização in Situ Fluorescente/métodos , Microscopia de Fluorescência/métodos , Proteína da Leucemia Promielocítica , Proteínas Supressoras de TumorAssuntos
Cromátides/fisiologia , Proteínas Nucleares/fisiologia , Ciclo Celular , Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Cromossomos Humanos Par 6 , Replicação do DNA , Proteínas Fúngicas , Humanos , Hibridização in Situ Fluorescente , Complexo Principal de Histocompatibilidade/genética , CoesinasRESUMO
Phosphorylated serine- and arginine-rich (SR) proteins play an important role in the formation of spliceosomes, possibly controlling the regulation of alternative splicing. Enzymes that phosphorylate the SR proteins belong to the family of CDC2/CDC28-like kinases (CLK). Employing nucleotide sequence comparison of human expressed sequence tag sequences to the murine counterpart, we identified, cloned, and recombinantly expressed the human orthologue to the murine CLK4 cDNA. When fused to glutathione S-transferase, the catalytically active human CLK4 is able to autophosphorylate and to phosphorylate myelin basic protein, but not histone H2B as a substrate. Inspection of mRNA accumulation demonstrated gene expression in all human tissues, with the most prominent abundance in liver, kidney, brain, and heart. Using fluorescence in situ hybridization, the human CLK4 cDNA was localized to band q35 on chromosome 5 [corrected].
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 4 , DNA Complementar/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Algoritmos , Animais , Northern Blotting , Encéfalo/metabolismo , Clonagem Molecular , Etiquetas de Sequências Expressas , Glutationa Transferase/metabolismo , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Rim/metabolismo , Fígado/metabolismo , Camundongos , Dados de Sequência Molecular , Miocárdio/metabolismo , Fosforilação , Proteínas Tirosina Quinases , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Distribuição TecidualRESUMO
The abundant chromosome abnormalities in most carcinomas are probably a reflection of genomic instability present in the tumor, so the pattern and variability of chromosome abnormalities will reflect the mechanism of instability combined with the effects of selection. Chromosome rearrangement was investigated in 17 colorectal carcinoma-derived cell lines. Comparative genomic hybridization showed that the chromosome changes were representative of those found in primary tumors. Spectral karyotyping (SKY) showed that translocations were very varied and mostly unbalanced, with no translocation occurring in more than three lines. At least three karyotype patterns could be distinguished. Some lines had few chromosome abnormalities: they all showed microsatellite instability, the replication error (RER)+ phenotype. Most lines had many chromosome abnormalities: at least seven showed a surprisingly consistent pattern, characterized by multiple unbalanced translocations and intermetaphase variation, with chromosome numbers around triploid, 6-16 structural aberrations, and similarities in gains and losses. Almost all of these were RER-, but one, LS411, was RER+. The line HCA7 showed a novel pattern, suggesting a third kind of genomic instability: multiple reciprocal translocations, with little numerical change or variability. This line was also RER+. The coexistence in one tumor of two kinds of genomic instability is to be expected if the underlying defects are selected for in tumor evolution.
Assuntos
Neoplasias Colorretais/genética , Translocação Genética , Neoplasias Colorretais/patologia , Humanos , Cariotipagem , Células Tumorais CultivadasRESUMO
The molecular events involved in pancreatic cancer are becoming increasingly well characterized, with mutations in the dominant oncogene KRAS and the tumour suppressor genes TP53, CDKN2A and MADH4 being typically observed. However, other genetic abnormalities remain to be identified and molecular cytogenetics may be useful to detect chromosomal loci involved in recurrent rearrangements. We have used spectral karyotyping to characterize cytogenetic aberrations in a panel of 20 human pancreatic carcinoma cell lines and confirmed their identities by dual and triple color fluorescence in situ hybridization. The most common partial or whole-arm gains involved 5p, 7q, 12p, 1q, 7p, 5q, 9p, 9q and 11p. The most common partial or whole-arm losses affected 9p, 11q, 18q, 3p, 2q and 1p, as well as the short arms of the acrocentric chromosomes. Spectral karyotyping allowed us to identify a number of recurrent structural aberrations, all of them unbalanced: most frequently i(5)(p10), del(11)(q23), i(12)(p10), i(1)(q10), del(7)(q22) and del(10)(p11). Spectral karyotyping mapped the complex aberrations occurring in pancreatic cancer cell lines and identified non-random patterns of chromosomal rearrangement. This comprehensive characterization should be useful to direct future investigation. The observation that loss at 11q and gains at 5p with i(5)(p10) and 12p with i(12)(p10) are more frequent changes than previously reported would justify more intensive investigation of these chromosomal regions.
Assuntos
Aberrações Cromossômicas/genética , Cariotipagem , Neoplasias Pancreáticas/genética , Inversão Cromossômica , Mapeamento Cromossômico , Coloração Cromossômica , Deleção de Genes , Humanos , Hibridização in Situ Fluorescente/métodos , Ploidias , Translocação Genética , Células Tumorais CultivadasRESUMO
Neuroblastoma is a childhood tumour of the sympathetic nervous system that demonstrates striking clinical heterogeneity. In order to determine which genes are abnormally expressed in neuroblastoma, we screened regions of amplification from the short arm of chromosome 2 in the neuroblastoma cell line IMR-32 and found that the homeobox gene, myeloid ecotropic integration site 1 (MEIS1), is highly amplified. MEIS1 normally maps to chromosome band 2p14. High expression of MEIS1 without amplification was also found in other neuroblastoma cell lines, with and without MYCN amplification, and in medulloblastoma and crythroleukaemia cell lines. MEIS1 is highly expressed in cerebellum and ubiquitously expressed in normal immunohaematopoietic tissues and is thought to be important in cell proliferation and differentiation. While several lines of evidence point towards a role for homeobox genes in the development of other malignancies, this is the first report showing the amplification of a homeobox gene in neuroblastoma.
Assuntos
Genes Homeobox/genética , Proteínas de Homeodomínio/genética , Proteínas de Neoplasias/genética , Neuroblastoma/genética , Northern Blotting , Amplificação de Genes , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Proteína Meis1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
A key feature of interphase chromosomes is their compaction into discrete "territories" in the nucleus. In this review, we focus on the compartmentalization of the genome conferred by this organization and evaluate our current understanding of the role of large-scale chromatin folding in the regulation of gene expression. We examine evidence for the hypothesis that transcription occurs at the external surfaces of chromosomes and follow its evolution to include transcription at the surfaces of chromatin-rich domains within chromosomes. We also present prevailing views regarding the details of large-scale chromatin folding and the functional relationship between chromatin and the enigmatic nuclear matrix.
Assuntos
Cromossomos Humanos/química , Cromossomos Humanos/metabolismo , Interfase/genética , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Bandeamento Cromossômico , Cromossomos Humanos/genética , Fibroblastos , Humanos , Hibridização in Situ Fluorescente , Conformação Molecular , Matriz Nuclear/química , Matriz Nuclear/metabolismo , Transcrição GênicaRESUMO
Neuroblastoma is the commonest extracranial solid tumour in children. There are a number of molecular genetic features known which are of prognostic importance and which are used to direct therapy. Identification and targeting of high-risk individuals with intensive therapeutic regimens may allow an improvement in survival rates. The most powerful biological parameters associated with prognosis in this malignancy are chromosomal changes, especially MYCN amplification, deletion of chromosome 1p and aneuploidy. Rapid characterization of these aberrations at the time of diagnosis is paramount if stratification according to risk group is to be achieved. This paper describes the rapid detection of del(1p), MYCN amplification and trisomy using interphase fluorescence in situ hybridization on imprints from fresh tumour biopsies. The results are related to those obtained by standard molecular methods and karyotyping.
Assuntos
Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 8/genética , Amplificação de Genes , Genes myc , Hibridização in Situ Fluorescente/métodos , Neuroblastoma/genética , Trissomia , Aneuploidia , Biópsia , Southern Blotting , Medula Óssea/patologia , Núcleo Celular/patologia , Centrômero/genética , Criança , Cromossomos Humanos Par 1/ultraestrutura , Cromossomos Humanos Par 8/ultraestrutura , Predisposição Genética para Doença , Humanos , Cariotipagem , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Prognóstico , Sensibilidade e EspecificidadeRESUMO
Juvenile polyposis syndrome (JPS; Online Mendelian Inheritance in Man2 174900) is a rare Mendelian disorder in which individuals have typical hamartomatous polyps within the gastrointestinal tract. The stromal element of the polyps has classically been thought to be the proliferative component, although epithelial malignancies (largely gastrointestinal cancers) occur more frequently than expected in JPS patients. Germ-line mutations in SMAD4 (DPC4) account for about a third of JPS cases. It has been postulated that the apparent paradox of a stromal lesion predisposing to epithelial malignancy can be resolved by the "landscaper" effect: an abnormal stromal environment affects the development of adjacent epithelial cells, and the resulting regeneration of damaged epithelium leads to an increased risk of cancer. We have found allele loss at the SMAD4 locus on 18q in polyps from JPS individuals with a germ-line SMAD4 mutation, showing that SMAD4 is acting as a tumor suppressor gene in JPS polyps, as it does in sporadic cancers of the gastrointestinal tract. Interphase fluorescence in situ hybridization showed deletion of one copy of SMAD4 in the epithelial component of JPS polyps, but not in the inflammatory infiltrate. Fluorescence in situ hybridization also suggested that a single copy of SMAD4 was present in stromal fibroblasts of JPS polyps. Thus, biallelic inactivation of SMAD4 occurs in both the epithelium and some of the stromal cells in these lesions, suggesting a common clonal origin. Epithelial malignancies almost certainly develop in juvenile polyposis through direct malignant progression of the epithelial component of the hamartomas. SMAD4/DPC4 probably acts as a "gatekeeper" tumor suppressor in juvenile polyps, and there is no need to invoke a "landscaper hypothesis."
Assuntos
Proteínas de Ligação a DNA/genética , Gastroenteropatias/genética , Perda de Heterozigosidade , Pólipos/genética , Transativadores/genética , Polipose Adenomatosa do Colo , Cromossomos Humanos Par 18 , Epitélio/metabolismo , Mutação em Linhagem Germinativa , Homozigoto , Humanos , Hibridização in Situ Fluorescente , Repetições de Microssatélites , Proteína Smad4 , SíndromeRESUMO
The approximately 1-Mb leukocyte receptor complex at 19q13.4 is a key polymorphic immunoregion containing all of the natural killer-receptor KIR and related ILT genes. When the organization of the leukocyte receptor complex was compared from two haplotypes, the gene content in the KIR region varied dramatically, with framework loci flanking regions of widely variable gene content. The ILT genes were more stable in number except for ILT6, which was present only in one haplotype. Analysis of Alu repeats and comparison of KIR gene sequences, which are over 90% identical, are consistent with a recent origin. KIR genesis was followed by extensive duplication/deletion as well as intergenic sequence exchange, reminiscent of MHC class I genes, which provide KIR ligands.
Assuntos
Cromossomos Humanos Par 19 , Família Multigênica , Receptores Imunológicos/genética , Elementos Alu , Mapeamento Cromossômico , Primers do DNA , Éxons , Variação Genética , Haplótipos , Humanos , Células Matadoras Naturais , Repetições de Microssatélites , Reação em Cadeia da Polimerase , Receptores KIRRESUMO
The large-scale chromatin organization of the major histocompatibility complex and other regions of chromosome 6 was studied by three-dimensional image analysis in human cell types with major differences in transcriptional activity. Entire gene clusters were visualized by fluorescence in situ hybridization with multiple locus-specific probes. Individual genomic regions showed distinct configurations in relation to the chromosome 6 terrritory. Large chromatin loops containing several megabases of DNA were observed extending outwards from the surface of the domain defined by the specific chromosome 6 paint. The frequency with which a genomic region was observed on an external chromatin loop was cell type dependent and appeared to be related to the number of active genes in that region. Transcriptional up-regulation of genes in the major histocompatibility complex by interferon-gamma led to an increase in the frequency with which this large gene cluster was found on an external chromatin loop. Our data are consistent with an association between large-scale chromatin organization of specific genomic regions and their transcriptional status.
Assuntos
Núcleo Celular/efeitos dos fármacos , Cromatina/genética , Cromossomos Humanos Par 6 , Interferon gama/farmacologia , Complexo Principal de Histocompatibilidade/genética , Linhagem Celular , Humanos , Interfase/efeitos dos fármacos , Masculino , Transcrição GênicaRESUMO
We examined 42 fresh non-small cell lung carcinomas for allelic loss using 4 microsatellite markers located in a 4.5 Mb region in 21q11-21, a gene-poor interval recently found by others to be homozygously deleted and exhibiting frequent allelic loss in lung cancer. We found allelic loss across the entire segment in 13/34 informative squamous carcinomas, with 2 cases showing loss in only part of the region. Analysis by fluorescence in situ hybridization of P1-derived artificial chromosomes from the region directly on paraffin sections of the tumor is in concordance with the loss of heterozygosity (LOH) results, and tentatively excludes a 2 Mb segment bearing 2 of the only 3 known genes in the area. Exon trapping in the remaining segment of loss led to identification and cloning of a novel gene spanning 150 kb within the deletion. The full-length gene encodes a protein of 1,055 amino acids with homology to ubiquitin-specific proteases across the eukaryotic evolutionary spectrum. The expressed protein acts as a de-ubiquitinating enzyme as proved by the ability to cleave ubiquitin from a model fusion protein. We found no mutations in the sequence of the functional domains of this gene in any of the LOH-exhibiting tumor DNA samples. It is, however, interesting that genes of the same superfamily have been reported on 3p21, a locus showing the most frequent allelic instability and deletions in lung cancer. Genes Chromosomes Cancer 27:153-161, 2000.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 21/genética , Endopeptidases/genética , Neoplasias Pulmonares/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA/química , DNA/genética , Endopeptidases/metabolismo , Éxons , Vetores Genéticos , Humanos , Hibridização in Situ Fluorescente , Perda de Heterozigosidade , Repetições de Microssatélites , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Proteases Específicas de Ubiquitina , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences
Assuntos
Cromossomos Humanos Par 3/genética , DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Biblioteca Gênica , Animais , Linhagem Celular , Mapeamento Cromossômico , DNA/química , DNA/metabolismo , Bases de Dados Factuais , Etiquetas de Sequências Expressas , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Camundongos , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
APC is often cited as a prime example of a tumor suppressor gene. Truncating germline and somatic mutations (or, infrequently, allelic loss) occur in tumors in FAP (familial adenomatous polyposis). Most sporadic colorectal cancers also have two APC mutations. Clues from attenuated polyposis, missense germline variants with mild disease and the somatic mutation cluster region (codons 1,250-1,450) indicate, however, that APC mutations might not result in simple loss of protein function. We have found that FAP patients with germline APC mutations within a small region (codons 1,194-1,392 at most) mainly show allelic loss in their colorectal adenomas, in contrast to other FAP patients, whose 'second hits' tend to occur by truncating mutations in the mutation cluster region. Our results indicate that different APC mutations provide cells with different selective advantages, with mutations close to codon 1,300 providing the greatest advantage. Allelic loss is selected strongly in cells with one mutation near codon 1,300. A different germline-somatic APC mutation association exists in FAP desmoids. APC is not, therefore, a classical tumor suppressor. Our findings also indicate a new mechanism for disease severity: if a broader spectrum of mutations is selected in tumors, the somatic mutation rate is effectively higher and more tumors grow.
Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Genes APC/genética , Mutação em Linhagem Germinativa/genética , Modelos Genéticos , Mutação/genética , Adenoma/genética , Adenoma/patologia , Polipose Adenomatosa do Colo/patologia , Alelos , Sequência de Bases , Códon/genética , Neoplasias Colorretais/patologia , Análise Mutacional de DNA , Éxons/genética , Saúde da Família , Fibromatose Agressiva/genética , Fibromatose Agressiva/patologia , Mutação da Fase de Leitura/genética , Deleção de Genes , Frequência do Gene , HumanosRESUMO
How does breast cancer progress? There is evidence both to support (S. W. Duffy et al., Br. J. Cancer, 64: 1133-1138, 1991; R. Rajakariar et al., Br. J. Cancer, 71: 150-154, 1995) and refute (M. Hakama et al., Lancet, 345: 221-224, 1995; R. R. Millis et al., Eur. J. Cancer, 34: 548-553, 1998) the hypothesis of dedifferentiation; the theory that as breast cancers grow they evolve from well differentiated (grade I) to poorly differentiated (grade III) tumors. We provide evidence to support the view that the majority of grade I tumors do not progress to grade III tumors. Comparative genomic hybridization was used to screen entire genomes of a large sample (40 grade I and 50 grade III) of invasive ductal breast carcinomas, stratified by grade. We found distinct genetic differences between grade I and grade III tumors. Significantly, we found that 65% of grade I tumors lost the long arm of chromosome 16 compared with only 16% of grade III tumors. This pattern of loss leads us to conclude that the majority of grade I tumors do not progress to grade III tumors. These findings have important implications because they suggest that different breast tumor grades may have distinct molecular origins, pathogenesis, and behavior and, therefore, potentially present distinct molecular targets for research and treatment.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Aberrações Cromossômicas , Cromossomos Humanos Par 16 , Feminino , Humanos , Hibridização de Ácido NucleicoRESUMO
We have examined the genetic aberrations in two near-diploid glioblastoma multiforme cell lines that appear to have arisen from different glial lineages. One cell line, Hu-O-2A/Gb1, expresses antigens and metabolic profiles characteristic of the oligodendrocyte-type-2 astrocyte (0-2A) lineage of the rat central nervous system. This line generates, in vitro, cells with characteristics of 0-2A progenitor cells, oligodendrocytes and astrocytes. The second cell line, IN1434, is derived from an astrocyte or a precursor cell restricted to astrocytic differentiation. In Hu-O-2A/Gb1 the sole homologue of chromosome 10 is disrupted at band 10p11-12.1 by translocation with chromosomes X and 15. The translocation breakpoint is localized between genetic markers D10S2103 and [D10S637, D10S1962, D10S355]. Other aberrations include a 5;14 translocation, deletion of the long and short arms of chromosome 16 and loss of one copy of the CDKN2 gene. IN1434 cells share some cytogenetic abnormalities with Hu-O-2A/Gb1 cells, despite their apparent derivation from a different biological origin, but also have translocations involving the long and short arms of chromosome 1 and the long arm of chromosome 7, and deletion of chromosome 13 at bands 13q12-21.
Assuntos
Neoplasias Encefálicas/genética , Aberrações Cromossômicas , Cromossomos Humanos Par 10 , Glioblastoma/genética , Translocação Genética , Animais , Astrócitos/metabolismo , Neoplasias Encefálicas/cirurgia , Linhagem Celular , Mapeamento Cromossômico , Glioblastoma/cirurgia , Humanos , Cariotipagem , Masculino , Repetições de Microssatélites , Pessoa de Meia-Idade , Oligodendroglia/metabolismo , Ratos , Células Tumorais CultivadasRESUMO
The chromosomal localization of the human CD83 gene was determined using somatic cell hybrids, a radiation hybrid mapping panel and FISH analysis on human metaphase chromosomes. PCR-based analysis of a single chromosome hybrid panel identified the presence of the CD83 gene on human chromosome 6 and subsequent analysis of the Genebridge4 radiation panel located the gene between AFMa192wg9 and AFMb322wd1 with a lod score of 9.2. Finally, using FISH analysis the CD83 gene was localized to chromosome 6 band p23.
Assuntos
Cromossomos Humanos Par 6/genética , Células Dendríticas/metabolismo , Imunoglobulinas/genética , Glicoproteínas de Membrana/genética , Antígenos CD , Biomarcadores , Bandeamento Cromossômico , Mapeamento Cromossômico , DNA/química , DNA/genética , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Análise de Sequência de DNA , Antígeno CD83RESUMO
High-performance liquid chromatography (HPLC) combined with mass spectrometry has become the method of choice for identifying and characterizing cell-expressed biomolecules. This technology has evolved so rapidly that efficient sample preparation in a high-throughput mode has become a rate-limiting step. Applications using C18 resin, 200 A pore size, and 15-microm bead size silica and sulfonated divinylbenzene particles were tested. The convenient, solvent-resistant, 96-well MultiScreen filter plates with the Multiscreen Column Loader provided efficient removal of salts and detergents and excellent sample recovery for small volumes. The 96-well simultaneous, uniform loading of dry powders, beads, or resins in 45-, 80-, or 100-microL volumes easily accommodates various media capacities and elution volumes. Recovered eluates demonstrated high well-to-well reproducibility during analyte adsorption, washing, and elution. Sample recovery was analyzed by HPLC for a variety of proteins, peptides, and proteolytic digests. The utility of MultiScreen 96-well mini-columns in performing sample clean-up was also demonstrated for HPLC and mass spectroscopy.