RESUMO
Analysis of the telomeric region of chromosome 14q has enabled us to complete a map of the immunoglobulin VH locus which accounts for almost all VH segments known to rearrange in B-lymphocytes. The human germline VH repertoire consists of approximately 50 functional VH segments--the exact number depending on the haplotype--spanning 1,100 kilobases upstream of the JH segments. A yeast artificial chromosome used to map these segments was isolated by its ability to provide telomere activity in yeast, suggesting that the VH locus may be located within a few kilobases of the 14q telomere. The limited structural diversity encoded by the functional VH segments demonstrates the importance of combinatorial diversity produced by VDJ joining and the association of heavy and light chains in producing the human antibody repertoire.
Assuntos
Cromossomos Humanos Par 14 , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Diversidade de Anticorpos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 8 , DNA/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Haplótipos , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Translocação Genética , Células Tumorais CultivadasRESUMO
A gene (ESS1) predisposing to the development of multiple invasive but self-healing skin tumours (squamous cell epitheliomata) is tightly linked to the polymorphic DNA marker D9S53 (9q31) with a maximum lod score of 9.02 at a recombination fraction of 0.03. Multipoint linkage analysis demonstrates that the disease locus is most likely to lie between D9S58 (9q22.3-31) and ASSP3 (9q11-q22). Comparison of markers associated with ESS1 in independently ascertained families suggests a common origin of the disease and defines the location of ESS1. Haplotype studies indicate that the disease locus is most likely to lie between D9S29 (9q31) and D9S1 (9q22.1-q22.2).
Assuntos
Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 9 , Regressão Neoplásica Espontânea/genética , Neoplasias Cutâneas/genética , Alelos , Sequência de Bases , Mapeamento Cromossômico , DNA/genética , Sondas de DNA , Feminino , Ligação Genética , Marcadores Genéticos , Haplótipos/genética , Humanos , Hibridização in Situ Fluorescente , Masculino , Dados de Sequência Molecular , Oncogenes , Linhagem , Reação em Cadeia da PolimeraseRESUMO
A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.
Assuntos
Cromossomos Artificiais de Levedura , Genoma , Camundongos/genética , Mapeamento Físico do Cromossomo , Animais , Mapeamento Cromossômico , Mapeamento de Sequências Contíguas , Marcadores Genéticos , Modelos GenéticosRESUMO
Chromosome 9 is highly structurally polymorphic. It contains the largest autosomal block of heterochromatin, which is heteromorphic in 6-8% of humans, whereas pericentric inversions occur in more than 1% of the population. The finished euchromatic sequence of chromosome 9 comprises 109,044,351 base pairs and represents >99.6% of the region. Analysis of the sequence reveals many intra- and interchromosomal duplications, including segmental duplications adjacent to both the centromere and the large heterochromatic block. We have annotated 1,149 genes, including genes implicated in male-to-female sex reversal, cancer and neurodegenerative disease, and 426 pseudogenes. The chromosome contains the largest interferon gene cluster in the human genome. There is also a region of exceptionally high gene and G + C content including genes paralogous to those in the major histocompatibility complex. We have also detected recently duplicated genes that exhibit different rates of sequence divergence, presumably reflecting natural selection.
Assuntos
Cromossomos Humanos Par 9/genética , Genes , Mapeamento Físico do Cromossomo , Composição de Bases , Eucromatina/genética , Evolução Molecular , Feminino , Duplicação Gênica , Genes Duplicados/genética , Variação Genética/genética , Genética Médica , Genômica , Heterocromatina/genética , Humanos , Masculino , Neoplasias/genética , Doenças Neurodegenerativas/genética , Pseudogenes/genética , Análise de Sequência de DNA , Processos de Determinação SexualRESUMO
The finished sequence of human chromosome 10 comprises a total of 131,666,441 base pairs. It represents 99.4% of the euchromatic DNA and includes one megabase of heterochromatic sequence within the pericentromeric region of the short and long arm of the chromosome. Sequence annotation revealed 1,357 genes, of which 816 are protein coding, and 430 are pseudogenes. We observed widespread occurrence of overlapping coding genes (either strand) and identified 67 antisense transcripts. Our analysis suggests that both inter- and intrachromosomal segmental duplications have impacted on the gene count on chromosome 10. Multispecies comparative analysis indicated that we can readily annotate the protein-coding genes with current resources. We estimate that over 95% of all coding exons were identified in this study. Assessment of single base changes between the human chromosome 10 and chimpanzee sequence revealed nonsense mutations in only 21 coding genes with respect to the human sequence.
Assuntos
Cromossomos Humanos Par 10/genética , Genes , Mapeamento Físico do Cromossomo , Animais , Composição de Bases , Mapeamento de Sequências Contíguas , Ilhas de CpG/genética , Evolução Molecular , Éxons/genética , Duplicação Gênica , Variação Genética/genética , Genética Médica , Genômica , Humanos , Pan troglodytes/genética , Proteínas/genética , Pseudogenes/genética , Análise de Sequência de DNARESUMO
Chromosome 13 is the largest acrocentric human chromosome. It carries genes involved in cancer including the breast cancer type 2 (BRCA2) and retinoblastoma (RB1) genes, is frequently rearranged in B-cell chronic lymphocytic leukaemia, and contains the DAOA locus associated with bipolar disorder and schizophrenia. We describe completion and analysis of 95.5 megabases (Mb) of sequence from chromosome 13, which contains 633 genes and 296 pseudogenes. We estimate that more than 95.4% of the protein-coding genes of this chromosome have been identified, on the basis of comparison with other vertebrate genome sequences. Additionally, 105 putative non-coding RNA genes were found. Chromosome 13 has one of the lowest gene densities (6.5 genes per Mb) among human chromosomes, and contains a central region of 38 Mb where the gene density drops to only 3.1 genes per Mb.
Assuntos
Cromossomos Humanos Par 13/genética , Genes/genética , Mapeamento Físico do Cromossomo , Mapeamento Cromossômico , Genética Médica , Humanos , Pseudogenes/genética , RNA não Traduzido/genética , Análise de Sequência de DNARESUMO
Chromosome 6 is a metacentric chromosome that constitutes about 6% of the human genome. The finished sequence comprises 166,880,988 base pairs, representing the largest chromosome sequenced so far. The entire sequence has been subjected to high-quality manual annotation, resulting in the evidence-supported identification of 1,557 genes and 633 pseudogenes. Here we report that at least 96% of the protein-coding genes have been identified, as assessed by multi-species comparative sequence analysis, and provide evidence for the presence of further, otherwise unsupported exons/genes. Among these are genes directly implicated in cancer, schizophrenia, autoimmunity and many other diseases. Chromosome 6 harbours the largest transfer RNA gene cluster in the genome; we show that this cluster co-localizes with a region of high transcriptional activity. Within the essential immune loci of the major histocompatibility complex, we find HLA-B to be the most polymorphic gene on chromosome 6 and in the human genome.
Assuntos
Cromossomos Humanos Par 6/genética , Genes/genética , Mapeamento Físico do Cromossomo , Animais , Éxons/genética , Doenças Genéticas Inatas/genética , Antígenos HLA-B/genética , Humanos , Pseudogenes/genética , RNA de Transferência/genética , Análise de Sequência de DNARESUMO
Chromosome microdeletions or duplications are detected in 10-20% of patients with mental impairment and normal karyotypes. A few cases have been reported of mental impairment with microdeletions comprising tumor suppressor genes. By array-CGH we detected 4 mentally impaired individuals carrying de novo microdeletions sharing an overlapping segment of approximately 180 kb in 17p13.1. This segment encompasses 18 genes, including 3 involved in cancer, namely KCTD11/REN, DLG4/PSD95, and GPS2. Furthermore, in 2 of the patients, the deletions also included TP53, the most frequently inactivated gene in human cancers. The 3 tumor suppressor genes KCTD11, DLG4, and GPS2, in addition to the GABARAP gene, have a known or suspected function in neuronal development and are candidates for causing mental impairment in our patients. Among our 4 patients with deletions in 17p13.1, 3 were part of a Brazilian cohort of 300 mentally retarded individuals, suggesting that this segment may be particularly prone to rearrangements and appears to be an important cause (approximately 1%) of mental retardation. Further, the constitutive deletion of tumor suppressor genes in these patients, particularly TP53, probably confers a significantly increased lifetime risk for cancer and warrants careful oncological surveillance of these patients. Constitutional chromosome deletions containing tumor suppressor genes in patients with mental impairment or congenital abnormalities may represent an important mechanism linking abnormal phenotypes with increased risks of cancer.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 17/genética , Genes Supressores de Tumor , Deficiência Intelectual/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Proteínas Reguladoras de Apoptose , Proteínas de Ciclo Celular , Criança , Pré-Escolar , Mapeamento Cromossômico , Hibridização Genômica Comparativa , Proteína 4 Homóloga a Disks-Large , Feminino , Dosagem de Genes , Genes p53 , Humanos , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Fenótipo , Canais de Potássio/genética , TransferasesRESUMO
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.
Assuntos
Quebra Cromossômica , Mapeamento Cromossômico/métodos , Coloração Cromossômica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Translocação Genética , Adulto , Pré-Escolar , Cromossomos Humanos/genética , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente , Lactente , Masculino , Dados de Sequência MolecularRESUMO
Deletions of chromosome 6 are a common abnormality in diverse human malignancies including astrocytic tumours, suggesting the presence of tumour suppressor genes (TSG). In order to help identify candidate TSGs, we have constructed a chromosome 6 tile path microarray. The array contains 1,780 clones (778 P1-derived artificial chromosome and 1,002 bacterial artificial chromosome) that cover 98.3% of the published chromosome 6 sequences. A total of 104 adult astrocytic tumours (10 diffuse astrocytomas, 30 anaplastic astrocytomas (AA), 64 glioblastomas (GB)) were analysed using this array. Single copy number change was successfully detected and the result was in general concordant with a microsatellite analysis. The pattern of copy number change was complex with multiple interstitial deletions/gains. However, a predominance of telomeric 6q deletions was seen. Two small common and overlapping regions of deletion at 6q26 were identified. One was 1,002 kb in size and contained PACRG and QKI, while the second was 199 kb and harbours a single gene, ARID1B. The data show that the chromosome 6 tile path array is useful in mapping copy number changes with high resolution and accuracy. We confirmed the high frequency of chromosome 6 deletions in AA and GB, and identified two novel commonly deleted regions that may harbour TSGs.
Assuntos
Astrocitoma/genética , Deleção Cromossômica , Cromossomos Humanos Par 6/genética , Glioblastoma/genética , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Astrocitoma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Cromossomos Artificiais Bacterianos , DNA de Neoplasias/análise , Dosagem de Genes , Glioblastoma/patologia , Humanos , Repetições de Microssatélites , Telômero/genéticaRESUMO
BACKGROUND: Karyotype analysis has been the standard method for prenatal cytogenetic diagnosis since the 1970s. Although highly reliable, the major limitation remains the requirement for cell culture, resulting in a delay of as much as 14 days to obtaining test results. Fluorescent in situ hybridisation (FISH) and quantitative fluorescent PCR (QF-PCR) rapidly detect common chromosomal abnormalities but do not provide a genome wide screen for unexpected imbalances. Array comparative genomic hybridisation (CGH) has the potential to combine the speed of DNA analysis with a large capacity to scan for genomic abnormalities. We have developed a genomic microarray of approximately 600 large insert clones designed to detect aneuploidy, known microdeletion syndromes, and large unbalanced chromosomal rearrangements. METHODS: This array was tested alongside an array with an approximate resolution of 1 Mb in a blind study of 30 cultured prenatal and postnatal samples with microscopically confirmed unbalanced rearrangements. RESULTS: At 1 Mb resolution, 22/30 rearrangements were identified, whereas 29/30 aberrations were detected using the custom designed array, owing to the inclusion of specifically chosen clones to give increased resolution at genomic loci clinically implicated in known microdeletion syndromes. Both arrays failed to identify a triploid karyotype. Thirty normal control samples produced no false positive results. CONCLUSIONS: Analysis of 30 uncultured prenatal samples showed that array CGH is capable of detecting aneuploidy in DNA isolated from as little as 1 ml of uncultured amniotic fluid; 29/30 samples were correctly diagnosed, the exception being another case of triploidy. These studies demonstrate the potential for array CGH to replace conventional cytogenetics in the great majority of prenatal diagnosis cases.
Assuntos
Aberrações Cromossômicas , Doenças Fetais/diagnóstico , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Diagnóstico Pré-Natal/métodos , Feminino , Doenças Fetais/genética , Genoma Humano , Humanos , Gravidez , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Autism spectrum disorders (ASD) refer to a broader group of neurobiological conditions, pervasive developmental disorders. They are characterised by a symptomatic triad associated with qualitative changes in social interactions, defect in communication abilities, and repetitive and stereotyped interests and activities. ASD is prevalent in 1 to 3 per 1000 people. Despite several arguments for a strong genetic contribution, the molecular basis of a most cases remains unexplained. About 5% of patients with autism have a chromosome abnormality visible with cytogenetic methods. The most frequent are 15q11-q13 duplication, 2q37 and 22q13.3 deletions. Many other chromosomal imbalances have been described. However, most of them remain undetectable using routine karyotype analysis, thus impeding diagnosis and genetic counselling. METHODS AND RESULTS: 29 patients presenting with syndromic ASD were investigated using a DNA microarray constructed from large insert clones spaced at approximately 1 Mb intervals across the genome. Eight clinically relevant rearrangements were identified in 8 (27.5%) patients: six deletions and two duplications. Altered segments ranged in size from 1.4 to 16 Mb (2-19 clones). No recurrent abnormality was identified. CONCLUSION: These results clearly show that array comparative genomic hybridisation should be considered to be an essential aspect of the genetic analysis of patients with syndromic ASD. Moreover, besides their importance for diagnosis and genetic counselling, they may allow the delineation of new contiguous gene syndromes associated with ASD. Finally, the detailed molecular analysis of the rearranged regions may pave the way for the identification of new ASD genes.
Assuntos
Transtornos Globais do Desenvolvimento Infantil/diagnóstico , Aberrações Cromossômicas , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Adolescente , Adulto , Criança , Transtornos Globais do Desenvolvimento Infantil/genética , Cromossomos Humanos , Feminino , Testes Genéticos/métodos , Genômica/métodos , Humanos , Masculino , SíndromeRESUMO
BACKGROUND: The underlying causes of mental retardation remain unknown in about half the cases. Recent array-CGH studies demonstrated cryptic imbalances in about 25% of patients previously thought to be chromosomally normal. OBJECTIVE AND METHODS: Array-CGH with approximately 3500 large insert clones spaced at approximately 1 Mb intervals was used to investigate DNA copy number changes in 81 mentally impaired individuals. RESULTS: Imbalances never observed in control chromosomes were detected in 20 patients (25%): seven were de novo, nine were inherited, and four could not have their origin determined. Six other alterations detected by array were disregarded because they were shown by FISH either to hybridise to both homologues similarly in a presumptive deletion (one case) or to involve clones that hybridised to multiple sites (five cases). All de novo imbalances were assumed to be causally related to the abnormal phenotypes. Among the others, a causal relation between the rearrangements and an aberrant phenotype could be inferred in six cases, including two imbalances of the X chromosome, where the associated clinical features segregated as X linked recessive traits. CONCLUSIONS: In all, 13 of 81 patients (16%) were found to have chromosomal imbalances probably related to their clinical features. The clinical significance of the seven remaining imbalances remains unclear. The limited ability to differentiate between inherited copy number variations which cause abnormal phenotypes and rare variants unrelated to clinical alterations currently constitutes a limitation in the use of CGH-microarray for guiding genetic counselling.
Assuntos
Desequilíbrio Alélico/genética , Rearranjo Gênico/genética , Hibridização in Situ Fluorescente , Deficiência Intelectual/genética , Criança , Cromossomos Humanos Par 2/genética , HumanosRESUMO
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.
Assuntos
Anormalidades Congênitas/genética , Translocação Genética , Linhagem Celular , Aberrações Cromossômicas , Cromossomos Artificiais Bacterianos , Clonagem Molecular , Feminino , Rearranjo Gênico , Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Incidência , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , FenótipoRESUMO
Microscopic karyotype analysis of cultured cells has been regarded as the gold standard for prenatal diagnosis for over 30 years. Since the first application of this technique to prenatal testing in the early 1970's, this procedure has proved to be highly reliable for identifying chromosome copy number abnormalities (aneuploidy) and large structural rearrangements in foetal cells obtained invasively by either amniocentesis or chorionic villus sampling (CVS). Recognising the need for more rapid testing methods which do not require cell culture, fluorescence in situ hybridisation (FISH) and quantitative fluorescence PCR (QF-PCR) have been introduced to this field in order to answer specific diagnostic questions. However, both FISH and QF-PCR suffer the disadvantage in that they are difficult to scale to a comprehensive, genome-wide screen. Array-comparative genomic hybridisation (array-CGH) in contrast is a comprehensive, genome-wide screening strategy for detecting DNA copy number imbalances which can be rapid, less labour-intensive than karyotype banding analysis and is highly amenable to automation. Array-CGH has the potential to be used for prenatal diagnosis and may address many of the limitations of both conventional microscopic cytogenetic analyses and the more recently employed rapid-screening strategies.
Assuntos
Dosagem de Genes , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Diagnóstico Pré-Natal/métodos , Cromossomos Humanos/genética , DNA/análise , Feminino , Genômica/métodos , Humanos , Hibridização de Ácido Nucleico/métodos , GravidezRESUMO
The underlying causes of learning disability and dysmorphic features in many patients remain unidentified despite extensive investigation. Routine karyotype analysis is not sensitive enough to detect subtle chromosome rearrangements (less than 5 Mb). The presence of subtle DNA copy number changes was investigated by array-CGH in 50 patients with learning disability and dysmorphism, employing a DNA microarray constructed from large insert clones spaced at approximately 1 Mb intervals across the genome. Twelve copy number abnormalities were identified in 12 patients (24% of the total): seven deletions (six apparently de novo and one inherited from a phenotypically normal parent) and five duplications (one de novo and four inherited from phenotypically normal parents). Altered segments ranged in size from those involving a single clone to regions as large as 14 Mb. No recurrent deletion or duplication was identified within this cohort of patients. On the basis of these results, we anticipate that array-CGH will become a routine method of genome-wide screening for imbalanced rearrangements in children with learning disability.
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos/genética , Análise Citogenética/métodos , Deficiência Intelectual/genética , Deficiências da Aprendizagem/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Deleção Cromossômica , Feminino , Humanos , MasculinoRESUMO
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.
Assuntos
Aberrações Cromossômicas , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Adulto , Linhagem Celular , Criança , Pré-Escolar , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/patologia , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 22/genética , Feminino , Citometria de Fluxo , Humanos , Hibridização in Situ Fluorescente/métodos , Cariotipagem/métodos , Masculino , Translocação GenéticaRESUMO
Flow cytometry has been examined as a method for quantitative measurement of the accumulation in macrophages of ceroid, an autofluorescent polymer composed of oxidised protein and lipid. Murine peritoneal macrophages were cultured in the presence of cholesteryl linoleate- or arachidonate-bovine serum albumin (CL/BSA or CA/BSA) complexes. Ceroid accumulation was greater from CA/BSA than from CL/BSA and was dependent upon both time and cell plating density. Inclusion of vitamin E with the complexes diminished the accumulation of ceroid fluorescence after exposure to either CL/BSA or CA/BSA. Controls included exposure of macrophages to BSA, alone and with vitamin E, both of which led to some fluorescence at a similar wavelength to that used to monitor ceroid accumulation (Ex: 351.1-363.8 nm/Em: 490 nm and upwards). Ceroid accumulation can be monitored semi-quantitatively by staining techniques. However, such methods are relatively crude and give little information about the amount of ceroid within cells. Flow cytometry, on the other hand, can give a quantitative assessment of cellular ceroid accumulation, provided experiments are conducted with appropriate controls. The findings are discussed in the context of human atherosclerosis and of future investigation of cell-mediated lipid oxidation and its potential antagonists.
Assuntos
Ácidos Araquidônicos , Ceroide/metabolismo , Macrófagos/metabolismo , Animais , Células Cultivadas , Ésteres do Colesterol/metabolismo , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Soroalbumina Bovina/metabolismo , Vitamina E/metabolismoRESUMO
As with many human cancers, canine tumors demonstrate recurrent chromosome aberrations. A detailed knowledge of such aberrations may facilitate diagnosis, prognosis and the selection of appropriate therapy. Following recent advances made in human genomics, we are developing a DNA microarray for the domestic dog, to be used in the detection and characterization of copy number changes in canine tumors. As a proof of principle, we have developed a small-scale microarray comprising 87 canine BAC clones. The array is composed of 26 clones selected from a panel of 24 canine cancer genes, representing 18 chromosomes, and an additional set of clones representing dog chromosomes 11, 13, 14 and 31. These chromosomes were shown previously to be commonly aberrant in canine multicentric malignant lymphoma. Clones representing the sex chromosomes were also included. We outline the principles of canine microarray development, and present data obtained from microarray analysis of three canine lymphoma cases previously characterized using conventional cytogenetic techniques.
Assuntos
Cães/genética , Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/veterinária , Genes Neoplásicos/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Animais , Cromossomos Artificiais Bacterianos/genética , DNA de Neoplasias/genética , Doenças do Cão/genética , Feminino , Perfilação da Expressão Gênica/estatística & dados numéricos , Regulação Neoplásica da Expressão Gênica/genética , Hibridização in Situ Fluorescente/métodos , Hibridização in Situ Fluorescente/estatística & dados numéricos , Hibridização in Situ Fluorescente/veterinária , Linfoma de Células B/genética , Linfoma de Células B/veterinária , Linfoma não Hodgkin/genética , Linfoma não Hodgkin/veterinária , Masculino , Metáfase/genética , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/estatística & dados numéricos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/veterináriaRESUMO
Platelets, expressing only class I antigens on their surface, have been shown to improve renal allograft survival in some experimental models but do not lead to humoral sensitization. In this pilot study, 24 previously untransfused patients were given 3 platelet transfusions at 2-week intervals before renal transplantation. Twelve patients (group 1) received 200 X 10(8) platelets contaminated by 15 X 10(6) leukocytes in each transfusion, and 42% developed cytotoxic antibodies; 12 further patients (group 2) received a purified preparation containing 200 X 10(8) platelets and less than 5 X 10(6) leukocytes, and no cytotoxic antibodies were detected. Using a more sensitive flow cytometry technique, 83% of group 1 but only 17% of group 2 (P less than 0.004) responded to their platelet donors. From each group 9 patients have been transplanted. Four grafts in group 1 and 6 in group 2 have failed, yielding a 1-year graft survival rate of 44%, compared with 69% in blood-transfused patients (P = 0.03). Pure platelet transfusions, using our protocol, do not appear to improve renal allograft survival, and minimal contamination by leukocytes can lead to humoral sensitization.