RESUMO
UNLABELLED: The phenotypic and functional characteristics of immune cells of osteoporotic women compared to healthy controls similar for age and estrogen level showed for the first time significant changes in several B lymphocytes populations in postmenopausal osteoporosis, related to bone mineral density (BMD) and fractures, and a significant lower basal secretion of interferon-gamma (IFN-gamma) by CD4(+). INTRODUCTION: To investigate the interactions between bone and immune system, we studied the phenotypic and functional characteristics of immune cells of 26 postmenopausal women with osteoporotic (OP) fractures compared to 24 healthy controls. METHODS: We analyzed surface markers of peripheral B, CD4(+) and CD8(+) lymphocytes and cytokine secretion in supernatants of these cells cultured with or without stimulation. Body composition was assessed by dual energy X-ray absorptiometry. RESULTS: The two groups were similar for age and estrogen level. OP women had a significantly lower body mass index, fat mass, and lean mass. The number of CD19(+), CD19(+)/CD27(+), CD19(+)/CD27(+)/CD5(-)/CD38(+) and CD19(+)/CD27(+)/RANK(+), CD4(+)/CD27(+)/CD45RA(-)/RANK(+), and CD4(+)/CD27(+)/CD45RA(-)/CD28(+) was lower in OP women and positively correlated to BMD. In OP women, under basal conditions, CD4(+) secreted less IFN-gamma and B lymphocytes more granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF was positively correlated to fracture rate and negatively to BMD. CONCLUSIONS: Our results suggest that, regardless of age and estrogen status, postmenopausal OP is associated with immune changes, highlighting a possible role of IFN-gamma in the pathophysiology of OP and reporting, for the first time, changes in several B lymphocyte populations. These alterations may reflect the frailty observed after fracture, providing new insight into the mechanisms of morbidity and mortality associated with OP fractures.
Assuntos
Osteoporose Pós-Menopausa/imunologia , Idoso , Idoso de 80 Anos ou mais , Subpopulações de Linfócitos B/imunologia , Composição Corporal/fisiologia , Estudos de Casos e Controles , Células Dendríticas/imunologia , Estrogênios/sangue , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Humanos , Imunidade Celular , Imunofenotipagem , Interferon gama/biossíntese , Pessoa de Meia-Idade , Osteoporose Pós-Menopausa/sangue , Osteoporose Pós-Menopausa/fisiopatologia , Fraturas por Osteoporose/imunologia , Fraturas por Osteoporose/fisiopatologia , Projetos Piloto , Subpopulações de Linfócitos T/imunologiaRESUMO
In gel electrophoresis, nucleic acids and protein-detergent complexes larger than a threshold size all migrate at the same rate. For DNA molecules, this effect can be overcome by the simple procedure of periodically inverting the electric field. Tuning the frequency of the field inversions from 10 to 0.01 hertz, makes it possible to resolve selectively DNA's in the size range 15 to greater than 700 kilobase pairs.
Assuntos
DNA/isolamento & purificação , Eletroforese/métodos , Peso Molecular , Relação Estrutura-AtividadeRESUMO
Fragments of exogenous DNA that range in size up to several hundred kilobase pairs have been cloned into yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. Individual clones of yeast and human DNA that have been analyzed by pulsed-field gel electrophoresis appear to represent faithful replicas of the source DNA. The efficiency with which clones can be generated is high enough to allow the construction of comprehensive libraries from the genomes of higher organisms. By offering a tenfold increase in the size of the DNA molecules that can be cloned into a microbial host, this system addresses a major gap in existing experimental methods for analyzing complex DNA sources.
Assuntos
Clonagem Molecular , Vetores Genéticos , Saccharomyces cerevisiae/genética , Cromossomos , Enzimas de Restrição do DNA , Engenharia Genética/métodos , Humanos , Plasmídeos , Tetrahymena/genéticaRESUMO
The objective of bone tissue engineering is to reconstruct bone stock using matrix structures, osteoinductor factors and osteogenic cells. Different types of natural or synthetic biomaterials are available or under development. The objective of recent work is to optimize matrix materials, particularly with better cell adhesion to the surface and better osteoconduction. For osteoinductors, most research is currently focused on bone morphogenetic protein (BMP) and angiogenic factors such as vascular endothelial growth factor (VEGF). Concerning the nature of the cells to be implanted, there is a clear dissociation between fundamental and clinical studies. Many clinical studies have demonstrated the strong osteogenic potential of fresh harvested total bone marrow. There has been nevertheless little fundamental work on the use of total bone marrow as a source of cells for bone tissue engineering. Most of the fundamental work has been focused on the use of mesenchymatous stromal cells selected from bone marrow and cultivated ex vivo. This approach which was first developed more than fifteen years ago has shown that the adjunction of these cells can improve the osteoformative capacity of bone substitutes. This strategy has, however, had almost no clinical impact to date since only two studies involving four patients have been reported. The purpose of this article is to review current research concerning bone tissue engineering using total bone marrow and mesenchymatous stromal cells.
Assuntos
Osso e Ossos , Engenharia Tecidual/métodos , Animais , Células da Medula Óssea , HumanosRESUMO
Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.
Assuntos
Cromossomos , DNA Fúngico/genética , Histoplasma/genética , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Ágar , Histoplasma/análise , Polimorfismo Genético , Especificidade da EspécieRESUMO
In the course of our study on the amplification of 11q13 sequences in human breast cancer, we have investigated the amplification status of the anonymous DNA fragment D11S97 in a series of 125 mammary tumors. Our results indicate that, as with bladder carcinomas, D11S97 can be amplified separately from BCL1. In addition, we have shown that D11597 is physically linked to both D11S146 and BCL1, and is less than 100 kb centromeric to the D115146. These results indicate that, in addition to other 11q13 loci, sequences located approximately 500 kb centromeric from BCL1 could contribute to carcinogenesis of epithelial cells in vivo.
Assuntos
Neoplasias da Mama/genética , Cromossomos Humanos Par 11 , Amplificação de Genes , Mapeamento Cromossômico , Ciclina D1 , Humanos , Proteínas Proto-Oncogênicas/genética , Mapeamento por RestriçãoRESUMO
We have determined the genomic structure of the mouse fra-1 gene, which consists of four exons and three introns at positions also found in the other members of the fos gene family. Fra-1 is expressed rather highly in the brain and testes of adult mice, and at low levels in most other tissues. Absence of c-Fos leads to significantly reduced serum stimulation of fra-1 expression in gene targeted mouse fibroblasts, demonstrating that mitogen induction of fra-1 is partially mediated by c-Fos/AP-1. A polymorphic (CA)n microsatellite marker was found in intron 2 of fra-1 and used to map the gene to the centromeric region of mouse chromosome 19. Since fra-1 maps to the same genomic region as oc (osteosclerosis), an autosomal recessive disorder leading to the bone remodelling disease osteopetrosis, we tested it as a candidate gene for oc. The segregation of fra-1 in two different crosses of mice carrying oc and an allelism test between oc and a targeted disruption of fra-1 demonstrate that fra-1 and oc are two distinct genes rather than oc being a mutant allele of fra-1.
Assuntos
Genes , Osteosclerose/genética , Proteínas Proto-Oncogênicas c-fos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Éxons , Expressão Gênica , Masculino , Camundongos , Repetições de Microssatélites , Dados de Sequência Molecular , RNA Mensageiro/genética , Mapeamento por Restrição , Distribuição TecidualRESUMO
The gene responsible for multiple endocrine neoplasia type 1 (MEN1), a heritable predisposition to endocrine tumours in man, has recently been identified. Here we have characterized the murine homologue with regard to cDNA sequence, genomic structure, expression pattern and chromosomal localisation. The murine Men1 gene spans approximately 6.7 kb of genomic DNA and is comprised of 10 exons with similar genomic structure to the human locus. It was mapped to the pericentromeric region of mouse chromosome 19, which is conserved with the human 11q13 band where MEN1 is located. The predicted protein is 611 amino acids in length and overall is 97% homologous to the human orthologue. The 45 reported MEN1 mutations which alter or delete a single amino acid in human all occur at conserved residues, thereby supporting their functional significance. Two transcripts of approximately 3.2 and 2.8 kb were detected in both embryonal and adult murine tissues, resulting from alternative splicing of intron 1. By RNA in situ hybridization and Northern analysis the spatiotemporal expression pattern of Men1 was determined during mouse development. Men1 gene activity was detected already at gestational day 7. At embryonic day 14 expression was generally high throughout the embryo, while at day 17 the thymus, skeletal muscle, and CNS showed the strongest signal. In selected tissues from postnatal mouse Men1 was detected in all tissues analysed and was expressed at high levels in cerebral cortex, hippocampus, testis, and thymus. In brain the menin protein was detected mainly in nerve cell nuclei, whereas in testis it appeared perinuclear in spermatogonia. These results show that Men1 expression is not confined to organs affected in MEN1, suggesting that Men1 has a significant function in many different cell types including the CNS and testis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Camundongos/genética , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas , Proto-Oncogenes , Sequência de Aminoácidos , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Mapeamento Cromossômico , DNA Complementar/genética , Feminino , Biblioteca Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos/embriologia , Camundongos/crescimento & desenvolvimento , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos , Splicing de RNA , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Testículo/embriologia , Testículo/metabolismoRESUMO
Sequences near the ends of the insertion element IS50 are essential for its transposition, probably because they serve as sites upon which the IS50-encoded transposase protein acts. To determine if these essential sequences include the first base-pair at each end of IS50 we generated 5'C to 5'G transversions at these positions. Each mutation reduced the transposition frequency to 1% to 2% of wild-type. DNA sequence analyses showed that the mutant 5'G is preserved during transposition.
Assuntos
Bacteriófago lambda/genética , Elementos de DNA Transponíveis , DNA Viral , Sequência de Bases , Enzimas de Restrição do DNA , Mutação , PlasmídeosRESUMO
Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for three-dimensional amplification and transfer of chondrocytes in cartilage tissue engineering.
Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Metilcelulose/análogos & derivados , Silanos/química , Animais , Cartilagem/metabolismo , Cartilagem Articular/citologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Dimerização , Glicosaminoglicanos/química , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Derivados da Hipromelose , Metilcelulose/química , Microscopia de Fluorescência , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Engenharia TecidualRESUMO
Infantile malignant osteopetrosis (IMO) is a rare and lethal disease characterized by an absence of bone resorption due to inactive OCLs. Affected patients display an increased bone mass and hematological defects. The osteopetrotic oc/oc mouse displays a bone phenotype similar to the one observed in IMO patients, and the same gene, Tcirg1, is mutated in this model and in the majority of these patients. Therefore, we explored in oc/oc mice the consequences of the perturbed bone microenvironment on hematopoiesis. We show that the myelomonocytic differentiation is increased, leading to an elevated number of OCLs and dendritic cells. B lymphopoiesis is blocked at the pro-B stage in the bone marrow of oc/oc mouse, leading to a low mature B-cell number. T-cell activation is also affected, with a reduction of IFNgamma secretion by splenic CD4(+) T cells. These alterations are associated with a low IL-7 expression in bone marrow. All these data indicate that the lack of bone resorption in oc/oc mice has important consequences in both myelopoiesis and lymphopoiesis, leading to a form of immunodeficiency. The oc/oc mouse is therefore an appropriate model to understand the hematological defects described in IMO patients, and to derive new therapeutic strategies.
Assuntos
Reabsorção Óssea , Hematopoese/fisiologia , Linfopoese/fisiologia , Osteopetrose/patologia , Animais , Linfócitos B/citologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Diferenciação Celular , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Modelos Animais de Doenças , Hematopoese/genética , Interferon gama/metabolismo , Interleucina-7/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/metabolismo , Osteopetrose/metabolismo , Baço/imunologia , Linfócitos T/citologiaRESUMO
We have recently discovered a new class of potassium channels with two pore-forming domains and four membrane-spanning domains. When heterologously expressed, these channels produce time- and voltage-independent currents that classify them as background or leak channels. TWIK (for tandem of P domains in a weak inwardly rectifying K+ channel) was the first member of this family to be cloned. Here, we describe the genomic organization of TWIK in the mouse. The coding sequence as well as the untranslated sequences are contained in three exons. The twik gene (or KCNK1) has been mapped to chromosome 8, consistent with its localization to 1q42-43 in human. The twik gene is expressed in virtually all mouse tissues. It is most abundantly expressed in brain and moderately in other organs such as kidney. The level of expression is increased in brain and kidney from neonate to adult animals, but the TWIK message is also detected during embryogenesis, as early as day 7 post conception.
Assuntos
Mapeamento Cromossômico , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/genética , Animais , Sequência de Bases , Sítios de Ligação , DNA Complementar , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Iniciação Traducional da Cadeia Peptídica , Canais de Potássio/metabolismo , RNA Mensageiro , Distribuição Tecidual , Transcrição GênicaRESUMO
Bone hybrids made of bioceramics seeded with mesenchymal or osteoblastic cells are very promising alternatives to autologous bone graft. Along this line, the development of in vitro models, dedicated to analyze the influence of these biomaterials on osteogenic cells, will help to improve the performance of these bone substitutes. In the present work we analyzed the effects of a macroporous biphasic calcium phosphate ceramic (BCP, Triosite) on three different human osteosarcoma cell lines and on human primary osteogenic cells and compared this culture substratum to traditional culture on plastic. We showed that all these osteoblastic cells adhere and proliferate on the trabecular BCP blocks, with a different spatial organization for osteosarcoma cells compared to normal osteogenic cells. We also demonstrated that osteoblastic marker genes such as Cbfa1, type I collagen, osteonectin, osteopontin, and osteocalcin were expressed at similar levels by these cells cultured on either substratum, suggesting that adhesion to BCP does maintain the osteoblastic phenotype of these cells. Next, we provided the first evidence of differences of cytokine expression profiles revealed on this Ca-P ceramic as compared to expression in classical culture. These modifications affected the expression of cytokines such as TGF-beta1, G-CSF, and IL-3 and were quantitatively different between osteosarcoma cells and normal osteogenic cells. Given the role of these cytokines in bone biology and in hematopoiesis, these results obtained in vitro suggest that the BCP ceramic studied here could stimulate osteogenesis in vivo by activating cellular processes during bone formation and healing. This study highlights the notion that the nature of the culture substratum must be taken into account when studying bone cell biology in vitro. Owing to the nature and spatial organization of the BCP, our hypothesis is that culture on BCP is closer to the physiological situation than culture on plastic.
Assuntos
Neoplasias Ósseas/metabolismo , Substitutos Ósseos/farmacologia , Fosfatos de Cálcio/farmacologia , Osteogênese/efeitos dos fármacos , Osteossarcoma/metabolismo , Adolescente , Adulto , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Osteogênese/fisiologia , Osteossarcoma/genética , Células Tumorais CultivadasRESUMO
Osteosclerosis (oc) is an autosomal recessive lethal mutation that impairs bone resorption by osteoclasts, and induces a general increase of bone density in affected mice. Genetic mapping of the oc mutation was used as a backbone in a positional cloning approach in the pericentromeric region of mouse chromosome 19. Perfect cosegregation of the osteopetrotic phenotype with polymorphic markers enabled the construction of a sequence-ready bacterial artificial chromosome (BAC) contig of this region. Genomic sequencing of a 200-kb area revealed the presence of the mouse homologue to the human gene encoding the osteoclast-specific 116-kDa subunit of the vacuolar proton pump. This gene was located recently on human 11q13, a genomic region conserved with proximal mouse chromosome 19. Sequencing of the 5' end of the gene in oc/oc mice showed a 1.6-kb deletion, including the translation start site, which impairs genuine transcription of this subunit. The inactivation of this osteoclast-specific vacuolar proton ATPase subunit could be responsible for the lack of this enzyme in the apical membranes of osteoclast cells in oc/oc mice, thereby preventing the resorption function of these cells, which leads to the osteopetrotic phenotype.
Assuntos
Mutação , Osteoclastos/enzimologia , Osteosclerose/genética , ATPases Translocadoras de Prótons/genética , Deleção de Sequência , ATPases Vacuolares Próton-Translocadoras , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromossomos Artificiais de Levedura , Primers do DNA , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
Among the techniques to separate large DNA fragments, field-inversion gel electrophoresis (FIGE, 1)is probably the easiest to perform with a minimum of special equipment. Indeed, the only requirement besides a regular gel electrophoresis box and a power supply is a device enabling the periodic inversion of the electric field direction over the course of the experiment. This method was derived from experiments done on a modified orthogonal-field-alternation gel electrophoresis (OFAGE) apparatus (2) based on the observation that obtuse angles lead to a better separation. The widest angle being 180°, the four electrode pulsed-field gel electrophoresis (PFGE) system was reduced to a standard submarine gel electrophoresis box with only two electrodes. This simple configuration generates a highly uniform electric field across the gel, making the lane to lane comparison very easy (Fig. 1). Two basic electrophoretic modes can be used in order to achieve a net migration in this configuration:
RESUMO
We had previously reported that gallium (Ga) inhibited both the differentiation and resorbing activity of osteoclasts in a dose-dependent manner. To provide new insights into Ga impact on osteoclastogenesis, we investigated here the molecular mechanisms of Ga action on osteoclastic differentiation of monocytes upon Rankl treatment. We first observed that Ga treatment inhibited the expression of Rankl-induced early differentiation marker genes, while the same treatment performed subsequently did not modify the expression of late differentiation marker genes. Focusing on the early stages of osteoclast differentiation, we observed that Ga considerably disturbed both the initial induction as well as the autoamplification step of Nfatc1 gene. We next demonstrated that Ga strongly up-regulated the expression of Traf6, p62 and Cyld genes, and we observed concomitantly an inhibition of IκB degradation and a blockade of NFκB nuclear translocation, which regulates the initial induction of Nfatc1 gene expression. In addition, Ga inhibited c-Fos gene expression, and subsequently the auto-amplification stage of Nfatc1 gene expression. Lastly, considering calcium signaling, we observed upon Ga treatment an inhibition of calcium-induced Creb phosphorylation, as well as a blockade of gadolinium-induced calcium entry through TRPV-5 calcium channels. We identify for the first time Traf6, p62, Cyld, IκB, NFκB, c-Fos, and the calcium-induced Creb phosphorylation as molecular targets of Ga, this tremendously impacting the expression of the master transcription factor Nfatc1. In addition, our results strongly suggest that the TRPV-5 calcium channel, which is located within the plasma membrane, is a target of Ga action on human osteoclast progenitor cells.
Assuntos
Gálio/farmacologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Osteoclastos/citologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The chromosomal DNA molecules of a standard laboratory strain of Saccharomyces cerevisiae have been separated into 12 well-resolved bands by orthogonal-field-alternation gel electrophoresis. DNA X DNA hybridization probes derived from cloned genes have been used to correlate this banding pattern with yeast's genetically defined chromosomes. The 12 bands are shown to represent 9 singlets and 3 comigrating doublets, thereby accounting for 15 chromosomes that were identified as I-XI and XIII-XVI. Because the three comigrating doublets could be readily resolved in certain laboratory yeast strains that contain chromosome-length polymorphisms relative to our standard strain, all 15 of these chromosomes could be displayed as a single band in at least one of four strains that were studied. A 16th chromosome (number XII), which is known to contain the genes for rRNA, does not reproducibly enter the gels. By making use of the band identifications, the previously unmapped fragment F8 was assigned to chromosome XIII. With the possible exception of chromosomes that differ greatly in size or electrophoretic behavior from all the known chromosomes, the results appear to define a complete "electrophoretic karyotype" for yeast.
Assuntos
Saccharomyces cerevisiae/genética , Eletroforese em Gel de Ágar , Cariotipagem/métodos , Polimorfismo GenéticoRESUMO
We have developed a genetic screen for the isolation of larger or smaller recombinant yeast artificial chromosomes derived from overlapping YACs. Integration plasmids were used to modify the TRP1 and URA3 auxotrophic markers present respectively on the left and right vector arms of one of the parental YACs. Diploids containing the two parental YACs were studied through meiosis and mitosis. Tetrad analysis revealed the presence of meiotic recombinant YACs at a frequency comparable with what is expected for yeast DNA (about 3 kb/cM). More direct genetic selection of diploids on -TRP-LYS synthetic media in the presence of 5-fluoro-orotic acid (5-FOA), led to the isolation of mitotic recombinant YACs at a high frequency. Analysis of these yeast cells by pulsed-field gel electrophoresis, confirmed the loss of both parental artificial chromosomes, and the specific retention of a larger or smaller recombinant YAC.
Assuntos
Cromossomos Artificiais de Levedura , Clonagem Molecular/métodos , Seleção Genética , Animais , Southern Blotting , Caenorhabditis elegans/genética , Eletroforese em Gel de Campo Pulsado , Marcadores Genéticos , Meiose , Mitose , Recombinação GenéticaRESUMO
A simple agarose-gel apparatus has been developed that allows the separation of DNA molecules in the size range from 50 kb to well over 750 kb, the largest size for which size standards were available. The apparatus is based on the recent discovery that large DNA molecules are readily fractionated on agarose gels if they are alternately subjected to two approximately orthogonal electric fields. The switching time, which was on the order of 20-50 sec in our experiments, can be adjusted to optimize fractionation in a given size range. The resolution of the technique is sufficient to allow the fractionation of a sample of self-ligated lambda DNA into a ladder of approximately 15 bands, spaced at 50 kb intervals. We have applied the technique to the fractionation of yeast DNA into 11 distinct bands, several of which have been shown by DNA-DNA hybridization to hybridize uniquely to different chromosome-specific hybridization probes. In this paper, we describe the design of the apparatus, the electrophoretic protocol, and the sample-handling procedures that we have employed.