RESUMO
Macrophage scavenger receptors class A (MSR) are thought to play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins by macrophages in the vessel wall leading to foam cell formation. To investigate the in vivo role of the MSR in this process, a transgenic mouse model expressing both isoforms of the human MSR was generated. A 180-kb yeast artificial chromosome (YAC) containing the human MSR gene (MSR1) with 60- and 40-kb flanking sequence at the 5' and 3' end, respectively, was obtained by reducing the size of a 1050-kb YAC by homologous recombination. This 180-kb YAC was microinjected into mouse oocytes. In the resulting transgenic mice, high levels of mRNA for both type I and type II human MSR1 were detected in peritoneal macrophages and trace levels in other organs, known to contain macrophage-derived cells. Using an antibody against the human MSR, the Kupffer cells in the liver were shown to contain the MSR protein. In vivo clearance of acetyl-LDL was not changed in the MSR1-transgenic mice. However, in vitro studies using peritoneal macrophages from the transgenic mice showed a two-fold increased degradation of acetyl-LDL and cholesterolester accumulation concomitant with a four-fold increase in foam cell formation, as compared to wild-type macrophages. Thus, macrophage specific overexpression of the MSR may lead to increased foam cell formation, which is one of the initial and crucial steps in atherogenesis.
Assuntos
Cromossomos Artificiais de Levedura/química , Células Espumosas/metabolismo , Macrófagos Peritoneais/metabolismo , Receptores Imunológicos/genética , Animais , Sequência de Bases , Células Cultivadas , Cromossomos Artificiais de Levedura/genética , Modelos Animais de Doenças , Células Espumosas/patologia , Expressão Gênica , Humanos , Células de Kupffer/química , Lipoproteínas LDL/metabolismo , Lipoproteínas LDL/farmacocinética , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Receptores Imunológicos/análise , Receptores Depuradores , Receptores Depuradores Classe A , Sensibilidade e Especificidade , Especificidade da Espécie , Distribuição TecidualRESUMO
The disease loci for X-linked Retinoschisis (RS), Keratosis follicularis spinulosa decalvans (KFSD), and Coffin-Lowry syndrome (CLS) have been localized to the same, small region in Xp22 on the human X Chromosome (Chr). To generate a high-resolution map of the available contig in this area, we have used the YAC fragmentation vectors pBP108/ADE2 and pBP109/ADE2 and generated fragmented YACs from a 2.5-Mb YAC (y939H7) spanning the mentioned disease gene candidate regions. Forty-seven fragmented YACs were generated and analyzed, ranging in size from 170 kb to over 2400 kb. The resulting YAC fragmentation panel was used to construct a detailed restriction map of the region and has been used to bin clones and markers. As a deletion panel, it will present a valuable resource for further mapping.
Assuntos
Doenças do Desenvolvimento Ósseo/genética , Mapeamento Cromossômico/métodos , Doença de Darier/genética , Degeneração Retiniana/genética , Cromossomo X , Anormalidades Múltiplas/genética , Southern Blotting , Cromossomos Artificiais de Levedura , Clonagem Molecular , Cosmídeos , Fragmentação do DNA , Eletroforese em Gel de Campo Pulsado , Marcadores Genéticos , Humanos , Deficiência Intelectual/genética , Mapeamento por Restrição , SíndromeRESUMO
We have constructed a set of fragmentation vectors for the truncation of either the centromeric or the noncentromeric end of YACs containing a human DNA insert. These vectors carry ADE2 or HIS5 as the selectable marker, enabling direct use in AB1380, the host strain of most publicly available YAC libraries. Centromeric fragmentation vectors for AB1380 have not been reported previously; the noncentromeric vectors show high frequencies of fragmentation.
Assuntos
Cromossomos Artificiais de Levedura/genética , Vetores Genéticos/genética , Carboxiliases/genética , Fragmentação do DNA , Marcadores Genéticos , Humanos , Mutagênese Insercional , Recombinação Genética , Transformação GenéticaRESUMO
Rieger syndrome (RGS) is an autosomal dominant disorder of morphogenesis affecting mainly the formation of the anterior eye chamber and of the teeth. RGS has been localized to human chromosome 4q25 by linkage to epidermal growth factor (EGF). We have constructed a detailed physical map and a YAC contig of the genomic region encompassing the EGF locus. Using FISH, several YACs could be shown to cross the breakpoint in two independent RGS patients with balanced 4q translocations. Alu- and LINE-fragmentation of a 2.4-Mb YAC generated a panel of shorter YACs ranging in size from 2.4 Mb to 75 kb. Several fragmentation YACs were subcloned in cosmids, which were mapped to specific subregions of the original YAC by hybridization to the fragmentation panel to further refine the localization of the translocation breakpoints, allowing mapping of the breakpoints to within the most-telomeric 200 kb of the original 2.4-Mb YAC. FiberFISH of cosmids located in this 200-kb region mapped the two translocation breakpoints within a 50-kb region approximately 100-150 kb centromeric to D4S193, significantly narrowing down the candidate region for RGS. The mapping data and resources reported here should facilitate the identification of a gene implicated in Rieger syndrome.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 4/genética , Anormalidades Craniofaciais/genética , Glaucoma/genética , Anormalidades Dentárias/genética , Translocação Genética/genética , Umbigo/anormalidades , Southern Blotting , Linhagem Celular , Humanos , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase , SíndromeRESUMO
The centromere of Kluyveromyces lactis was delimited to a region of approximately 280 bp, encompassing KlCDEI, II, and III. Removal of 6 bp from the right side of KlCDEIII plus flanking sequences abolished centromere function, and removal of 5 bp of KlCDEI and flanking sequences resulted in strongly reduced centromere function. Deletions of 20-80 bp from KlCDEII resulted in a decrease in plasmid stability, indicating that KlCDEII must have a certain length for proper centromere function. Centromeres of K. lactis do not function in Saccharomyces cerevisiae and vice versa. Adapting the length of KlCDEII to that of ScCDEII did not improve KlCEN function in S. cerevisiae, while doubling the ScCDEII length did not improve ScCEN function in K. lactis. Thus the difference in CDEII length is not in itself responsible for the species specificity of the centromeres from each of the two species of budding yeast. A chimeric K. lactis centromere with ScCDEIII instead of KlCDEIII was no longer functional in K. lactis, but did improve plasmid stability in S. cerevisiae, although to a much lower level than a wild-type ScCEN. This indicates that the exact CDEIII sequence is important, and suggests that the flanking AT-rich CDEII has to conform to specific sequence requirements.
Assuntos
Centrômero/fisiologia , DNA Fúngico/genética , Kluyveromyces/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Análise Mutacional de DNA , Dados de Sequência Molecular , Especificidade da EspécieRESUMO
We have investigated the chromatin structure of Kluyveromyces lactis centromeres in isolated nuclei of K. lactis and Saccharomyces cerevisiae by using micrococcal nuclease and DNAse I digestion. The protected region found in K. lactis is approximately 270 bp long and encompasses the centromeric DNA elements, KlCDEI, KlCDEII, and KlCDEIII, but not KlCDE0. Halving KlCDEII to 82 bp impaired centromere function and led to a smaller protected structure (210 bp). Likewise, deletion of 5 bp from KlCDEI plus adjacent flanking sequences resulted in a smaller protected region and a decrease in centromere function. The chromatin structures of KlCEN2 and KlCEN4 present on plasmids were found to be similar to the structures of the corresponding centromeres in their chromosomal context. A different protection pattern of KlCEN2 was detected in S. cerevisiae, suggesting that KlCEN2 is not properly recognized by at least one of the centromere binding proteins of S. cerevisiae. The difference is mainly found at the KlCDEIII side of the structure. This suggests that one of the components of the ScCBF3-complex is not able to bind to KlCDEIII, which could explain the species specificity of K. lactis and S. cerevisiae centromeres.
Assuntos
Cromatina/ultraestrutura , Kluyveromyces/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Centrômero/ultraestrutura , Cromossomos Fúngicos/ultraestrutura , DNA Fúngico/genética , Kluyveromyces/ultraestrutura , Dados de Sequência Molecular , Saccharomyces cerevisiae/ultraestrutura , Deleção de Sequência , Especificidade da EspécieRESUMO
The nucleotide sequences of five of the six centromeres of the yeast Kluyveromyces lactis were determined. Mutual comparison of these sequences led to the following consensus: a short highly conserved box (5'-ATCACGTGA-3') flanked by an AT-rich (+/- 90%) stretch of +/- 160 bp followed by another conserved box (5'-TNNTTTATGTTTCCGAAAATTAATAT-3'). These three elements were named KlCDEI, KlCDEII, and KlCDEIII respectively, by analogy with the situation in Saccharomyces cerevisiae. In addition, a second 100 bp AT-rich (+/- 90%) element, named KlCDE0, was found +/- 150 bp upstream of KlCDEI. The sequences of both KlCDEI and KlCDEIII are highly conserved between K. lactis and S. cerevisiae; however, centromeres of K. lactis do not function in S. cerevisiae and vice versa. The most obvious differences between the centromeres of the two yeast species are the length of the AT-rich CDEII, which is 161-164 bp in K. lactis versus 78-86 bp in S. cerevisiae and the presence in K. lactis of KlCDE0, which is not found in S. cerevisiae.
Assuntos
Centrômero , DNA Fúngico/genética , Kluyveromyces/genética , Sequência de Bases , Cromossomos Fúngicos , Clonagem Molecular , Sequência Consenso , Dados de Sequência Molecular , Plasmídeos/genética , Mapeamento por Restrição , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Transformação GenéticaRESUMO
A direct selection method was used to isolate centromeres from a genomic library of the yeast Kluyveromyces lactis. The method is based on the lethality at high copy number of the ochre-suppressing tRNA gene SUP11. Five different chromosomal fragments were found that confer mitotic stability to plasmids containing a replication origin of K. lactis (KARS). In addition, KARS plasmids containing these fragments have a copy number of approximately one, and each of the five fragments hybridizes to a different chromosome of K. lactis. From these results we conclude that five of the six centromeres of K. lactis have been isolated. These centromeres do not function in S. cerevisiae.
