RESUMO
Psoriasin is an abundant low molecular weight protein in keratinocytes from psoriatic lesions. Because of similarities in gene structure and expression to other genes on human chromosomal band 1q21, we hypothesized that psoriasin might also map to this region. To test this hypothesis, we identified and used a genomic lambda clone (lambda 9.2) as a probe for fluorescent in situ hybridization. lambda 9.2 detected the 1q21 region in 81% of 52 chromosomes 1 examined, although it also hybridized to acrocentric chromosomes. lambda 9.2 DNA yielded polymerase chain reaction amplification of 121-bp sequence colinear with psoriasin cDNA, as did genomic DNA from hybrid cell lines containing all or part of chromosome 1. The psoriasin gene was present on a 380-kb yeast artificial chromosome clone that was previously mapped to 1q21 and shown to contain calcyclin; here it is also shown to contain MRP8 and CaN19. Psoriasin and several other tightly linked 1q21 genes were markedly overexpressed in psoriatic lesions, suggesting a role for these clustered genes in the regulation of epidermal proliferation.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Cromossomos Humanos Par 1 , Família Multigênica , Psoríase/genética , Sequência de Bases , Mapeamento Cromossômico , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Proteína A7 Ligante de Cálcio S100 , Proteínas S100RESUMO
Calcyclin is a member of the S100 family of proteins, many of which are encoded by genes that have been localized to the proximal long arm of human chromosome 1 (bands q21-q22). A 450-kb yeast artificial chromosome clone containing the human calcyclin gene was identified by PCR screening and used as a probe for fluorescence in situ hybridization (FISH). Along with the expected hybridization to 1q21, simultaneous, specific hybridization to the centromeric region of the short arm of chromosome 1 was also observed. An identical pattern of hybridization was observed when microdissected 1q21 DNA sequences were used as a probe for FISH, confirming the presence of homologous sequences flanking both sides of the centromere of human chromosome 1. These results are consistent with a model in which human chromosome 1 arose by insertion of the centromere and heterochromatin into an ancestral chromosome containing chromosome-specific repetitive sequences.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular , Centrômero , Cromossomos Humanos Par 1 , Proteínas S100 , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Primers do DNA , Fator de Crescimento Epidérmico/genética , Humanos , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Proteína A6 Ligante de Cálcio S100 , Homologia de Sequência do Ácido NucleicoRESUMO
In human epidermis, expression of CD1a is confined to Langerhans cells (LC), whereas CD1c expression has been observed in dendritic cells of the dermis, as well as the epidermis. In transfected fibroblasts, expression of CD1c at the cell surface appears to exclude expression of either CD1b or CD1a, despite continued transcription of the latter genes. In order to determine whether this mechanism might be operative in human skin, we have compared the expression of CD1a and CD1c on the surface of dermal and epidermal dendritic cells to their expression at the level of mRNA using a combination of dual-label immunofluorescence microscopy, northern blot hybridization, and reverse transcriptase-polymerase chain reaction (RT-PCR). By both immunofluorescence and Northern blotting, CD1c expression was observed in both dermal and epidermal cells, whereas expression of CD1a was confined largely to the epidermis. Moreover, as shown by immunomagnetic bead selection and RT-PCR, CD1a and CD1c were both expressed on epidermal LC, but were absent from other epidermal cell types. These results argue against cell surface exclusion as a mechanism for selective expression of CD1c in human dermis.
Assuntos
Antígenos CD/genética , Expressão Gênica/genética , Pele/química , Antígenos CD/análise , Antígenos CD1 , Sequência de Bases , Biópsia , Northern Blotting , Células Dendríticas/química , Células Dendríticas/citologia , Imunofluorescência , Humanos , Células de Langerhans/química , Células de Langerhans/citologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Biossíntese de Proteínas , RNA Mensageiro/análise , RNA Mensageiro/genética , Pele/imunologia , Pele/patologiaRESUMO
The most commonly used DNA transfection method, which employs the calcium phosphate co-precipitation of the donor DNA, involves several discrete steps (1,2). These include the uptake of the donor DNA by the recipient cells, the transport of the DNA to the nucleus, transient expression prior to integration into the host cell genome, concatenation and integration of the transfected DNA into the host cell genome and finally the stable expression of the integrated genes (2,3). Both the concatenation and the integration of the donor DNA into the host genome involve the formation and ligation of DNA strand-breaks. In the present study we demonstrate that the nuclear enzyme, adenosine diphosphoribosyl transferase (ADPRT, E.C. 2.4.2.30), which is dependent on the presence of DNA strand breaks for its activity (4,5) and necessary for the efficient ligation of DNA strand-breaks in eukaryotic cells (4,6), is required for the integration of donor DNA into the host genome. However, ADPRT activity does not influence the uptake of DNA into the cell, its episomal maintenance or replication, nor its expression either before or after integration into the host genome. These observations strongly suggest the involvement of ADPRT activity in eukaryotic DNA recombination events.