RESUMO
Genes of human neurotrophins NGF, BDNF, NT-3 were cloned, and the corresponding proteins and their fragments were expressed in Escherichia coli BL-21 (DE3lambda) cells. Their intracellular localization was determined. The conditions for isolation and purification of the target recombinant proteins and for folding of BDNF and NT-3 precursors were selected. The recombinant proprecursors of human neurotrophines have been shown to possess complex oligomeric structure.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/química , Escherichia coli/metabolismo , Fator de Crescimento Neural/química , Neurotrofina 3/química , Biopolímeros , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Fator Neurotrófico Derivado do Encéfalo/genética , Clonagem Molecular , Escherichia coli/genética , Humanos , Fator de Crescimento Neural/biossíntese , Fator de Crescimento Neural/genética , Neurotrofina 3/biossíntese , Neurotrofina 3/genética , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Desnaturação Proteica , Dobramento de Proteína , Precursores de Proteínas/biossíntese , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Expression of certain neurotrophin genes and their receptors, as well as NGF-induced gene EGRI was studied in human normal lung, squamous cell lung cancer, and adenocarcinoma tissues. Differential expression pattern of NGF, BDNF, and NT-3 mRNA was established by RT-PCR in normal human lung. NGF expression level varying from minor to significant was demonstrated in double specimens (histologically diagnosed human lung cancer and appropriate adjacent tissue). Interestingly, a half of the double specimens studied demonstrated the differential expression pattern in both cancer and adjacent tissues, whereas in other cases no difference in the NGF expression between these pair of tissues was observed. In the majority of the double specimens, we detected low levels of NT3 and BDNF expression for both cancer and adjacent tissue. No expression of TrkA, TrkB, p75 was found in double specimens and normal tissues. Differential expression patterns of TrkC were observed in normal tissues as well as in certain double specimens. High levels of EGR1 expression were detected in normal tissues. No EGRI expression was observed in cancer tissue compared to its high expression level in adjacent tissue in the majority of double specimens.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Proteínas de Neoplasias/biossíntese , Fatores de Crescimento Neural/biossíntese , Receptores de Fator de Crescimento Neural/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/genética , Humanos , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Fatores de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/genéticaRESUMO
Brain-specific human genes were studied over the recent years in the Department of Molecular Bases of Human Genetics, Institute of Molecular Genetics. Clones Hfb1, Hmob3, and Hmob33 were selected from human brain cDNA libraries by differential screening. The clones were sequenced, mapped, and tested for expression in various human tissues. In vitro and in silico experiments identified Hfb1 as an earlier unknown complexin 2 gene (CPLX2) fragment, which codes for the large 3'-untranslated region of the CPLX2 mRNA. Hmob3 proved to correspond to an earlier unknown fragment of the large 3'-untranslated region of the human MAP1B mRNA. With Hfb1 and Hmob3, new terminal exons were revealed and exact structures established for CPLX2 and MAP1B. Hmob33 was identified as a fragment of the 3'-terminal exon of a new gene, MOB, which codes for a thus far unknown evolutionarily conserved transmembrane protein. The structure of the deduced protein product was analyzed.
Assuntos
Encéfalo/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Membrana , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas , Proteínas Adaptadoras de Transporte Vesicular , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Clonagem Molecular , Sequência Conservada , Proteínas de Ligação a DNA/química , Evolução Molecular , Éxons , Sequências Hélice-Alça-Hélice , Humanos , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , RNA Mensageiro , Análise de Sequência de DNA , Fatores de Transcrição/química , Transferases (Outros Grupos de Fosfato Substituídos)RESUMO
A genomic clone hybridizing with brain-specific sequence Hfb1 was isolated from a chromosome 5 consmid library. Hfb1 proved to correspond to a new gene exon which codes for a large 3'-untranslated region of the mRNA for synaptic protein complexin 2. Together with the 985-nt Hfb1 cDNA (EMBL Y15167) isolated previously from a cDNA library of the frontal cerebral cortex, the primary structure was established for genomic clone Ghfb sized more than 4 kb. A GenBank search revealed complete identity of the 5' end of Ghfb and the 3'-untranslated region (878-933) of the human complexin 2 mRNA. Large transcripts with the 5' end corresponding to the complexin 2 mRNA and the 3' end to Ghfb were detected in total mRNA of the human brain by means of RT-PCR. The size of the 3'-untranslated region of the human complexin 2 mRNA was estimated at 4 kb.
