RESUMEN
A polymorphism in the genes encoding alpha- and beta-adducin (ADD) was described as being associated with blood-pressure variation in a genetically hypertensive strain of rats (MHS). ADD is a cytoskeletal heterodimeric protein which may be involved in cellular signal transduction and interacts with other membrane skeleton proteins which affect ion transport across the cell membrane. The cDNA encoding the alpha subunit of rat ADD was isolated using PCR methods. The cDNA consists of about 3900 bp and encodes a protein of 735 amino acids (aa) which shows 91% aa identity with the human counterpart. In spleen and kidney, three alternative spliced exons were found by PCR amplification and confirmed by RNase protection analysis. 17 inbred rat strains were genotyped for the polymorphism in the alpha- and beta-ADD genes. Chromosomal localisation mapped rat alpha-ADD on chromosome 14 and rat beta-ADD on chromosome 4.
Asunto(s)
Proteínas de Unión a Calmodulina/genética , Alelos , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Genes , Hipertensión/genética , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas/genéticaRESUMEN
Adducin (ADD) is a heterodimeric protein of the membrane skeleton with subunits of 103 (alpha) and 97 kDa (beta). It promotes the assembly of the spectrin-actin network. We have previously shown that one point mutation in each of the alpha and beta rat ADD-encoding genes is associated with blood pressure variation in an animal model for hypertension, the Milan hypertensive strain of rats, probably due to a change in the phosphorylation pattern. In fact, the rat mutations, Y to F for alpha and R to Q for beta, are located, respectively, in a tyrosine kinase and a protein kinase A phosphorylation site. We have now determined, for the human beta-ADD-encoding gene, its chromosomal localisation, exon-intron organisation and alternative splicing patterns. We report here that human beta-ADD is localised on chromosome 2 and we also show a characteristic 3' end alternative splicing of the beta-ADD RNA that generates two distinct beta-ADD families, namely ADD 63 and 97; both of them in turn present a very complex differential splicing pattern in the internal exons.
Asunto(s)
Proteínas de Unión a Calmodulina/genética , Proteínas del Citoesqueleto/genética , Empalme Alternativo , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 2 , Expresión Génica , Genes , Humanos , ARN Mensajero/genética , Mapeo RestrictivoRESUMEN
Data are presented from a comparative research on expression of epidermal growth factor (EGF) receptors and response to EGF of six independently established cell lines derived from human hepatoma. These lines differ in terms of the degree of differentiation, presence of hepatitis B virus (HBV) DNA copies in integrated form and expression of HBV genes. Our results indicate differential expression of membrane EGF receptors and differential response to EGF under serum- and hormone-free culture conditions. Furthermore, a significant difference in affinity could be detected between EGF receptors of the two highly dedifferentiated cell lines (HA22T/VGH and Li7A) whose replication is inhibited by EGF concentrations capable of stimulating more differentiated phenotypes.
Asunto(s)
Carcinoma Hepatocelular/genética , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Regulación de la Expresión Génica , Neoplasias Hepáticas/genética , Southern Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , División Celular , ADN Viral/genética , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Genes Virales , Virus de la Hepatitis B/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
The 6 x His affinity tags have proved invaluable for the exclusive purification of proteins, in Escherichia coli, of genes cloned in frame with a 6 x His tag and a strong inducible promoter. Here, we demonstrate that the system can be extended to Gram-negative bacteria other than E. coli, by the use of compatible broad-host-range plasmids. As an example, the inducible synthesis and specific purification of the Pseudomonas 6 x His-PfrA siderophore regulatory protein in Pseudomonas putida WCS358 is demonstrated.
Asunto(s)
Proteínas Bacterianas , Clonación Molecular/métodos , Escherichia coli/genética , Regiones Promotoras Genéticas , Pseudomonas putida/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/aislamiento & purificación , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Histidina , Datos de Secuencia Molecular , Plásmidos , Pseudomonas putida/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Mapeo Restrictivo , Factores de Transcripción/genéticaRESUMEN
Under iron limiting conditions, Pseudomonas putida WCS358 produces and secretes a fluorescent siderophore called pseudobactin 358 which consists of a nonapeptide linked to a fluorescent dihydroxy quinoline moiety. Previous studies have identified a major gene cluster involved in pseudobactin 358 biosynthesis and several regulators responsible for the activation of biosynthetic genes under iron starving conditions. In this study, we identified the promoter transcribing the pseudobactin 358 synthetase gene. Promoter deletion experiments have demonstrated that the DNA region downstream of the initiation of transcription site is necessary for proper promoter functioning. This promoter controls the expression of a gene designated ppsD which encodes a 2,247-residue protein, PpsD, which has a predicted molecular weight of 247,610 Da and contains two highly homologous domains of approximately 1000 amino acids each. ppsD::Tn5 mutants of strain WCS358 are unable to synthesise pseudobactin 358 and can be complemented when ppsD is provided in trans. It is concluded that ppsD is a peptide synthetase involved in the biosynthesis of the peptide moiety of pseudobactin 358. PpsD displays a very high degree of similarity (52% aa identity) with PvdD from P. aeruginosa, a non-ribosomal peptide synthetase involved in the biosynthesis of pyoverdine, the fluorescent siderophore produced by P. aeruginosa. It also displayed homology with other peptide synthetases from other micro-organisms involved in the biosynthesis of siderophores and peptide antibiotics.
Asunto(s)
Genes Bacterianos , Péptido Sintasas/genética , Pseudomonas putida/genética , Sideróforos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Familia de Multigenes , Regiones Promotoras Genéticas , Sideróforos/biosíntesisRESUMEN
Pseudomonas mediterranea strain CFBP 5447(T) is a phytopathogenic bacterium isolated from tomato plants affected by pith necrosis disease. Moreover, its ability to produce medium-chain-length polyhydroxyalkanoates (mcl-PHAs) in culture from different carbon sources and valuable microbial products, such as cyclic lipopeptides, has been well documented. Here, we report the first draft genome sequence of this species.
RESUMEN
The establishment of a new, differentiated, hepatitis B virus DNA-negative, human hepatoma cell line (named PLC/AN/2) is described. Neoplastic liver tissue was obtained during hepatectomy in an HBsAg-negative man. The established cell line is negative for alpha-fetoprotein and carcinoembryonic antigen; it has retained in vitro some of the differentiated functions of normal hepatocytes. Additionally, it presents a distinctive rearrangement (translocation) at the long arm of chromosome 4. The high degree of independence from serum growth factor requirements appears to be a major in vitro characteristic of PLC/AN/2 cells, making them a suitable model system for the more precise definition of the human hepatocellular carcinoma phenotype, including mechanisms of growth control.