RESUMEN
Black aspergilli, and particularly Aspergillus carbonarius, are responsible for ochratoxin A production in grapes. Correct identification of these species is essential for toxicological risk assessment in grape and wine. A low-complexity oligonucleotide microarray (OLISA, Apibio, F) based on DNA oligonucleotides probes, obtained from sequences of the calmodulin gene, was set up in order to detect A. carbonarius, A. japonicus/A. aculeatus and A. ibericus isolated from grape. The designed microarray distinguished all Aspergillus species and the detection limit for A. carbonarius was 3.2 pg of DNA as a template for the PCR reaction. This microarray offers a quick and parallel analysis to detect individual Aspergillus species in pure cultures and in naturally contaminated grape samples.
Asunto(s)
Aspergillus/aislamiento & purificación , Ocratoxinas/biosíntesis , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Vitis/microbiología , Aspergillus/clasificación , Aspergillus/genética , ADN de Hongos/genética , Contaminación de Alimentos/análisis , Microbiología de Alimentos , Expresión Génica , Reacción en Cadena de la Polimerasa/métodos , Especificidad de la EspecieRESUMEN
The EP4 prostaglandin receptor (EP4R) is a member of the seven transmembrane receptor superfamily. We have obtained the human EP4 receptor gene sequence and determined its structure relative to EP4R cDNA synthesized from peripheral blood lymphocytes. The EP4R gene spans approximately 22 kb and consists of three exons separated by two introns. The first exon (530 bp) is noncoding. After an intron of 472 bp, the second exon contains a short (43 bp) 5' sequence before a 289-amino-acid open reading frame (ORF). An 11.5-kb intron is found at the end of transmembrane 6, and the rest of the ORF is in exon 3. The gene structure is analogous to those of the thromboxane, PGI, and PGD receptors. The deduced initiation site does not contain a conventional TATA box but is 70% GC-rich and contains CCAAT boxes, SP1 and AP2 motifs, and motifs consistent with activation by proinflammatory cytokines. Southern blot analysis of human genomic DNA shows two genes with homology to the EP4R gene. Both appear to be pseudogenes with 70% amino acid identity to the EP4R up to the "ERY" sequence at the end of transmembrane 3, where an Alu-like repetitive sequence element was found. The ORF sequence is also interrupted by a stop codon. The pseudogenes differ in that one contains a second "repetitive element" (a line 1 repeat) in the 5' end of the ORF. Northern blot analysis of human mRNA using a pseudogene probe showed hybridization only to the EP4 receptor transcript. PCR also failed to detect expression of either pseudogene. This study defines the gene structure of EP4R and suggests the existence of two related pseudogenes.