RESUMO
Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized them to two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B.
Assuntos
Resistência à Doença/genética , Biblioteca Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Triticum/genética , Triticum/microbiologia , Ascomicetos , Cromossomos de Plantas/genética , DNA de Plantas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Marcadores Genéticos , Anotação de Sequência Molecular , Especificidade de Órgãos/genética , Doenças das Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Deleção de Sequência , Triticum/imunologiaRESUMO
Escherichia coli has been recognized as the principal indicator of fecal contamination of water. Indeed, E. coli is the only species in the coliform group found in relationship with gastrointestinal tract of human and warm-blooded animals and subsequently excreted in large numbers in the human feces. To obtain a complete picture of water quality and therefore, a better protection of public health, different techniques for water analysis have been proposed. In this article, we describe an alternative method that uses SYBR green real time-polymerase chain reaction (RT-PCR) technology to identify and quantify all E. coli biotypes in a group of wastewater samples collected from a wastewater depurator located in South of Italy. This new RT-PCR protocol is accurate in measuring the concentration of chromosomal E. coli DNA using the amplification of three new specific fragments of the following bacteria genes: CadC, HNS, and Allan whose sequence is specific for E. coli family and conserved in all E. coli subtypes. This method allowed us to detect the presence of all E. coli biotypes directly in wastewater samples and estimated the correspondence between colony forming units and bacterial DNA concentrations. The availability of a rapid and sensitive method may be useful to monitor the persistence of E. coli in water, to evaluate the efficiency of wastewater purification treatments and the possible recycle for agricultural use. Furthermore, the development of a simple and routine method to monitor water quality with RT-PCR analysis can encourage the testing of a higher number of samples.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Escherichia coli/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Águas Residuárias/microbiologia , Técnicas de Tipagem Bacteriana/instrumentação , Benzotiazóis , Contagem de Colônia Microbiana , Primers do DNA/genética , Diaminas , Escherichia coli/classificação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Compostos Orgânicos/química , Quinolinas , Reação em Cadeia da Polimerase em Tempo Real/instrumentaçãoRESUMO
BACKGROUND: Polyamine metabolism has a critical role in cell death and proliferation representing a potential target for intervention in breast cancer (BC). This study investigates the expression of spermine oxidase (SMO) and its prognostic significance in BC. Biochemical analysis of Spm analogues BENSpm and CPENSpm, utilized in anticancer therapy, was also carried out to test their property in silico and in vitro on the recombinant SMO enzyme. METHODS: BC tissue samples were analyzed for SMO transcript level and SMO activity. Student's t test was applied to evaluate the significance of the differences in value observed in T and NT samples. The structure modeling analysis of BENSpm and CPENSpm complexes formed with the SMO enzyme and their inhibitory activity, assayed by in vitro experiments, were examined. RESULTS: Both the expression level of SMO mRNA and SMO enzyme activity were significantly lower in BC samples compared to NT samples. The modeling of BENSpm and CPENSpm complexes formed with SMO and their inhibition properties showed that both were good inhibitors. CONCLUSIONS: This study shows that underexpression of SMO is a negative marker in BC. The SMO induction is a remarkable chemotherapeutical target. The BENSpm and CPENSpm are efficient SMO inhibitors. The inhibition properties shown by these analogues could explain their poor positive outcomes in Phases I and II of clinical trials.