Survey sequencing and radiation hybrid mapping to construct comparative maps.

In MURPHY WJ (ed.) Phylogenomics, Humana Press. (Methods in Molecular Biology, 422)International audienceRadiation hybrid (RH) mapping has become one of the most well-established techniques for economically and efficiently navigating genomes of interest. The success of the technique relies on random chromosome breakage of a target genome, which is then captured by recipient cells missing a preselected marker. Selection for hybrid cells that have DNA fragments bearing the marker of choice, plus a random set of DNA fragments from the initial irradiation, generates a set of cell lines that recapitulates the genome of the target organism several-fold. Markers or genes of interest are analyzed by PCR using DNA isolated from each cell line. Statistical tools are applied to determine both the linear order of markers on each chromosome, and the confidence of each placement. The resolution of the resulting map relies on many factors, most notably the degree of breakage from the initial radiation as well as the number of hybrid clones and mean retention value.A high-resolution RH map of a genome derived from low pass or survey sequencing (coverage from 1 to 2 times) can provide essentially the same comparative data on gene order that is derived from high-coverage (greater than x7) genome sequencing. When combined with fluorescence in situ hybridization, RH maps are complete and ordered blueprints for each chromosome. They give information about the relative order and spacing of genes and markers, and allow investigators to move between target and reference genomes, such as those of mouse or human, with ease although the approach is not limited to mammal genomes

Two Different Aspects of Genomics: the Construction of a High-Density Radiation Hybrid Map and the Study of the Involvement of Mirnas in the Mammary Gland

In questa tesi vengono affrontati due diversi tipi di studio. Il primo tratta della costruzione di una mappa di ibridi bovino-criceto di radiazione di seconda generazione tramite la tipizzazione di un pannello RH, fornito dal Roslin Institut, con un set di Est non ridondanti provenienti da una libreria di cloni a cDNA di cervello bovino. Il secondo soggetto è il coinvolgimento dei microRNA, una nuova classe di piccoli RNA regolatori non codificanti, nello sviluppo della ghiandola mammaria. E' stata analizzata l'espressione di un set di microRNA noti in letteratura nei diversi stadi dello sviluppo dell'organo e sono state costruite librerie di cloni a cDNA di potenziali microRNA a partire da diversi stadi del ciclo dell'organo.In this thesis two different subjects have been studied. The first is the construction of a second generation high-density RH map of the bovine using the RH panel of the Roslin Institute. The panel have been characterized by PCR with a set of non-redundant EST chosen from a cDNA library of bovine brain. The second work treats about the involvement of miRNAs in the development of mammary gland. A set of 25 known miRNAs have been chosen and their expression have been examined in the different stages of mammary gland development. Libraries of potential miRNAs have been constructed from different stages of mammary gland development and some miRNAs have been validated

Cardiovascular candidate genes within the oxidative stress pathway: rat and human studies

Combined congenic breeding strategy and microarray expression profiling studies from our group previously identified rat glutathione s-transferase mu type-1 (rGstm1) as a functional and positional candidate gene implicated in hypertension. In the previous studies, expression of rGstm1 was lower in the kidney from the hypertensive rat model, stroke-prone spontaneously hypertensive rat (SHRSP0, compared to the normotensive Wistar Kyoto (WKY) and a chromosome 2 congenic strain (SP. WKYG1a2c*). This project aims to characterise Gstm gene family in rat and human tissues. Results from this thesis demonstrate reduced mRNA expression of several members of rGstm gene family, in vascular and renal tissues. Most notably, expression of vascular and renal rGstm1 mRNA and protein, are lower in the SHRSP compared to WKY and SP.WKYG1a2c*. Vascular mRNA expression of rGstm2, rGstm5 and rGstm7 were reduced in the SHRSP males compared to the WKY males and SP.WKYG1a2c* males but not in the females. The expression profile of the human GSTM (hGSTM) gene family in vascular tissue from varicose vein ad saphenous vein have also been characterised in an attempt to identify the true human orthologue of rGstm1. There are five known members in the hGSTM gene family, all of which were expressed in vascular tissues. Results from characterisation of the vascular hGSTM gene family show that four hGSTM members are homologous to rGstm1. These four hGstm genes remain as potential orthologue for rGstm1. Functional roles of vascular and renal rGstm genes have been investigated using two pharmacological intervention studies. Olmesartan (AT1R antagonist) or hydralazine plus hydrochlorothiazide (direct vasodilator and diuretic) have been used to evaluate the oxidative stress status and gene expression of rGstm genes, in the reversal and prevention studies. Both drug treatments improved the BP of SHRSP rats in reversal study and prevented the rise in BP in the prevention study. Olmesartan-treated SHRSP rats demonstrated reduced superoxide (O2-) and hydrogen peroxide (H2O2) production in both reversal and prevention studies. Minor effects by both drug treatments were observed for the rGstm gene family in the prevention but not the reversal study. Both drug treatments did not influence vascular of renal rGstm1 expression in either reversal or prevention studies. Improvement in BP did not improve rGstm1 gene expression. The rGstm1 was not responsive to pharmacological interventions due to strain-dependent genetic abnormalities. Functional polymorphisms in two key enzymes involved in ROS and NO balance were investigated for association with CAD and vascular compliance as single polymorphism and as haplotypes. There was an association between CYBA A-930G with CAD, with the A allele being recessive. There was also an association between NOS3 G894T with CAD, only when G allele was dominant. The T-786C of NOS3 was associated with small artery compliance index (C2), in both CAD and control groups