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dc.contributor.advisor Siam, Rania Mohammed, Mahera 2014-05-22T20:57:56Z 2016-05-21T22:00:19Z 2014 Spring en 2014-05-22
dc.description.abstract Red sea, described as one of the unique marine ecosystems, incorporates up to 25 deep-sea brine pools. These pools posses multiple extreme conditions influencing the evolution and survival of their inhabiting microbial community. The combination of maximum depth (2194 m), high temperature (68 ◦C), anoxia, high salinity (26%), high pressure and high concentrations of heavy metals in the lower convective layer (LCL) of the Atlantis II brine pool makes it an ideal environment for identification of novel enzymes with unique characteristics and potential biotechnological applications. Here we describe the identification and the preliminary in vivo functional investigation of the ligase domain of an ATP-dependent DNA ligase from the DNA of the prokaryotic community extracted from water samples of the LCL of Atlantis II brine pool. Previously, these water samples were serially filtered on different membranes and the DNA isolated from the 0.1m filter was subjected to 454 pyrosequencing. A metagenomic dataset was initiated and used in this study to mine for genes encoding DNA ligases through Pfam search of conserved domains. The search and subsequent bioinformatic analysis resulted in the identification of a contig harboring an ORF of 915 bp (305 amino acids) that encodes a putative DNA ligase (LigATII). Homology search of the putative DNA ligase showed highest similarity to Erysiopelotrichaceae Bacterium (39% identity, 54% positive). LigATII displays modular architecture that is similar to two distinct domains-(the adenylation domain of LigD and the oligonucleotide binding (OB) fold domain)-that are conserved in ATP-dependent DNA ligases. Functional annotation of the LigATII ORF, identification of the functional conserved amino acids by the Consurf tool, 3D modeling and comprehensive phylogenetic analysis were conducted. These analyses have revealed the relatedness of LigATII to the family of ATP-dependent DNA ligases that has been recently identified through computational studies to exist in prokaryotes. This family is expected to be involved in the specialized form of genomic DNA repair through the non-homologous end joining pathway which acts to join double-stranded breaks (DSBs) or to promote genetic diversity under conditions of selection pressures. Accordingly, the putative LigATII was amplified from the whole genome DNA amplification of LCL. Sanger sequencing confirmed the sequence of the gene before cloning into pET100 Topo directional expression vector. The cloned LigATII was transformed into a temperature sensitive mutant strain of Escherichia coli; strain GR501, with mutation in the DNA ligase gene. LigATII complemented the temperature sensitive strain at the non-permissive temperature (43◦C) verifying the in vivo functional activity. The biochemical characteristics of the novel LigATII protein will be described. en
dc.description.sponsorship First the person who influenced me the most in my graduate career has been my advisor Dr. Rania Siam, Associate Professor and Chair of the Biology Department in the School of Science and Engineering at the American University in Cairo. I am thankful to my thesis committee members. Thanks for Dr. Wael Mohammed, Visiting Assistant Professor in the Biology Department at the American university in Cairo, Dr. Ramy Aziz, Assistant Professor in the Microbiology and Immunology Department at the Faculty of Pharmacy Cairo University and Dr. Ahmed Abdellatif, Visiting Assistant Professor in the Biology Department at the AUC, Thanks to all of them for agreeing to serve in my committee. I would like to thank all the professors in the department who taught me through my master program and also I would like to thank the administrators in our school division for providing any assistance requested. I acknowledge the King Abdullah University of Science and Technology (KAUST) for funding the Red Sea Metagenomics project and for the graduate research fellowship that provided the necessary financial support for this research. I am grateful to Nahla Hussien her for helping me to learn that there is more than one way to approach a problem. I would also like to give a special thanks to my colleagues and friends in the American University in Cairo. I am especially grateful to Hadeel El-Bardisy and Aya Medhat who I had the great fortune of becoming close friends. I thank my lifetime friends outside the AUC for being truly amazing friends. Last, but by no means least, to thank the people who made me into the person who I am now, my Mom and Dad, I love you both and I wish you all the happiness. My sister and my best friend Maram, thank you for your support through my endless favors. Finally, My dearest husband Mohammed, you are my one and only, I could not have done all of this without you by my side and my precious daughter Lana, I have done this for you to be proud of your mother. Both of you and your father were my best cheerleaders :) I love you. en
dc.format.extent 79 p. en
dc.format.medium theses en
dc.language.iso en en
dc.rights Author retains all rights with regard to copyright. en
dc.subject Red Sea en
dc.subject Atlantis II (Ship : 1963-1996) en
dc.subject.lcsh Thesis (M.S.)--American University in Cairo en
dc.subject.lcsh Biotechnology -- Red Sea -- Egypt.
dc.subject.lcsh Molecular biology -- Red Sea -- Egypt.
dc.subject.lcsh Microbiology -- Red Sea -- Egypt.
dc.subject.lcsh Geochemistry -- Red Sea -- egypt.
dc.subject.lcsh Chemistry, Analytic -- Red Sea -- egypt.
dc.title Functional identification of a Ligase in the Red Sea Atlantis II deepest Layer en
dc.type Still Image en
dc.type Text en
dc.subject.discipline Biotechnology en
dc.rights.access This item is restricted for 2 years from the date issued en
dc.contributor.department American University in Cairo. Dept. of Biology en
dc.description.irb American University in Cairo Institutional Review Board approval is not necessary for this item, since the research is not concerned with living human beings or bodily tissue samples. en
dc.contributor.committeeMember Aziz, Ramy
dc.contributor.committeeMember Mohammed, Wael

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    This collection includes theses and dissertations authored by American University in Cairo graduate students.

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