To determine if the disruption of the MMR pathway results in the reduced conservation of methylated adenines as well as an increased tolerance for mutations that result in the loss or gain of new GATC sites, we surveyed individual clones isolated from experimentally evolving wild-type and MMR-deficient (mutL- ;conferring an 150x increase in mutation rate) populations of E. coli with whole-genome sequencing. Initial analysis revealed a lack of mutations affecting methylation sites (GATC tetranucleotides) in wild-type clones. However, the inherent low mutation rates conferred by the wild-type background render this result inconclusive, due to a lack of statistical power, and reveal a need for a more direct measure of changes in methylation status. Thus as a first step to comparative methylomics, we benchmarked four different methylation-calling pipelines on three biological replicates of the wildtype progenitor strain for our evolved populations.
While it is understood that these methylated sites play a role in the MMR pathway, it is not fully understood the full extent of their effect on the genome. Thus the goal of this thesis was to better understand the forces which maintain the genome, specifically concerning m6A within the GATC motif.
Multiplex Automated Genome Engineering, or MAGE, is a genome editing technique that enables scientists to quickly edit an organism’s DNA to produce multiple changes across the genome. In 2009, two genetic researchers at the Wyss Institute at Harvard Medical School in Boston, Massachusetts, Harris Wang and George Church, developed the technology during a time when researchers could only edit one site in an organism’s genome at a time. Wang and Church called MAGE a form of accelerated evolution because it creates different cells with many variations of the same original genome over multiple generations. MAGE made genome editing much faster, cheaper, and easier for genetic researchers to create organisms with novel functions that they can use for a variety of purposes, such as making chemicals and medicine, developing biofuels, or further studying and understanding the genes that can cause harmful mutations in humans.
George McDonald Church studied DNA from living and from extinct species in the US during the twentieth and twenty-first centuries. Church helped to develop and refine techniques with which to describe the complete sequence of all the DNA nucleotides in an organism's genome, techniques such as multiplex sequencing, polony sequencing, and nanopore sequencing. Church also contributed to the Human Genome Project, and in 2005 he helped start a company, the Personal Genome Project. Church proposed to use DNA from extinct species to clone and breed new organisms from those species.
Francis Sellers Collins helped lead the International Human Genome Sequencing Consortium, which helped describe the DNA sequence of the human genome by 2001, and he helped develop technologies used in molecular genetics while working in the US in the twentieth and twenty-first centuries. He directed the US National Center for Human Genome Research (NCHGR), which became the National Human Genome Research Institute (NHGRI), of the US National Institutes of Health (NIH), located in Bethesda, Maryland, from 1993 to 2008. Collins led teams of researchers to use data on human genomes to investigate the genetic aspects of diseases and treatments, the variations among people in terms of their DNA sequences, and the evolution of humans. Collins became director of the NIH in 2009. Some criticized him for his Christian faith and its possible impacts on science funding through the NIH, such as for stem cell research, cloning, and embryonic genetic testing. As a director of the NHGRI and the NIH, Collins helped shape the structures and aims of projects in biology that pursue what he called big science, and he helped relate those projects to federal governments and to private companies.
The Human Genome Project (HGP) was an international scientific effort to sequence the entire human genome, that is, to produce a map of the base pairs of DNA in the human chromosomes, most of which do not vary among individuals. The HGP started in the US in 1990 as a public effort and included scientists and laboratories located in France, Germany, Japan, China, and the United Kingdom. Scientists hypothesized that mapping and sequencing the human genome would facilitate better theories of human development, the genetic causes and predispositions for a number of diseases, and individualized medicine. The HGP, alongside the private effort taken up by the company Celera Genomics, released a working draft of the human genome in 2001 and a complete sequence in 2003. The history of the HGP ripples beyond biomedical science and technology into the social, economic, and political.
John Craig Venter helped map the genomes of humans, fruitflies, and other organisms in the US in the late 1990s and early 2000s, and he helped develop an organism with a synthetic genome. In February 2001, Venter and his team published a human genome sequence after using a technique known as Expressed Sequence Tags, or ESTs. Venter worked to bridge commercial investment with scientific research. Venter founded a number of private companies, including the for-profit Celera Genomics, headquartered in Alameda, California, as well as research institutes, such as the not-for-profit J. Craig Venter Institute, located in Rockville, Maryland, and La Jolla, California.