While the fundamental unit of life is the cell, it is the cellâs DNA which instructs how a cell is to function. These microscopic blueprints can be read. Reading DNA sequences has enabled genes to be identified and revealed insights about the causes of many human diseases such as Huntington’s and numerous cancers. Reading and understanding how DNA functions, however, is only half the challenge in genetics research. To correct genetic errors accumulated in diseased genes, it is necessary to also write in DNA. But while DNA is essential in all genetics research, it is difficult to produce. Methods to rewrite or create new DNA sequences from scratch are limited. Regions of DNA can be copied, but there are relatively few methods of generating new fragments. Nucleotides are the characters of the DNA language. A 1,000 nucleotide gene costs approximately $1,000 to construct. Given that the average human gene is more than 3,000 nucleotides long, and that regulatory regions of DNA can be tens of thousands of nucleotides long, the cost of producing DNA becomes daunting. Daniel Horspool is researching a new laboratory technique for DNA construction. While the primary method involves adding one nucleotide at a time chemically to the growing sequence, the new technique relies on stitching multi-nucleotide fragments together in parallel. This process could be much faster and less error prone than the conventional method, and by using microfluidic devices, DNA could be produced at a much lower cost. The research could lead to an approach for constructing complete genes, which would be an important new tool for basic and applied biomedical research. The research could ultimately contribute to efforts to realize the promise of genomic and personalized medicine.
Program: Trainee
Computational simulation of transcription factor binding for the prediction of regulatory regions in DNA sequences
The regulation of when and where a gene is turned on (gene expression) is a complex process, fundamental to how a cell behaves and interacts with the environment around it. Abnormal changes in gene regulation are associated with many diseases, including cancer, asthma, and obesity. One class of proteins involved in the regulation of genes are transcription factors (TFs). TFs recognize and bind to short sequences of DNA near the genes they regulate and act to increase or decrease the expression of their target gene. The binding interaction between TF proteins and DNA is affected by an array of biophysical factors in the cell nucleus, making this complicated process a good candidate for computational modelling through bioinformatics. Bioinformatics is a relatively new field in which computational approaches are used to study biological problems; a field that unites computer science, statistics and the life sciences. Rebecca Hunt Newbury is developing a software simulator to model the TF binding process in a dynamic, interactive setting, with the intent of predicting the locations in a DNA sequence at which TFs will bind and regulate genes. From there, she will begin to incorporate the spatial relationships and combinatorial interactions between TFs that result in the different expression responses of genes. Hunt Newburyâs research will contribute to clearly defining the regions of DNA that participate in regulating a gene. Her work may ultimately contribute to new approaches for combating diseases caused by abnormal gene regulation.
Global view of pre-mRNA splicing in Saccharomyces cerevisiae
Proteins, the molecules that carry out most cellular functions, are synthesized according to information contained in DNA sequences. Converting information from DNA into a protein requires an intermediate step in which the DNA sequence is copied into a molecule called mRNA. In humans, there is an essential biochemical process called pre-mRNA splicing, in which certain (non-coding) portions of the sequence are removed and the remaining protein-coding portions are joined together to form a template for protein synthesis. This is a complex process with multiple steps, and even small errors can be dangerous. Many diseases, such as some cancers, Alzheimer’s disease, and Parkinson’s syndrome, can be attributed to defects in the pre-mRNA splicing machinery. Perhaps due to the complexity and requirement for absolute precision in splicing, the molecular machine that carries out splicing â termed the spliceosome â is enormous. For both humans and yeast, its components number well over 100. However, several splicing proteins likely remain unidentified. A thorough understanding of splicing will require a complete inventory of its parts. Paul Kahlke is identifying novel splicing factors in yeast, which serves as a laboratory model for human splicing. His objective is to uncover previously unknown splicing factors and to determine whether existing candidate proteins are indeed integral to splicing. His studies take a whole genome approach, testing many genes one by one to see which ones are involved in splicing. By screening a large number of genes, Kahlke hopes to identify several new splicing factors and gain insight into the function of known pre-mRNA splicing factors.
Structure and RNA-cleaving function of apurinic/apyrimidinic endonuclease 1 (APE1)
Cancer occurs when cells wonât stop growing. The source of this malfunction is often an alteration in DNA, the genetic instructions for making different proteins inside a cell. To make proteins, a gene is copied out from the DNA into a molecule called mRNA. The mRNA travels out of the cell nucleus and into the cytoplasm, where its genetic instructions are used as a template for synthesizing proteins. C-myc mRNA serves as a template for synthesizing the c-Myc protein. This protein plays fundamental roles in regulating growth, differentiation, and cell death in virtually all mammalian cells, and it is implicated in diverse human cancers. In fact, it has been estimated that c-Myc dysfunction contributes to one-seventh of all cancer deaths. One way that cells regulate their level of proteins is by destroying mRNA in the cytoplasm by chemically cutting, or cleaving, its molecular structure. Recently, the enzyme APE1 was discovered to cleave c-myc mRNA. This opens the potential for using APE1 to reduce or eliminate levels of c-Myc protein in the cytoplasm as a potential treatment for cancer. Wan Kim is exploring the function of APE1 as an mRNA destroyer. He is identifying the key amino acids and mechanisms APE1 uses to cleave c-myc mRNA, and determining whether the enzyme cleaves any other types of mRNA. Kim hopes to generate valuable insights into how APE1 can degrade c-myc mRNA and influence gene expression. Also, the study will provide useful information on the potential design of novel approaches in cancer treatment.
Integrated microfluidic technologies for optimization of hematopoietic stem cell expansion
Blood contains different types of specialized cells. Red cells are responsible for oxygen transport, white cells ensure body’s defense against infections, and platelets initiate clotting to limit the loss of blood after an injury. These cells are constantly renewed, and are manufactured in the centre of the bones in a sponge-like tissue called bone marrow. Hematopoietic stem cells are a small subset of cells found in bone marrow that have the astounding ability to self-renew and divide, and to differentiate into a variety of mature blood cells. They are often used to treat blood-related diseases or given after cancer treatment. Although stem cells have great potential for regenerative medicine, they are extremely rare and they are difficult to expand in the lab, because they very readily differentiate into other cell types. The multiple factors that influence their self-renewal are poorly understood. VĂ©ronique Lecault is exploiting the potential of microfluidic technology, an engineering advance that allows thousands of different experiments to be performed in tandem upon a device the size of a microscope slide. Across rows and rows of miniature cell culture chambers, individual hematopoietic stem cells can each be exposed to different chemical conditions and tracked over time. This makes determining the specific environments that will allow the cells to be expanded much more efficient. This technology could lead to the ability to produce more hematopoietic stem cells for use in disease therapies. It could also help researchers gain a better understanding of stem cell biology, perhaps leading to the discovery of new ways to identify and purify these rare cells.
Vascular Endothelial Growth Factor Signalling in Cardiac Allograft Vasculopathy
Atherosclerosis, also known as hardening of the arteries, is a common vascular disease caused by the buildup of a waxy plaque on the inside of blood vessels. This narrowing of blood vessels can cause blood clots, leading to heart attack or stroke. In almost half of all heart transplant patients, an accelerated form of hardening of the arteries, known as Transplant Vascular Disease (TVD), occurs in the transplanted heart. In fact, TVD is a leading cause of death one year after transplantation. The exact mechanisms behind this process remain unclear. Blood vessels are lined with endothelial cells, specific cells that create a barrier between blood and the artery. An important factor in TVD is damage to endothelial cells. This damage increases the size of gaps between cells, allowing fats to accumulate in artery walls. One protein that causes endothelial “”leakiness”” is called Vascular Endothelial Growth Factor (VEGF). VEGF is also important in many other serious diseases, such as cancer and degenerative eye diseases. David Lin is expanding on previous research that showed that VEGF is increased in the muscle cells in arteries of transplanted hearts. He is studying in detail the mechanisms by which VEGF alters the function and structure of endothelial cells. By learning how VEGF works in transplanted hearts, Lin hopes his research will lead to the development of new ways to maintain the health of heart blood vessels following transplantation.
Investigation of genetic networks involving genes that confer chromosome stability in S. cerevisiae and C. elegans
Chromosomes are found in all organisms that have a cell nucleus, and carry the organismâs hereditary material. During cell division, chromosomes divide and distribute equally to the daughter cells. Errors in the division process can result in daughter cells that contain an incorrect number of chromosomes. Known as aneuploidy, this state is responsible for many genetic diseases and is characterized by a specific type of genome instability known as Chromosome Instability (CIN). Because chromosome stability is a fundamental requirement across all organisms, it can be studied using simpler organisms, such as bakerâs yeast. Genome wide screens on yeast have identified approximately 300 genes important for maintaining chromosome stability (CIN genes). A subset of these genes has also been found to be mutated at an elevated rate in some human cancers, which suggests that these genes contribute to tumour progression and development. Mutations in these key genome stability genes may also represent an Achillesâ heel for tumours. Enhancing chromosome instability to a point where tumour cells can no longer function and reproduce could halt their division or lead to cell death. Jessica McLellan is studying the subset of CIN genes mutated in colon cancer. She specifically aims to identify genes that, when mutated in combination with a CIN gene mutation, lead to cell death. By exploiting the mutations seen in many types of cancers, this project could lead to the development of novel cancer therapeutics that are less harmful to non-cancer cells than current treatments. McLellanâs research will increase our understanding of the complex biological processes that ensure genome stability and the mechanisms by which these processes can become deregulated.
The Role of beta-catenin Stabilization in the Synaptic Pathology of Alzheimerâs Disease
Alzheimer’s disease (AD) is a devastating neurological disorder characterized by the loss of cognitive function and an inability to process and store new memories, caused by the progressive death of neurons (brain cells). It is becoming increasingly evident that before neurons die, changes can be observed at their synapses â the junction between neurons across which information is transmitted. Deficits in synaptic function, loss of synapses, and a reduced ability to form new synapses are the major correlates of dementia. It is therefore crucial to understand the basic biology of synapses, and how these processes are affected in AD. The cadherin family of cell adhesion molecules and their intracellular partner, b-catenin, play a critical role in regulating the formation and remodelling of synapses. Both molecules also associate with presenilin-1 (PS1), a protein that normally degrades (breaks down) b-catenin. Mutations in the PS1 gene account for nearly 70 per cent of early-onset familial AD cases. Fergil Mills is investigating the effects on neurons when b-catenin is not normally degraded by PS1. Using isolated neurons and a mouse model, he is characterizing the synaptic consequences of stabilizing (maintaining) b-catenin in neurons, and determining the molecular mechanisms of b-catenin in the development of synaptic structures. These studies will help determine whether b-catenin stabilization leads to the synaptic pathology and cognitive deficits seen in AD. Millsâ studies will further our understanding of synapse pathology and cognitive deficits, and could lead to new treatments for patients with AD or other neurological disorders.
The role of SHIP's C2 and PH domains in regulating hematopoietic cell growth and function
Various cancers and inflammatory diseases occur as a result of inappropriate activation of the bodyâs blood-forming hematopoietic cells. Normally, cellular activation, growth and survival in hematopoietic cells are regulated by the phosphoinositide 3-kinase (PI3K) pathway, which drives a wide range of cellular processes. Keeping tight control on this pathway is SHIP (SH2 domain-containing inositol 5′ phosphatase), a counteracting enzyme that inhibits PI3K action. SHIP is found only in blood and immune system cells and is the major restraining mechanism in these cell types. Loss or impaired activity of SHIP â in effect, removing the brakes on the PI3K pathway â has been implicated in certain leukemias and in inflammatory disease. Recently, researchers discovered small molecules that are capable of enhancing SHIP activity, resulting in both the inhibition of immune cell activation and the death of hematopoietic cancer cells. This represents a previously unknown mode of regulating SHIP enzyme activity. Andrew Ming-Lum is determining the significance of this novel type of regulation of SHIP function. Using cell lines and mouse models, he is focusing on a previously unrecognized domain on the enzyme, upon which the small molecules are believed to act. These studies will provide greater insight into how this mechanism affects the function, growth and survival of hematopoietic cells. It will also provide insight into the dysregulation that occurs in certain cancers and inflammatory diseases.
Unraveling transcriptional regulatory networks governing mouse development
The genome of each cell within an organism contains hereditary information. Among other features, the genome contains genes â DNA sequences that specify the genetic code (encode) for functional products such as proteins. The product of a gene is produced when the gene is turned on (expressed). The process of turning certain genes on or off is governed by regulatory proteins called transcription factors (TFs). While cells of different tissues within an organism contain the same genome, their function may be different due to the fact that they express different genes. While in simpler organisms gene expression is regulated by individual TFs, in more complex organisms such as mammals, several TFs may work together to control the expression of a gene. Many complex diseases such as cancer are at least partially due to improper expression of certain genes. Understanding how gene expression is regulated in an organism, including identifying what TFs work together to regulate particular genes, is instrumental to understanding the biology of numerous health conditions. Olena Morozova is working as part of a large research project aimed at identifying genetic networks governing development. Using the mouse as a model system to study the regulation of gene expression, she is focusing on how different TFs interact to regulate the development of mouse pancreas, heart, and liver, and how these interactions can be used to identify master regulators and the main control nodes in the development of these organs. Overall, this project will provide invaluable insights into mammalian gene regulation and will ultimately help to understand the biology of diseases resulting from errors in the regulation of gene expression. The identified TFs that work together with many other TFs are potentially useful targets for effective disease therapy.