Synthetic chromosomes to decipher requirements for optimal transmission of DNA in yeast

细胞分裂是生物体自我繁殖的过程。在这个过程中，染色体上携带的基因组DNA必须复制并均匀分布到子细胞中。这一过程中的错误会产生染色体数目错误的细胞。这与癌症有关，并导致出生缺陷和不孕症。因此，了解这个过程是如何工作的，在医学上是重要的。染色体分离的许多特征在人类和非常简单的单细胞生物（如面包酵母）之间是共同的。酵母作为染色体分离机制的发现工具具有悠久的历史，因为它易于生长和遗传操作。此外，酵母在某些工业应用中具有优于其他微生物如细菌的优势。酵母是无毒的，易于操作，由于细胞机制比细菌更接近人类，酵母细胞更有可能重现人类生物分子的生产。为了实现这一点，酵母必须忠实地传递携带指令的DNA，以在许多代中产生这些生物分子。然而，我们还没有完全了解DNA在细胞分裂过程中如何准确地传递到子细胞。这些知识不仅有助于我们了解人类疾病（如癌症）的问题所在，还有助于我们设计更好的DNA载体进行生物合成。本项目将采取“合成生物学”方法来解决这一问题。天然的酵母染色体密集地排列着细胞生长和繁荣的重要特征。这些包括基因转录和翻译，使蛋白质产品，建立染色体分离的机制。因此，很难剖析出DNA序列本身的特性，促进染色体分离，本研究的目的。为了避免这些问题，我们将化学合成DNA，从头开始构建染色体，首先在计算机中，然后在试管中，最后在酵母中。这些染色体将不需要细胞生长，也不会携带任何基因，使我们能够检查DNA序列和活性的作用。我们已经知道，染色体长度和环化影响其分离。我们将建立不同长度的染色体在两个线性和圆形的形式。然后，我们将检查这些合成染色体招募一种称为粘着蛋白的关键因子的能力，该因子将染色体对粘在一起。我们预期分离较差的染色体募集较少的粘着蛋白。接下来，我们将测试基因表达本身有助于招募粘着蛋白和改善染色体分离的想法。我们将在微型染色体上添加编码无细胞功能的蛋白质产物的基因，并测试其对粘着蛋白募集和染色体分离的影响。内源性染色体在成对染色体附着于纺锤体的点周围组装了一个富含内聚蛋白的区域，称为近着丝粒，纺锤体将染色体分开。我们将使用合成的微型染色体，以确定重要的特征，为pericentromere的形成。我们的最终目标是利用本研究中获得的知识构建一个具有“完美”染色体分离的“设计者”染色体。总之，本研究将提供DNA序列特征的基本生物学知识，以确保其在细胞分裂过程中的准确传输。这项研究在未来工业应用DNA载体的设计中将非常有用。此外，该项目的一个重要优先事项是与公众接触，鼓励围绕合成生物学新科学领域及其潜力进行讨论。

Cell division is the process by which organisms reproduce themselves. During this process, the genomic DNA, carried on chromosomes, must be replicated and equally distributed to the daughter cells. Errors in this process produce cells with the wrong number of chromosomes. This is associated with cancer and causes birth defects and infertility. Understanding how this process works is therefore medically important. Many of the features of chromosome segregation are common between humans and very simple single-celled organisms, such as baker's yeast. Yeast has a long history as a discovery tool in mechanisms of chromosome segregation since it is easy to grow and manipulate genetically. Furthermore, yeast has advantages over other microorganisms such as bacteria for some industrial applications. Yeast is non-toxic, easy to manipulate and, since the cellular machinery is closer to that of humans than bacteria, yeast cells are more likely to recapitulate the production of human biomolecules. To achieve this, yeast must faithfully transmit the DNA carrying the instructions to produce these biomolecules over many generations. However, we do not yet have a complete understanding of how DNA is transmitted accurately to daughter cells during cell division. This knowledge will not only help us understand what goes wrong in human diseases, such as cancer, but will also help us design better DNA carriers for biosynthesis.This project will take a "synthetic biology" approach to address this problem. Natural yeast chromosomes are densely packed with important features for cells to grow and flourish. These include genes which are transcribed and translated to make protein products that build the machinery for chromosome segregation. Therefore, it is difficult to dissect out the properties of the DNA sequence itself that promote chromosome segregation, the aim of this study. To avoid these problems, we will chemically synthesise DNA and build chromosomes from scratch, first in a computer, then in a test tube and finally in yeast. These chromosomes will not be required for cell growth and will not carry any genes, allowing us to examine the role of DNA sequence and activity. We know already that chromosome length and circularization influences its segregation. We will build chromosomes of different lengths in both linear and circular form. We will then examine the ability of these synthetic chromosomes to recruit a key factor, called cohesin, that sticks chromosome pairs together. We expect chromosomes with poorer segregation to recruit less cohesin. Next, we will test the idea that the act of gene expression itself helps to recruit cohesin and improve chromosome segregation. We will add genes encoding protein products without a cellular function onto the minichromosomes and test the effect on cohesin recruitment and chromosome segregation. Endogenous chromosomes assemble a cohesin-rich domain, called the pericentromere, around the point at which pairs of chromosomes are attached to the spindle that will pull them apart. We will use the synthetic minichromosomes to identify the features important for pericentromere formation. Our final objective is to use the knowledge gained in this study to build a "designer" chromosome with "perfect" chromosome segregation.Overall, this study will provide fundamental biological knowledge of the sequence features of DNA which ensure its accurate transmission during cell division. This research will be useful in the future in the design of DNA carriers for industrial applications. Furthermore, an important priority for this project is engagement with the public to encourage discourse around the new scientific area of synthetic biology and its potential.