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必须被复制并均匀地分布到子细胞中。这个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很重要的。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由来已久，因为它易于生长和遗传操作。此外，在某些工业应用中，酵母比其他微生物（如细菌）具有优势。酵母是无毒的，易于操作，并且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。为了实现这一目标，酵母必须忠实地将携带指令的DNA传递给许多代，以产生这些生物分子。然而，我们还没有完全了解在细胞分裂过程中DNA是如何准确地传递到子细胞的。这些知识不仅将帮助我们了解人类疾病（如癌症）的问题所在，而且还将帮助我们设计更好的生物合成DNA载体。这个项目将采用“合成生物学”的方法来解决这个问题。天然酵母染色体密集地包裹着细胞生长和繁荣的重要特征。这些基因包括转录和翻译成蛋白质产物的基因，这些蛋白质产物构建了染色体分离机制。因此，很难解剖出促进染色体分离的DNA序列本身的特性，这是本研究的目的。为了避免这些问题，我们将化学合成DNA并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。这些染色体不需要细胞生长，也不携带任何基因，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的长度和环状化影响它的分离。我们将以线形和圆形的形式构建不同长度的染色体。然后，我们将检查这些合成染色体招募一种叫做内聚蛋白的关键因子的能力，这种因子将染色体对粘在一起。我们预计分离性较差的染色体募集的黏结蛋白较少。接下来，我们将测试基因表达本身的行为有助于招募黏结蛋白和改善染色体分离的想法。我们将在小染色体上添加编码无细胞功能蛋白产物的基因，并测试其对粘聚蛋白募集和染色体分离的影响。内源性染色体在染色体对附着在纺锤体上的点周围聚集了一个内聚蛋白丰富的区域，称为中心粒，纺锤体将它们分开。我们将使用合成的小染色体来识别对着丝粒形成重要的特征。我们的最终目标是利用本研究中获得的知识来构建具有“完美”染色体分离的“设计师”染色体。总的来说，这项研究将提供DNA序列特征的基础生物学知识，以确保其在细胞分裂过程中准确传递。本研究对今后工业应用的DNA载体设计具有重要意义。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的新科学领域及其潜力的讨论。

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.