Diamond professorial fellowship for imaging chromosomes by coherent X-ray diffraction

染色体是真核生物所有遗传物质的储存库。人类的核型中有22条独立的染色体，加上一条特殊的决定X/Y性别的染色体。染色体生命周期的三分之一，即中期，与有丝分裂有关，在此过程中，所有遗传物质安全传递给后代细胞；剩下的时间，即间期，是基因被拆解并转录以操作细胞机制并复制产生子染色体的时间。有丝分裂是一个复杂的细胞过程组合，将DNA保护性地包装成安全有序的束——染色体——是基本步骤之一。染色质的第一级组织，双螺旋，核小体（dna -组蛋白复合物）和“串珠”（BoS），与组蛋白H1一起包裹在30纳米的纤维中，代表了生物学上的重大突破，从而获得了诺贝尔奖。这些层次的结构，总结在图1中，现在已经被很好地理解了，尽管仍然有几个可能的模型讨论纤维，可能还有一些有趣的多样性。这是中期染色体结构的下一个层次，在那里组织变得复杂，这里提出的研究将开始产生影响。为了保护在有丝分裂过程中的基因，它们被紧密地包裹在一起，形成熟悉的x形染色体对，一旦细胞分裂开始就会分开。这些30纳米的纤维大概是在染色单体的下一层有规则的上层结构中盘绕起来的；这个线圈就是我们打算用x射线方法成像的结构。这种包装是由支架蛋白实现的，支架蛋白也可以保护DNA免受可能破坏基因的力的影响。这个密集复合体的光学折射率足够高，染色体在中期最容易被看到。高密度也意味着中期染色体可以在光学显微镜下用细胞遗传学家熟悉的显微操作工具进行处理。我们计划将这些工具用于x射线成像实验的样品制备。该提案计划在30纳米分辨率水平上对染色体进行完整的3D成像。中期染色体必须是一个紧凑的物体，没有会干扰有丝分裂的悬挂链或环。在PI部分开发的分阶段方法中使用“支持”约束可以很好地工作。对于生物物质来说，染色体的电子密度很高，因为磷（以及与DNA相关的反离子）被紧密地包裹着。染色方法甚至可以用来增强x射线的对比。良好定义的边界将使寻求支持的方法，如“shrinkwrap”有效地发挥作用。因为它们很容易操作，如上所述，单个染色体可以被分离并安装在针或基化膜上进行测量。然而，染色体并不像核糖体或某些病毒那样是高度可复制的结构，可以用MX或x射线自由电子激光连续晶体学的新方法来解决。事实上，对细胞遗传学的兴趣主要在于个体染色体之间的差异以及来自同一个体的拷贝之间的差异。所有这些都可以在人权理事会的范围内加以考虑和尝试。

The chromosome is the repository of all genetic material in eukaryotes. Humans have 22 separate chromosomes in their karyotype plus the special X/Y sex-determining chromosome. One third of the life cycle of the chromosome, metaphase, is involved with mitosis, whereby the safe transmission of all the genetic material to progeny cells is undertaken; the remainder of the time, interphase, is when the genes are are unpacked and transcribed to operate the cellular machinery and copied to make the daughter chromosomes. Mitosis is an elaborate combination of cellular processes of which the protective packaging of the DNA into safe organized bundles - the chromosomes - is one of the fundamental steps. The first levels of organization of chromatin, the double helix, the nucleosome (DNA-histone protein complex) and the 'beads on a string' (BoS), packed into 30nm fibres with histone H1, have represented major breakthroughs in biology resulting in Nobel prizes. These levels of structure, summarized in Fig 1, are now relatively well understood, although there are still several possible models discussed for the fibre, and possibly some interesting diversity also. It is the next level of structure of the metaphase chromosome where the organisation becomes complicated and the research proposed here will begin to have impact. To protect the genes in transit though mitosis, they are packed together tightly into the familiar X-shaped chromosome pairs that separate once the cell division begins. The 30nm fibres are presumably coiled up in a regular superstructure at the next level within the chromatids; this coiling is the structure we intend to image by X-ray methods. This packing is achieved by scaffolding proteins which also protect the DNA mechanically from forces that could damage the genes. The optical refractive index of this dense complex is high enough that chromosomes are most easily visualized in metaphase. The high density also means that metaphase chromosomes can be handled under the optical microscope with micromanipulation tools that are familiar to cytogeneticists. We plan to use these same tools for sample preparation for the X-ray imaging experiments. This proposal plans to undertake a full 3D imaging of the chromosome at the 30nm resolution level. The metaphase chromosome is of necessity a compact object devoid of hanging strands or loops that would interfere with mitosis. This works well with the use of a 'support' constraint in phasing methods partly developed by the PI. The chomosome electron density is high for a biological substance because of the tightly-packed phosphorus (and counterions associated with the DNA). Staining methods might even be developed to enhance the X-ray contrast. The well-defined boundary will enable support-seeking methods such as 'shrinkwrap' to work effectively. Because they are readily manipulated, as described above, individual chromosomes can be isolated and mounted on pins or fiducialised membranes for measurement. Yet the chromosome is not expected to be a highly reproducible structure like the ribosome or certain viruses that can be solved by MX or the newer method of serial crystallography with an X-ray free-electron laser. In fact, much of the interest in cytogenetics lies in the differences between chromosomes of individuals and between copies from the same individual. All this can be contemplated and attempted in the context of the HRC.