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在活体组织中观察基因激活

在一个温暖的实验室隔间中孵育的果蝇幼虫非常兴奋，它们给康奈尔大学的研究者指出了条康庄大道，让他们可以观察活动中的染色体并且可以实时看到基因在活体组织中是如何表达的。

利用康奈尔大学物理学家Watt W. Webb开发的前沿技术——多光子荧光显微术，研究者第一次能够观察到染色体形态的改变，这一改变是为了激活果蝇卵中一些基因的表达以合成相关的关键蛋白。这项技术在理解基因表达背后的基本过程方面是一次显著的进步。

这项发现是多学科交叉的结果，由Webb和康奈尔大学分子生物学和遗传学专业的Barbara McClintock教授以及Jie Yao合作完成，其中后者最近刚刚获得了康奈尔大学授予的博士学位，参与了这项研究的启动和促进工作。

“这项技术革命性得改变了我们观察生物体基因表达的方法，”Lis说道，“我们目前正在实时观察活体细胞的转录过程。”

这项研究报告发表在今年8月31出版的《Nature》上。

研究小组的实验主要关注基因调控的机理：尤其是当一个外部的刺激（比如热刺激）促进某一特定基因激活时，细胞核里发生了什么事情，以及这些被激活的基因是如何指导蛋白产物保护果蝇免遭热刺激的影响。

“无论细胞何时受到刺激，总会有相应的蛋白产生来帮助细胞对抗这些刺激”康奈尔大学应用物理教授Webb和工程学教授S.B. Eckert说道。这一过程被一个名为热激活因子（HSF）的分子所触发，它与相关基因相互作用，发出合成新蛋白的信号，但这一广为人知的过程还从没有在活细胞中观察到。

Yao利用多光子显微镜（MPM）来为果蝇的活体唾液腺组织成像。其它方法缺乏穿透力，并且会损害标本，MPM不同，它可以呈现出锐利清晰的图像，即使像果蝇唾液腺组织这样较薄的标本也是这样。

这项研究可能最终还要感谢果蝇与众不同的染色体组成——巨大、多链状染色体，和一般细胞两套染色体不同，果蝇染色体有数百套之多。这比寻常的细胞核要大上10倍之多，足以呈现图像的细节。

结果好得让人眩晕。Lis说：“两个星期内，我们就得到了壮观的照片。”图像包括了保护果蝇免遭极热效应影响的基因（hsp70）。通过增大热量，研究者可以激活这些基因。科学家构建了一种HSF基因上携带游荧光蛋白的果蝇。利用这种果蝇，他们能够观察到转录因子的活动。

Webb说“这是第一次人们观察到活体内转录因子如何打开，如何激活一个原位基因的这一过程的细节”。

利用Webb在康奈尔大学构建的另一种方法：光脱色荧光恢复技术。研究者还发现了HSF激活因子在被新的HSF替代之前与hsp70结合的时间比以往认为的更长，这就提出了一些新的关于基因转录机制问题。

这项技术也可以为生物科学的研究者提供一种新工具。Webb说，它标志了交叉科学发展趋势的成功，这一发展趋势为多个领域的研究者提供了新的潜能。

“物理科学与生命科学的相互交叉展现非一般的威力，”Webb说。“在推进我们对生命科学的理解方面，它将变得更加有力”

对低等生物转录过程的更好理解讲帮助我们理解高等生物中的这一过程，Yao补充说。“我们希望这项技术能用于人类细胞，这是未来20年的目标。”

图释
果蝇幼虫的唾液腺包含了巨大的多线染色体，具有带状结构，易于在光学显微镜下观察到。通常观察这些染色体需要核崩解，然后染色体在二维尺度上延展开来。使用双光子激光扫描显微镜，在活体唾液腺组织中可实时的在三维尺度上观察这些染色体，并具有较高的解析度。这幅图像展示了一系列光学切片，其中染色体使用Hoechst来着色。

Hot flies produce cool results -- the ability to watch genes activating in live tissue

Feverish fruit fly larvae, warmed in a toasty lab chamber, are giving Cornell researchers a way to watch chromosomes in action and actually see how genes are expressed in living tissue.

Using multiphoton fluorescence microscopy, a technique pioneered at Cornell by physicist Watt W. Webb, researchers have for the first time been able to watch chromosomes change their form in order to activate their genes to synthesize key proteins in fruit fly cells. The advance could be a significant step toward understanding the basic processes that underlie gene expression.

The discovery was the result of cross-disciplinary collaboration between Webb and John Lis, Cornell's Barbara McClintock Professor of Molecular Biology and Genetics. Jie Yao, who recently earned his Ph.D. at Cornell, initiated and facilitated the work.

"This technology will revolutionize the way we see gene expression in organisms," said Lis. "We're watching transcription in real time in living cells."

The research was described in the Aug. 31 issue of the journal Nature.

The team's experiments focused on gene regulatory mechanisms: specifically, what happens in a cell's nucleus when an external stimulus (heat) prompts specific genes to activate, and how those activated genes direct the production of proteins that protect the fly against the stress of heating.

"Whenever a cell is stressed -- bingo, it will produce proteins that will help the cell resist stress," said Webb, Cornell professor of applied physics and the S.B. Eckert Professor in Engineering. The process is triggered by a molecule called heat shock factor (HSF), which interacts with genes to cue the synthesis of new proteins. But this well-known process had never been seen in living cells.

Yao used multiphoton microscopy (MPM) to image living salivary gland tissue of Drosophila (fruit flies). Unlike other methods, which lack penetrating power and can damage the specimen, MPM delivers crisp, clear images, even in thicker tissue samples like Drosophila salivary glands.

The research was ultimately possible thanks to the unique composition of the fruit flies' polytene cells -- giant, multistranded chromosomes with hundreds of sets of the genome instead of the usual two sets in conventional cells. This enlarges the usual nuclear dimensions by about 10 times, making them large enough to image the detail.

The results were stunning. "Within two weeks we had spectacular pictures," said Lis. The images included pictures of the genes (hsp70 genes) that protect flies from the effects of extreme heat. By cranking up the heat, the researchers could activate these genes, and by using fruit flies specifically bred to carry fluorescent proteins on HSF, they could watch the transcription factors in action.

"This is the first time ever that anyone has been able to see in detail, at native genes in vivo, how a transcription factor is turned on, and how it then is activated," said Webb.

Using another method that Webb engineered at Cornell, called fluorescence recovery after photobleaching, the researchers also discovered that HSF activators bind to hsp70 genes much longer than previously thought before being replaced with new HSFs, which raises new questions about the mechanisms of gene transcription.

The technique also may offer a new tool for researchers across the biological sciences. Webb says it marks the success of an interdisciplinary trend that offers new potential for researchers in a variety of fields.

"Interaction between the physical sciences and the life sciences is very powerful," said Webb. "And it's becoming more powerful as a tool for advancing our understanding of the life sciences."

Better understanding transcription in lower organisms will help understand the processes in higher organisms, Yao added. "We hope to push the limits to human cells. That's the goal in the next 20 years."

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Salivary glands of Drosophila larvae contain giant polytene chromosomes whose characteristic banded structure is readily visible by light microscopy. Usually these chromosomes are visualized by breaking the nuclei and "spreading" the chromosomes in two dimensions. Using two-photon laser-scanning microscopy, these chromosomes can be examined in living salivary gland tissue in 3D and in real time with similar high resolution. This image shows a series of optical sections where the DNA is stained with Hoechst (pseudo-colored red). Copyright © Cornell University


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