Half a century of locating DNA and RNA in cells

Among his many wise statements, the late Sydney Brenner (1) emphasized that methods innovations drive discovery. Fifty years ago this year, the field of cell biology saw the discovery by Joseph Gall and Mary Lou Pardue that specific RNA and DNA sequences could be located in cells by a technique called in situ hybridization. That denatured strands of DNA could, in the test tube, reassociate was discovered in the mid-1950s, and subsequent work soon revealed that synthetic polymers of RNA and DNA could similarly associate into DNA-RNA hybrid duplexes. The notion that these in-solution molecular associations could be turned to the detection of specific DNA and RNA sequences inside cells began to be discussed in the early to mid-1960s. There were considerations such as, in the case of RNA molecules, how their packaging proteins might limit accessibility and, in the case of DNA, how its double-stranded structure could be opened in a cytologic preparation. In 1969, Joseph Gall and Mary Lou Pardue, at Yale, published a technique by which they were able to detect a specific set of DNA sequences in frog oocyte nuclei by incubating the preparation with radioactive RNA complementary to the target sequences (2). I vividly recall their prior presentation of this at the 1968 meeting of the American Society for Cell Biology meeting. Pardue gave the talk, and although many speakers left the slides to a hired projectionist, as she began the talk Dr. Gall was at the projector, making sure that their amazing discovery would not succumb to any projection gaffes. As I and others have noted, Gall has been an insightful master of perceiving how a particular biologic material might be exploited to for a certain purpose, perhaps most exemplified by his career-long, prescient use of the frog Xenopus oocyte's so-called lampbrush chromosomes to make numerous seminal discoveries about chromosome architecture and function. The 1969 publication introducing the method of in situ hybridization is another case in point. He had previously discovered that the genes that produce ribosomal RNA become amplified into tens of thousands of extrachromosomal copies in these oocytes and thus would be a highly abundant DNA target. Only a few months after their seminal paper, Pardue and Gall published a variation of the method in which specific DNA sequences were detected (3), adding to the vast number of applications of this technique. As in situ hybridization quickly became adopted, many cell biologists were becoming increasingly aware that molecular detection was now at hand for almost every domain within this science. This was a paradigm shift and led to the term “molecular cell biology” and isoforms thereof, soon to become textbook titles. This was not just a nomenclature revision; it was an epistemological transition. I was asked by the journal Cell to review the first two of these new textbooks and still recall how strongly I sensed that they were messengers of change. The Czech revolutionist Václav Havel once wrote an engaging op-ed piece in the New York Times that he titled “The End of the ModernEra” (4). (I would recommend that all of us read it again, for many current reasons.) For cell biology, the 1960s might have been dubbed the “beginning of the postmodern era,” in which everything became “molecularized.” Embryonic development became known via signaling molecules and gene regulatory networks of transcription factors, and cellular processes such as protein traffic and apoptosis were reduced to molecules, earning the deserved attention of Stockholm. The latest incarnation in this transition is single-molecule cell biology and its sibling field of systems biology. But there can be no doubt that in situ hybridization was one of the transformative catalysts in this multidecade epistemological transition. In due course the method adopted fluorescent instead of radioactive probes, adding feasibility and sensitivity. Beyond its employment with fixed cytologic preparations, it even became possible to conduct in situ hybridization in living cells, to thus track the dynamic movements of RNA [e.g.,(5)]. The use of in situ hybridization probes that are chromosome-specific has powerfully expanded human genome science, including the three-dimensional organization of chromosomes with the nucleus, now a vibrant field. It was a bold step for Gall and Pardue to try their idea. At the 50th anniversary of their success, the field of molecular cell biology salutes them.

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