Short Stories & Essays

Pure Genes, Pure Genius

As a physical substance, deoxyribonucleic acid (DNA) was first purified more than 140 years ago by Friedrich Miescher. Some 40 years later, Avery, McCarty, and McLeod utilized purified bacterial DNA to demonstrate it as the transforming substance, thus proving the chemical substance of genes to be composed of DNA. A decade later, Watson and Crick resolved the double-helical nature of DNA. The culminating event in the evolution of this science came roughly two decades later in the form of experiments that facilitated the isolation of purified, single genes. Whereas many scientists contributed to this watershed accomplishment, Don Brown and Tom Maniatis stand in unique positions at or near the apex of the list.

Why would one want to purify a single gene, and what could one do with a purified gene in hand? In retrospect, the answers to these simple questions are obvious. What must be remembered, however, is the fact that, when Brown, Maniatis, and others first accomplished the objective of gene isolation and purification, we did not know how to sequence a gene, we did not know how to express a gene, we did not know where regulatory sequences might be located, and we did not know whether the coding regions of genes would be co-linear with that of their mRNA products. As such, it was an inspired adventure for these scientists to purify genes before we even knew quite what to do with them.

The accomplishments of Brown and Maniatis sit on either side of what may arguably be deemed the most transformative event in biological sciences over the past century, the advent of gene cloning. This happened in the mid-1970s as a result of the science of Daniel Nathans, Hamilton Smith, Tom Kelly, Werner Arber, Herbert Boyer, Stanley Cohen, Paul Berg, and Dale Kaiser. These and other icons arranged the tool kit and provided the assembly directions, facilitating the molecular cloning of single, purified segments of DNA. In the decade before, Donald Brown began to show us what could be done with purified genes. And in the decade after that, Maniatis refined the techniques of gene cloning such that even the least skilled among us could exploit the technology.

Don Brown’s Magic

For historical purposes, I will start with a review of Don Brown’s magic. How could one possibly purify a gene before molecular cloning? Knowing that the density of DNA is proportional to its guanine and cytosine (GC) content, Brown used cesium chloride equilibrium centrifugation to purify the DNA encoding ribosomal RNA. Aside from having a higher GC content than bulk chromosomal DNA, ribosomal genes offered two additional advantages favoring purification. First, the genomes of almost all organisms encode multiple ribosomal RNA genes. Second, as independently demonstrated by Joseph Gall and Brown, ribosomal RNA genes are hugely amplified in number during formation of amphibian oocytes (Brown and Dawid, 1968; Gall, 1968). The combination of these quirky features—high GC content, repetitive nature, and amplification—allowed the independent achievements of Brown and Max Birnstiel to purify ribosomal RNA genes (Brown and Weber, 1968; Birnstiel et al., 1971).

Before DNA sequencing and all of the other capabilities evolving from the field of molecular biology, what could one do with a purified gene? Here is where Brown and others began to show us the way. He began to painstakingly dissect gene anatomy, initially using electron microscopy. By partially denaturing the DNA duplex with a combination of temperature and denaturants, Brown discovered that ribosomal genes are strung along the chromosome in repetitive fashion, with transcribed regions being separated by nontranscribed “spacers.” This discovery was confirmed in spectacular fashion by the elegant electron micrographs taken by Oscar Miller showing actively transcribed ribosomal RNA genes (Miller and Beatty, 1969). Subsequent evolution of this anatomical approach to gene structure employed R looping as a means of showing where on a gene its RNA product belonged and physical mapping via the use of restriction endonuclease enzymes. Although seemingly crude and rudimentary, it was the pioneering work of Brown, Birnstiel, and others that taught us the concept of using physical methods to dissect the anatomy of purified genes.

Brown also exploited the unusually high GC content of the gene encoding silk fibroin of silk moth larvae to purify that gene despite the fact that it was neither repetitive nor amplified. Yet what eventually evolved as Brown’s most rich vein of research came from his purification of the amphibian genes encoding 5S ribosomal RNA. Although repetitive in nature, like the genes encoding the larger 18S and 28S ribosomal RNAs, the 5S ribosomal RNA genes were small and ultimately suited for an unprecedented accomplishment beyond gene anatomy: gene expression. During a decade-long period straddling the advent of gene cloning, Brown and colleagues were able to develop a test tube system that accurately recapitulated 5S ribosomal RNA gene expression (Birkenmeier et al., 1978). This paved the way for the mapping of the regulatory sequences that facilitated 5S gene transcription, which, to the surprise of all, were located right in the middle of each gene (Bogenhagen et al., 1980). Finally, this path of research led to the first discovery of a eukaryotic transcription factor, designated TF3A (Pelham and Brown, 1980). In turn, studies of TF3A led to the discovery of the first zinc finger (Klug and Rhodes, 1987). It can safely be concluded that Don Brown’s path of research laid the groundwork for the entire field of eukaryotic gene regulation, all of which has relied most vitally on purified genes.

Meteoric Rise of Maniatis

With Brown’s pioneering research approaching logarithmic momentum just on the south side of the advent of gene cloning, that of Tom Maniatis was perched just on the north side of the divide. Liberated by cloning, Maniatis did not have to restrict his sights to genes that were repetitive, amplified, or differentiated by unusual GC content—he could study any gene of choice. Despite this liberation, Maniatis employed Brown-like focus in choosing to demystify anything and everything associated with the mammalian genes encoding hemoglobin protein. Maniatis was first to clone a full-length cDNA—that encoding β-globin protein (Efstratiadis et al., 1976) —and first to deduce the structure and sequence of this cDNA (Efstratiadis et al., 1977). These seminal accomplishments laid the groundwork for subsequent studies revealing the identities of human mutations that cause β-thalassemia and methodologically instructed the field as to how to synthesize, clone, sequence, and characterize cDNAs encoding specific gene products. Like Brown, Maniatis taught us how to do it.

The next step in the meteoric rise of “Tom terrific” was his construction of libraries of genomic DNA (Maniatis et al., 1978). Although not alone in accomplishing this feat, few would argue that anyone was more responsible for transforming gene cloning into what we now call genomics. Just the idea that a small test tube could contain recombinant bacteriophage housing each and every segment of DNA in the entire genome of an organism—a virtual library—remains astounding to this day. The value of a library is of course explicitly dependent upon one’s ability to access the individual books or genes. Together with others, Maniatis taught us how to fish out the one-in-a-million recombinant phage that contained our gene of interest—not just the synthetic cDNA copy of a specific mRNA, but also the genomic copy of the very same gene (Fritsch et al., 1980).

It was at this point that the field began to erupt with totally unanticipated surprises. I can vividly remember the very first scientific meeting I ever attended, the 1977 Cold Spring Harbor Symposium on Quantitative Biology. Although nominally organized around the field of chromatin, it was at that meeting where we first learned from Phil Sharp, Rich Roberts, Rich Gelinas, and others that mRNA sequences were not co-linear with their encoding genes, the key observation that led to the discovery of pre-mRNA splicing. Once the dust settled and the symposium volume was published, Maniatis’ influence was pervasive. The way to resolve gene anatomy was simple: clone the cDNA, clone the genomic copy, sort out the relationship between the two, and the task was done.

Like Brown, Maniatis took a love to gene expression. He worked collaboratively with Richard Axel to develop cotransfection methods to stably move purified, recombinant genes into mammalian cells, showing how these methods could facilitate the expression of any protein of choice (Wigler et al., 1979). Boy did this change the world! It fundamentally created the biotechnology industry, allowing Genentech, Amgen, and other companies to industrialize the production of recombinant proteins as therapeutic medicines. Not only did Maniatis facilitate this transformation, but he, like Brown, dug deep into the processes responsible for regulating gene expression. He studied regulatory DNA sequences, including promoters and enhancers. He discovered transcription factors that bound to these sequences. He studied pre-mRNA splicing—anything and everything to do with the enabling technology of having purified genes in hand.

Molecular Recipes that Work

In addition to their profound records of scientific accomplishment, both Brown and Maniatis selflessly contributed to the broad community of biomedical sciences in special ways. Starting with its first edition in 1982, Maniatis teamed up with Ed Fritsch and Joe Sambrook to assemble “Molecular Cloning: A Laboratory Manual” (Maniatis et al., 1982). This and subsequent editions constituted comprehensive cookbooks for the deployment of all methodologies essential for even the most novice of scientists to productively use the techniques of molecular biology. Although it is a testament to the science of molecular biology itself that it can be utilized in so facile a manner worldwide, there is no doubt that the Maniatis, Fritsch, and Sambrook cloning manual liberally greased the wheels for the dissemination of the technology. Just like the cookbooks of Betty Crocker and Fannie Farmer, the molecular cloning manual was chock full of recipes that worked. It was endowed with easily deciphered indices and was likewise updated through the years to incorporate the ever-expanding and improving technologies of the field. It can be argued that no other printed treatise has had more value and impact to biomedical sciences than the molecular cloning manual produced by Maniatis and colleagues.

Creating Something from Nothing

Don Brown’s extracurricular contributions to science come in a very different flavor. Back in the early 1980s Brown founded the Life Science Research Foundation (LSRF). With not a dime in his pocket, Brown decided to create the LSRF as a postdoctoral fellowship foundation. His concept was simple: gather a bunch of friends to offer gravitas (Don Seldin, Jim Watson, Bruce Alberts, Sol Snyder, Phil Sharp, Paul Berg, David Baltimore, Joe Goldstein, Mike Brown, Al Gilman, Shirley Tilghman, etc.), assemble a review team composed of the very best scientists (long headed by Tom Silhavy and Jim Broach), and then badger the profit-making pharmaceutical and biotechnology companies to donate the funds necessary to provide fellowship stipends to 10–20 young biologists every year. The LSRF has now been in operation for a quarter of a century and has funded the postdoctoral training of more than 400 young scientists. From 500–1,000 applications submitted each year, the advisory committee selects LSRF finalists solely on merit—no politics at all. It was Brown’s vision to have the LSRF burdened with almost no restrictions with respect to the citizenship of the applicants and no restrictions as to whether the award would be for a first or second postdoctoral fellowship; the applicants could also work in literally any field of biology or medicine. The legacy of this selfless act of community service is the training and allegiance of hundreds of young scientists—each and every one to whom Don Brown is godfather.

Personal Reflections

Having known and admired Don Brown and Tom Maniatis for more than 30 years, I close with several personal reflections. Both Brown and Maniatis are serious and ambitious men. Their seriousness, however, never succeeded in masking their open and vibrant personalities. Both are people who, upon entry, light up the room. Likewise, their ambition never masked their humanity. Both Brown and Maniatis contributed to the scientific community in ways that matched or exceeded their personal achievements as scientists. Every institution where Maniatis has been employed—from Cold Spring Harbor, to California Institute of Technology, to Harvard, to Columbia—has gained more from Tom than he has from them. It was Maniatis who founded the molecular cloning course at Cold Spring Harbor Laboratory, a course that I had the privilege of teaching and a course that has helped scientists young and old to learn the new techniques of molecular biology. Maniatis did this selflessly—this is Tom Maniatis.

It can likewise be said that the fingerprints left by Don Brown on the Department of Embryology of the Carnegie Institution of Washington remain indelible. From the early 1970s to near the end of the century, Brown created and directed the institutional paradigm of using pure genes to pry apart complex biology. Along with Gerry Rubin, Allan Spradling, Joe Gall, Andy Fire, Nina Fedoroff, Alejandro Sanchez-Alvarado, Doug Koshland, and many others, I was one to profit from Don Brown’s inspirational leadership. Offering one small personal reflection, Don let me work as a nominally “independent” postdoctoral fellow. During a 3 year period, I endeavored to pick apart the promoter of the herpes simplex virus thymidine kinase (TK) gene via a process that I dubbed “linker scanning mutagenesis.” Brown coached me all the way, but when it came time to publish the work (McKnight and Kingsbury, 1982), Don refused to be listed as an author. That single act of generosity affected me more profoundly than all of the technical experience garnered during my postdoctoral training.

Through their science, Don Brown and Tom Maniatis taught us how to use and deploy pure genes. Through their commitment to the scientific community, Don and Tom taught us the pure genius of giving back. It is hard to conceive of a better duo to receive the 2012 Albert Lasker Special Achievement Award in Medical Science.

Acknowledgments

I thank Drs. Joseph Goldstein and Joseph Gall for editorial comments helpful to the revision of this essay.

References

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