lasker~koshland special achievement ESSAY in medical science award

Developmental biology using purified

Donald D Brown

Some history Control Anucleolate Magnesium From the NIH I went to the Pasteur Institute mutant deficient After three years of college I entered the in Paris to study bacterial regulation in University of Chicago Medical School in the 1959, the year after the Lac repressor had been fall of 1952 and discovered biochemistry and discovered. Before leaving Bethesda, by the research. Lloyd Kozloff, a member of the greatest luck I learned about a small research bacteriophage group in the Department of institution in Baltimore that was associated Biochemistry, guided my research. While in at that time with the Johns Hopkins Medical medical school I began searching for a future School called the Department of Embryology of research subject, thinking it should be an the Carnegie Institution of Washington. I con- important medically related problem but unex- tacted Jim Ebert, the director, and arranged an plored by what were then the modern methods advanced postdoctoral fellowship after my year of biochemistry. in Paris. It is hard to imagine two more diverse The field of embryology, newly named research institutions. ‘developmental biology’, caught my attention. The Pasteur Institute was at the forefront of Reproductive biology was barely discussed, biology, involved in the founding of molecular and descriptive embryology was taught in two biology. Every day at lunch Jacques Monod, lectures as a part of gross anatomy. In 1953, I François Jacob and André Lwoff presided attended a biochemistry journal club discus- over an exciting discussion usually augmented Figure 1 Comparison of control (left), anucleolate sion of the Watson-Crick Nature paper describ- by a prominent visitor. The Department of mutant (center) and magnesium-deficient (right) ing the structure of DNA. I knew immediately Embryology was just phasing out 50 years of embryos of X. laevis after four days of that my future would involve some merger of describing the anatomical development of the development. Magnesium deficiency arrests DNA research, biochemistry and the field of human embryo. The faculty was principally embryos at the same stage of development as the embryology. engaged in experimental embryology. I was the anucleolate mutant. From ref. 2. After an internship at Charity Hospital in first biochemist. This is how I came to spend By the early 1960s, sucrose-gradient centrifuga- New Orleans and my wedding, my wife and I 50 years pursuing the topic of gene regulation tion had been devised to separate by size three moved to Bethesda, Maryland. My two years in development at the Carnegie Institution’s general classes of RNA, the 18S and 28S rRNA, of service were fulfilled as a member of the first Department of Embryology in Baltimore. I low molecular weight RNAs and a very hetero- class of ‘research associates’ at the US National cannot imagine a more ideal place. geneous DNA-like RNA that was presumed to Institutes of Health (NIH). I spent two wonderful include the newly discovered mRNA. I found years in the Institute of Mental Health as part of The isolation of genes before the that the three size classes of RNA were synthe- a project organized by Seymour Kety to investi- recombinant DNA era sized at vastly different rates during develop- gate systematically the metabolism of molecules I choose embryos of the frog Rana pipiens to ment. Even though the unfertilized frog egg is a that had been implicated in schizophrenia. I study because the eggs could be fertilized in single cell, it has as many ribosomes as the same was assigned to study the urinary metabolites vitro and the embryos developed synchronously weight of a multicellular somatic tissue such as of the amino acid histidine because it was the and provided unlimited amounts of material the liver. New rRNA was not synthesized until precursor of one such compound—histamine. I for biochemistry. While I was at the Pasteur the embryo had developed to 10,000 cells, and ended my two years by feeding 10 microCuries Institute, two separate groups discovered mes- new ribosomes did not contribute substantially of 14C-histidine to ten healthy controls and ten senger RNA (mRNA), and it had become clear to the total content of ribosomes until several people with schizophrenia, collecting their urine from studies with bacteria that ribosomal RNA days later. and analyzing the radioactive products. Then I (rRNA) and transfer RNAs were direct gene My first discovery at Carnegie was that frog published one of the few negative studies on the products. Previously, ‘differential gene action’ embryos do not develop in distilled water. They subject of the biochemical basis of schizophrenia. had been studied in embryos as fluctuations need four cations—calcium, sodium, potassium in enzymatic activity that was assumed to and magnesium, in that order during develop- Donald D. Brown is in the Department of represent changes in the amount of a protein. ment. Magnesium deficiency was especially Embryology, Carnegie Institute for Science, I realized that, as RNA was closer to the gene interesting because, without it, embryos develop Baltimore, Maryland, USA. than protein was, its analysis would be a more for several days and arrest as early swimming e-mail: [email protected] reliable reflection of gene expression changes. tadpoles at the same stage that a frog embryo

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different base composition for the next decade. was the first example of a class of transcription It led to the purification of X. laevis ribosomal factors called zinc finger proteins15. DNA (rDNA) by Birnstiel. This was the first iso- lation of a gene from any organism. In 1966, at Silk fibroin mRNA and its gene a symposium on the in Montevideo, The discovery of gene amplification in 1968 Uruguay, Oscar Miller showed electron micro- encouraged us to ask whether this mechanism graphs of genes in the nuclei of amphibian is used more broadly when a cell synthesizes a oocytes transcribing RNA4. These genes were large amount of a gene product. I had read about the size predicted for the rRNA genes, and they the unusual amino acid sequence and the huge came from the thousands of nucleoli in each size of the protein silk fibroin from the silkworm oocyte nucleus (Fig. 2). As the oocyte is a tetra- Bombyx mori. Most of the protein consists of ploid cell and should have just four nucleoli, we repeats of the peptide glycine-alanine-glycine- predicted that these extra nucleoli must contain alanine-glycine-serine, which predicts an many extra genes encoding rRNA. Igor Dawid mRNA and gene with a very high guanine and and I5 and, independently, Joe Gall6 showed the cytosine content. At the end of larval develop- Figure 2 An isolated nucleus from a mature ‘specific amplification’ of the genes encoding the ment, most of the protein synthesized by the oocyte of X. laevis. The diameter of the nucleus is 18S and 28S rRNAs in amphibian oocyte nuclei. posterior end of the silk gland is this single pro- about 400 µm. Cresyl violet stain accentuates the tein. At that time, only hemoglobin mRNA in thousands of nucleoli. From ref. 5. We learned that the genes encoding the 5S rRNAs are not linked to the rRNA genes but reticulocytes had been identified on a sucrose begins to increase its total amount of rRNA. It comprise an even greater fraction of the genome gradient as a discrete mRNA. Yoshiaki Suzuki was well known that magnesium holds ribo- of X. laevis DNA than does the rDNA. We puri- came to my lab as a postdoctoral fellow, and we somes together. In 1962, I read an article about fied and studied the 5S rRNA genes (5S DNA) began to raise silk worms. He isolated a large a mutant of the South African clawed toad from the X. laevis genomic DNA in 1971 (ref 7), 32P-labeled mRNA from the posterior silk gland Xenopus laevis that had an altered number of taking advantage of the unusual base composi- by sucrose-gradient centrifugation. Partial nucleoli1. The wild type had two nucleoli in tion of the many tandemly repeated 5S rRNA sequencing identified the predicted unusual each nucleus; the heterozygote had one nucleo- genes. These small genes were especially use- sequence of the silk fibroin mRNA16. The lus and was perfectly normal. Crossing two het- ful for detailed characterization and functional radioactive mRNA was pure enough to hybrid- erozygotes yielded a typical Mendelian ratio of studies in the days before DNA sequencing and ize with DNA from the posterior silk gland and offspring in which one-quarter of the progeny recombinant DNA methods were available. We from control silkworm tissues to show that there were anucleolate. These homozygous mutants learned that spacer DNA separated tandem is a single fibroin gene in the DNA of all cell arrested development exactly at the stage of a genes (Fig. 3). In different species of Xenopus the types. This ruled out DNA amplification as an normal embryo deprived of magnesium (Fig. 1), spacers were very different, whereas the genes explanation for the large and specific synthesis suggesting to me that the mutation affected the were highly conserved. Yet the spacers within a of silk fibroin mRNA by the posterior gland. synthesis of new ribosomes. species were remarkably similar, a phenomenon Silk protein synthesis is an example of extreme The only source of this mutant was the that we called horizontal evolution. Within each translational amplification. In a period of only Oxford lab where it had been discovered. 5S rRNA gene repeat there is a pseudogene. two to three days, each fibroin gene guides the Meanwhile, I learned that X. laevis was The first eukaryotic gene to be completely synthesis of 104 copies of mRNA, each of which, imported from South Africa for use as a preg- sequenced was one repeat of genomic 5S DNA in turn, synthesizes about 105 copies of the pro- nancy test by an entrepreneur who lived just ten by Nina Fedoroff and George Brownlee8. We tein. One gene makes 109 protein molecules. miles away in a suburb of Baltimore. I quickly learned that there are multiple families of 5S switched from R. pipiens to X. laevis. John rRNA genes in the X. laevis genome. The larg- Amphibian metamorphosis Gurdon at Oxford and I teamed up to show that est one is expressed only in oocytes. We called With the discovery of recombinant DNA tech- the anucleolate mutant synthesized no 18S or this the ‘dual 5S rRNA gene system’, a develop- nology in the late 1970s, the study of any gene 28S rRNA2. These mutant embryos synthesized mental problem that we pursued for a decade9. became possible. In 1990, after a decade of ana- the other two general classes of RNA normally. 5S DNA is a template for accurate transcription Max Birnstiel and his colleagues in Edinburgh in crude extracts of oocyte nuclei10, providing a took the next step3. It was well known that the simple assay for the cis-acting DNA sequences base composition of rRNA has a much higher required for accurate initiation and termina- guanine and cytosine content than the bulk of tion of transcription. Once genes could be the DNA. Using the method of equilibrium cloned, we made successive deletions around CsCl centrifugation developed by Jerome and into the 5S rRNA gene and then tested the Vinograd, Birnstiel and his colleagues showed ability of each mutant to transcribe 5S rRNA in that the genes encoding rRNA separated from the crude nuclear extract. To our amazement, the bulk of the DNA and were absent in the the gene transcribed properly even when we anuceolate mutant. By molecular hybridization had deleted one-third of either the 5ʹ or the they showed that there are several hundred cop- 3ʹ ends of the gene11,12 (Fig. 4). We called this ies of rRNA genes per haploid complement of the ‘internal control region’. Bob Roeder and Figure 3 Electron micrograph of a partially X. laevis DNA and that they must be clustered, his colleagues purified the first eukaryotic denatured molecule with repeating units of 5S 13 as a single mutation deletes all of them. transcription factor from X. laevis oocytes . DNA. The gene for 5S rRNA comprises one-third Equilibrium CsCl centrifugation became the This protein, called TFIIIA, binds the internal of the native region. The average repeat length is most important method for separating DNAs of control region of the 5S rRNA gene14. TFIIIA about 700 base pairs. From ref. 7.

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+ solicit sponsors every year. We are exceedingly + proud of the distinction of our 456 alumni and + + current fellows. In 2012, our distinguished peer- + review team selected 50 finalists from 900 appli- + + cations and awarded 24 fellowships. This fall we + are having a celebration of alumni marking our – thirtieth year. – 5S RNA gene ACKNOWLEDGMENTS I owe a great deal to the Carnegie Institution of –80 –40 0 +40 +80 +120 Washington, now called the Carnegie Institute for Science. It has supported my research for 50 years. I – have had generous grants from the US National Institute – + of General Medical Science since 1975. The G. Harold & + Leila Y. Mathers Foundation supported my research in + amphibian metamorphosis. Many wonderful graduate + students, postdoctoral fellows and other colleagues have contributed to these studies. It is a special pleasure Figure 4 A transcriptional-control region within the 5S rRNA gene. The horizontal bars indicate the extent to share the responsibility of running the LSRF with of each deletion. Mutants that still support accurate initiation of transcription are indicated as +. The D. Koshland. He and I are responsible for finding region delineated by vertical dashed lines is the internal control region of the gene. From refs. 11 and 12. sponsors who will support postdoctoral fellows. C. Pratt carries out the day-to-day decisions with the highest lyzing the structure and regulation of the simple post-doc. The key to these positions is that the efficiency and intelligence. S. DiRenzo, T. Silhavy and 5S rRNA genes, I decided to change projects. I investigator is completely independent. A staff J. Broach administer LSRF’s peer review process at chose amphibian metamorphosis—a wonder- associate works full time in the lab with the aid Princeton University, which this year reached its highest application number ever of 900. ful biological problem that had fascinated every of a technician. These are term positions so that succeeding biological discipline but had been staff associates know that they must leave after COMPETING FINANCIAL INTERESTS ignored by molecular biologists. five years. These independent young investiga- The author declares no competing financial interests.

The question was simple. How can one tors have added great diversity and energy to 1. Elsdale, T.R., Fischberg, M. & Smith, S. A mutation that molecule—thyroid hormone—change the fate our department over the years. Most of them reduces nucleolar number in Xenopus laevis. Exp. Cell of so many different cell types? The discovery of have moved to excellent jobs with great distinc- Res. 14, 642–643 (1958). 2. Brown, D.D. & Gurdon, J.B. Absence of ribosomal RNA the thyroid receptors as transcription factors by tion. Several other research universities and synthesis in the anucleolate mutant of Xenopus laevis. Ron Evans and Björn Vennström in 1986 con- institutions have initiated similar programs. Proc. Natl. Acad. Sci. USA 51, 139–146 (1964). 3. Wallace, H. & Birnstiel, M. Ribosomal cistrons and the vinced me that the control of metamorphosis nucleolar organizer. Biochim. Biophys. Acta 114, 296– by thyroid hormone must occur by changes in The Life Sciences Research Foundation 310 (1966). the expression of many genes. The simple lesson that research is best done 4. Miller, O.L. & Beatty, B.R. Visualization of nucleolar genes. Science 164, 955–957 (1969). The technical advantages of the system had by young people is partly responsible for 5. Brown, D.D. & Dawid, I.B. Specific gene amplification in been well explored. Tadpoles are exquisitely my founding the Life Sciences Research oocytes. Science 160, 272–280 (1968). 6. Gall, J.G. Differential synthesis of the genes for ribosomal sensitive to exogenous thyroid hormone. The Foundation (LSRF; http://www.lsrf.org/). By RNA during amphibian oogenesis. Proc. Natl. Acad. Sci. chemicals that inhibit synthesis of the hor- 1980, it had become clear that biology was USA 60, 553–560 (1968). mone by the thyroid gland in mammals work beginning to replace chemistry in the pharma- 7. Brown, D.D., Wensink, P.C. & Jordan, E. Purification and some characteristics of 5S DNA from Xenopus laevis. well in amphibians. The pituitary-thyroid axis ceutical industry. , inspired Proc. Natl. Acad. Sci. USA 68, 3175–3179 (1971). is similar in all vertebrates. The disadvantages by the new recombinant DNA technology, was 8. Miller, J.R. et al. The nucleotide sequence of oocyte 5S DNA in Xenopus laevis. II. The GC-rich region. Cell 13, of the system were the absence of traditional poised to revolutionize how drugs are made. 717–725 (1978). genetics and the long life cycle of frogs. When Furthermore, these discoveries were not made 9. Brown, D.D. The role of stable complexes that repress and transgenesis was discovered in X. laevis in at companies, but at nonprofit institutions paid activate eucaryotic genes. Cell 37, 359–365 (1984). 10. Birkenmeier, E.H., Brown, D.D. & Jordan, E. A nuclear 1996, we could overexpress transgenes driven for by the government through the extramural extract of Xenopus laevis oocytes that accurately tran- by cell-specific promoters. Among other dis- grant programs of the NIH and the National scribes 5S RNA genes. Cell 15, 1077–1086 (1978). coveries, this enabled us to prove that the thy- Science Foundation. University scientists had 11. Sakonju, S., Bogenhagen, D.F. & Brown, D.D. A control region in the center of the 5S RNA gene directs specific roid hormone receptors are responsible for all begun to start companies. initiation of transcription. I. The 5ʹ border of the region. of the many changes induced by the hormone I sought a way to further this interaction, Cell 19, 13–25 (1980). 17 12. Bogenhagen, D.F., Sakonju, S. & Brown, D.D. A control in metamorphosis. In a recent review I sum- expecting these companies to have a new region in the center of the 5S RNA gene directs specific marized the current state of the field. interest in the biological research carried out initiation of transcription. II. The 3ʹ border of the region. in universities and research institutions. After Cell 19, 27–35 (1980). 13. Engelke, D.R. et al. Specific interaction of a purified The Department of Embryology of the some thought and consultation, I selected a transcription factor with an internal control region of 5S Carnegie Institution for Science simple postdoctoral program that I hoped RNA genes. Cell 19, 717–728 (1980). Our Department has eight permanent ‘staff would be sponsored by the major pharmaceu- 14. Sakonju, S. et al. The binding of a transcription factor to deletion mutants of a 5S ribosomal RNA gene. Cell 23, member’ slots. These positions do not confer tical companies. In addition to these compa- 665–669 (1981). tenure, but are renewed competitively every five nies, the Howard Hughes Medical Institute, 15. Miller, J., McLachlan, A. & Klug, A. Repetitive zinc- binding domains in the protein transcription factor IIIA years. One of the changes that I made when I a number of foundations, one government from Xenopus oocytes. EMBO J. 4, 1609–1614 (1985). became director of our department in 1976 agency (Department of Energy–Division of 16. Suzuki, Y. & Brown, D.D. Isolation and identification of was to establish an independent junior faculty Biosciences) and several philanthropic scien- the messenger RNA for the silk fibroin from Bombyx mori. J. Mol. Biol. 63, 409–429 (1972). program. A ‘staff associate’ position is avail- tists have sponsored LSRF fellowships. Alas, we 17. Brown, D.D. & Cai, L. Amphibian metamorphosis. Dev. able right after graduate school or as a second have never had endowment and must therefore Biol. 306, 20–33 (2007).

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