Fundamental biomolecular techniques

During the first few days of development, frog embryos don’t make ribosomes; rather, they use the stockpile that the egg endowed to them. No one knew how a single cell could churn out so many protein-making factories. Brown and Igor Dawid (and, independently, Joseph Gall; Lasker Special Achievement Award, 2006) showed that frog eggs create extra rRNA genes. Thus, the researchers had unveiled the first example of gene amplification, a process that underlies events outside of embryonic development as well; for instance, it fosters runaway growth of drug-resistant cancer cells.

These amplified genes provided a plentiful source of rRNA genes that Brown could isolate and study. He supplied purified 18S and 28S rRNA genes to Herbert Boyer and Stanley Cohen for use in their classic 1974 work that opened the study of eukaryotic genes to recombinant DNA technology (Lasker Basic Medical Research Award, 1980).

When this new era arrived, Brown used the methods of recombinant DNA to analyze a small rRNA, 5S RNA. He discovered a region in the middle of its gene that unexpectedly governs production of the RNA — the first known ‘internal control region’. This observation led to Robert Roeder’s (Lasker Basic Medical Research Award, 2003) purification of a protein that binds to this sequence and thus allows the enzyme that copies DNA into RNA to choose its target — the inaugural example of a gene-specific eukaryotic transcription factor.

Brown’s isolation and purification of the 5S RNA gene relied upon the fact that it is repeated many times. In contrast, no tools existed for separating a single-copy protein-coding gene — or its corresponding messenger RNA (mRNA) — from the rest of the genome or the bulk of mRNAs.

Maniatis took key next steps to solve this problem. One way to save a genetic sequence — whether it’s RNA or DNA — is to put it into a form that can be introduced into a living cell and reproduced there. In the mid 1970s, Maniatis and collaborators Argiris Efstratiadis and Fotis Kafatos tackled this challenge. They chose the mRNA of the rabbit β-globin gene because they knew that most mRNAs in red blood cells encode this hemoglobin subunit. Then they devised ways to perform all of the enzymatic manipulations necessary to make a collection — or library — of DNA copies of mRNA molecules, called cDNAs, from the red blood cells and fish out the globin cDNAs. Unlike today, when researchers order enzymes from catalogs and they are delivered a few days later, the team had to purify all of the necessary reagents from raw materials.

Their β-globin gene was the first full-length cDNA molecule isolated. They sequenced it, and their results established that the process of cDNA cloning did not introduce genetic errors, an essential feature for using the new recombinant DNA technology in the ways scientists imagined. Maniatis had thus established generally applicable methods for constructing cDNA libraries and for retrieving any sequence of interest.

He subsequently made the first complete human ‘genomic’ DNA library — containing all of the genes in an organism — whose sequences included regulatory sequences and other DNA regions that do not provide a template for protein. In addition to supplying genomic clones for the entire biomedical research community, this monumental task yielded a standard technique that laboratories everywhere used for many years.

Maniatis then isolated the first eukaryotic genomic DNA for a protein-coding gene and identified numerous genetic defects that underlie the inherited human illness β thalassemia. Some of the changes in single DNA letters cause the cell to splice out entire chunks of protein-coding regions. He had exposed — for the first time — disease-associated ‘point’ mutations that cause aberrant splicing.

Maniatis devised many additional innovations that have driven key advances in molecular biology and has used them to make numerous landmark discoveries.

Exemplary citizenship

Brown’s and Maniatis’s contributions to the research enterprise have extended far beyond their seminal scientific findings. In 1979, James Watson (Lasker Basic Medical Research Award, 1960) asked Maniatis to bring his techniques to the community by teaching a course at Cold Spring Harbor Laboratory — and Maniatis generously agreed. Its tremendous success spurred Maniatis and postdoctoral fellow Edward Fritsch to turn the course manual into a book. With Joseph Sambrook, they did so. Soon people all over the globe who studied myriad cellular processes were using it. Their Molecular Cloning manual, first published in 1982, sold 62,000 copies, and that number jumped to 95,000 in the second edition.

In different ways, Brown stimulated and bolstered biological research. He conceived and founded the Life Sciences Research Foundation (LSRF), an organization that honors excellence and potential in young investigators by awarding them prestigious postdoctoral fellowships. With the advent of recombinant DNA technology, Brown realized that biology would play a central role in pharmaceutical research and reasoned that companies would want to give something back to the system that enabled their success. Through his relentless efforts on an annual basis over the past 30 years, Brown has persistently tapped into this funding source as well as foundations and philanthropists to sustain the program. LSRF boasts 450 current fellows and alumni, many of whom have gone on to highly successful scientific careers.

In addition to helping seed the world with bright young scientists, Brown built a top-notch biology research program at his home institution. As Director of the Department of Embryology at the Carnegie Institution between 1976 and 1994, he created a scientifically diverse and stimulating environment, the quality of which is especially impressive given the department’s small size. When Brown left the directorship, five of eight laboratory heads were members of the US National Academy of Sciences. Two of the remaining three would later join that society and one of them would win the Nobel Prize.

by Evelyn Strauss

Key publications of Donald D. Brown

Brown, D.D. and Dawid, I.B. (1968). Specific gene amplification in oocytes: Oocyte nuclei contain extrachromosomal replicas of the genes for ribosomal RNA. Science. 160, 272-280.

Brown, D.D., Wensink, P.C., and Jordan, E. (1971). Purification and some characteristics of 5S DNA from Xenopus laevis. Proc. Natl. Acad. Sci. USA. 68, 3175-3179.

Brown, D.D. (1973). The isolation of genes. Sci. Am. 229, 21-29.

Sakonju, S., Bogenhagen, D.F., and Brown, D.D. (1980). A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5′ border of the region. Cell. 19, 13-25.

Pelham, H.R.B. and Brown, D.D. (1980). A specific transcription factor that can bind either the 5S RNA gene or 5S RNA. Proc. Natl. Acad. Sci. USA. 77, 4170-4174.

Brown, D. D. and Cai, L. (2007). Amphibian metamorphosis. Dev. Biol. 306, 20-33.

Key publications of Tom Maniatis

Maniatis, T., Kee, S.G., Efstratiadis, A., and Kafatos, F.C. (1976). Amplification and characterization of a β-globin gene synthesized in vitro. Cell. 8, 163-182.

Maniatis, T., Hardison, R.C., Lacy, E., Lauer, J., O’Connell, C., Quon, D., Sim, G.K., and Efstratiadis, A. (1978). The isolation of structural genes from libraries of eucaryotic DNA. Cell. 15, 687-701.

Ruskin, B., Krainer, A.R., Maniatis, T., and Green, M.R. (1984). Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 38, 317-331.

Palombella, V.J., Rando, O.J., Goldberg, A.L., and Maniatis, T. (1994). The ubiquitin-proteasome pathway is required for processing the NF-κB1 precursor protein and the activation of NF-κB. Cell. 78, 773-785.

Thanos, D. and Maniatis, T. (1995). Virus induction of human IFN-β gene expression requires the assembly of an enhanceosome. Cell. 83, 1091-1100.

Book:
Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.