Nucleic Acids, the Genetic Code, and Transposable Genetic Elements: A Life in Research

During Maxine Singer's forty-year career as a bench scientist, biology was transformed from a predominantly phenomenological science based on observation, description, and measurement, to an experimental one based on investigation of chemical and physical mechanisms on a molecular level. Singer was at the forefront of this rise of molecular biology, starting with her research on enzymes that catalyze and regulate the synthesis of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), the chemical substances of genes in inheritance. The artificial RNA polymers and trinucleotides she produced with Leon Heppel as a byproduct of their work on RNA structure and synthesis enabled Marshall Nirenberg and Heinrich Matthaei to decipher the genetic code in the early 1960s. Her scientific research subsequently expanded to the structure and evolution of defective viruses, chromatin structure, and mobile genetic elements.

Although Singer's dissertation was on protein chemistry, she soon began investigating the chemistry of another group of biologically active molecules: nucleic acids. Only a handful of laboratories worldwide were studying these complex macromolecules when Singer began her research career in 1956. Although Singer had studied no genetics and little biology, her dissertation adviser, the biochemist Joseph S. Fruton, urged her to enter the nascent field. She soon realized that it held the key for understanding heredity, evolution, and the origins of many diseases.

In 1956, it had only been a dozen years since Oswald Avery demonstrated that DNA was the chemical basis of genes in higher organisms, and only three years since James Watson and Francis Crick had unraveled its double-helical structure. Even more recently, scientists had proposed that RNA was a messenger in the transfer of genetic information from the DNA in the cell nucleus to the site of protein synthesis in the cell's cytoplasm. But how RNA was synthesized from individual bases in the cell nucleus, and how it coded for proteins, remained mysteries.

As a new postdoctoral researcher in Leon Heppel's laboratory at the National Institute of Arthritis, Metabolism, and Digestive Diseases (NIAMDD) at the National Institutes of Health, Singer participated in research into the role of enzymes that regulate the synthesis of nucleic acids. Heppel was one of the first scientists to investigate the synthesis of RNA and DNA in vitro by using polynucleotide phosphorylase, an enzyme discovered by Severo Ochoa and Marianne Grunberg-Manago. This enzyme strings together individual nucleotides into random strands of RNA, known as polyribonucleotides. In particular, he had developed techniques of electrophoresis and paper chromatography for analyzing the base compositions--the sequence of the four chemical bases of RNA--of the polyribonucleotides he was producing with the help of polynucleotide phosphorylase. Knowledge of the sequence of these polyribonucleotides enabled Heppel and Singer not only to understand how the enzyme catalyzed their synthesis, but also to make artificial RNAs of different defined compositions. Over several years Singer and Heppel accumulated a library of artificial polyribonucleotides: strings of RNA in which all the bases were identical--all uracil, for example--or in which two bases, such as uracil and cytosine, alternated in random order, or in which the end of the string had a different composition from the rest.

At the same time, Heppel and a few other scientists at NIAMDD, in particular the biochemist Marshall Nirenberg, were adding RNA extracted from cells to solutions containing free amino acids, the chemical components of proteins. The RNA then strung these amino acids together into a polypeptide chain, the precursor of a protein. This result lent support to the thesis that RNA plays a key role in using genetic information from DNA to direct the synthesis of proteins. This cell-free system enabled Nirenberg and his coworker Heinrich Matthaei to show in 1961 that an RNA molecule made up entirely of triplets of the base uracil--polyuridylic acid, or poly-U--spurred the synthesis of a polypeptide chain composed entirely of phenylalanine, one of the twenty common amino acids that make up proteins. Nirenberg's finding that triplets of uracil coded for phenylalanine represented the first step in cracking the genetic code. Singer played a key role in these experiments, producing RNA molecules with specific, predetermined base sequences that over the next four years Nirenberg and others showed could specify all of the twenty amino acids. By matching each amino acid to a particular triplet of RNA bases, these scientists laid open the dictionary of three-letter words in which the genetic code is written. Throughout the 1960s, Singer continued her work in nucleic acid enzymology, specifically on the action of polynucleotide phosphorylase.

During a sabbatical in 1971-72, spent in the laboratory of Ernest Winocour at the Weizmann Institute of Science in Rehovoth, Israel, Singer began a new line of work on the DNA of simian virus 40, a polyomavirus isolated from the kidney tissue of Rhesus monkeys. SV40 was of interest to scientists both because of its structural simplicity--it was the first microorganism that scientists learned to assemble from its DNA and protein components--and because it transforms mouse cell cultures in vitro, turning them into cancer cells. Singer showed that through a process of genetic recombination, SV40 can acquire DNA sequences from infected host cells that make the virus defective in certain biological functions, such as plaque formation, and that such "substituted" DNA can replicate even though it no longer codes for plaque formation. Singer's discovery helped explain how defective viruses evolved and reproduced.

Later in the 1970s, Singer took up yet another new line of investigation in DNA biochemistry: the interaction of histone H1, one of a large group of simple proteins, and the DNA with which it is associated in chromatin, the highly coiled DNA-protein complex that is the carrier of genes on the chromosomes of higher organisms. Singer determined that histone H1 played a role in forming superhelical DNA, the tight coiling of the double helix of DNA that makes it possible to fit a six-foot long strand of DNA into the nucleus of each human cell.

Singer's most important discovery since her contribution towards deciphering the genetic code came in the 1980s, even though, as head of the large Laboratory of Biochemistry at the National Cancer Institute, she was spending more than half of her time on administration and support of her laboratory's 15 research groups. Singer had begun to focus on a large family of repeated stretches of mammalian DNA called LINEs, or long interspersed nucleotide elements, that are present with very little variation in the genomes of all mammals. Focusing on LINE-1, a DNA sequence repeated and interspersed thousands of times in human chromosomal DNA, Singer concluded that it is capable of transposition, or movement and insertion into new places on chromosomal DNA. (It is, in fact, to this point the only known human transposable element.) She studied the precise mechanism whereby LINE-1 replicates and disperses copies to new locations along the genome. She also posited that the insertion of transposable genetic elements into new genomic locations can induce mutations in nearby genes, and that LINE-1 transposition played an important role in genetic diseases. Researchers later confirmed Singer's suspicion when they found that LINE-1 insertions into a gene whose protein product is required for blood clotting are associated with hemophilia.

Singer's contribution to molecular biology did not lie in a single, seminal discovery. Instead, she worked diligently over the course of four decades to detail the chemical structure and regulation of genomes in bacterial, viral, and, ultimately, human cells. Her research helped elucidate the crucial role of RNA in the transfer of genetic messages, the action of proteins on human chromosomal DNA, and the presence of transposable elements in the human genome. Her work on genetic recombination in animal viruses in the early 1970s also drew her into the growing controversy over recombinant DNA research and genetic engineering.