Captivated by Chromosomes
Peering through a microscope since age 14, Joseph Gall, now 89, still sees wonder at the other end.
December 1, 2017|
Cell biologist Joseph Gall, who was born in 1928, grew up spending lots of time outside, observing and collecting frogs, butterflies, and other insects. “There was no television when I was younger. After school, I roamed around the neighborhood and the nearby woods,” says Gall, now a staff scientist at the Carnegie Institution for Science in Baltimore, Maryland. “My mother used to make me dozens of butterfly nets and made sure I always had science books.” Gall attributes his lifelong interest in science to her. “She was the first person in her family to go to college. This was in the 1920s and was rare for a woman. After college, she immediately married my father, a lawyer, had my older brother, and became a homemaker. That was the pattern in those days. Today she would have been a professional of some sort.”
When Gall was 14, his father bought a 500-acre cattle farm in Virginia and hired a farm manager. Gall helped bale hay and did other farm chores in the summer, when not at boarding school, but his real love was science. Through a work connection, Gall’s father got him a professional Bausch and Lomb microscope. “I can’t remember a time when I wasn’t interested in looking through a microscope. I was completely self-taught. My mother got me the right books, including a copy of E.B. Wilson’s The Cell in Development and Heredity. It was the bible in cell biology for many years,” says Gall. “By the time I was 14, I had read that and other cell biology books and had set up a laboratory in my room. I made slides of everything—insects, the protozoa in our pond water—and then progressed to making slides of the organs of the farm animals.” Gall’s parents got him the tools he needed to fix tissues and make paraffin sections. “I learned this all myself and it made me really independent.”
Here, Gall recalls how he invented in situ hybridization, why he has always promoted women in science, and why he never “became” a biologist.
Gall Gets Going
Professional biologists. For three years, Gall attended a boarding school outside of Charlottesville, Virginia. While he enjoyed the regimented schedule and the language classes, he was less than inspired by the science curriculum. “But it didn’t do anything to my scientific interests,” he says. The headmaster decided that Gall should attend Yale University and “somehow it was all arranged and it happened. I don’t remember even applying.” He started at Yale as an undergraduate in 1945, when most colleges had been nearly emptied because of World War II and were looking for students. Gall chose a premed major only because he didn’t know that there was such thing as a professional biologist. “I thought that you had to be a doctor, and only in my junior year did I realize that there was graduate school and that the biology professors teaching me weren’t MDs. The lack of career counseling would be astounding to anyone today.”
“I have been credited, legitimately, with fostering women in the lab at a time when there were not many women in science. It was unusual for the time and it goes back to the fact that I learned science from my mother.”
Observing chromosomes. Gall graduated in 1949 and arranged with Donald Poulson, a Drosophila geneticist and cell biologist in the zoology department, to stay on at Yale as a graduate student. In his home laboratory, Gall had already been making mitotic spreads using fixed tissues, and he wanted to work on chromosomes for his PhD thesis. In a textbook, he came across an image of a lampbrush chromosome—a conformation formed by the unusually high transcription of the meiotic chromosomes in immature oocytes of amphibians and other animals, but not in mammals. Gall couldn’t believe the magnification scale on the image and wanted to see them for himself. He ended up analyzing these chromosomes—which had not been well characterized—in newt oocytes. “They are truly gigantic and one of the best-kept secrets in biology, up to 1 mm in length and can almost be seen with the naked eye,” says Gall. The phase-contrast microscope had recently been invented, and Yale had just purchased its first one. Gall published a 70-page paper describing lampbrush chromosomes in 1954.
One strand. After obtaining his PhD in 1952, Gall took an instructor position in the zoology department at the University of Minnesota. He was mostly expected to teach, but also was given a microscope and some lab space. “In 1952, the National Science Foundation (NSF) had just been formed and the NIH still only had a meager funding budget,” Gall recalls. He was among the first to receive grant funding from the NSF, as one of his colleagues in the department, H. Burr Steinbach, was an assistant director there and told him how to apply. Gall continued to study lampbrush chromosomes and began a decades-long collaboration with Harold “Mick” Callan, a professor at the University of St Andrews who was also studying them. An experiment by Callan’s graduate student Herbert Macgregor, using the enzyme DNase to cut lampbrush chromosomes into fragments, inspired Gall to perform a similar experiment, but to control the kinetics of the reaction in order to determine how many DNA molecules make up a chromosome. The experiment showed that there was only one DNA molecule per chromatid within the chromosome and that the brush analogy wasn’t really correct: the bristles of the brush were loops. “At the time, it was almost universally believed that chromosomes of higher organisms were multistranded and that larger genomes meant more strands in the chromosomes, even though there was no evidence for this. This is probably the most important early experiment I did, although it’s almost never cited,” Galls says. “Matthew Meselson and Franklin Stahl, who used the tiny E. coli circular chromosome, are given credit for showing that a chromosome is a single DNA strand. For higher organisms, Herbert Taylor used tritium-labeled incorporation into living chromosomes to demonstrate that the label distributed semi-conservatively during replication. Taylor’s paper is one of the most important of semi-forgotten experiments in cell biology.”
Gall Goes Higher
Poring over pores. Again following on Callan’s experiments, this time in flattening and laying out the nuclear envelope on a slide prior to electron microscopy, Gall showed in 1954 that the envelope is peppered with nuclear pore complexes; 13 years later, he showed that these complexes are octagonal rather than circular. “We thought that these pores were so big that anything could get in and out. I never thought at the time that there was regulated transport into and out of the nuclear envelope,” says Gall. (See “Nuclear Pores Come into Sharper Focus,” The Scientist, December 2016.)
Moving on. In 1964, Gall returned to his alma mater, Yale, where he became a professor in the biology department and in the newly formed Department of Molecular Biophysics and Biochemistry. “I realized that there was this new field beginning that would eventually be called molecular biology. It was clearly the future, but there was as yet no way to detect specific DNA or RNA sequences within cells,” he says. Researchers were already immobilizing nucleic acids onto nitrocellulose filters and using a radioactively labeled piece of RNA to detect the complementary sequence on the filter and quantitate it. The approach inspired Gall to develop a similar technique for identifying a specific nucleic acid sequence in DNA immobilized inside a tissue preparation.
Gall and others had been studying the phenomenon of “gene amplification”—specifically, the production of massive amounts of extrachromosomal DNA coding for ribosomal RNA that occurs in amphibian oocytes. “I realized that here was the perfect test material for developing a technique to detect specific DNA molecules in fixed tissues.” Because there was no cloning yet, Gall and his graduate student Mary-Lou Pardue used this naturally amplified DNA. In 1968, the two developed a method called in situ hybridization, using tritium-labeled RNA as a probe to target the many copies of ribosomal DNA in Xenopus oocytes and visualizing the hybridization with autoradiography. The technique worked beautifully.
Gall’s lab showed that in Drosophila and mouse the densely stained, highly concentrated DNA regions that were found to be free of genes actually corresponded to simple DNA repeats called satellite DNA. “Possibly the most important early discovery to come out of the in situ hybridization technique was the realization that satellite DNA corresponds to heterochromatin,” he says. A modified, more sensitive version of the technique, FISH (fluorescence in situ hybridization), now incorporates fluorescently labeled rather than radioactively labeled nucleic acids and employs fluorescence microcopy rather than autoradiography for visualization.
Telomere sequences before telomeres. Gall began to study the chromosomes of the ciliate Tetrahymena after he saw images of its multiple nucleoli. After extracting the Tetrahymena DNA, he used ultracentrifugation to separate out the multicopy extrachromosomal ribosomal DNA, and then, using electron microscopy, observed that the strands were either circularized or linear in form. “There was something funny about the ends that made them stick together sometimes. Elizabeth Blackburn, who had learned how to do DNA sequencing in Fred Sanger’s lab, joined my lab as a postdoc and decided to sequence these ends,” he says. Blackburn found that the ends all contained the same sequence, TTGGGG, repeated many times. “That was the discovery of the telomeric sequence, but not the discovery of the telomere because we had no idea at the time that all chromosomes have this sequence at their ends and that they form a specific structure,” says Gall. Blackburn, along with Carol Greider and Jack Szostak, went on to win the Nobel Prize in 2009 for research on how telomeres and telomerase work to protect the ends of linear chromosomes.
Nothing unusual. Greider, who was a graduate student in Blackburn’s lab, credits Gall with being a fantastic mentor and training many of the prominent female scientists who became leaders in the study of telomeres, among other fields. “I have been credited, legitimately, with fostering women in the lab at a time when there were not many women in science,” says Gall. “It was unusual for the time, and it goes back to the fact that I learned science from my mother. It was nothing unusual to me that women should be scientists. It was not that I was positively seeking women in my lab, but to those who wanted to join, I would say ‘Yes,’ and that wasn’t true for many other male professors.”
Bodies of confusion. In 1983, Gall moved from Yale to the Carnegie Institution for Science in Baltimore because at Yale he was fending off offers to become an administrator or a dean, and he wanted to remain focused on his lab. More recently, he has been studying nuclear bodies, subnuclear organelles whose functions are still poorly understood. One of these structures, which he named the Cajal body after its discoverer in the early 1900s, Santiago Ramón y Cajal, is thought to be involved in RNA splicing. Gall’s lab found Cajal bodies—which are typically identified by the presence of a protein called coilin—in Drosophila melanogaster in 2006. Further study of these organelles in Xenopus oocytes led Gall’s team to conclude in 2010 that a different type of nuclear body, which Gall named the histone locus body, had been confused with Cajal bodies in the literature because both are associated with coilin.
Mystery introns. The lab is currently focused on stable introns found in the cytoplasm of Xenopus oocytes. While most introns are spliced out of pre-messenger RNA and degraded within minutes, the stable, circular introns Gall and graduate student Gaëlle Talhouarne identified in 2014 persist and are transferred to the fertilized egg, suggesting a regulatory role in mRNA translation. (See “Uncovering Functions of Circular RNAs,” The Scientist, July/August 2017.)
Lab rat. “I still do experiments,” says Gall. “My name is not on the papers as a courtesy. I typically do the in situ hybridization experiments and someone else does the molecular biology and the bioinformatics. I’ve also done a lot of the Drosophila microscopy and immunostaining.”
Book-ish. Gall is an avid collector of biology books and texts, with an extensive library containing items that date back to the 17th century. The most prized part of his collection: “An original copy of the journal containing Mendel’s paper.” Gall also has most of Theodor Boveri’s original papers, and other important 19th-century cell biology books and papers.
Biologist by birth. “When people ask me, ‘When did you become a biologist?’ I always answer, ‘I never became a biologist, I just always was.’ I think I am one of those very lucky people who never had to do any soul searching. I always knew what I was from day one.”
Going strong. “I will retire when I can’t think of anything else to do! For now, I don’t have any plans to retire, but it all depends on health. Fortunately, I am quite healthy at this point, but I am not taking on new graduate students because at 89, I don’t want to make a five-, six-year commitment. I am just as anxious to come to the lab each morning as I ever was.”
- Using DNase kinetics, showed that amphibian lampbrush chromosomes are not multistranded, but consist of a single, extremely long DNA molecule
- With Mary-Lou Pardue, invented the in situ hybridization technique, which uses labeled RNA or DNA molecules to bind and visualize complementary DNA or RNA within fixed tissues or cells
- Showed that DNA-dense yet gene-free chromosomal regions in mouse and Drosophila corresponded to simple DNA repeats called satellite DNA
- With Elizabeth Blackburn, identified tandemly repeated sequences at the ends of ribosomal RNA genes (rDNA) in Tetrahymena that later turned out to be telomere sequences
- Characterized Cajal bodies and histone locus bodies, organelles in the nucleus