Read excerpts from :
The Mechanics of Falling
Curled in the Bed of Love
The End of the Class War
- from From Chapter 3 of Elizabeth Blackburn
Joe Gall’s lab at Yale differed dramatically from the virtually all-male environment at the MRC. Among Blackburn’s colleagues were Virginia (Ginger) Zakian, just completing her doctorate, and grad student Kathy Karrer. It made a difference for Blackburn to be working in a lab with other women. She recalled Zakian as “forthright about women’s issues, open, and clearly very good at science.” And Karrer took Blackburn under her wing, teaching this newly arrived Australian basic lessons on domestic life in the United States. Karrer explained to Blackburn that “when you need things for the house, you go to Sears,” and Blackburn was conscious that “women do this kind of thing more than men.” If she registered that feminism was in the air—Our Bodies, Ourselves, a feminist manual on women’s health and sexuality, had only recently been published—Blackburn didn’t see it as particularly relevant to her professional life: “In the lab, these things weren’t supposed to matter.” At Yale Blackburn came to know Joan Steitz, who, along with her husband, Tom, was now an assistant professor in Yale’s Molecular Biochemistry and Biophysics Department, but they never discussed the discrimination Steitz had encountered at the MRC and during her subsequent job search. (Before they took posts at Yale, Steitz’s husband, hoping they would both find posts at the University of California, Berkeley, was warned that a woman would be unlikely to obtain an academic position there.12) “We talked about science,” Blackburn said.
For Blackburn, preserving this cherished freedom to focus on the work meant resolutely shutting one’s eyes to evidence that gender could be a handicap, just as she denied other obstacles. In contrast to Blackburn’s perception that “in the lab, these things weren’t supposed to matter,” another female colleague at Yale in the 1970s portrayed the university as a tough place to be a woman. Diane K. Lavett (then Juricek), had taught briefly before coming to Yale for postdoctoral work, and she emphasized that discrimination was the norm:
My teaching position was temporary, and I was on the search committee for my replacement. The two final candidates were a man and a woman, and everyone agreed the woman was the better candidate. Yet the committee voted for a man, even though I said, ‘Let me explain about affirmative action,’ and pointed out that if the candidates were merely equal, under the law the woman should have been hired. When I was a doctoral candidate, I was advised by a guy on my thesis committee to use an initial, not my first name, when I tried to publish my dissertation. He told me the double whammy of being from the South and being female would ensure my papers did not get published. This was helpful—even accurate—advice. At that time in academia, there was no room for women to maneuver, and I’m not sure there’s any now. It wasn’t necessarily that these guys were intentionally sexist. They just didn’t know better.
As the mother of two children, Lavett could not stay till late at night doing research, and she noted that “Liz had a rightfully deserved reputation of being exceedingly hard working. I didn’t, because I didn’t stay till ten or eleven at night. If you were a married woman with kids, you were dead.” Lavett spoke of the atmosphere at Yale at this time as “contaminated”: “When we discovered that we would not have enough room at a conference to accommodate the audience, a primary investigator, a basically nice guy, said we should set up a TV in another room and have all the women watch the lectures on TV. In another instance, I had researched and written a paper on my own but discovered that a primary investigator had added the names of several other researchers as co-authors. When I told him that I felt I had been mugged, he answered, ‘Well, at least you haven’t been raped.’”13
Although sexism was not directly discussed in the lab, Blackburn told a story that revealed Gall’s concern with its impact on his students’ careers and betrayed her own inability to admit this: “One day Joe came into the lab waving a piece of paper and announcing that Mary Lou Pardue had just received tenure at MIT. I couldn’t quite make sense of his excitement, and I said, ‘Well, of course she should get it—she’s very good.’ The tenor of Joe’s reply suggested to me he well understood that for a woman, to be very good might not be enough, and so Mary Lou’s triumph was especially sweet. It was an important message to convey to the lab.” Gall had in fact worked hard to help his former student succeed against the odds, and in his opinion, some of the same difficulties hold true today: “Mary Lou Pardue was one of the first female faculty—one of few—at MIT in the seventies. It is unclear whether women were being discriminated against overtly, but clearly they were and still are being discriminated against in terms of promotion. Things have changed, but it is still not an even playing field.”14
The timing of Blackburn’s arrival in Gall’s laboratory could not have been more perfect. She had anticipated she could use her exciting new abilities to sequence DNA, and her passion for sequencing was so great (“sequences were cool—every one a treasure”) that determining what she might sequence came almost as an afterthought. But Blackburn had become curious about DNA at the chromosome end regions, specialized structures that capped both ends of a chromosome. And Gall had just discovered how to purify the linear minichromosomes in Tetrahymena, a single-celled protozoan, away from the rest of its cellular DNA. He had also determined that rDNA genes, which encode ribosomal RNA, were housed on these very short, linear minichromosomes. Each cell had tens of thousands of these minichromosomes, thus enabling the organism to produce enough ribosomes to provide protein synthesis for so large a cell.
Typical chromosomes are millions of bases long, and their end-region DNA constitutes too small a fraction of the whole to be purified for analysis by the methods then available. In contrast, Tetrahymena produces plentiful minichromosomes with two end regions for every twenty-thousand base pairs, a much larger proportion of the genetic material, thus making it possible, for the first time, to separate out the end regions for further study. In the 1970s Ray Wu and collaborators and Ken and Noreen Murray had sequenced a few bases at each end of the linear DNA in the lambda bacteriophage, far simpler DNA structures than the chromosomes of more complex organisms. So the genetic material of Tetrahymena offered a chance to explore a true eukaryotic organism, with potentially far-reaching implications. Though eukaryotic end regions had been defined cytologically (under the microscope), no one at the time was exploring their molecular nature. Although Blackburn first considered studying the very short minichromosomes of another type of ciliated protozoan, Gall pointed out that Tetrahymena would be easier to culture in the quantities needed for direct molecular analysis. Tetrahymena offered one too many advantages for Blackburn to pass up: “I was fresh from a world where we’d found all these new methods for sequencing DNA, and here were these juicy molecules, their end regions ripe for the picking.”
In the 1930s the natural ends of chromosomes had attracted the attention of Barbara McClintock and Hermann Muller, both Nobel Prize-winning geneticists. Working in advance of the 1944 discovery that DNA was the genetic material, Muller and McClintock had both found that the DNA at the chromosome end regions was specialized to prevent chromosomes from fusing end-to-end, which could cause the destruction of the cell. Muller, working with the Drosophila fruit fly, had shown that genetic mutation could be caused by X-rays, and he studied the broken chromosomes that resulted from irradiation, noting they could “reattach” to other broken chromosomes. McClintock had developed powerful cytological methods for observing the whole chromosomes of maize under a light microscope, focusing on how mutations in the genes were later reflected in the altered phenotypes of the maize plants. McClintock also discovered an interesting phenomenon that she had not set out to investigate. Blackburn had read an early report of hers, printed in 1931, in which McClintock noted that X-ray irradiation frequently produced translocations, deficiencies, and ring chromosomes. Like Muller, McClintock deduced that these alterations in the chromosomes resulted from the fusion of broken ends with each other. One statement leapt out for Blackburn: “No case was found of the attachment of a piece of one chromosome to the end of another [intact chromosome].”15 While Muller did not coin the term telomere until 1938, McClintock had already presciently recognized that the natural end of an intact chromosome had distinct functional properties that preserved the integrity of chromosomes. Her work on the fate of broken chromosomes underscored this mysterious property. Studying germ cells and endosperm in maize, she observed that even if broken chromosomes fused with each other, they were repeatedly broken each time such cells divided, a process she termed the breakage-fusion-bridge cycle.16
By the 1970s, when gene replication was more fully understood, speculation about the function of these end regions, or telomeres, grew intense as scientists puzzled over the problem posed by replication of the ends of linear DNA molecules. During cell reproduction, the complex array of protein enzymes that replicate DNA are unable to copy the DNA strand all the way out to the end, which in theory means that DNA will grow shorter and shorter each time a cell reproduces. Clearly, something in the cell compensates for this predicted loss. By this time biologists had determined how the problem of DNA replication was neatly sidestepped in the lambda phage. Prior to replication, its DNA molecules formed a ring, which presented no problem for DNA replication enzymes. Influenced by the current predilection for universal, minimalist solutions that would bear out the elegant simplicity of DNA itself, molecular biologists surmised the process might well work the same way in other organisms.
Blackburn now had a heady opportunity to explore the problem of DNA replication, though with no certainty that sequencing the genetic material would provide any answers. Looking through a microscope at Tetrahymena thermophila (T. thermophila) in Gall’s lab, Blackburn “fell in love at first sight.” Like its better-known relative Paramecium, T. thermophila is a ciliated protozoan that lives in pond water; some species of Tetrahymena can grow to four-fifths of a millimeter in size, relatively large for a protozoan. The laboratory species of T. thermophila, only a tenth of that size, is still visible to the naked eye and looks like a speck of dust in water. Poetically named “swirlers,” these single-celled organisms swim in pond water in a corkscrew path by rhythmically beating rows of hairlike cilia that cover the plump, pear-shaped cell. With their chubby cells and lively, graceful motions, T. thermophila, informally referred to as Tetrahymena, are visually appealing to humans. Blackburn noted that “they tend to inspire affection from their laboratory handlers, who have been known to refer to them as ‘cute,’ and in my lab at Berkeley, it wasn’t unheard of to sing Ravel’s Bolero to them to encourage them to mate.”
Blackburn raved about the fascinating traits of her organism: “Tetrahymena has seven sexes. And one cell can be up to four of them at a time. After mating, it has to go through a number of cellular divisions before it reaches sexual maturity and is able to mate again, and it even has a recognizable adolescent period. The last, somehow to me endearing, accomplishment I will mention is that when it is starved, it reabsorbs its cilia, becomes long and lithe in body shape, puts out its cilia in new rows in a racing configuration, and grows a long caudal (back-end) flagellum so it can cover great distances swimming fast to look for food. Need I say more?” Biologists often cherish the idiosyncratic qualities of a favorite experimental organism, and ultimately idiosyncrasy (of organism and observer) can prove valuable to scientific endeavor. As Blackburn noted, “Biology sometimes reveals its general principles through what appears to be the arcane and even bizarre. But in evolution most things have stayed essentially the same mechanistically at the molecular level, and what changes is the extent and setting in which the various molecular processes are played out. So what looks like an exception often turns out to be a manifestation of a molecular process that is actually fundamentally well conserved. This is the case with the ciliated protozoa.”
Tetrahymena’s distinctive properties were perfectly suited to Blackburn’s purposes. In Gall’s lab, Karrer had shown that the entire rDNA minichromosome was a palindrome—its bases were arranged in an order that reversed itself at midpoint, as in “madam I’m adam.” Karrer had also found that these minichromosomes could be cut with a restriction endonuclease that separated a large central fragment from its two small end-region fragments. Not only did Blackburn have a source of purified DNA to analyze, she could also conjecture—rightly, but for the wrong reason—that the two end regions on a palindromic molecule would be alike (although in inverted orientation). Using an electron microscope, Joe Gall had observed that the minichromosomes sometimes formed circles, providing an important clue that these end regions might resemble those of the lambda phage, sequenced in the early 1970s. Consequently, Blackburn could start off on her quest by applying end-labeling techniques similar to those Wu and the Murrays had used for the lambda phage.
Just as Gall’s shared excitement had launched her on her project, the technical expertise of other molecular biologists at Yale often helped Blackburn to carry it out. Before she could begin sequencing, Blackburn had to learn from her peers in the lab, from Karrer in particular, how to cultivate Tetrahymena, purify the rDNA minichromosomes, and examine them by electron microscopy, a process still new to her. Thanks to new methods, including those developed by Sedat and others at the MRC, Blackburn could now sequence DNA directly, though it was not yet possible to clone a gene to make greater amounts of DNA. For access to technology necessary for analyzing nucleic acids, she relied on Sid Altman, a Yale faculty member who had been at the MRC a few years before Blackburn; Altman had set up the sequencing equipment Blackburn would use at Yale and could provide occasional pointers. Blackburn again drew on her MRC connections to obtain help from Joan Steitz, and she was also aided by a younger faculty member, Alan Weiner.
Because sequencing was such a new field, Blackburn had to jury rig some of the equipment she would need for her experiments. Martha Truett, intrigued by this “new and different” equipment, still recalled much of it years later. Blackburn installed heavy glass tanks, two to three feet high, to hold organic solvents for separating oligonucleotides. She fitted these tanks with grooves to hold glass rods, over which she draped the blotting paper that wicked up oligonucleotides. “Scientists had to be creative in terms of the equipment,” Truett said. “Only later did it become more of a production line, with specialized companies producing scientific lab equipment. For the early DNA-sequencing efforts, the shop at the lab had to make the glass plates to which you attached the gels.”17
For her part, Blackburn was also being exposed to a new way of working. In a cell biology lab, visual observation, as opposed to biochemical analysis, played a crucial role, even when, as in Gall’s lab, researchers were exploiting a variety of approaches. The diverse organisms studied in the lab were chosen based on their suitability for observation under a microscope—Drosophila, Tetrahymena, Paramecium, Physarum (slime mold) and Xenopus (African clawed frogs). Each organism lent itself to certain experimental questions, as Martha Truett explained: “Xenopus had nice, fat eggs (oocytes) that at certain stages in development have big chromosomes that you can spread on a slide for microscopic viewing, and you can actually see the RNA being transcribed off the DNA in the lampbrush chromosome—the whole structure looks like a Christmas tree. That was one of the first things Joe showed me, and I thought, wow, what a fantastic structure! And you’re seeing what it really looks like, at once molecular and structural and unique to that organism.”18 Gall’s enthusiasm and curiosity extended to many species, so long as they could be viewed under a microscope. Blackburn recalled that once, after an outbreak of head lice at a local school, a lab member collected a louse from her daughter’s hair and delivered it to Gall, confident he would happily identify the species by microscopy. Gall had a particularly aesthetic appreciation for his work; his Pictorial History: Views of a Cell is a compilation of slides of stained cells viewed under the microscope—colonies of algae as beautifully tinted and intricately patterned as Moroccan tiles, the spirals of chromosomes in an onion root cell reminiscent of a mobile by Miro.