Letter to Max Perutz from Sydney Brenner
5 June, 1963
These notes record and extend our discussions on the possible expansion of research activities in the Molecular Biology Laboratory.
First, some general remarks. It is now widely realized that nearly all the “classical” problems of molecular biology have either been solved or will be solved in the next decade. The entry of large numbers of American and other biochemists into the field will ensure that all the chemical details of replication and transcription will be elucidated. Because of this, I have long felt that the future of molecular biology lies in the extension of research to other fields of biology, notably development and the nervous system. This is not an original thought because, as you well know, many other molecular biologists are thinking in the same way. The great difficulty about these fields is that the nature of the problem has not yet been clearly defined, and hence the right experimental approach is not known. There is a lot of talk about control mechanisms, and very little more than that.
It seems to me that, both in development and in the nervous system, one of the serious problems is our inability to define unitary steps of any given process. Molecular biology succeeded in its analysis of genetic mechanisms partly because geneticists had generated the idea of one gene-one enzyme, and the apparently complicated expressions of genes in terms of eye color, wing length and so on could be reduced to simple units which were capable of being analyzed. Molecular biology succeeded also because there were simple model systems such as phages which exhibited all the essential features of higher organisms so far as replication and expression of the genetic material were concerned, and which simplified the experimental work considerably. And, of course, there were the central ideas about DNA and protein structure.
In the study of development and the nervous system, there is nothing approaching these ideas at the present time. It is possible that the repressor/operator theory of Jacob and Monod will be the central clue, but there is not very much to suggest that this is so, at least in its simple form. There may well be insufficient information of the right kind to generate a central idea, and what we may require at the present is experimentation into these problems.
The experimental approach I would like to follow is to attempt to define the unitary steps of development using the techniques of genetic analysis. At present, we are producing and analyzing conditional lethal mutants of bacteria. These are mutants which are unable to grow at 44C but do grow normally at 37C. The mutations affect genes controlling the more sophisticated processes of the bacterial cell, and some work which we have already done indicates that it will be possible to dissect the process of cell division into its unitary steps. We have mutants in which neither a cell membrane septum nor a cell wall is made, others in which a septum is made but not a cell wall septum and so on. We have mutants in which the control of DNA replication is affected. I intend to expand this research activity in the near future.
Our success with bacteria has suggested to me that we could use the same approach to study the specification and control of more complex processes in cells of higher organisms. As a first stage, I would like to initiate studies into the control of cell division in higher cells, in particular to try to find out what determines meiosis and mitosis. In this work there is a great need to “microbiologize” the material so that one can handle the cells as one handles bacteria and viruses. Hence, like in the case of replication and transcription, one wants a model system. For cell division, in particular meiosis, the ciliates seem the likely candidates. Already, in these cells, the basic plan of meiosis is present and there is no doubt that the controlling elements must be the same in ciliates as they are in the oocytes of mammals.
Another possibility is to study the control of flagellation and ciliation. This again is a differentiation in higher cells and its control must resemble the control in amoebo-flagellates.
As a more long term possibility, I would like to tame a small metazoan organism to study development directly. My ideas on this are still fluid and I cannot specify this in greater detail at the present time.
As an even more long term project, I would like to explore the possibilities of studying the development of the nervous system using insects…
From The Nematode Caenorhabditis elegans, WB Wood and the community of C elegans researchers, eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988.
Why Caenorhabditis elegans? Why has it been so important in biology? Interestingly, Sydney Brenner was turned on to this nematode as a model system by Dr. Ellsworth C. Dougherty, then in the Department of Nutritional Sciences at the University of California at Berkeley.
Actually, Dougherty first recognized the potential of C. briggsae, which had been found by Margaret Briggs on the campus of Stanford University in Palo Alto, California, in 1944 and used in her MS studies (Briggs, 1946). Briggs studied the lifecycle of what she identified as Rhabditis sp. in association with bacteria and in various culture media devoid of other organisms. She showed that the population could not be sustained in the absence of bacteria or even on dead bacterial cells, living bacteria were a necessary food source. However, survival of individuals was greater on some bacteria-free media than others.
Later (1949), Dougherty and Victor Nigon of France described the nematode used by Briggs as Rhabditis briggsae (Dougherty and Nigon, 1949) and later as Caenorhabditis briggsae. Dougherty wanted to culture nematodes in defined media, and was almost successful except for the necessary inclusion of a liver extract. The liver extract probably provided cholesterol which was later found to be necessary.
Dougherty was a medical doctor with the Permanente Medical Group who began to work on nematode nutrition in 1947, initially with the sexually-reproducing Rhabditis pellio. When he became aware that the nematode studied by Margaret Briggs was a self-fertilizing hermaphrodite, he realized that the impact of genetic variability could be reduced; he switched to that nematode. Briggs had attempted to culture the nematode on 12 gram -ve bacteria and 10 that were gram +ve. Six of the gram -ve bacteria, but only one of the gram +ve, supported the nematode monoxenically. Dougherty wondered what the differences among these bacteria were. Margaret Briggs later married and became Margaret Briggs Gochnauer. She published (Gochnauer and McCoy, 1954) on responses of R. briggsae to antibiotics.
The potential value of Rhabditis species for genetic research was pointed out by Dougherty and Calhoun (1948). Caenorhabditis elegans was initially described and named Rhabditis elegans by Maupas (1900); it was subsequently placed in the subgenus Caenorhabditis by Osche (1952) and then raised to generic status by Dougherty (1955). The name is a blend of Greek and Latin (Caeno, recent; rhabditis, rod; elegans, elegant).
Two strains of C. elegans have historical importance. One strain, Bergerac, was collected from the soil near Bergerac, France, by Victor Nigon of the Universite de Lyon (Nigon 1949), and the other strain, Bristol, was isolated by L.N. Staniland (National Agricultural Advisory Service, London) from mushroom compost near Bristol, England (Nicholas et al. 1959). Staniland was an applied nematologist; he published extensively on the nature and control of a variety of nematode problems between 1926 and 1967, including rhabditid swarming in mushroom beds
The Bristol isolate was sent to Gunther Osche of Friederich Alexanders Universitat, Erlangen, Germany. I’m not sure about the date, I assume 1947 or 48 because Nigon certainly had it prior to 1949. Osche confirmed that it was a Rhabditis. It was later sent to Nigon who identified the species as R. elegans, conforming with the original description by Maupas (1900). Osche (1952) placed R. elegans and R. briggsae (and maybe others) in subgenus Caenorhabditis; Dougherty (1955) elevated the subgenus to a genus. (see Andrássy, 1983).
Nigon and Dougherty (1949) did some classic mating studies with C. briggsae and C. elegans. Among other things, I think that is where they found that hermaphrodites mated with males produced more male offspring. Also, they began to realize that these were prime candidates for genetic studies (Nigon, 1949, Dougherty and Calhoun, 1948, etc), and described the first morphological mutant of C. briggsae (Nigon and Dougherty, 1950).
The Bergerac strain of C. elegans could not be cultured at temperatures above 18C; at that temperature it became infertile. The Bristol strain can be cultured at temperatures up to 25C, though males will not copulate below 20C (Fatt and Dougherty, 1963; Nicholas, 1975). In several reported cases, rhabditid nematodes seem to be adversely affected by higher temperatures. For example, embryogenesis fails at temperatures of 25C and higher in Rhabditis cucumeris isolated from soil in the Central Valley of California (Venette and Ferris, 1997).
Through his work with Nigon, Dougherty obtained a culture of the Bristol strain of C. elegans. Dougherty who was then at the Kaiser Foundation Research Institute in Richmond, California. Warwick Nicholas was Lecturer in the Department of Zoology, University of Liverpool from 1955 until 1960. After that he went to Australian National University. In 1956, Nicholas established the first axenic cultures of both Bristol and Bergerac strains of C. elegans (Fatt, 1961; Nicholas and McEntegard, 1957). In 1957 and 1958 he was on leave from University of Liverpool and was a Traveling Fellow of the British Medical Research Council (MRC) funded by a Rockefeller grant. During the tenure of the fellowship, he worked with Dougherty and Dr. Eder Hansen in the Lab of Comparative Biology at the Kaiser Foundation Research Inst. in Richmond CA. Among other things, they were trying to determine the nature of undefined components Rb and Cb needed for axenic culture of C. briggsae. I think those components were provided by bovine liver extract and I assume Rb and Cb are the initial letters of R. and later C. briggsae) (Dougherty et al, 1959; Nicholas et al, 1959).
Consider the importance of the Nicholas MRC connection. Sydney Brenner was the mover and shaker at MRC and was debating the next steps in translating the successes of Watson and Crick into a greater understanding of “life”. See his proposal and letter to Max Perutz. Some ideas must have been transferred to Brenner through Nicholas. When Brenner visited our department (Nematology, UC Davis) in about 1987, he talked about visiting Dougherty (then at UC Berkeley) and discussing C. elegans as the candidate worm. Before that he had been thinking about C. briggsae as he began to learn about nematodes. He obtained his culture of the Bristol strain of C. elegans from Dougherty (Brenner, 1974).
In 1961, Dougherty moved to the Department of Nutritional Sciences at the UC Berkeley and continued to study the nutritional requirements and axenic cultivation of Caenorhabditis species, particularly C. briggsae, until his death in 1965. Although permanent cultures were maintained on nutrient agar slants inoculated with E. coli, an axenic medium with chemically undefined supplements was developed in 1954 (Dougherty et al. 1959).
In his studies, Dougherty recognized the potential for using C. briggsae in studies of genetics and developmental biology (Dougherty and Calhoun,1948) because of ease of culture and maintenance, different reproductive patterns (hermaphroditism and sexual), few chromosomes, less than 1000 cells, etc. and sold the idea to Brenner. Nigon’s original C. elegans isolate from France (Bergerac) strain is infertile above 18C, so the Bristol strain has been very important. Virtually all C. elegans genetics has been done with the Bristol strain, more specifically with the N2 line that Sydney Brenner derived from the Bristol culture he obtained from Ellsworth Dougherty.
Back in Cambridge, after his visit with Dougherty, Brenner extracted nematodes from his own garden and liked what he saw. Cultures of those worms became the N1 strain, but the worms on which were used in the genetics and development studies were the N2 strain from Dougherty. Dougherty and colleagues had found a mutation that would be useful in Brenner’s studies, an autosomal dominant gene for heat tolerance (Fatt and Dougherty, 1963; Brown, 2003).
Interestingly, until the mid 1970s, people working in developmental biology frequently confused C. briggsae and C. elegans and many of the cultures being used were mis-identified. In the mid 1970s, graduate student Paul Friedman working with Ed Platzer at UC Riverside developed the diagnostic criteria for separating the two species and resolved the confusion (Friedman et al, 1977). Friedman and Platzer were testing antibiotics on C. briggsae and C. elegans cultures and getting inconsistent results. They decided that their cultures were genetically different. The two species are distinguished by the pattern of rays in the male bursa; a difficult feature when males are rare. Friedman and Platzer used SDS electrophoresis and also separated isozymes of malate dehydrogenase. Their results were consistent with those of morphological characters and mating tests.