Eduardo CésarIt was with the Rhynchosciara that Pavan discovered the phenomenon of gene amplification (left). Drosophilae, fruit flies: model organisms for genetic research (right)Eduardo César
As is very often the case with scientific discoveries, the most important one made by Crodowaldo Pavan was by chance. On one of his outings to collect drosophilae (fruit flies, which are a model organism for genetics research) in the late 1940s, at a banana plantation in Mongaguá, on the southern coast of the state of São Paulo, Pavan kicked a banana tree that was on the ground and, underneath it, discovered a ball of what looked like larvae. His scientist’s habit and instinct made him take some larvae back to the laboratory. He discovered that they were indeed the larvae of a fly of the genus Rhynchosciara, which would later enable him to discover the phenomenon of gene amplification, which overturned one of biology’s dogmas, namely that DNA was constant.
The larvae were of the species Rhynchosciara angelae (which is nowadays known as R. americana). According to Luiz Edmundo Magalhães, they turned out to be excellent research material for several reasons. “The larvae have large chromosomes in the cells of their saliva glands,” he explains. “Moreover, all the larvae in each so-called ‘cake’ are the offspring of a single female; they have gregarious habits and develop simultaneously.” André Perondini recalls that because of the last characteristic Pavan used to say that analyzing samples from one group of them on successive days was like watching the film of a phenomenon.
The subsequent research undertaken by Pavan and his collaborators with the Rhynchosciara angelae yielded a series of scientific papers that were published over the course of the next few years. The most important of these, co-authored by Pavan and his collaborator Marta Breuer, was published in 1955. The text threw light on a revolutionary discovery, a historic landmark in biology, as it overturned a paradigm of science.
Up until then, there was a general belief that the number of genes and, consequently, the amount of DNA in the cells of each living species was constant. In the case of the Rhynchosciara Pavan noted the appearance of certain formations at specific points of the polytenic chromosomes (giant chromosomes that appear in the cells of the fly’s saliva glands), which registered significant growth, and called them puffs. “Studying the formation of these puffs by means of a special technique, he declared that there was multiplication of the genes in this region, with DNA synthesis,” explains Magalhães. “This discovery led to the downfall of the dogma that the quantity of DNA in a cell was constant.”
This is a mechanism that is nowadays known as gene amplification, whereby, in certain cells and at specific points of development, some genes produce extra copies, in addition to the simple duplication of the chromosome filament, such as occurs in the case of normal cellular division. When he described the phenomenon of gene amplification, Pavan also came up with an interpretation for it. The interpretation mentions the well-known fact that in order for the cells to function, each gene is responsible for the production of a specific product, and that this is carried out, at a certain point in time, by the transcription of specific RNAs.
Culmination Point
According to Perondini, this process takes place at a specific rate per unit of time and it is unlikely that the pace at which this occurs could be increased under the individual body’s normal conditions. “Therefore, suppose that at specific moments in the life of an organism there was a need for a very large quantity of a specific gene product,” he says. “How could this production be increased? It could take place either by increasing the amount of time that the process lasted or by increasing the gene’s rate of transcription. It is extremely difficult for either of these situations to occur. Another way would be to increase the number of copies of the gene (amplification), thus ensuring that the necessary RNA was produced in larger quantities, during the same unit of time.”
Even though it was revolutionary – or perhaps because of this very fact –, it took eight years for Pavan’s discovery to be accepted by the scientific community. “During this period I presented the data and people would say: ‘Your data is valid, but this is an exception. It’s an insect’”, Pavan explained in an interview that is included in the book Cientistas do Brasil [Scientists of Brazil] (CNPq, 1998). “Up until the point at which they confirmed that certain genes multiply more than others in the chromosome, and that this was not an exception and even occurred in human beings.”
The research with the Rhynchosciara was the culmination of Pavan’s scientific career. Prior to this, he had carried out research into other species of animals. The first of these was the blind cavefish, Typhlobagrus kronci, which lives in the caves of Iporanga (state of São Paulo), on which he wrote his doctoral thesis, completed in 1944.
After obtaining his doctorate, the geneticist devoted himself to research with drosophilae, influenced by Theodosius Dobzhansky, who came to Brazil for the first time in 1943. “The group that Pavan belonged to carried out a survey of the native species of drosophilae throughout almost all of Brazil,” recalls Perondini. “It was a very important project at the time as the population study of drosophilae was still in its very early stages.”
In the following years, Pavan’s work initially centered basically on the collection, cataloguing and description of the Brazilian species of this fly. “This resulted in a very detailed description of the chromosomes, genitals and body features of a large number of tropical species,” explains Perondini. “Afterwards the work progressed with analyses of correlations between species environment, population size and geographical distribution of the different groups of species.”
In the mid 1970s, Pavan became interested in a new area of research. He suggested to some scientists who were developing research in basic genetics that they should start to study insects that were of financial interest, in other words, those that cause harm to human beings or losses to crop and livestock farming. Therefore, up until the end of his life he maintained his links with the laboratory. During his last few years, for instance, he switched his attention to another very important biological problem, the role of bacteria in nitrogen fixation.
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