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Knocking out 19,000 worm genes, one by one
Researchers report data on functional analyses of the C. elegans genome
By Bijal P. Trivedi

A microscopic worm is being used to decipher the function of hundreds of genes, many of which are present in humans. Two teams of researchers, one in the UK and one in Germany, are knocking out genes one at a time to understand the role each gene plays in building a worm. The UK team knocked out a gene and watched each worm under the microscope to see the effect. The German team made thousands of time lapse films of the worm's embryonic development to see how each gene loss affected early growth. Together the two groups have determined the function of 472 genes.

Time-lapse photograph of a worm embryo. About 10 minutes after fertilization, the embryo divides for the first time, forming two cells.

"The Caenorhabditis elegans worm is so well understood, that when things go wrong during development it is very easy to notice," says Julie Ahringer, of the University of Cambridge in the UK. The transparent, one-millimeter long worm is one of the most extensively studied creatures on earth and a favorite among geneticists. The fate of each of its 959 cells is known from birth to death—302 nerve cells, 95 muscle cells, 20 intestinal cells, etc. It goes from egg to adult in three days, and its entire genetic sequence containing more than 19,000 predicted genes has been deciphered.

The method used by Ahringer's group helped them identify broad categories of genes on chromosome I. Some affect the movements of chromosomes as the cell grows and divides. Many genes are required to build and maintain the cell's structure and all the compartments within. Others are essential for communicating with other cells, producing energy, or manufacturing proteins.

Ahringer finds Unc genes the most interesting. When these genes are blocked, the muscles and nerve cells are affected and the worms become uncoordinated. "Normally these worms move with a graceful sinusoidal wave-like motion," says Ahringer. "But when an Unc gene is blocked the worms move too quickly or too slowly or not at all."

Another class of genes that caught Ahringer's attention led to a high incidence of males (Him) being born. C. elegans worms are in most cases hermaphrodites, with two X chromosomes, but there is a low frequency of males born that have only one X chromosome. When any of the Him genes were blocked, there was a huge increase in the number of males born. These sex-determining genes could play a role in separating the chromosomes, says Ahringer.

The Cambridge team knocked out genes by feeding the worms genetically engineered bacteria. The researchers produced close to 2500 strains of bacteria, each of which produces a double stranded RNA (dsRNA) molecule that is specifically tailored to block the action of one gene. When the worm eats the bacteria, the dsRNA is absorbed through the worms' intestine and disrupts the role of a single specific gene throughout its body. Ahringer and her colleagues then observed the worms through a microscope to see the effects of blocking a specific gene. The UK team assigned a role to about 14 percent of the genes on chromosome I.

The German team took a different approach. "Our goal was to find every single gene on chromosome III that was required for a cell to divide," says Anthony Hyman, of the Max-Planck-Institute for Cell Biology and Genetics, in Dresden, Germany. "We wanted to concentrate on this one process and study it exhaustively."

Hyman's team injected the dsRNA directly into the gonads of an adult worm using a tiny glass needle. This ensures that the embryonic worm will lack whatever gene corresponds to the dsRNA. About 24 hours after the injection, the embryo, which is a single cell, is removed from the adult worm and placed under a microscope. The microscope was hooked up to a special camera that took a picture of the worm embryo every five seconds until the worm had divided twice to form a bundle of four cells.

Hyman's team injected over 12,000 worms and made a 20-minute movie of each worm embryo growing from the one to four cells. "The injections were tedious but fairly quick. What took a huge amount of time was watching and analyzing the movies," says Hyman.

"The whole process of cell division hardly ever varies. When it does, the eye is incredibly good at picking up these very small changes," says Hyman. Hyman and his colleagues watched movies showing how a normal healthy worm embryo goes from one to four cells. The scientists contrasted this process with worm embryos that lacked a specific gene and watched to see whether the process was disrupted. Of the 2,174 genes on chromosome III, 133 were necessary for cell division.

In a span of 20 minutes, a normal worm embryo reaches the early four-cell stage (left). During the same period, a worm lacking a gene involved in energy production only reaches the first cell division (right). View movies of normal and mutant worm embryos at the Hyman laboratory's data page.

Of these 133 genes, almost half are also found in yeast and the fruit fly. "The fact that these genes are so highly conserved suggests that cell division is a very ancient process," says Hyman.

Knocking out some genes produced intriguing results. When Hyman knocked out a gene critical for energy production, he expected the embyro to die. Instead, the growth of the embryo slowed as if responding directly to the energy shortage but was still able to divide and reach the four cells.

Both Ahringer's and Hyman's team want to continue their gene by gene analysis for the remaining 5 chromosomes, each sticking to their own method. Hyman has founded a company called Cenix Biosciences that has scaled up the movie-making to analyze the roughly 17,000 genes remaining on chromosomes I, II, IV, V, and X. Cenix will focus on genes involved in cell division.

Ahringer intends to make bacterial strains corresponding to each gene on the other chromosomes.

The library of bacterial strains that Ahringer's team has produced will be useful for the whole community of worm researchers, says Hyman. Researchers wanting to study the effect of a particular gene will be able to take the bacteria corresponding to their gene of interest and do their own experiments. Ahringer has been approached by several pharmaceutical companies that would like to use her system for drug testing.

"The most exciting part of the study was that it worked," says Ahringer. "A lot of people tried to talk us out of doing this project, but we found the function of 14 percent of the genes we analyzed. No one has done this on a large scale before—it is really a technological feat."

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Fraser, A. et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408, 325-330 (November 16, 2000).
G÷nczy, P. et al. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III. Nature 408, 331-336 (November 16, 2000).
To view movies from the entire chromosome III screen visit:

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