Box 7.1Analysis of chromosome I of Caenorhabditis elegans by RNA interference

Functions have been assigned to 339 genes on C. elegans chromosome I after individual inactivation by the RNA interference technique.

C. elegans is a tiny nematode worm (see Figure 12.22) which has been extensively used as a model for the development of multicellular eukaryotes (Section 12.3.2). It has many advantages for this type of research, being easy to grow in the laboratory and having a generation time of just a few days. The outer surface of the worm is transparent, which means that its internal development can be followed by microscopy. Every cell division in the developmental pathway from fertilized egg to adult worm has been charted, and every point at which a cell adopts a specialized role has been identified. In addition, the entire connectivity of the 302 cells that comprise the worm's nervous system has been mapped.

The draft sequence of the 97-Mb C. elegans genome was completed in 1998 (see Table 2.1). This was an important step forward because many molecular biologists believe that analysis of the C. elegans genome will eventually enable a link to be established between the worm's developmental pathway and the activities of its genes. For this goal to become a reality it will first be necessary to identify the functions of all, or at least the majority, of the genes in the C. elegans genome. Genetic analysis had identified a few important genes even before the genome sequence was available, and a detailed homology analysis enabled functions to be tentatively assigned to a few more. But over 60% of the 19 099 predicted genes in the draft genome sequence were unidentified. Understanding the genome sequence, and establishing the link between the genome and the worm's developmental pathways, will clearly require a substantial amount of experimental work. During the years since the draft sequence was published, various techniques have been used in attempts to determine the functions of the unidentified C. elegans genes. One of these techniques has been RNA interference.

RNA interference with C. elegans

The basis of RNA interference is shown in Figure 7.16. The key step is the introduction into a worm of a double-stranded RNA molecule that will give rise to single-stranded interfering RNAs specific for a particular gene. The easiest approach is simply to feed the RNA to the worms. C. elegans eats bacteria, including Escherichia coli, and is often grown on a lawn of bacteria on an agar plate. If the bacteria are synthesizing a double-stranded RNA with the same sequence as a C. elegans gene then, after ingestion, the RNA interference pathway begins to operate.

Cloning techniques (Section 4.2) can be used to prepare an E. coli strain that makes a double-stranded RNA specific for a C. elegans gene. To begin the procedure, the C. elegans gene is amplified by PCR (Section 4.3). The PCR product is then ligated into a special cloning vector, called L4440, which possesses two short DNA sequences, called promoters (Section 9.2.2), that initiate RNA synthesis by the highly active RNA polymerase of the T7 bacteriophage. One of these sequences is located to the right of the ligation site and one to the left, in opposite orientations, so that the RNA that is synthesized is a copy of both strands of the inserted PCR product (see Figure below). A strain of E. coli that contains the T7 RNA polymerase is transformed with the recombinant L4440 molecule, which is copied into two complementary RNA strands, which link together by base-pairing to produce a double-stranded RNA. The bacteria are grown in the well of a microtiter tray and three C. elegans worms are added. The worms eat the bacteria, ingesting the double-stranded RNA. This initiates the RNA interference process, which subsequently inactivates the target gene in the worms' genomes.

Inactivation of genes on chromosome I

How successful has RNA interference been with C. elegans? A study of the 2769 predicted genes on chromosome I provides a good illustration of what has been achieved. This study made use of a library of 2445 bacterial clones, targeting 2416 different genes. In 339 cases, gene inactivation by RNA interference led to a detectable change in the phenotype of the treated worms. The commonest change was ‘embryonic lethal’, meaning that embryos produced by the treated worms died at an early stage in their development. Other worms became sterile after gene inactivation, or failed to develop beyond the larval stage. Phenotypes that were only detectable in the adult worm were less common, but these included interesting defects such as ‘uncoordinated’, caused by abnormalities in the neuromuscular system, and ‘high incidence of males’, which occurs when these normally hermaphroditic organisms develop male characteristics at a higher frequency than usual. The table below lists some of the phenotypes that were observed (note that many worms displayed more than one phenotype).

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Many of the genes whose inactivation led to a detectable change in phenotype were ones that had previously been unidentified, but a few had already been assigned biochemical functions by genetic studies or homology analysis. It was therefore possible to begin to examine the relationship between the specific biochemical activities coded by individual genes and the more general phenotype changes observable in the living worm. As expected, inactivation of genes that specified enzymes involved in central metabolic pathways often resulted in one of the more drastic phenotype changes, such as sterility or death of embryos. The less dramatic phenotypes, such as ‘uncoordinated’, were more frequently associated with genes whose products play a role in cell structure or organization. These comparisons also showed that most of the unidentified genes give rise to a subtle change in phenotype when inactivated, suggesting that they have specialized biochemical functions, rather than coding for important enzymes. To identify these genes it might therefore be necessary to go beyond simple inactivation studies: we really need to find out more about the biochemistry of development before the link can be made with the genome.

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Lessons for the functional analysis of other genomes

Only 14% of the genes on C. elegans chromosome I could be assigned a function after inactivation by RNA interference. This success rate reflects, to a certain extent, the limited nature of the analysis that was adopted in this particular study. For example, no attempt was made to assess the effect of gene inactivation on the ability of the worms to respond to environmental stress. But the relatively low success rate underlines how difficult it can be to detect a change in phenotype after inactivation of a single gene (see Box 7.1). The implication is that substantially less than half of the 19 099 genes of C. elegans will be identifiable by straightforward phenotype analysis, and that more extensive and time-consuming tests will be needed to assign functions to the remainder. If this view turns out to be correct then the complete annotation of gene functions for the human genome, whether by direct analysis of those genes or by studies of homologous genes in other organisms, will take a great deal of time.

References

  1. Timmons L. Fire A. Specific interference by ingested dsRNA. Nature. (1998); 395:854. [PubMed: 9804418]
  2. Fraser AG,, Kamath RS,, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature. (2000);408:325–330. [PubMed: 11099033]
Phenotypes that were observed.

Table

Phenotypes that were observed.

Box Icon

Box 7.1

The phenotypic effect of gene inactivation is sometimes difficult to discern. Once a gene-inactivated yeast strain, knockout mouse, or equivalent with any other organism has been obtained, the next stage is to examine the phenotype of the mutant in order (more...)

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From: Chapter 7, Understanding a Genome Sequence

Cover of Genomes
Genomes. 2nd edition.
Brown TA.
Oxford: Wiley-Liss; 2002.
Copyright © 2002, Garland Science.

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