Download C. elegans

IB404 - Caenorhabditis elegans 1 – Feb 8
1. C. elegans was being studied by a few UK naturalists in
the 1960s when South African Sydney Brenner at
Cambridge, England decided to leave bacterial molecular
genetics for a simple animal that would allow detailed
developmental and neurobiological/behavioral studies.
He is now retired at the Salk Institute in San Diego.
2. He recruited John Sulston to join him, and Sulston
undertook the remarkable serial EM sectioning that
allowed identification of every one of the 996 cells in the
hermaphrodite (males have a few more cells). Sulston
went on to run half of the genome project and later the
British component of the human genome at Sanger Center.
3. With a Harvard PhD, Robert Horvitz joined Sulston’s
effort and discovered apoptosis (programmed cell death) in
worms, then moved back to MIT. They received the
Physiology/Medicine Nobel Prize in 2002, understood as a
prize to the C. elegans field in general as a model animal.
4. The worm community is largely derived from
Brenner’s postdocs and students, including Bob
Waterston, who ran the WashU genome center.
5. They sequenced the genome using a clone-by-clone approach, with a
physical map of ±17,500 cosmid clones with inserts of 20-40 kb in
about 700 clusters. Gaps were spanned by an overlapping map of
±3,500 YACs with 150-500 kb inserts. This map was organized using
fingerprinting of restriction enzyme digest patterns of these clones,
looking for overlaps.
Lines at the top are YAC clones (names start with a Y). Below are
cosmid clones. The yellow boxed cosmids are those sequenced.
6. A draft genome was published
in 1998, and finished to the last
base pair in 2001.
7. The genome is about 100 Mbp
in six roughly equal size
chromosomes, I-V and X.
8. Genes are evenly spread across
the chromosomes (light blue), but
highly conserved genes (those
with yeast matches – dark blue)
tend to be in the middle of
chromosomes, while various
repeats and transposons are near
the ends. This is the opposite of
other animals, perhaps related to
their distributed centromeres
(worms are holocentric, with no
single centromere).
9. Genes are generally reasonably small,
with generally short introns, with a clear
peak around 60 bp, but some introns
contain transposons, and a few genes are
in large introns of other genes.
10. Initial annotation using various
automated methods came up with
~19,000 proteins-coding genes, and
refinement has brought that near to
~20,000.
11. Initial analyses could only compare
proteins with E. coli, yeast, and a subset
of available human genes. Remarkably,
about 36% of the predicted C. elegans
genes had confident human matches,
supporting model status for this worm.
12. Worm/yeast comparisons allow identification of basic eukaryotic
cell proteins, as well as those specific to multicellular worms. Most
fundamental proteins are simple orthologs, but cytoskeleton proteins
have many paralogs in worms (red), having duplicated within animals.
13. Worms have many classes of proteins absent from yeasts, as well as
many highly expanded protein families containing conserved domains.
I worked up their ~1800 putative chemoreceptors (7TM proteins)!
14. About 15% of nematode genes are organized into operons of 2-8
genes, analogous to the operons of bacteria. The genes in these operons
are of course transcribed together as a polycistronic transcript and hence
are coordinately expressed, but the downstream genes are translated as a
result of a trans-splicing event in which a short RNA leader is spliced
onto the front of the internal gene mRNAs (SL in figure below).
Some classes of genes are commonly in operons, e.g. those involved in
RNA degradation, the basic machinery of transcription, RNA splicing
and translation, as well as those that encode mitochondrial proteins;
generally ancient conserved genes. Other gene classes are never in
operons, e.g. the ~1800 chemoreceptor genes.
The 5'-most gene is mitochondrial ATPase inhibitor-1 (mai-1). This gene is not TRANS-SPLICED.
The two downstream genes, glyceraldehyde 3-phosphate dehydrogenase-2 (gpd-2) and
glyceraldehyde 3-phosphate dehydrogenase-3 (gpd-3), encode isoforms of a glycolytic enzyme.
15. WormBase is a remarkable database of all features of C. elegans
biology, largely centered around the genome sequence and annotation.
Many “tracks” can be viewed, including here the gene name, model of
exons/introns, alternative splicing, cDNA and EST sequence evidence, C.
briggsae alignments, RNAi experiments, and ORFeome project, e.g.
synaptotagmin is an 8 exon gene, with alternative splicing of exon 6.
In lieu of generating mutants of every gene,
screens of knockdowns of gene expression can
be conducted using RNAi in worms. The neat
trick is that one can feed worms bacteria
expressing double-stranded RNA and RNAi
against that gene is induced. So a region of
each gene is amplified by PCR from worms,
cloned into a special plasmid vector (right), and
these recombinant plasmids are transformed
into a mutant strain of E. coli that does not
have much RNase activity. A library of ~18,000
such bacterial strains was generated and then
worms fed each of these and phenotypes
examined as in a mutant screen. E.g. a GFP
transgene (a) was knocked down by feeding a
RNAi construct for 24 (b) or 48 (c) hours.
About 10% of genes knocked
down had obvious phenotypes
such as lethality, sterility, or
growth defects. Most of these
genes encode ancient conserved
proteins involved in fundamental
cellular processes. Genes yielding
viable phenotypes were more
likely involved in signaling and
other functions. ±700 genes were
lethals or just 5% of those tested,
which seems somewhat low to me,
probably because the knockdowns
are not complete; they are leaky.
Lethal
Other phenotype
Another RNAi screen looked for
worms that stored more or less fat,
indicated by a red fat-binding dye.
Major players in this pathway were
identified, such as the insulin-like
protein (daf-2) and transcription factors
that regulate its expression (daf-16),
and various enzymes involved in fat
metabolism.
For example, in daf-2 mutants that
accumulate fat, RNAi against a fatty
acid elongase counteracts that - last
two panels.
Remarkably about 20 of these ±400
genes are 7TM chemoreceptors,
perhaps indicating importance of
detection of fats either during feeding
or during accumulation.
Another RNAi screen searched for genes that when knocked down led to
increased lifespan. It was already known that a remarkable variety of
mutant genes can lead to increased lifespan in worms, up to about 50%
per gene and much more for combinations of mutants. The inevitable
conclusion is that much of lifespan regulation involves evolved shortness
of lifespan. This RNAi screen detected numerous mitochondrial function
genes, e.g. cytochrome oxidase subunits.
The interpretation is
that some, but not
complete, knockdown
of mitochondrial
function reduces
oxidative stress on the
organism, much as
restricted calorie diets
in mice, monkeys,
and other animals
increases lifespan.