Short Take
Dr. Clean Genes
Sorting out the genome exposes vital DNA sequences
By Amy Adams
Photograph by Bryce Duffy
Neat freaks be warned -- the human genome is a very untidy place. It
is strewn with random DNA and dotted with redundant genes. Viral sequences
are wedged between human genes. Replicates of single genes have scattered
throughout the chromosomes. That's in addition to the mutations that
crop up in each generation. And it's not just us -- the genomes of all
vertebrates have become increasingly cluttered and mutated with each
evolutionary step.
Except, that is, for some isolated genetic sequences that have remained
virtually untouched by evolutionary debris. Despite millions of years
of accumulated change, these genetic snippets are still nearly identical
even in far distant species. According to geneticists such as Arend Sidow,
PhD, these unaltered regions are no mere evolutionary oversights -- they
are regions too important to change. Any stray mutations, inserted sequences
or genomic rearrangements that cropped up must have so ruined the gene's
function that the organism died, taking its problematic DNA with it.
Sidow, an assistant professor in Stanford's pathology and genetics departments,
suspects that locating these unchanged sequences could guide researchers
to the most important parts of the genome -- prime hunting grounds for
disease-causing mutations or sequences that control when and how a gene
is used. "They have enormous predictive value for mutations in humans," Sidow
says. The more conserved the region the more deadly any mutation may
be. But so far finding them has been difficult.
It turns out that software Sidow developed to help deduce evolutionary
relationships between genes can be put to use in finding conserved DNA
sequences. The software, called ProPhylER (short for Protein Phylogenies
and Evolutionary Rates), is particularly adept at seeing through accumulated
genetic clutter to work out how genes are related. It figures out when
the genes duplicated and which members of a gene family in one species
are related to members of similar gene families in another species. "The
initial goal was to find out what the gene relationships were," Sidow
says, adding that this goal of tidying up gene family trees has been
with him since his graduate school days. "I wanted to put the genes in
a rational framework," he says. Once he works out these gene family trees,
he can turn ProPhylER's power to picking out similarities in the related
genes.
To run a ProPhylER analysis, Sidow selects gene sequences from among
those housed in online databases of publicly available genes. These include
sequences from biology's usual suspects such as humans, mice, rats and
frogs, as well as a few sequenced genes from animals such as chickens
or fish. ProPhylER then plots the degree of similarity across the gene's
length, with spikes showing where evolution has caused the sequences
to diverge and dramatic dips where the genes are nearly identical. Many
such dips exactly correlate with regions that are known to be important
to a protein's function -- regions that recognize a hormone or bind two
proteins together. Any evolutionary change to this region would have
rendered the protein useless, so the gene sequence for that region stayed
the same while neighboring sequences accumulated changes and rearrangements.
Revealing the Jewels Amid the Junk
As an example of ProPhylER's use, Sidow points to the gene that's involved
in cystic fibrosis. The ProPhylER (pronounced "profiler") analysis for
this gene highlights a region just before the gene begins that's similar
in several species. Because this region has changed very little while
the bases around it have followed their own evolutionary paths, that
region is bound to be important for the gene to work correctly. This
conserved section lines up with a known binding site for a protein that
latches onto the DNA and regulates when and where the gene gets used.
A mutation here prevents the protein from binding and turning this gene
on in the appropriate tissue, leading to cystic fibrosis.
That ProPhylER identifies regions of known importance is reassuring
to Sidow, but the software is only truly useful if it can guide researchers
through the genetic jumble to regions of hitherto unrecognized importance.
To that end, Sidow has begun collaborations with two Stanford graduate
students who are using ProPhylER to speed their search for conserved
sequences. Although for now Sidow and ProPhylER-adept members of his
lab have to run the analysis, he hopes one day to have the software available
online in a user-friendly format that researchers worldwide could access.
One of Sidow's collaborations is with Shelley Force Aldred, a graduate
student in the lab of genetics professor Richard Myers, PhD. Force Aldred
entered graduate school interested in understanding how stretches of
DNA that come as much as 80,000 base pairs before or after a gene can
regulate when and where that gene is used. Traditionally, the only way
to find these regions is a patience-testing technique called bashing
that requires making mutations along that enormous DNA landmass and hoping
to find something interesting – a process akin to poking random
holes in the ground in the hopes of finding oil. "If you wanted to understand
that region you had to bash the heck out of it," Force Aldred says.
Force Aldred decided to speed up this tedious process with a two-pronged
approach. One of those prongs uses ProPhylER to search for stretches
of DNA that are virtually unchanged in the DNA regions surrounding the
gene of interest in humans and mice. "If you find noncoding DNA that's
highly conserved it must be something important," she says. It's almost
impossible to find these conserved regions without ProPhylER because
they may be at dramatically different locations before the human and
mouse genes. "Even if I had sequence data, I don't have the skills to
do the alignment," Force Aldred says.
The other prong involves developing a new bench science to pull out
DNA chunks that regulate where genes are turned on and off in tissue
culture. In theory, the regions Force Aldred pulls out experimentally
should be those same regions that show a dramatic dip in Sidow's plot
of DNA variability. "It's like a validation of what we're doing," Force
Aldred says. It both substantiates her time-saving approach so others
can use it with confidence and shows the value of Sidow's data. "We hope
it will shave years off of bashing experiments," Force Aldred says.
Picking Out Key Protein Parts
Force Aldred stresses that although she's using Sidow's data and tools
to examine gene regulation, a very important use for ProPhylER will be
what it tells researchers about the proteins fashioned by the genes.
One project that takes advantage of this capability is Sidow's collaboration
with another graduate student, Dennis Ko, who works in the lab of developmental
biology professor Matthew Scott, PhD.
Ko began his graduate career studying the childhood neurodegenerative
disease Niemann-Pick C. Although other researchers had found that two
genes called NPC1 and NPC2 are mutated in children with the disease,
no one knew much about these proteins' normal roles. Ko heard Sidow talk
at a genetics department retreat and realized that ProPhylER could speed
up his studies. "I thought I could apply his tools to point out regions
of importance," Ko says.
Sidow used ProPhylER to analyze NPC2 in six different vertebrate species.
First he worked out which proteins from the other species are directly
related to NPC2. He then compared those evolutionarily related genes
for similarities. From this, ProPhylER zeroed in on several regions that
were remarkably consistent in each species. "Because nothing is known
about how parts of NPC2 are involved in fulfilling the protein's molecular
function, this was the first glimpse of which regions are important," Ko
says.
Ko made mutations in the conserved regions and tested the modified proteins
in tissue culture. Sure enough, mutations in each of the conserved regions
prevented the NPC2 protein from functioning normally in the cells. Some
mutations blocked NCP2 from binding cholesterol -- one known function
of the protein -- but others had no effect on cholesterol binding, telling
Ko that the protein has another role in the cell. Ko said that without
Sidow's analysis he would have either made mutations blindly or guessed
which regions were likely to be important based on similarities to other
known cholesterol-binding proteins.
Speeding up genetic research such as Ko's and Force Aldred's looks like
ProPhylER's most immediate use. But Sidow sees a day when ProPhylER could
perform the ultimate in genomic spring cleaning -- pinpointing all the
most conserved regions of the human genome. "Maybe we can narrow the
genome down to a much smaller fraction that's important," Sidow says.
With that roadmap to the genome's most conserved sequences, researchers
seeking disease-causing mutations need only look in isolated spots rather
than sequencing the clutter of the entire genome. "You give me a genotype
and I can say 'this person has a mutation at this position.' " From there,
Sidow says, ProPhylER could predict just how bad any mutations might
be -- if the mutation alters a highly conserved region it could spell
trouble. How and when such an analysis could become part of medicine
Sidow leaves for others to decide.
"More information has never been bad," he says, "It's a matter of how
that information is used."
My Kingdom for a Cow
Cow genome data would strengthen analysis
Forget sequencing the genome of monkeys and apes. Sidow would like science
to turn its attention to the cow.
That's because the more distant the evolutionary relationship between
the vertebrates used in a ProPhylER analysis, the greater the accuracy
of the results. Think of it this way: How surprising would it be that
humans and our close relatives the apes share many genetic sequences?
Not very.
But if humans, cows and dogs all have a similar sequence wedged amidst
accumulated mutations, then that sequence must have been preserved for
a reason. "The more different species you have the better the statistics
are," Sidow says.
With that in mind, Sidow says that the cow and the dog genomes would
do nicely for his purposes, or the pig and the cat. Anything, really,
so long as it comes from a far-flung branch of the vertebrate evolutionary
tree.
Comments? Contact Stanford Medicine at
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