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| DATE | 2014-05-28 |
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| SUBJECT | Subject: [NYLXS - HANGOUT] interesting read
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30
Apr
The Man Who Rewrote the Tree of Life
By Carrie Arnold on Wed, 30 Apr 2014
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Carl Woese may be the greatest scientist you’ve never heard of.
“Woese is to biology what Einstein is to physics,” says Norman
Pace, a microbiologist at the University of Colorado, Boulder. A
physicist-turned-microbiologist, Woese specialized in the fundamental
molecules of life—nucleic acids—but his ambitions were hardly
microscopic. He wanted to create a family tree of all life on Earth.
Woese certainly wasn’t the first person with this ambition. The
desire to classify every living thing is ageless. The Ancient Greeks and
Romans worked to develop a system of classifying life. The Jewish
people, in writing the Book of Genesis, set Adam to the task of naming
all the animals in the Garden of Eden. And in the mid-1700s, Swedish
botanist Carl von Linné published Systema Naturae, introducing the
world to a system of Latin binomials—Genus species—that
scientists use to this day.
CarlWoese
Carl Woese in his later years
What Woese was proposing wasn’t to replace Linnaean classification,
but to refine it. During the late 1960s, when Woese first started
thinking about this problem as a young professor at the University of
Illinois, biologists were relying a lot on guesswork to determine how
organisms were related to each other, especially microbes. At the time,
researchers used the shapes of microbes—their morphologies—and
how they turned food into energy—their metabolisms—to sort them
into bins. Woese was underwhelmed. To him, the morphology-metabolism
approach was like trying to create a genealogical history using only
photographs and drawings. Are people with dimples on their right cheeks
and long ring fingers all members of the same family? Maybe, but
probably not.
“If you wanted to build a tree of life prior to what Woese did,
there was no way to put something together that was based upon actual
data,” says Jonathan Eisen, an evolutionary microbiologist at the
University of California Davis.
Just as outward appearances aren’t the best way to determine family
relations, Woese believed that morphology and metabolism were inadequate
classifiers for life on Earth. Instead, he figured that DNA could sketch
a much more accurate picture. Today, that approach may seem like common
sense. But in the late 60s and early 70s, this was no easy task. Gene
sequencing was a time-consuming, tedious task. Entire PhDs were granted
for sequencing just one gene. To create his tree of life, Woese would
need to sequence the same gene in hundreds, if not thousands, of
different species.
“When Woese first announced his results, I thought he was
exaggerating at first.”
So Woese toiled in his lab, sometimes with his postdoc George Fox but
often alone, hunched over a light box with a magnifying glass,
sequencing genes nucleotide by nucleotide. It took more than a decade.
“When Woese first announced his results, I thought he was
exaggerating at first,” Fox recalls. “Carl liked to think big,
and I thought this was just another of his crazy ideas. But then I
looked at the data and the enormity of what we had discovered hit
me.”
Woese and Fox published their results in 1977 in a well-respected
journal, the Proceedings of the National Academy of Science. They had
essentially rewritten the tree of life. But Woese still had a problem:
few scientists believed him. He would spend the rest of his life working
to convince the biological community that his work was correct.
Animal, Vegetable, Mineral
Following the publication of Linnaeus’s treatise in the 18th
century, taxonomy progressed incrementally. The Swedish botanist had
originally sorted things into three “kingdoms” of the natural
world: animal, vegetable, and mineral. He placed organisms in their
appropriate cubbyholes by looking at similarities in appearance. Plants
with the same number of pollen-producing stamens were all lumped
together, animals with the same number of teeth per jaw were grouped,
and so on. With no knowledge of evolution and natural selection, he
didn’t have a better way to comprehend the genealogy of life on
Earth.
Woese believed that DNA could unlock the hidden relationships between
different organisms.
The publication of Darwin’s On the Origin of Species in 1859,
combined with advances in microscopy, forced scientists to revise
Linnaeus’s original three kingdoms to include the tiniest critters,
including newly visible ones like amoebae and E. coli. Scientists
wrestled with how to integrate microbial wildlife into the tree of life
for the next 100 years. By the mid-20th century, however, biologists and
taxonomists had mostly settled on a tree with five major branches:
protists, fungi, plants, animals, and bacteria. It’s the
classification system that many people learned in high school biology
class.
Woese and other biologists weren’t convinced, though. Originally a
physics major at Amherst College in Massachusetts and having received a
PhD in biophysics from Yale in 1953, Woese believed that there had to be
a more objective, data-driven way to classify life. Woese was
particularly interested in how microbes fit into the classification of
life, which had escaped a rigorous genealogy up until that point.
He arrived at the University of Illinois Urbana-Champaign as a
microbiologist in the mid-1960s, shortly after James Watson and Francis
Crick won the Nobel prize for their characterization of DNA’s
double-helix form. It was the heyday of DNA. Woese was enthralled. He
believed that DNA could unlock the hidden relationships between
different organisms. In 1969, Woese wrote a letter to Crick, stating
that:
…this can be done by using the cell’s ‘internal fossil
record’—i.e., the primary structures of various genes.
Therefore, what I want to do is to determine primary structures for a
number of genes in a very diverse group of organisms, on the hope that
by deducing rather ancient ancestor sequences for these genes, one will
eventually be in the position of being able to see features of the
cell’s evolution.
This type of thinking was “radically new,” says Norman Pace, a
microbiologist at the University of Colorado, Boulder. “No one else
was thinking in this direction at the time, to look for sequence-based
evidence of life’s diversity.”
Evolution’s Timekeeper
Although the field of genetics was still quite young, biologists had
already figured out some of the basics of how evolution worked at the
molecular level. When a cell copies its DNA before dividing in two, the
copies aren’t perfectly identical. Mistakes inevitably creep in.
Over time, this can lead to significant changes in the sequence of
nucleotides and the proteins they code for. By finding genes with sites
that mutate at a known rate—say 4 mutations per site per million
years—scientists could use them as an evolutionary clock that would
give biologists an idea of how much time had passed since two species
last shared a common ancestor.
To create his evolutionary tree of life, then, Woese would need to
choose a gene that was present in every known organism, one that was
copied from generation to generation with a high degree of precision and
mutated very slowly, so he would be able to track it over billions of
years of evolution.
“This would let him make a direct measure of evolutionary
history,” Pace says. “By tracking these gene sequences over
time, he could calculate the evolutionary distance between two organisms
and make a map of how life on Earth may have evolved.”
The choice was especially fortuitous.
Some of the most ancient genes are those coding for molecules known as
ribosomal RNAs. In ribosomes, parts of the cell that float around the
soupy cytoplasm, proteins and ribosomal RNA, or rRNA, work together to
crank out proteins. Each ribosome is composed of large and small
subunits, which are similar in both simple, single-celled prokaryotes
and more complex eukaryotes. Woese had several different rRNA molecules
to choose from in the various subunits, which are classified based on
their length. At around 120 nucleotides long, 5S rRNA wasn’t big
enough to use to compare lots of different organisms. On the other end
of the spectrum, 23S rRNA was more than 2300 nucleotides long, making it
far too difficult for Woese to sequence using the technologies of the
time. The Goldilocks molecule—long enough to allow for meaningful
comparisons but not too long and difficult to sequence—was 16S rRNA
in prokaryotes and its slightly longer eukaryotic equivalent, 18S rRNA.
Woese decided to use these to create his quantitative tree of life.
His choice was especially fortuitous, Eisen says, because of several
factors inherent in 16S rRNA that Woese couldn’t have been aware of
at the time, including its ability to measure evolutionary time on
several different time scales. Certain parts of the 16S rRNA molecule
mutate at different speeds. Changes to 16S rRNA are, on the whole, still
extremely slow (humans share about 50% of their 16S rRNA sequence with
the bacterium E. coli), but one portion mutates much more slowly than
the other. It’s as if the 16S rRNA clock has both an hour hand and a
minute hand. The very slowly evolving “hour hand” lets
biologists study the long-term changes to the molecule, whereas the more
quickly evolving “minute hand” provides a more recent history.
“This gives this gene an advantage because it lets use ask questions
about deep evolutionary history and more recent history at the same
time,” Eisen says.
Letter by letter
Selecting the gene was just Woese’s first challenge. Now he had to
sequence it in a variety of different organisms. In the late 60s and
early 70s, when Woese began his work, DNA sequencing was far from
automated. Everything, down to the last nucleotide, had to be done by
hand. Woese used a method to catalog short pieces of RNA developed in
1965 by British scientist Frederick Sanger, which used enzymes to chop
RNA into small pieces. These small pieces were sequenced, and then
scientists had to reassemble the overlapping pieces to determine the
overall sequence of the entire molecule—a process that was tedious,
expensive, and time-consuming, but that was seen as a minor annoyance to
a workhorse like Woese, Fox says. “All he cared about was getting
the answer.”
Woese started with prokaryotes, the single-celled organisms that were
his primary area of interest. He and his lab started by growing bacteria
in a solution of radioactive phosphate, which the cells incorporated
into backbones of their RNA molecules. This made the 16S rRNA
radioactive. Then, Woese and Fox extracted the RNA from the cells and
chopped it into smaller pieces using enzymes that acted like scissors.
The enzymatic scissors would only cut at certain sequences. If a
sequence was present in one organism but missing in a second, the
scissors would pass over the second one’s sequence. Its fragment
would be longer.
“To Carl, each spot was a puzzle that he would solve.”
Since RNA’s sugar-phosphate backbone is negatively charged, the
researchers could use a process known as electrophoresis to separate the
different length pieces. As electricity coursed through gels containing
samples, it pulled the smaller, lighter bits farther through the gels
than the longer, heavier chunks. The result was distinct bands of
different lengths of RNA. Woese and Fox then exposed each gel to
photographic paper over several days. The radioactive bands in the gel
transferred marks to the paper. This created a Piet Mondrian-esque
masterpiece of black bands on a white background. Each different
organism left its own mark. “To Carl, each spot was a puzzle that he
would solve,” Fox says.
After developing each image, Woese and Fox returned to the gel and
neatly cut out each individual blotch that contained fragments of a
certain length. They then chopped up these fragments with another set of
enzymes until they were about five to 15 nucleotides long, a length that
made sequencing easier. For some of the longer fragments, it took
several iterations of the process before they were successfully
sequenced. The sequences were then recorded on a set of 80-column IBM
punch cards. The cards were then run through a large computer to compare
band patterns and RNA sequences among different organisms to determine
evolutionary relationships. At the beginning, it took Woese and Fox
months to obtain a single 16S rRNA fingerprint.
“This process was a huge breakthrough,” says Peter Moore, an RNA
chemist at Yale University who worked with Woese on other research
relating to RNA’s structure. “It gave biologists a tool for
sorting through microorganisms and giving them a conceptual way to
understand the relationship between them. At the time, the field was
just a total disaster area. Nobody knew what the hell was going on.”
RNA is so fundamental to life that some scientists think it's the spark
that started it all. To learn more about RNA, visit NOVA’s RNA Lab.
By the spring of 1976, Woese and Fox had created fingerprints of a
variety of bacterial species when they turned to an oddball group of
prokaryotes: methanogens. These microbes produce methane when they break
down food for energy. Because even tiny amounts of oxygen are toxic to
these prokaryotes, Woese and Fox had to grow them under special
conditions.
After months of trial and error, the two scientists were finally able to
obtain an RNA fingerprint of one type of methanogen. When they finally
analyzed its fingerprint, however, it looked nothing like any of the
other bacteria Woese and Fox had previously analyzed. All of the
previous bacterial gels contained two large splotches at the bottom.
They were entirely absent from these new gels. Woese knew instantly what
this meant.
To fellow microbiologist Ralph Wolfe, who worked in the lab next door,
Woese announced, “I don’t even think these are bacteria,
Wolfe.”
He dropped the full bombshell on Fox. “The methanogens didn’t
have any of the spots he was expecting to see. When he realized this
wasn’t a mistake, he just went nuts. He ran into my lab and told me
we had discovered a new form of life,” Fox recalls.
The New Kingdom
The methanogens Woese and Fox had analyzed looked superficially like
other bacteria, yet their RNA told a different story, sharing more in
common with nucleus-containing eukaryotes than with other bacteria.
After more analysis of his RNA data, Woese concluded that what he was
tentatively calling Archaea (from Latin, meaning primitive) wasn’t a
minor twig on the tree of life, but a new main branch. It wasn’t
just Bacteria and Eukarya any more .
To prove to their critics that these prokaryotes really were a separate
domain on the tree of life, Woese and Fox knew the branch needed more
than just methanogens. Fox knew enough about methanogen biology to know
that their unique RNA fingerprint wasn’t the only thing that made
them strange. For one thing, their cell walls lacked a mesh-like outer
layer made of peptidoglycan. Nearly every other bacterium Fox could
think of contained peptidoglycan in its cell wall—until he recalled
a strange fact he had learned as a graduate student—another group of
prokaryotes, the salt-loving halophiles, also lacked peptidoglycan.
SONY DSC
Grand Prismatic Spring in Yellowstone National Park is home to many
species of thermophilic archaea.
Fox turned to the research literature to search for other references to
prokaryotes that lack peptidoglycan. He found two additional examples:
Thermoplasma and Sulfolobus. Other than the missing peptidoglycan, these
organisms and the methanogens seemed nothing alike. Methanogens were
found everywhere from wetlands to the digestive tracts, halophiles
flourished in salt, Thermoplasma liked things really hot, and Sulfolobus
are often found in volcanoes and hot, acidic springs.
Despite their apparent differences, they all metabolized food in the
same, unusual way—unlike anything seen in other bacteria—and the
fats in the cell membrane were alike, too. When Woese and Fox sequenced
the 16S rRNA of these organisms, they found that these prokaryotes were
most similar to the methanogens.
“Once we had the fingerprints, it all fell together,” Fox says.
Woese believed his findings were going to revolutionize biology, so he
organized a press conference when the paper was published in PNAS in
1977. It landed Woese on the front page of the New York Times, and
created animosity among many biologists. “The write-ups were
ludicrous and the reporters got it all wrong,” Wolfe says. “No
biologists wanted anything to do with him.”
It wasn’t just distaste for what looked like a publicity stunt that
was working against Woese. He had spent most of the last decade holed up
in his third floor lab, poring over RNA fingerprints. His reclusive
nature had given him the reputation of a crank. It also didn’t help
that he had single-handedly demoted many biologists’ favorite
species. Thanks to Woese, Wolfe says, “Microbes occupy nearly all of
the tree. Then you have one branch at the very end where all the animals
and plants were. And the biologists just couldn’t believe that all
the plants and all the animals were really just one tiny twig on one
branch.”
“He was a brash, iconoclastic outsider, and his message did not go
down well.”
Although some specialists were quick to adopt Woese’s new scheme,
the rest of biology remained openly hostile to the idea. It wasn’t
until the mid-1980s that other microbiologists began to warm to the
idea, and it took well over another decade for other areas of biology to
follow suit. Woese had grown increasingly bitter that so many other
scientists were so quick to reject his claims. He knew his research and
ideas were solid. But he was left to respond to what seemed like an
endless stream of criticism. Shying from these attacks, Woese retreated
to his office for the next two decades.
“He was a brash, iconoclastic outsider, and his message did not go
down well,” says Moore, the Yale RNA chemist.
Woese’s cause wasn’t helped by his inability to engage critics
in dialogue and discussion. Both reticent and abrupt, he preferred his
lab over conferences and presentations. In place of public appearances
to address his detractors, he sent salvos of op-eds and letters to the
editor. Still, nothing seemed to help. The task of publicly supporting
this new tree of life fell to Woese’s close colleagues, especially
Norman Pace.
But as technology improved, scientists began to obtain the sequences of
an increasing number of 16S rRNAs from different organisms. More and
more of their analyses supported Woese’s hypothesis. As sequencing
data poured in from around the world, it became clear to nearly everyone
in biology that Woese’s initial tree was, in fact, been correct.
Now, when scientists try to discover unknown microbial species, the
first gene they sequence is 16S rRNA. “It’s become one of the
fundamentals of biology,” Wolfe says. “After more than 20 years,
Woese was finally vindicated.”
Woese died on December 30, 2012, at the age of 84 of complications from
pancreatic cancer. At the time of his death, he had won some of
biology’s most prestigious awards and had become one of the
field’s most respected scientists. Thanks to Woese’s legacy, we
now know that most of the world’s biodiversity is hidden from view,
among the tiny microbes that live unseen in and around us, and in them,
the story of how life first evolved on this planet.
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