"The New York Times"
 July 26, 2001

 Not life as we know it
 DNA in all living creatures consists of four bases, known by their
initials, A, C, G and T. Scientists are trying to move beyond this limit
to create, essentially, aliens

 Andrew Pollack

 Scientists are taking the first steps toward creating alternative life
forms -- organisms that use a genetic code different from the one used by
all other creatures on Earth.

 Such organisms, bacteria to start with, would have novel chemical units
in their DNA and synthetic building blocks in their  proteins. Scientists
hope such organisms can be used to study biochemical processes in new ways
and to produce new  medical or electronic materials that cannot now be
made by living things.

 The research goes well beyond current genetic engineering, which involves
reshuffling the ordinary components of DNA or  proteins into new
combinations or moving DNA from one organism to another.

 Adding completely new elements to DNA and proteins is essentially
rewriting the genetic code, the fundamental language of life. As such, it
is likely to raise new ethical and safety issues, although there has been
no controversy yet because the work is still five to 10 years from any
practical use.

 "We're not trying to imitate nature; we're trying to supplement nature,"
said Floyd E. Romesburg, an assistant professor of chemistry at Scripps
Research Institute. "We're trying to expand the genetic code."

 So far, scientists are nowhere near creating truly novel life forms. They
have been able to get only one unnatural protein building block at a time
substituted for a natural one. And no one has been able to get unnatural
DNA to function in living cells, although progress has been made in test
tubes.

 Despite life's vast diversity, all creatures -- from yeast to humans,
from microbes that live in near-boiling water to those that tolerate
freezing temperatures -- spell out their genetic instructions using the
same four DNA chemical units, known as bases, which are represented by the
letters A, C, G and T.

 Different three-letter combinations specify amino acids, which are strung
together like beads to make up the proteins that carry out most functions
in a cell. With rare exceptions, all living things use the same 20 amino
acids.

 The genetic code, then, is a language of four letters used to make 20
words. Despite the limited vocabulary, those words can be used to make the
huge variety of sentences and paragraphs that characterize life.

 But what if there could be additional genetic letters and words? That,
scientists say, would allow organisms to be more versatile, just as some
languages have sounds or express concepts not found in English.

 David A. Tirrell, chairman of chemistry and chemical engineering at the
California Institute of Technology, got bacteria to make a protein with
the non-stick properties of Teflon by having the microbes substitute an
unnatural amino acid for one of the 20 natural ones. He said such a
protein might one day be used to make artificial blood vessels. Teflon is
currently used to make them.

 Tirrell and others also imagine incorporating fluorescent amino acids
into proteins. That would allow proteins to be studied in finer detail.
And synthetic DNA units are already being used in at least one genetic
test.

 Scientists say creatures with a truly different genetic code would
essentially be alien life forms. Indeed, one of the aims of the research
is to see what kinds of life may be possible outside Earth.

 "We can't think of any transparent reason that these four bases are used
on Earth," said Steven A. Benner, a professor of chemistry at the
University of Florida, "and it wouldn't surprise me in the slightest if
life on Mars used different letters."

 The scientists working on the creation of novel organisms say that for
now at least, there is no chance the microbes will run  amok. The bacteria
created so far that use an unnatural amino acid have to pick up the
synthetic component from the medium in which they grow. If they escaped
into the wild, they would die or revert to using a natural amino acid.

 Still, safety questions will no doubt be raised. "It's a powerful
technology," said Jonathan King, a professor of molecular biology at the
Massachusetts Institute of Technology, "and like all powerful
technologies, it needs appropriate oversight and  regulation."

 King said, for instance, that proteins with artificial amino acids might
elicit allergic reactions if they were used as drugs or in food.

 While proteins rarely contain amino acids beyond the normal 20, it is not
hard to come up with more. Chemists can synthesize dozens of amino acids.
And some organisms create such amino acids for purposes other than making
proteins. But these other amino acids are not incorporated into proteins
-- except when the cell makes a mistake.

 So most efforts to have bacteria use synthetic amino acids have hinged on
encouraging such mistakes. When bacteria are fed a diet rich in an
unnatural amino acid that closely resembles a natural one, they may evolve
to prefer the new amino acid, even to the extent that they cannot live
without it.

 Andrew Ellington, a professor of chemistry and biochemistry at the
University of Texas, has used such forced evolution, completely
substituting an unnatural amino acid for one of the 20 natural ones.

 Efforts to expand the genetic code have drawn attention with the
publication of two papers in the journal Science last April. Both are from
scientists at the Scripps Research Institute.

 August Bock, chairman of the Institute of Genetics and Molecular Biology
at the University of Munich in Germany, commented in Science that the two
papers pointed to "a new realm of biology, bordering the world of
chemistry, which will allow experimenters to explore ideas about
completely new proteins that were once inconceivable."

 One of the papers presented a variation of the error-causing theme.
Scientists introduced a genetic change that crippled an enzyme involved in
correcting errors in protein formation. That allowed an unnatural amino
acid to be taken up at 24% of the locations in all the bacteria's proteins
where the amino acid called valine was supposed to go. The work was led by
Paul Schimmel at Scripps and Philippe Marliere of Genoscope, a French
research institute, and Evologic, a biotech company.

 The second Scripps paper presented a different approach. Instead of
substituting a new amino acid for one of the 20, the scientists introduced
a 21st amino acid. And instead of widespread substitution, they put the
new amino acid in a specific spot of their choosing. They did this by
creating special molecules to deliver this amino acid to the cell's
protein-making  machinery.

 This work was led by Peter G. Schultz, a chemistry professor who is also
director of the Genomics Institute of the Novartis Research Foundation.
Some experts said the work paved the way for introducing more than one new
amino acid into bacteria and doing so with a precision previously
unobtainable.

 "I would say it's a major, major advance," said Uttam Rajbhandary, a
molecular biologist at MIT, who is doing similar work.

 But if scientists are going to add new amino acids this way, they have to
specify where in the proteins these amino acids should go. So they must
put the genetic code for the new amino acids into the bacteria's DNA at
the right spots.

 There is only one problem: There is no sequence of DNA letters that
encode for amino acids that nature has not encountered before. With four
DNA bases, there are 64 possible three-letter combinations, called codons,
which can specify an amino acid. But 61 of them are already used for the
20 natural amino acids. (There are duplications; for instance, six
different codons specify leucine.)

 When the cell encounters one of three remaining codons that do not
specify an amino acid, it stops building the protein. Schultz picked one
of those codons as the code for his new amino acid.

 But this approach has an obvious problem. What if the bacteria's DNA
naturally contains this codon at spots where protein formation really is
supposed to stop? If the 21st amino acid was inserted instead at such
spots, erroneous proteins would be  made that could kill the organisms.

 Schultz said the codon he chose was rarely used by this bacterium, so
this would not be a significant problem. Still, there are only three
codons that do not already code for an ordinary amino acid, limiting the
number of new amino acids that can be  introduced this way.

 So if scientists want to introduce many new amino acids, new codons will
be needed. That is why they are trying to add letters to the genetic
alphabet. If DNA consisted of six bases -- say, A, C, G, T, X and Y --
there could be 216 codons instead of 64.

 Such artificial DNA bases have been made by Benner in Florida, Romesburg
at Scripps and Dr. Eric T. Kool, a chemistry professor at Stanford.

 Besides fitting into the double helix of DNA, each artificial base must
pair with only one artificial counterpart, just as A always pairs with T,
and C with G. Such pairing is essential for accurate DNA replication.

 Benner in one case managed to use an artificial DNA base to produce a
protein with an unnatural amino acid -- but only in a test tube. It has
been extremely difficult to use natural enzymes to replicate DNA that
contains artificial bases, even in the test tube. And when artificial DNA
is introduced into organisms, the organisms
invariably die.

 But Kool is confident he will achieve replication, at least in the test
tube. "In five to 10 years, we'll have an alien-replicating system," he
said. Romesburg of Scripps said he had achieved test-tube replication of
DNA containing one extra base that pairs with itself.

 Still, while they cannot yet be used in living cells, Benner's artificial
bases are already being used in tests that read DNA sequences. The tests
are sold by EraGen Biosciences of Madison, Wis., which calls the
technology Aegis, for "an expanded  genetic information system."

 Besides answering questions about how life could have evolved elsewhere
in space, the research might shed light on evolution on Earth.

 Schimmel at Scripps said there might have been a stage in evolution when
the genetic code was not as precise as it is now. His work, in which the
protein-proofreading enzyme was disabled, was an attempt to recreate that
earlier, sloppier stage.

 Schultz wants to subject bacteria with extra synthetic amino acids to
such stresses as heat or poison to see if they evolve and adapt faster
than natural bacteria. "Will those forms of life with a bigger
building-block set be superior to the ones who have 20?" he asked.

 Schultz often says living things have only 20 amino acids because God
rested on the seventh day.

 "If he worked on Sunday," he said, "what would we look like?"
 

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