PDF link:  

http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6WK7-457D7X4...

If you had read this paper yourself, you may have noticed several
interesting things about the study.  For one thing, this study was an
"in vitro" study.  The E. coli themselves did not "evolve" the changes
in this enzyme here.  Besides this, the wild-type genes products of
both the lacZ as well as the gusA genes (beta-galactosidases and
beta-glucuronides) hydrolysis similar glycoside substrates that differ
only at the C4 and C5 positions. And, the beta-glucuronidase enzyme
(gus) already has some selectable (to the scientists)
beta-galactosidase activity to begin with. Each of the four subsequent
point mutations enhanced the beta-galactosidase efficiency of this
enzyme, in a stepwise manner, while reducing the beta-glucuronidase
efficiency of this enzyme.

What is especially interesting here is that this potential lactase
evolution of the gus gene did not happen in the "in vivo" experiments
done by Barry Hall and others - even over the course of tens of
thousands of generations.  In any case, the point of my argument and
use of experiments like Hall's is not to show that there is only one
or two potential lactase enzymes in sequence space.  I believe that
there are literally trillions upon trillions of potential lactase
sequences out there.  The point is that even this seemingly large
number of potential lactase enzymes and/or other beneficial sequence
of at this level of complexity (i.e., at least 400aa in length), is
nothing compared to the number of meaningless nonfunctional sequences
that occupy the sequence space at this level of complexity.  The ratio
is what is important here.  What is the ratio of potential benefit
compared with potential junk?  Ask Ian Musgrave how many potential
lactase enzymes there are in sequence space.  If he tells you the same
thing that he told me, he will give you 10e90 as the total number of
lactase enzymes in sequence space.  The problem is that the lactase
function seems to require a chain of at least 400 amino acids to
achieve the lactase function. Of course, this translates into a
sequence space of over 10e520 different sequences.  So tell me, what
is 10e90 when compared to 10e520? If Ian is right in this his
estimates, each functional lactase enzyme would be surrounded, on
average, by 10e430 non-lactase sequences.  This is an absolutely
miniscule ratio.  I believe that it is this ratio that limited Hall's
mutant E. coli from evolving the lactase function without the lacZ or
ebg genes - regardless of the presence of the gus gene.  Even if the
gus gene had evolved the lactase function to a beneficial level in
vivo, this would not help you.  Many other bacterial do not have the
lacz, gus, or ebg genes, or any other genetic sequences close enough
to evolve the lactase function even given millions of observed
generations.  We have hospital records going back over 50 years with
many species of bacteria remaining lac negative that entire time.  Why
do these bacteria have such apparently "limited evolutionary
potential" if not for the relatively low ratio of lactase enzymes in
sequence space?

This is the first time I've seen it.  I don't read everything that is
addressed to me in this forum you know . . .

The reason why Matsumura did not need to knock out the ebg gene was
that this study was an "in vitro" study, not an "in vivo" study.  The
mutations were introduced into the wild-type gusA "via mutagenic PCR".

Most likely there are trillions of potential lactase genes out there
in sequence space.  A demonstration of two of them is nothing.  The
ratio of beneficial sequences vs. non-beneficial sequences is the
issue here.  What is the density of beneficial sequences in sequence
space *at a given level of functional complexity*?  A lower levels of
functional complexity,   such as the level of antibiotic resistance
and the like, the beneficial density of sequences is relatively high.
At the higher level of single protein functions the density of
beneficial functions becomes much much less.  This is evident from the
fact that the evolution of single protein enzymes is much harder to
achieve than the evolution of antibiotic resistance in these same
bacteria.  Even those bacteria that cannot evolve the lactase function
are easily able to evolve antibiotic resistance to all kinds of
different antibiotics.  Moving up one more level to multi-protein
functions were all the proteins work together at the same time in a
specific orientation with each other, there simply are no examples of
evolution in action - period.  Now, why is that?

Sean

www.naturalselection.0catch.com