From: toms@fcs260c2.ncifcrf.gov (Tom Schneider)
Newsgroups: bionet.molbio.genbank,bionet.info-theory
Subject: Re: Author errors and omissions.
Date: 17 Apr 92 00:54:50 GMT
Article-I.D.: fcs280s.3013
Followup-To: bionet.molbio.genbank
Distribution: bionet
Organization: NCI Supercomputer Facility, Frederick, MD

In article <1992Apr15.152911.18229@gpu.utcs.utoronto.ca> lamoran@gpu.utcs.utoronto.ca
(L.A. Moran) writes:

>In an earlier posting I (Larry Moran) said
>
>      "Unfortunately it is not so easy for a computer to update old 
>       entries so the task falls to someone who is knowledgeable and 
>       willing to spend enough time to do the job correctly."
> 
>Keith Robison replied,
>
>      "I disagree entirely. It is all too EASY to update them 
>       automatically -- you just have to unconvince yourself that it 
>       is difficult."
>
>Keith, I just spent several hours meditating and trying to unconvince myself.
>Do you know what I discovered when I returned to my computer? It still 
>requires someone knowledgeable to do the job! Maybe I didn't I didn't work
>hard enough at "unconvincing" myself! (-:

Unfortunately, I agree with Larry.  The only people who can say exactly which
points of the sequence are bound by GAL4 is an expert on GAL4 who is familiar
with the experiments done on that system.  There is no way to get around the
hard work needed to put the proper annotation together.  Fortunately there are
tons of such data, it's just not getting into GenBank and so is effectively
buried!

>In defense of computers Keith says,
>
>      "If you can state a rule, then you can write a program to test 
>       whether any entries break that rule. Most of the rule-breakers 
>       will be errors, and those which aren't are likely to hold some 
>       interesting biology."
>
>What an interesting proposition. Here are a couple of "rules" that we can look
>at. I'm certain that we will all agree that these rules define information
>that we would like to see added to the database. I maintain that computers
>can't do this.
>
>       A GAL4 binding site is defined as a piece of DNA to which
>       GAL4 has been shown to bind by direct experiment.
(I dropped the rest)

Keiths statement is indeed interesting.  I have a quote by my desk:

  All science is either physics or stamp collecting.
   --- E. Rutherford

We all spend a lot of time stamp collecting in biology - GenBank is
an enormous example!  Here's a rule for you:

      If there is no funny business going on, then we expect Rsequence
      to be approximately equal to Rfrequency for a set of binding sites.

This rule, unlike the list Larry proposed, is mathematically precise and quite
robust.  (If you don't know what Rsequence and Rfrequency are, see NAR 18:
6097, 1990 for starters; then JMB 188:415, 1986; email me for papers; or join
us on bionet.info-theory.)

Keith understood that bacteriophage T7 promoters APPEAR to violate this rule.
This led to an interesting experiment (NAR 17:659, 1989).  Another case of an
interesting apparent violation will appear this summer in J. Bact (I'll
announce the exact reference in bionet.info-theory when I have it).

All this stuff is based on information theory, and so is related directly
to entropy.  Physics!  See, Rutherford was right!  :-)

TO DO THIS KIND OF ANALYSIS REQUIRES THAT WE HAVE COMPLETE AND CORRECT
ANNOTATION IN GENBANK, OR THAT WE OBTAIN THE DATA FROM THE LITERATURE
OURSELVES.  WE CANNOT EASILY DO THE PHYSICS OF BIOLOGY WITH THE CURRENT STATE
OF THE DATABASE.

Please understand that I much appreciate the efforts of the GenBank staff
so far as they have gone.

>It seems to me that it would be difficult to write a computer program that
>could examine DNA sequences to see if these "rules" were followed because the
>information cannot be deduced from the sequence alone! Even in the case of 
>interesting DNA sequences such as similarity to GAL4 consensus binding sites 
>it is difficult to formulate the "rule" in a meaningful manner that would 
>eliminate considerable human intervention. In this case the search would 
>presumably be limited to yeast sequences and furthermore to regions upstream 
>of a known promoter. Would that suffice, or are there still too many "hits" 
>that are quite irrelevent? And what do we do when the consensus changes? 
>(This has happened with heat shock elements and others.)

Read the papers mentioned above for starters, or write to me if you would
like me to send you copies.  The fact that you are worried about 'the consensus
sequence changing' is a reflection of the fact that

     ****************************************************************
     * CONSENSUS SEQUENCES ARE A VERY POOR MODEL FOR BINDING SITES! *
     ****************************************************************

I understand that you don't know what else to do so you keep using this utterly
horrible stinky model.  Do this:  go look at the figures in that 1990 NAR
paper.  You will see some "sequence logos" which are a mathematically sensible
replacement to the consensus sequences everybody is using.  (If you are inclined,
the figures are also available by anonymous ftp to ncifcrf.gov in pub/delila
in the files 

  globin.logo.Z lambcro.logo.Z ribo.logo.Z t7.logo.Z

(use binary mode for transfer then uncompress and send to your favorite
---color if available---PostScript printer.)

>I have trouble understanding how one can prepare a meaningful dataset from 
>GenBank over coffee.

IF GenBank did not have duplications (or had a mechanism for ignoring them)
   and
IF GenBank were not riddled with errors (as I find every time I work with it)
   and
IF binding sites were properly annotated in GenBank

THEN I could do hundreds of useful information analyses 'over coffee'.  At this
point I and my students have to spend weeks reading the literature and working
our way through the mess to figure out a good data set.  The actual computer
time for an information analysis is trivial (seconds to minutes).  To do it
properly, the human time is enormous (months to years).  Just ask that brave
soul, Owen White.

We KNOW what to do with the data about binding sites once it is in nice form.
The problem is getting it into nice form.

You really have to know what a sequence logo looks like to see what I mean.

>How would you implement scans for consensus site agreement?

There is an information theory based method for this.  The best publication
is by Berg and von Hippel.

> Do you want to
>search every eukaryotic sequence for CCAAT and annotate every hit?

NO!  That's trivial and meaningless.  We don't keep tables of logs anymore
because we can calculate them on the fly.  Likewise for searches.

> What consensus sequences do you think that the database managers should look
> for?

None.  Just put the experimental data that exists in the literature into the
database.  Don't put guesses in.  I would like to see every binding site
annotated with a data item (NOT A COMMENT!!!) that says how it is known that
this is a site (hydroxy radical footprinting?  co-crystal? etc)  At this point
I'd just settle for seeing that there was experimental support.  It can't be
guesses or we just will end up chasing our tails trying to figure out what the
sites really are.

>Can you give specific examples that can be automated?

Nope, since to do it right you have to use experimental data from the
literature.

> Should the database be re-analyzed every time a new "consensus" sequence is
> proposed?

No, because consensus is a stupid way to analyze the data.  The information
theory methods converge to a single solution (the sequence logo doesn't change
with time) if one is collecting data without a sudden bias change.

For example, suppose a position in a binding site can be either A or T but not
G or C.  Then the consensus base - the most frequent base - will change in time
depending on the order you add sites to the set.  A sequence logo shows both,
and so converges to a single image.

>Why can't you just look for these consensus sequences yourself if that is what
>interests you?

There are thousands of genetic control systems.  We already look for ourselves
and find that the database as it stands does not support the research.  Why
shouldn't all our hard work be used by others and vice versa?  We certainly
won't have the last word on T7 promoters or GAL4 just because we were the first
to analyse them with information theory.

  Tom Schneider
  National Cancer Institute
  Laboratory of Mathematical Biology
  Frederick, Maryland  21702-1201
  toms@ncifcrf.gov