BIONET

National Computer

   Resource

     for

Molecular Biology

  Grant No. P4lRRO168506
  Annual Progress Report
March 1,1988 - February Z&l989

The BIONETTM Resource is funded through a cooperative Agreement with IntelliGenetics, Inc., by the
Biomedical Research Technology Program, Division of Research Resourses, National Institutes of Health

BIONET Resource
c/o IntelliGenetics
700 E. El Camino Real
Mountain View, CA 94040
   415-324-GENE
    4159627300


P41BBO1685-66

1. Title Page

Table of Contents

2. Description of Program Activities
2.1 Scientific Subprojccts
    2.1.1 Collaborative Research and Service
   2.12 Technological Research and Development
    2.13 Training

2.2 Books, Papers, Abstracts
2.3 Resource Summary Table
3. Narrative Description
3.1 Summary of Research Progress
    3.1.1 Service

  3.1.1.1 Scientific Case Studies Using BIONET
  3.1.1.2 Scientific Consulting: BIONET User Support
  3.1.15 Service
  3.1.1.4 PWBIONET Communications - Distribution of the Resource
  3.1.1.5 FASTA-MAIL program
  3.1.1.6 New User Manual
  3.1.1.7 Revision of thelntroduction to BXONET
  3.1.1.8 Additional On-line HELP EXAMPLES
  3.1.1.9 BIONET Newsletter
  3.1.1.10 IDEAS programs
3.19 Collaborative Research
  3.1.2.1 Collaborative Efforts by the BIONET research staff
  3.1.2.2 DEC-2965 Software Contributors
  3.19.3 PC and Minicomputer-based Software
  3.1.2.4 Data Contributors
  3.1.2.5 Liaison with Other Resources
  3.1.2.6 Bulletin Boards and Leaders
3.1.3 Technological Research
  3.1.3.1 Research Ffforts by the BIONET Scientist
  3.1.3.2 FASTA-MAIL
  3.1.3.3 Development work on the new Sun central computing facility
  3.1.3.4 BIONET Satellite Software for VAXWMS systems
  3.1.3.5 The RICH program
  3.1.3.6 BioCard - a prototype menu-driven interface for BIONET
   3.1.3.7 XGENPUB

3.1.4 BIONET Training Program
3.1.5 Resource Facilities

    3.1.5.1 BIONETMJN Agreement
    3.1.5.2 Computer Hardware and Telecommunication Networks
    3.1.5.3 Summary Statistics on Machine Use
    3.1.5.4 Computer Software - Core Library
    3.1.5.5 Computer Software - System Library
    3.1.5.6 Computer Software - Contributed Library
    3.1.5.7 Database Library
3.2 Highlights

3.3 Administrative Changes
3.4 Resource Advisory Committee and Allocation of Resources
3.5 Dissemination of Information of Resource's Capabilities
    3.5.1 Community Interactions and Awareness
    3.53 Electronic Communications
3.6 IMPORTANT Suggestions and Comments
I. BIONET Research Publications
II. BIONET Training Publications

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P41RRO1685-06

III. BIONET Newsletters
IV. BIONET Software Lending Library Catalog
V. BIOSCI Bulletin Board Network Information
VI. BIONET Training Schedules
VII. BIONET Computer Facilities
VIII. Testimonials

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P41RRO1685-06

List of Figures

Figure 3-1: Actual use of the DEC-2065 for the Month of November, 1988    51
Figure 3-2: BIONET's Percentage of Total System Use, 12187 - 11188       52
Figure 3-3:  BIONET's Prime Time Use of the DEC.2065 12/87 - H/88       54
Figure 3-4: BIONET's Non-Prime Time Use of the DEC-2065,12/87 - 11188   56
Figure 3-5: BIONET's Total Use of the DEC-2065,12/87 - ll/88           58
Figure 3-6: Total Telenet and Compuserve Network Use, 12/87-11/88      60

. . .
111


P41RRO1685-06

                List of Tables
Table 2-1: Summary of the BIONET User Community                 2
Table 3-1: Summary of Monthly Rates of Inquiries                  21
Table 3-2: Summary of Monthly Rates of Questions for Programs and   22
       Databases.

Table 3-3:  BIONET Prime Time CPU Minutes
Table 34  BIONET Prime Time Connect Hours        :i
Table 3-5: BIONET Non-Prime Time CPU Minutes                 55
Table 3-6: BXONET Non-Prime Time Connect Hours                 55
Table 3-7:  BIONET Total CPU Minutes                          57
Table 3-8:  BIONET Total Connect Hours                        57
Table 3-9: BIONET Network Usage, Connect Hours                 59

iv



2.  TITLSOFCZRAB7k  BIONET, National Computer Resource for

  Molecular Biology

3.NAmclFRDcIpmnINsrmnz~  IntelXiGenetics, Inc.

4.  lzEAmi PWIu4ALs&caa (If ag@cable):

6.  P-AL -GXOR:
16a. M: Dr. Michael J.  Kelly

President, IntelliGenetics
                 *,

7.  D?xESI~(~,r)ay,Year):    December 12, 1988

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P41R.R01685-06

2. Description of Program Activities
This section of our Annual Report provides statistical information on the use of the BIONETtm
Resource. The period covered is 12/87 - 11/88, to coincide with the dates of preparation of our Report
and to follow our procedure of providing a full year's statistical information to compare with previous
years' Reports.

Individual sections are prepared under guidelines discussed previously with BRTP staff and used in
our previous Reports.  We use a format for reporting the hundreds of individual Principal
Investigator's use that is easy for us to generate while retaining the critical information necessary
for BRTP in its internal and governmental reporting requirements. Complete research abstracts are
kept at IntelliGenetics and are available upon request if needed.

The BIONET User community is divided into different classes, representing different levels of use of
the computer system and staf'fresources, as follows:
  o CIase I.  Class I users represent the Service component of the scientific community.
  They participate in the electronic communications facilities of BIONET (bulletin boards
  and electronic mail), and use the Core and Contributed Software libraries to pursue
   their research;

o Class II. Class II users represent the Collaborative component of the user community.
Scientists in Class II enjoy all benefits of Class I use, and in addition contribute software
and expertise to BIONET, working closely with BIONET staff. This category also
includes bulletin board leaders, accounts for other related Resources (GenBank,
NBRFLPIR, Dana Farber, etc.>, and National Advisory Committee members.

o Class III. Class III accounts are for BIONET satellite communications. These accounts
have the same system privileges as Class IV below but are provided free of charge.
o Class IV. Class IV users consist of those scientists who wish access only to the
electronic communication facilities of BIONET. They are given access to the electronic
mail and bulletin board facilities.

o Class V. Class V was implemented in response to a decision of our National Advisory
Committee. Class V accounts are similar to Class IV but are for use by industrial
scientists. Class V users sign an agreement stating that their communications account
will be used only for scientific purposes and not for commercial advertising or other
promotional purposes. Because of our ARPANET connection restrictions, users in this
class do not have access to the TELNET and FTP programs which allow direct contact to
other computers on the ARPANET.

Information on the number of PI's by Class is summarized in Table 2-1.
          Table 2-1: Summary of the BIONET User Community

                     Class I         813

Class II         26

Class III

7

Class IV

19

Class V          2
                 -v-

  Total        867

2


P41RRO1685-06

The total number of laboratories with access to BIONET, 867, is a 31 percent increase over the total
of 660 presented in our last annual report! This clearly demonstrates the continuing demand for and
the quality of the BIONET service.  Besides having a small rate of discontinued accounts (9.6%,
below) the resource has continued to grow at a remarkable rate. Between 12/87 - lYf88, 270 new
labs opened accounts on BIONET, and 63 (9.6%) labs discontinued their subscriptions.

The current number of BIONET PI's (867) represents about 14% of all the NIH extramural
investigators (some 6100 grants total). Actually, the total number of NIH-funded investigators may
be significantly less than 6100 since many investigators hold more than one grant. Since only about
half of the NIH grantees mention DNA sequence, cloning, or recombinant DNA in their grants, we
would estimate that the 14% of NIH grantees that are on BIONET represents between 25 and 30%
of all the scientists who could possibly make use of the BIONET facility. BIONET has considerable
potential for further growth. A major factor in this continued demand is the increasing recognition
by the scientific community of the need for a facility which provides rapid access to molecular biology
databases and which also can serve as a hub for electronic communications. As the amount of
sequence data continues to expand, the central role of BIONET will increase in value.

Considerable time and effort has been expended in developing the BIONET resource to its current
state. We trust that the NIH recognizes the value of this resource to the community and is aware of
the dislocation that would occur in a significant number of important research projects if any
disruption occurred in the service.

2.1 Scientific Subprojects

2.1.1 Collaborative Research and Service
In the following section we report the use of the BIONET Resource for Class I-V users. The "Usage
Factor" is reported as both central processor unit (cpu) time, in minutes, and connect time, in hours,
for each Principal Investigator on the BIONET DEC 2065 computer. These values are the sum of all
usage by the PI and his or her group members ("Sub-I's").

We note that, starting in August 1988, BIONET made available a Sun 3/280 computer to handle
database searches. This was necessary because of the tremendous user demands being made on the
DEC computer. Since user demand was affecting the response time on the latter system, we made
access to the Sun available despite its lack of accounting software. Actual user totals may therefore
be higher in many cases than listed below. We are currently working on implementing accounting
software on the Sun systems and statistics on its use will be available in the near future.

We report data only on those PI groups that have used the Resource during the past 12 months. Of
the 867 groups with active BIONET accounts, 693, representing about 2430 individual investigators,
have accessed BIONET. Last year's access total was 530 laboratories. Thus the number of groups
who have accessed the system increased by 31% as compared to last year.

If frequent users are defined as those laboratories utilizing 60 or more connect hours or 120 or more
cpu minutes during the year, there were 407 groups in the "frequent user" category this year (47% of
all current accounts). This represents an increase of 41% over this same category last year (288).

3


There are 174 accounts on the system that were inactive during the year. Most of the accounts in
this category were inactive either because they were created near the end of our accounting year and
not yet utilized, or because they fell into the category of complimentary (mainly foreign) accounts.
Foreign users are not charged an access fee but must pay their own telecommunications charges.
These accounts (now totaling 111) have been used infrequently because of the expense of
international telecommunications access.  However, since they utilize little of our resources, the
accounts have been maintained on the system and have accumulated over the past five years.

The summary usage statistics for each laboratory group follow below. Detailed usage statistics for
each individual user are maintained by the BIONET computer and are available to interested
parties.

We do not report Resource staff hours nor BRTP funds allocated for individual PI's because it is
impossible to allocate these rationally to such a large user community. Summary information on
allocation of staffhours is given in the Resource Summary Table.


P41R.R01685-06

2.1.2 Technological Research and Development
We report on the standard form summary information for our Technological Research projects.

In past years the Resource Technology used was the DEC-2065 computer. This year most work by
the staff has been performed on BIONETs new Sun computers, and accounting software for
reporting CPU minutes is not currently available on those machines.

The Usage Factor was previously reported as minutes of cpu time used for the project, but is not
provided this year due to the lack of usage statistics on the Sun computers.

Resource StafY Hours are based on time estimates of work reported on each project. For use in
further calculations (below), hours listed for each individual in the table are divided by total annual
hours per individual. This yields a fractional time or "FI'" for each individual on each project.

"BRTP Funds Allocated" are calculated as the sum of the following components:
  *Actual Personnel Costs.  The personnel costs for each project are derived by
  multiplying the above Fl' for each BIONET staff person's time spent on the project times
  their respective annual salary plus fringe benefits; the actual personnel cost is the sum
  of these individual figures.

o Consultant Costs. The FI' spent by a consultant involved in a project is multiplied
times the total consulting cost for the consultant; these are summed for each project
where appropriate.

o Fraction of Awarded Funds. The fraction of total awarded funds for each project is
derived by multiplying the fractional time spent on each project (defined below) by the
awarded funds (defined below). The fractional time is determined from the sum of
hours spent on the project by the investigators listed divided by the sum of hours spent
on BIONET by all investigators. For this calculation we have used the actual time spent
from 12/87 through 11/88. Although this period is three months out of phase with the
actual grant period, we do not think the fractional time spent will change significantly
during the next three months of the current period. In computation of awarded funds,
we include only the funds allocated in the grant categories of Supplies, Travel, and Other
Expenses. The categories of Personnet and Consultants are accounted for in the previous
two computations and the remaining grant category Equipment is accounted for in the
Resource Summary Table.

o Indirect Costa. Indirect costs are allocated by multiplying the total awarded indirect
costs for this reporting period by the fractional time devoted to each project. The
fractional time is determined from the sum of hours spent on the project by all
investigators divided by the sum of hours spent on BIONET by all investigators.

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2.1.3 Training

We report summary information for our Training program. The sites at which BIONET provided
some level of training are named here and are discussed in more detail in the Narrative Description
section.

The method for calculation of Usage Factors and BRTP Funds Allocated is the same as that
described above under Technological Research and Development.

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P41RRO1685-06

2.2 Books, Papers, Abstracts

We list on the next BRTP form the following research reports which detail some of BIONET's
progress during this last year. Copies of these reports are available in Appendix I.

The second form lists papers that have trained or informed scientists about the resource. Copies of
these papers are in Appendix II.

10


XI, SECTICNB                GRAmNmmER

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Fillautasqarate formforeachof the fallming~tegaries: chxkormz.

JII TECHNOiOCICAL RESEARCii
  X   & trEVELOP:lENT

Aehor(s)

Title uf Article, Jouxnal, Vol., Nuther
.Pagw(e.g., 44-483, Year Publbkd.

Jurka, J.  and T. Smith.   (1988). Proc. Natl. Acad. Sci. USA. 85,
    4775-4778.  A fundamental division inthe Alu family-repeated
    sequences.

I  Faulkner, D. V. and J. Jurka.   (1988).  Trends Biochem. Sci. 13,  321-322
    Multiple aligned sequence editor.    -- _--- __- ---  I

I  Jurka,  J.,T. F. Smith, and D. Labuda.   (1988).   NUCl.  Acids Res. 16,
      766.  Small cytoplasmic Ro RNA pseudogene andan Erepeat in
    the human &- 1 qlobinqene.       I

Jurka, J. and R. J. Britten.   (1988).  Cold Spr. Harb. Symp. abstr.
   Evolution of human Alu repeats: implicationsforqenome studies.

Holszt>nska, E., D. J. Waxman, and J. Jurka.   (1988).   Protein
   Society Mtq. abstr.  Studies on rat liver cytochromes P450
   using comparative sequence analysis.

             Protein Society Mtq. abstr.  Locating amino
   acid patterns in proteins by compcsition.


I

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Fillcut aseparate fomfor,each of the follcwbgcatqories: Cbckore.
                                                                           .
    TECHNOLOGICAL RESEARCH
     & DEVELOPYENT      I- -' El
                                    x TRAINIs

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Pag- (e.g., 44-481, Year F'ublished. I

I  MAillik. S_ (in nr~ss~.  Protein Seauence and Data Analysis.         I

LA&...--*-, I. ,-__ r----, -    - ~ - ---.~ ~~
   Protein Databases and Software on BI??%?T.


P4ll%RO1685-06

We report the publications by members of the BIONET scientific community on a version of the
special form provided by BRTP. These publications have ALL arisen from use of BIONET, although
support by BIONET and the NIH has not always been acknowledged.

The figures on Cumulative Number Published refer to the current year alone. We have received 44
papers that were published or are in press. The total for the three previous years was 250, bringing
the overall total to 294. We note that the actual number of publications which involved the use of
BIONET is undoubtedly higher because many investigators have not yet replied to our requests for
reprints, and the requirement to acknowledge BIONET is not strictly followed.

13


PAmIf, SEtTIcNB       - - IP1411l.RI RI@ 1116 1 8151-10 16
ma: IntelllGenetics              m PEmm:  3/l/88 to2,'28/189
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(, III TECHNOLOGICAL RESEARCH
     & DEVELOP~IENT      5
                                 x   -REsmRx r-p&am
                                                                           .

Aahor(s)                    Tia d Article, journal, vol., SUE&Z
                          Pages kg., 44-w ) Year Publisired.

Please see the following pages.


Part II Section B
Institution: IntelliGenetics

        Award Number P41 RR01 66506
Report Perii: March I, 1988 - February 28,1989

References

Akella, R., P. Arasu, and A. B. Vaidya. (1988) Molecular and Biochemical
   msitology. Vol. 30, pp. 165-174.  "Molecular clones of @-tubulin genes
   of Plasmodium yoelii reveal an unusual feature of the carboxy
    terminus".

Allison, L.A. , J. K.-C. Wong, V. D. Fitzpatrick, M. Moyle, and J. Ingels. (1988)
   Molecular and Cellular Bi&w. Vol. 8 pp. 321-329.  "The C-Terminal
  Domain of the Largest Subunit of RNA Polymerase II of Saccharomyces
   cerevisiae, Drosophila melanogaster,  and Mammal: a Conserved
    Structure with an Essential Function".

Auger, I. E. , and C. E. Lawrence. (1988) Society of Mathematical Biology, Vol.
   0092-8240, pp l-16.  " Algorithms For The Optimal Identification of
   Segment Neighborhoods".

Batter, D. K. , S. R. D'Mello, L. M. Turzai, H. B. Hughes III, A. E. Gioio, and B. B.
   Kaplan. (1988) The Journal of Neurosciences Research. Vol. 19, pp 367-
    376.  " The Complete Nucleotide Sequence and Structure of the Gene
   Encoding   Bovine Phenylethanolamine N-Methyltransferase".

Bray, S. J. , W. A. Johnson, 1. Hirsh, U. Heberlein, and R. Tijian. (1988) The
   EMBO Journal. Vol. 7, pp. 177-188.  "A cis-acting element and associated
   binding factor required for CNS expression of the Drosophila
   melanogaster dopa decarboxylase gene".

Brayton, K. A., J. Amim, H. Qui, R. Yazdanparast, M. A. Ghatei, J. M. Polak, S. R.
   Bloom, and J. E. Dixon. (submitted) DNA. "Cloning, Characterization,
   and Sequence of a Porcine cDNA Encoding a Novel Secreted Neuronal
    and Endocrine Protein".

Bums, G., T. Brown, K. Hatter, J . R. Sokatch. (1988) Eur. J. Biochem.. Vol. 176,
   pp. 165-169.  "Comparison of the amino acid sequences of the
   transacylase components of branched chain oxoacid dehydrogenase of
   Pseudomonas putida, and the pyruvate and 2-oxoglutarate
   dehydrogenases of Escherichia coli".

Bums, G. , T. Brown, K. Hatter, J. M. Idriss, and J. R. Sokatch. (1988) Eur. J,
   Biochem.. Vol. 176, pp. 31 l-317.  "Similarity of the El subunits of
   branched-chain-oxoacid dehydrogenase from Pseudomonas putida to
   the corresponding subunits of   mammalian branched-chain-oxoacid
   and pyruvate  dehydrogenases".

14a


Part II Section B
Institution: IntelliGenetics

        Award Number P41 RR01 686-06
Report Period: March I, 1988 - February 28,1989

Bums Jr. , J. M. , T. M. Daly, A. B. Vaidya, and C. A. Long. (1988) Proc. Natl.
      Sci. USA. Vol. 85 pp. 602606.  "The 3' portion of the gene for a
  Plasmodium yoelii mero'zoite surface antigen encodes the epitope
   recognized by a protective monoclonal antibody".

Cao, M., X. Xiao, B. Egbett, T. M. Darragh, and T. S. B. Yen. "Rapid Detection of
   Cutaneous Herpes Simplex Virus Infection with the Polymerase Chain
    Reaction".  (in press g/30/88)

Chang, J. H., C. Tamba, S. Dumbar, and M. 0. J. Olson. (1988) The!'
        . .

tolaprcal Chew. Vol. 763 pp. 12824-12827.  "cDNA and Deduced
Primary Structure of Rat Prot'ein B23, a Nucleolar Protein Containing
Highly Conserved Sequences*".

Cohen, J. I. , R. H. Miller, B. Rosenblum, K. Denniston, J. L. Get-in, and R. H.
   Purcell. (1988) Virology. Vol. 162 pp. 12-20.  :" Sequence Comparison of
  Woodchuck Hepatitis Virus Replica'tive Forms Shows Conservation of the
    Geneome".

Cooke, N. E., J. Ray, J. G. Emery, and S. A. Liebhaber. (1988) The Journal of
   Biolopical Chemistrv. Vol. 263 pp. 9001-9006.  "Two Distinct Species of
  Human Growth Hormone-variant mRNA in the Human Placenta Predict
   the Expression of Novel Growth Hormone Proteins".

Dickey, L. F. , E. C. Theil, Y. H. Wang, G. E. Shulls, and I. A. Wortman III. (1988)
               cal ChemtStEy, Vol. 263, pp 3071-3074. "The
   Importance of the 3' - Untranslated Region in the Translational Control
    of Ferritin mRNA*"

D'Mello , S. R. , E. P. Weisberg, M. K. Stachowiak, L. M. Turzai, A. E. Gioio, and
   B. B. Kaplan. (1988) Q
                                          v1 1
   pp. 440-449.  "Isolation and Nucleotide Sequence of a cDNA Clone
   Encoding Bovine Adrenal Tyrosine Hydreoxylase: Comparative Analysis
   of Tyrosine Hydroxylase Gene Products".

Jeppesen, C., B. Stebbins-Boaz, and S. A. Gerbi. (1988) Nucleic Acids Research,
   Vol. 16, pp. 2127-2148.  "Nucleotide sequence determination and
   secondary structure of Xenopus U3 snRNA".

Kokubu, F., K. Hinds, R. Litman, M. J. Shamblott, and G. W. Litman. (1988) The
   EMBO Journal. Vol. 7, pp. 1979-1988.  "Complete structure and
   organization of immunoglobulin heavy chain constant region genes in
   a phylogenetically primitive vertebrate".

Kokubu, F., R. Litman, M. J. Shamblott, K. Hinds, and G. W. Litman. (1988) m
   MB0 Journal. Vol. 7, pp. 3413-3422. "Diverse organization of
   immunoglobulin  VH gene loci in a primitive vertebrate".

14b


Part II Section 6
Institution: lntelliGenetic3

        Award Number P41 RR01 685-06
Report Perii: March 1,1988 - February 28.1989

Krawetz, S. A., W. Connor, and G. H. Dixon. (1988) DNA. Vol 6, DD. 47-57.
   "Cloning of Bovine Pl Protamine cDNA and the Evolution of Vertebrate
    Pl Protamines".

Krawetz, S. A., W. Connor, and G. H. Dixon. (1988) The Journal of BioloPical
       ry. Vol. 262 * pp. 32 l-326.  "Bovine Protamine Genes Cantain a
   Single Intron".

Krawetz, S. A., and G. A. Dixon. (1988) Journal of Molecular EvoluttQn, Vol. 22,
   pp. 291-297.  "Sequence Similarities of the Protamine Genes:
   Implications for Regulation and Evolution".

Linskens, M. H. , and J. A. Huberman. (1988) L
                                                  i 1
   Vol. 8, pp 4927-4935.  " Organization of Replication of Ribosomal DNA in
   Saccharomyces cerevisiae".

Manly, K. F. , G. R. Anderson, and D. L. Stoler. (1988) Journal of Virology, Vol.
   62, pp. 3540-3543.  " Harvey Sarcoma Virus Genome Contains No
   Extensive Sequences Unrelated to Those of Other Retroviruses except
    ras".

Mayfield, J. E., B. J. Bricker, H. Godfrey, R. M. Crosby, D. J. Knight, S. M. Halling,
   D. Balinsky, and L. B. Tabatabai. (1988)  Gene. vol 63, pp l-9.
   "The cloning, expression, and nucleotide sequence of a gene coding for
   an immunogenic Brucella abortus protein."

Miller, R. H. (1988) m. Vol. 239, pp. 1420-1422. "Human
   Immunodeficiency Virus May Encode a Novel Protein on the Genomic
    DNA Plus Strand".

Miller, R. H. (1988) Virology. Vol. 164, pp. 147-155.  "Close Evolutionary
   Relatedness of the Hepatitis B Virus and Murine Leukemia Virus
   Polymerase Gene Sequence".

Nagle, G. T., S. D. Painter, J. E. Blankenship, and A. Kurosky. (1988) m
                  . .
   Joumal of Blolwcal Chat&-v. Vol. 263, pp. 9223-9237. "Proteolytic
   Processing of Egg-laying Hormone-related Precursors in Aplysia" .

Nagle, G. T. , S. D. Painter, J. E. Blankenship, J. V. A. Choate, and A. Kurosky.
   (1988) Es&&& Vol. 9 pp. 867-872.  "The Bag Cell Egg-Laying Hormones
   of Aplysia  brasiliana `and Aplysia  californica are Indentical".

Nees, D. W. , P. A. Stein, and R. A. Ludwig. (1988) Nucleic Acids Research, Vol.
   Ld pp. 9839-9853.  "The Azorhizobium caulinodans nifA gene:
  dentification of upstream-activation sequences including a new
   element, the `anaerobo' "

14c


Part II Section B
Institution: IntelliGenetics

        Award Number P41 RROl685-06
Report Period: March 1,1988 - February 28,1989

Oka, Y., and C. A. Thomas, Jr. .  (1988) Nucleic Acids Research, Vol. 15, pg. 8877-
    8898.  "The cohering telomeres of Oxytricha".

Rimsky, L., J. Hauber. M. Dukovich, M. H. Malim, A. Langlois, B. R. Cullen, and
   W. C. Greene. (1988) Nature. Vol. 335, pp.738-740. "Functional
   replacement of the HIV-l rev protein by the HTLV-1 rex protein".

Singh, S. V., H. Ahrnad, A. Kurosky, and Y. C. Awasthi. (1988) Archives of
   Bioche&try a& Biophysics. Vol. 264 pp. 13-22.  "Purification and
  Characterization of Unique Glutathionk S-Transferases  from Human
    Muscle".

Suplick, K., R. Akella, A. Saul, and A. B. Vaidya. (1988) Molecular and
   mv. Vol. 30, pp. 289-290.  "Molecular cloning and
   partial sequence of a 5.8 kilobase pair repetitive DNA from Plasmodium
   falcparum" .

Sung, S. J., J. M. Bjomdahl, C. Y. Wang, H. T. Koa, and S. M. Fu. (1988) J. EXD.
   &I&.. Vol. 167, pp. 937-953.  "Production of Tumor Necrosis
   Factor/Cachectin by Human T Cell Lines and Peripheral Blood T
   Lymphocytes Stimulated By Phorbol Myristate Acetate and Anti-CD3
   Antibody".

Upton, C., J. L. Macen, R. A. Maranchuk, A. M. DeLange, and G. McFadden. (1988)
        .
     trol ogy. Vol. 0042 6822
                          -    pp. 229-239. Tumorigenic Poxviruses: Fine
  Analysis of the Recombinhtion Junction in Malignant Rabbit Fibroma
   Virus, a Recombinant between Shope Fibroma Virus and Myxoma Virus".

Vodkin, M. D. , and J. C. Williams. (1988) Journal of Bacteriology. Mar. 88, pp.
    1227- 1234.  " A Heat Shock Operon in Coxiella bumetii Produces a Major
   Antigen Homologous to a Protein in Both Mycobacteria and Escherichia
    coli"

Vold, B. S. , C. J. Green, N. Narasimhan, M. Strem, and J. N. Hansen. (1988) The
       al      . .
       of Btoloetcal -1stt-v. Vol. 263, pp. 14485-14490.
   "Transcriptional Analysis of Bacillus subtilis rRNA-tRNA Operons".

Wang, Y. H. , S. R. Sczekan, and E. C. Theil. (1988) Metal Ion Homeostasis:
   Molecular Biologvd Chemistry. UCLA Symposia on Molecular and
   Cellular Biology.  Vol. 98, D. Winge & D. Hamer, Editors, Alan R. Liss, Inc.,
    New York.

.

Weber, J. L. . (1988) Molecular and Btwal Parasitoloeu, Vol. 7.9 pp. 117-
    124. "Interspersed repetitive DNA from Plasmodium falciparuh" .

14d


Part II Section B
Institution: IntelliGenetics

        Award Number P41 RR01 685-06
Report Period: March 1,1988 - February 28,1989

Weber, J. L., J. A. Lyon, R. H. Wolff, T. Hall, G. H. Lowell, and J. D. Chulay. (1988)
   The Journal of Biological Chemistrv. Vol. 263, pp. 11421-11425. "Primary
   Structure of a Plasmodium falciparum Malaria Antigen Located at the
   Merozoite Surface and within the Parasitophorous Vacuole*".

Westaway, S. K., E. M. Phizicky, and J. Ableson. (1988) The Journal of
   Biolovical Chemistry. Vol. 263. pp. 3171-3176.  " Structure and Function
   of the Yeast tRNA Ligage Gene*".

Winkfien, R. J., R. D. Moir, S. A. Krawetz, J. Blanco, J. C. States, and G. H. Dixon.
   (1988) Fur. J. Biochem.. Vol. 176, pp. 255-264.  "A New family of
   repetitive, retroposon-like sequences in the genome of the rainbow
    trout".

Wohlrab, H., R. T. Bronson, R. C. Lu, and V. Nameth. (1988) Biomedical and
                            , pp. 1130-1136. "Towards

a Biomarker of Mammalian Senescence: Carbonic Anhydrase III".

Xiao, X., M. Cao. T. R. Miller, Z. Y. Cao, and T. S. B. Yen. The Lancet, October 15,
   1988 , p. 902.  Papillomavirus DNA in Cervical Carcinoma Specimens
    from Central China.  (abstract)

14e


P41RRO1685-06

2.3 Resource Summary Table

The Resource Summary Table includes the totals from the previous sections of Technological
Research and Development and Training.  The totals for Collaborative Research and Service are
derived from the following components:

o Usage Factor. The CPU Min. Used is the total for BIONET CPU use found in Table
III-7 since this year cpu use on the DEC was essentially entirely consumed by the user
community. Technological Research projects were performed on other hardware. The
total of staff hours is obtained from our accounting totals of hours spent on BIONET
projects and the value for Collaborative Research and Service is likewise the remainder
after subtracting time spent in other categories.

o BRTP Funds Allocated. This is computed as the difference between the total budget
for BIONET minus the categories of Technological R+D and Training, and minus the
capital  equipment  expenditures   for   the  year ($36,746) listed under
Administration/Miscellaneous.

The category of Collaborative Research and Service includes an entry of $169,182 in the column
Other Funds. This is the total money invoiced over the period 12l87 - 11188 for subscription fees
($252,932) minus outstanding receivables of $83,750. Each PI is asked to pay an access fee to help
defray the telecommunication costs for access to BIONET; this fee is currently $400/year. By
agreement with BRTP, these access fees are not grant related income.

The balance of these fees carried forward from the previous year (as of 12/l/86) was $156,523. After
twelve additional months of collecting subscription fees ($169,182 above) and disbursing them for
telecommunication expenses ($244,321), the balance is now $81,384.

No facility staff computer time, work hours, or BRTP funds are allocated to the category of
AdministrationlMisceZlaneous; we consider such time and funds to be an integral part of the support
of the other components of the Resource.  We do include as Funds Allocated the $36,746 used to
purchase items of capital equipment.

The category of down time includes the sum of scheduled and unscheduled maintenance on the
DEC-2065 computer. In the period 12/87 - 11/88, there was a total of 149 hours (8924 cpu minutes)
of downtime:

o 2545 cpu minutes of scheduled downtime for preventive maintenance and several
system-related tasks.

o 6379 minutes of downtime were due to unscheduled maintenance.

The downtime reported in the Summary Table (8032 cpu min) is 90% of the total, reflecting
BIONETs allocation of 90% of the machine. Note that the total unscheduled maintenance of 6379
cpu minutes is only 1.2% of the total cpu time available. Considering both categories of downtime,
the machine has been available for use by BIONET scientists, 98.3% of the time, 24 hours a day,
seven days a week. No funds have been allocated to this category.

15


c

    f









W
z
.
W
m

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P41RRO1665-06

3. Narrative Description

3.1 Summary of Research Progress

Although a very signficant fraction of staff time during our fifth year was involved with planning,
grant writing, and other obligations for our renewal, important progress was still made on the
Resource. The following sections describe in detail our accomplishments in the several components
of the BIONET Resource. Here, in brief, are some of the most notable.
  o The new computer network donated by Sun Microsystems is being prepared for direct
  access by the user community by the BIONET systems staff (Mr. Rob Liebschutz and
  Mr. Eliot Lear). As of August 1988 BIONET released the FASTA-MAIL program. This
  provided our users on the DEC with electronic mail access to high-speed database
  searches on our Sun 3/280 computer. Database search times were reduced from hours to
  tens of minutes or less, and the average mid-day user load on the DEC 2065 dropped by
  a factor of about three to five since it was no longer being used for these compute-
  intensive tasks. FASTA-MAIL used the FASTA program obtained from Dr. William
  Pearson, and the mail server portion was developed by Mr. Liebschutz, Mr. Lear, and
   Mr. Spencer Yeh.

o Dr. Jerzy Jurka, the BIONET Scientist, has published important work in the area of
repetitive DNA sequence analysis. In conjunction with this research, new functionality
has been added to the Multiple Aligned Sequence Editor (MASE) by the BIONET
applications programmer, Mr. Liang Jen Horng. This editor was originally developed by
Dr. Jurka and Donald Faulkner at Dana Farber's Molecular Biology Computer Research
Resource and then extended at BIONET over the past year. Work on the editor has also
involved collaborators from the machine learning group at the University of California at
Santa cnlz.

o Dr. Sunil Maulik, BIONETs Senior Scientific Consultant, has continued work on the
RICH program which performs database searches for sequences of defined percent
composition. Dr. Maulik has obtained some interesting preliminary results with the
program. He has also been involved in developing a new HyperCard tm -based user
interface for BIONET.

o The electronic communications network was significantly enhanced. The efforts of Dr.
David Kristofferson led to the formation of the international BIOSCI bulletin board
network. Because scientists work on a variety of computer networks around the world,
we recognized the necessity of developing a mechanism to allow all of them to
communicate without the necessity of learning the peculiarities of accessing each
network. We sought out computer sites on all major international networks and
arranged to have parallel copies of the original BIONET bulletin boards accessible from
these sites. Besides BIONET in the U.S., other major BIOSCI distribution sites are
situated in England, Ireland, Sweden, and Finland. Recipients of the bulletin boards
from these sites are located around the world from New Zealand and Australia, the Far
East, and Israel, throughout Europe, and back to North America. The bulletin boards
are available to users on the ARPANET, BITNET, EARN, Usenet, NSFnet, and JANET.
Users in any particular location need only post or receive messages from their closest
site. Any postings at any one site are automatically forwarded by the central BIOSCI
sites to all other participants on all of the above-listed networks.

o Finally, the research conducted by BIONETs 867 laboratory groups was made
significantly easier by a total revision of the BIONET documentation and the production
of a new User Manual. This involved major efforts by BIONET staffer's Ms. Vickie
Johncox, Mr. Spencer Yeh, and Ms. Kathryn Berg. The documentation was sent free of
charge to all users on the system this past summer.

17


P41RRO1685-06

3.1.1 Service

The Service component of BIONET includes primarily Class I investigators who use the BIONET
Core and Contributed program Libraries to support their research. BIONET user classes were
explained above under Description of Program Activities.  The BIONET consultants also provide
support as needed by the other classes of BIONET users, but these groups are much smaller than
the predominant class I group.

The computing assistance given by the staff can range from simply answering routine questions to
providing sophisticated help on sequence alignments or complex database searches. Especially in
the latter case the staff can make a significant contribution to the attainment of a BIONET user's
research goals. Viewed in this light the distinction between "service" and "research" may be hard to
discern.

Before going into the details of how the BIONET staff serves the user community we wish to provide
several examples of how BIONET Class I investigators utilize the resource. These "case studies" will
demonstrate the importance of the work being performed by the investigators which the BIONET
staff serves.

3.1.1.1 Scientific Case Studies Using BIONET
The success of BIONET can be measured in several ways. For example, one can count the number of
participating scientists, or count their publications. These numbers are interesting and impressive,
but do not convey the high quality of work that is being done. It is very difficult to measure this
quality objectively. We have examined the publications submitted to us, and have selected two that
we feel represent some of the quality research done on the system. We present these as "case
studies."

`Harvey sarcoma virus genome contains no extensive sequences unrelated to those of
other retroviruses except r-as."  Kenneth F. Manly, Garth R. Anderson, and Daniel
L. Stoler. Journal of Virology 62: 3540-3543 (1999).

Dr. Manly's laboratory has been studying the VL30 multigene elements present in rats and mice
with structures similar to those of retroviruses' and retrotransposons' genomes. Transcripts of these
elements are packaged as pseudotypes by type C retroviruses, and, when introduced into cells,
pseudotyped VL30 RNA can lead to integration at new sites in the cell genome, suggesting that they
are an entirely new class of transposable elements. Further, both the Rirsten and Harvey murine
sarcoma viruses are acute transforming viruses whose genomes comprise two distinct genomic
elements; a MS oncogene and a VL30 sequence, both of which appear to contribute directly to
oncogenic activity. VL30 element transcription is strongly induced as a cellular response to anoxic
stress, and studies in Dr. Manly's laboratory had previously suggested that the rat VI20 sequences
incorporated into Kirsten sarcoma virus genome might directly encode the major anoxic stress
protein ~34, lactate dehydrogenase k.

In order to characterise the VI20 domain from Harvey sarcoma virus (HaSV) and to evaluate its
similarity to other retroviral sequences, Dr. Manly and his collaborators compared translated HaSV
sequences with the entire NBRF-PIR database available on BIONET using the IFIND and XFASTP
database similarity searching software. Translations were done in all three reading-frames using

18


P41RRO1685-06

the PEP program. Selected sequences were evaluated for significance with the XRDF program,
which compares the observed similarity score with a group of similarity scores obtained by
randomizing one of the sequences many times.  Additionally, HaSV RNA subsequences which
correspond to the regions of peptide similarity were searched against the GenBank viral nucleic acid
sequences using the XFASTN program on BIONET. Terminator codons in the HaSV RNA sequence
were converted to X's to allow their acceptance by XFASTP. (The X character is treated by XFASTP
as an unknown residue and is given an intermediate relatedness score).

The results of the searches yielded eight major regions of sequence similarity with (optimized)
similarity scores ranging from 47 to 428 and z-scores (the alignment score for the sequence expressed
as the number of standard deviations above the mean of a set of scores from the randomized
sequences) ranging from 5 to 42. (Table I, pg. 3541). Three regions showed greatest similarity with
gag sequences of feline sarcoma virus, with the remaining 5 regions showing greatest similarity with
the gag and pal regions of murine leukemia viruses. Two of the regions were composites of more than
one reading frame. In these cases, searches showed immediately adjacent HaSV regions in different
frames matching immediately adjacent regions of viral sequences. This was interpreted to mean
that a mutation in the HaSV sequence had split the original coding sequence between two or more
reading frames. The sequences from the different reading frames were combined and searched
against the database again, treating the two combined sequences as one.

Residues 1160 to 1420 of the VI.30 showed the greatest similarity (110 residue identity out of 210) to
the C-terminal region of the retroviral pal polyprotein, which is cleaved to yield an endonuclease.
Confirmation of the amino acid sequences was found by searching for nucleic acid similarities. A 62%
identity was found over a 625-base overlap with Moloney leukemia virus sequences. In addition, the
nucleic acid alignments showed insertions or deletions corresponding in location to the frameshifts
introduced into the peptide sequences. The similarity includes an (imperfect) copy of the C-X2-C-X4-
H-X4-C motif, known to be conserved in these sequences.

Manly et al. conclude that the sequence comparisons suggest a distant evolutionary relationship
between rat VI.30 sequences and murine leukemia virus sequences. A relationship between the
VL30 sequences and sequences of the retrovirus group including feline sarcoma virus and baboon
endogenous virus are also suggested by the results of the database searches. Further, the likelihood
that VI.30 sequences directly code for an anoxic stress protein (lactate dehydrogenase k) is
considered unlikely, since the searches failed to find any similarity with dehydrogenase sequences.
However, their extensive similarity with endonuclease sequences suggests that they may code for a
protein with that function instead.

`Structure and Function of the Yeast tR.NA Ligaae Gene". Shawn K. Westaway, Eric
M. Phizicky, and John AJAson. Journal of Biological Chemistry 263: 3171-3176 (1988).

Dr. Abelson's laboratory has pioneered in the study of tRNA splicing in yeast, and in this paper they
describe the DNA sequence of the entire coding region of the Sacchuromyces cerevisae tRNA ligase
gene. tRNA ligase is one of two enzymes required for tRNA splicing in yeast. The substrates for
splicing are a subset of tR.NA precursors containing introns. There are no obvious conserved regions
at the splice junctions (unlike the case with introns in mRNA precursors) and the only common

19


feature is location, which is one based removed from the 3'-end of the anticodon.

The tRNA ligase molecule itself is a single -90-kDa polypeptide likely to contain the three separate
activities required for tRNA splicing - namely phosphorylation of the 5' terminus of the 3' half-tRNA
in the presence of ATP, opening of the 2',3'-cyclic phosphodiester bond of the 5' half-tRNA; and
ligation of the two tRNA halves. In order to study the functional domains of this protein the gene
was cloned from S. cerevisae and its DNA sequence determined. Westaway et al. cloned into Ml3
phage using four unique EcoRI restriction sites to subclone plasmid pUC12-RLG. Purified phage
DNA templates were sequenced by the Sanger dideoxy method by first using the Ml3 primer and
then by priming with synthetic oligonucleotides. The 4.2 kb EcoRI fragment containing the yeast
tRNA ligase gene was sequenced by obtaining a restriction map comprised of the Ml3 and synthetic
oligonucleotide primers. Sites used were: EcoRI (site A), HpaI, ScaI, BglII, HindIII, KpnI, EcoRV,
XbaI, and EcoRI (site B). Ml3 sequencing primer was used to obtain initial sequencing information.
Oligonucleotides corresponding to unique regions of nucleotide sequence were then used as primers
in directing continued synthesis along the same strand of each clone. Thus the sequence of both
strands of each restriction fragment was obtained.  To confirm the sequence at restriction site
junctions, separate overlapping clones which spanned the junctions were sequenced.

The sequences of each fragment were entered onto BIONET using the GENED sequence editor, and
the entire tRNA ligase sequence assembled using the GEL program. Prior knowledge of the initiator
codon allowed immediate recognition of the tRNA ligase open reading-frame. Two further open
reading-frames, ORFl and ORF2, were also discovered using the SEWRANSLATE software on
BIONET. The mature tRNA ligase molecule was found to be 827 amino acids long with a molecular
mass of 95.4 kDa. It is a basic protein (as expected for one involved in tRNA metabolism). Analysis
using the PEP program showed that greater than 10% of the amino acids are lysines, and the net
charge of the protein is +12.5 (counting histidine as +0.5). Codon usage frequencies, calculated using
SEQ, show that tRNA ligase uses a large percentage of rarely used codons, consistent with the
hypothesis that less abundant proteins (tRNA ligase is present in approximately 400 copies/yeast
cell) do not have the bias toward preferred codons seen in highly abundant proteins. Suspicions that
tRNA ligase expression might be controlled by levels of intron-containing tRNAs were proven
unfounded when the codon usage of tRNA ligase relative to codons in yeast which are specifically
translated by tRNAs (whose precursors contain introns) were compared using the SEQ program.

Despite the similarity in mechanism of action between yeast tRNA ligase and that of T4 RNA ligase
and T4 DNA ligase, no obvious sequence similarities could be detected between the yeast tRNA
ligase gene product and any of the other T4 ligases when compared with the IFIND program.
Further, a screening of the entire NBRF-PIR protein sequence database using both IFIND and
XFASTP revealed no significant similarities between T4 ligase and any database sequences. Other
data suggesting that tRNA ligase has clearly separable domains responsible for some or all of the
different activities observed during tRNA splicing should allow more subtle subsequence similarities
between tRNA ligase and other proteins of similar function to be discerned.

Westaway et al.  summarise the properties of the tRNA ligase gene product as:11 being able to
catalyze three different activities; 2) forming a splicing complex with endonuclease and tRNA
precursors; and 3) being localized to a specific site at or near the inner nuclear membrane. Further
studies of the gene and protein product will be neccessary to characterise how these different aspects

20


P41RBO1685-06

are embodied in a single polypeptide.

3.1.18 Scientific Consulting: BIONET User Support
The Service component of the BIONBT Resource is supported by a group of three BIONET staff
members as described below. The staff interacts with the community in a variety of ways, including
direct support via telephone calls, electronic mail and terminal links with individual investigators.
Support is also provided through staff participation at major meetings and trade shows, at trainings,
and through participation in providing on-line and printed documentation for User Manuals,
program descriptions and system procedures.

We currently have a full time BIONBT Scientific Consultant (Dr. Karen Davis), a full-time
Applications Analyst (Mr. Spencer Yeh), and a full time Senior Scientific Consultant (Dr. Sunil
Maulik). The Scientific Consultant provides direct support (telephone and e-mail assistance) to the
community 75% of her time on a rotating basis.  The other 25% of her time is devoted to the
development of the BIONET training program and other Service projects. Tha Applications Analyst
devotes 25% of his time to user support and the rest of his time to contributed software and database
development/maintenance tasks.  The Senior Scientific Consultant oversees the electronic mail
responses of the Consultant and Analyst, assists them in answering more complex questions, and
spends the remainder of his time participating in Technological and Collaborative Research.

3.1.1.3 Service

The Service component of the BIONET Resource includes primarily Class I investigators and takes
the form of answering questions by phone, by electronic mail, and by terminal links from
investigators to staff A survey of the monthly phone, mail, and terminal links for the past year
shows the different uses of the BIONET Resource by the user community. The monthly inquiry
rates broken down into several categories are given below.

Table 3-1: Summary of Monthly Bates of Inquiries

Category           Nuder of Inquiries    Percent of Total Inquiries

Programs and Databases        164                     52
TOPS20 System                 54                       11
Administration               28                       9
Electronic Mail               25                      8
PC and PC Software             20                      6
Telecommnications             14                        4
File Transfers                 14                        4
                                  ----                         -SW-

TOTALS                     319                    100

The yearly total is 3813 queries, or about 15 per day (based on a 260- day work year). As can be seen
from Table III-l, the largest number of inquiries--52%--concern the use of BIONET's programs and
databases. This Programs-and-Databases category has been subdivided into additional scientific
and program categories in Table 111-2.

21


P41RRO16S5-06

Table 3-2: Summary of Monthly Rates of Questions for Programs and
                    Databases.

Category

Number of Inquiries

Percent of Total Inquiries

Database Searches
and Database8

85                        52

DNA and Protein
Sequence Analysis

27                         16

Sequence and Gel Data
Entry and Manipulation

19                        12

Experiment Planning
and Analysis

Multi-Sequence
Alignment

TOPS20 System Program8        13                         8

Other

9                         5

TOTALS                     164                        100

As shown in Table 111-2, just over half of these questions about Programs and Databases concern
Databases and Database Searches. This undoubtedly reflects the convenient access to fast database-
searching programs and recent versions of sequence databases on BIONET. As mentioned below,
the FASTA-MAIL program, introduced this year to the BIONET system, has been especially popular
because of its speed and ease of use.

Table III-2 also shows that the breakdown of the other categories of Programs-and-Databases
questions is as follows: 16% on Sequence Analyses; 12% on Sequence and Gel Data Entry and
Manipulation; and less than 10% on each of the other categories. Inquiries about Multi-Sequence
Alignment mostly concerned the use of the IntelliGenetics' GENALIGN program and William Bains'
contributed XMULTAN program. Questions about TOPS20 System Programs concerned the use of
the FIND and XSEARCH programs to examine database-index files, and the use of the XGENPUB
program to submit sequences to the GENBANK, EMBL, and PIR databases. Inquiries about

22


P41RRO1685-06

Experiment Planning and Analysis concerned the use of the SIZER, MAP, and CLONER programs.
The category OTHER covers inquiries on the use of other contributed programs, such as Michael
Zuker's BIOFLD, and other miscellaneous questions.

Returning to Table III-l, the second largest category--17%-of questions listed includes those
concerning the TOPS20 operating system.  These are separate from the the TOPS20 system
programs listed in Table III-P. These questions concerned manipulating files and directories, using
the text editors, and logging in to one's BIONET account.

The third largest category (9%) comprised BIONET Administrative questions which came directly to
the consultants and were rerouted to the BIONET Administrator. The BIONET administration
category consisted mostly of application requests, training session information, and manual
requests.  In addition to the calls listed here there are many calls directly to the BIONET
Administrator which are not included in Table III-l.

The fourth largest category in Table III-1 is Electronic Mail at 8%. The Telecommunications
category, at 4%, includes both inquiries concerning the procedures involved in connecting to the
BIONET computer and the quality of the communications between remote users and BIONET. The
remaining categories are self-explanatory.

The number of inquiries this year (3813) and the rate per day (15) are only 4% higher than last year,
when there were 3700 queries, or about 14 per day. We believe that this constancy of rate reflects a
balance between several factors: (1) fewer inquiries per user, due to new documentation written this
year, but (2) counterbalanced by an increased number of users; and (3) a slightly greater increase in
the number of new accounts. This year 270 new labs opened accounts on the system versus 238 new
accounts last year, an increase of 13%. However, new documentation prepared this year includes
more on-line examples of the uses of the programs in the analysis of a research project and a major
revision of the Introduction to BIONET manual, which is sent to all new users. This documentation
is described below.

Not only was the total number of inquiries approximately the same this year as last year, but also
the relative ranking of the various categories of inquiries on the basis of percent of inquiries was
similar. The main difference is the higher proportion this year of questions on databases and
database searches.

All of the above data indicate that the consultant service is an extremely important component of the
BIONET Resource. There are so many features available on BIONET that the presence of a trained
expert can assist users in utilizing the resource efficiently and intelligently. In addition, the large
number of questions pertaining to the databases and the database searching programs point to a
major use of the Resource for large scale database searching and analyses.

3.1.1.4 PC/BIONET Communications - Distribution of the Resource
It has been clear from the beginning of operation of BIONET that the majority of the user
community had access to personal computers, and that they all were looking for ways to use the PC's
effectively in conjunction with BIONET. We have strongly supported this method of access, to the
extent of maintaining a lending (and on-line) library of software and documentation for file transfer
and terminal emulation programs. We have worked closely with the community in this way because

23


P41RRO1685-06

we recognize that distribution of the Resource would be required in order for the central DEC-2065
to support an ever-increasing number of users.

If the only use of PC's was as terminals and as a source or recipient of files transferred over the
network, the net burden on BIONET would probably increase, rather than decrease. The
availability of PC-based software for sequence analysis has provided scientists with a means for
performing many simple analyses locally. When they need access to BIONET for a more complicated
analysis, they merely log on and transfer any needed files to the DEC-2065. As this "style" of
computation increase in popularity, the burden on the DEC-2065 will be reduced significantly.

To facilitate use of PC's, our Consultants provide information on file formats and the use of editors
on BIONET to reformat sequence files uploaded from PCs.  In addition, several PC molecular
biology programs are distributed via the BIONET software lending library. These programs allow
users to perform many routine analyses locally. The programs are described below under Computer
Software - Contributed Library.

The IBM PC public domain version of BIONRTs on-line EMACS editor has been furnished via the
lending library. "MicroEMACS" is distributed free to users and has the virtues of producing ASCII
text files compatible with the BIONET software and of utilizing essentially the same command set
as the mainframe editor. As new versions become available BIONET has updated its lending library
diskettes and announced the availability to users via the electronic bulletin board system. Use of
MicroEMACS facilitates sequence entry on user PC's and reduces dependence on the mainframe.

BIONET has also sought to standardize file transfer protocols on the system by vigorously promoting
the use of the public-domain Kermit software. New versions of Kermit have been added to the
lending library as they have become available. This last year saw the inclusion of a new version of
Kermit for the IBM-PC which supported Tektronix 4014 emulation and automatic login macros. The
former feature allowed users to obtain graphic output of plasmid maps from the IntelliGenetics (IG)
CLONER program and dot-matrix plots from the IG DDMATRIX program. The latter feature (login
macros) automated the process of dialing in to the BIONET Resource over Telenet or CompuServe.

S.l.lb FASTA-MAIL program

On August 17, a new program was introduced on BIONET that has shortened the turnaround time
on database similarity searches by a factor of 20, increased the interactive response on the BIONET
DEC machine by a factor of 3, while at the same time maintaining excellent sensitivity to
biologically significant similarities. This was accomplished by utilizing BIONET's in-house network
capabilities to set up a remote database server on the new BIONET Sun 3/280 computer using the
FASTA contributed software program from David Lipman and William Pearson. The FASTA-MAIL
program was based on the earlier FASTP-MAIL project which allowed BIONET users to submit
remote FASTP database searches of the NBRF/PIR database on a Sun 3/280. The FASTA-MAIL
program incorporated the following enhancements: a simpler user interface, the ability to do nucleic
acid database searches of GenBank, the additional capability to search SWISS-PROT, the ability to
set the KI!UP parameter, the use of the more sensitive FASTA algorithm instead of the older FASTP
algorithm, and significantly expanded on-line documentation for using the program. Since its
introduction on BIONET, the FASTA-MAIL program has been extremely well-received and is used
950 times per month, one of the most popular programs on BIONET.


Extensive work was required by the BIONET st& on the FASTA-MAIL project. The FASTA
program itself had to be modified to accept IntelliGenetics' file format, to have a `brief output" option
to limit the alignment output to the aligned region only, and to improve the clarity and labeling of all
output. The `brief output" modification was necessary since without this modification, the program
frequently created output files that were larger than the BIONET mailer could handle (256 Kbytes)
in a single message. The actual user interface and remote mail server software was created by the
BIONET systems programmers Eliot Lear and Rob Liebschutz. This included a user interface on the
DEC to submit automatically a formatted mail message to the correct address on the Sun computer,
authorization software, queueing software to provide separate protein and DNA search queues on
the Sun, software for running the FASTA program non-interactively, and software for mailing the
search results to the correct user account on the BIONET DEC machine. Finally, two separate
on-line help topics were written to provide an explanation of the FASTA algorithm and to give a
step-by-step guide to using the program and accessing the output results through the MM mail
program on the DEC 2065.

The FASTA-MAIL program allows both nucleic acid searches and protein searches to be run with the
same program by using different scoring matrices and program switches for the two types of
searches. Since the FASTA program gains some of its speed through the use of a stripped version of
the databases, FASTA formatted versions of the GenBank, NBRF/PIR, and SWISS-PROT databases
were created. All together, supporting these three databases for FASTA searches currently requires
an additional 30 Mbytes of disk space beyond the normal requirements for these databases. We are
also planning to implement a FASTA-formatted version of the EMBL nucleic acid sequence Data
Library.

The user interface for the FASTA-MAIL program is designed to be extremely easy to use. The user
need only respond to four simple questions to set up the search; this only takes about fifteen seconds!
Since the job is run remotely, the user can log off as soon as the job is submitted. The results are
automatically deposited in his or her mail file when the search is completed. Average CPU times for
the searches are 1 minute for full protein databank searches and 20 minutes for full GenBank
searches. Actual turnaround times are determined by the number of jobs waiting to execute in the
queue. Typical turnaround times are about 5-20 minutes for full protein databank searches and 1-4
hours for full GenBank searches. Since these CPU intensive searches would otherwise have been
run directly on the BIONET DEC machine, the FASTA-MAIL program has had the positive effect of
dramatically increasing the responsiveness of the DEC machine. Typical mid-day user load levels on
the DEC-20 were in the range of 10 - 15 before FASTA-MAIL was implemented. Now the average is
3 - 4. This means that the BIONET DEC-20 machine appears to be running about 3 to 5 times faster
to the typical user.

Finally FASTA-MAIL, which although extremely fast, is still highly sensitive to biological
similarities. By using a PAM250 scoring matrix for proteins, functionally similar amino acids at
corresponding positions increase the score of an alignment, and by using a joining penalty in the
database scan step, sequences with insertion gaps in significant regions are still kept for optimal
alignment at a later stage. This last feature is the major improvement of the FASTA algorithm over
the FASTP and FASTN algorithms which sometimes dropped significant matches from consideration
because of the presence of small gaps. BIONET would like to thank the authors of FASTA, David
Lipman of the NIH and William Pearson of the Univ. of Virginia, for allowing the BIONET

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community access to their search program and for answering questions regarding the FASTA
algorithm.

3.1.1.6 New User Manual
A new User Manual was produced this year and distributed free of charge to all current BIONET
users at the end of June 1988. Unlike the old BIONET Training Manual which was organized by
program, the User Manual is organized around scientific tasks and contains examples which
demonstrate the integration of several programs on BIONET to accomplish the specified tasks. The
following sections are included in the manual:
  o Introduction

o Mechanics (Files Structure and Operating System topics)
o Sequence Entry and Editing
o Sequence Location
o Sequence and Map Display
o Sequencing Project Management
o Sequence Translation
o Sequence Composition
o Sequence Structure
o Restriction Analysis
o Cloning Simulation
o Sequence Comparison

The new manual has been favorably received by the community as demonstrated by many positive
comments received from the users by the BIONET consulting staff.

3.1.1.7 Revision of theZntro&ction to BZONET

In June, a completely revised Introduction to BIONET manual was sent to all subscribers along with
the new User Manual. While the User Manual covers the use of scientific programs on BIONET, the
Introduction to BZONET details aspects of accessing the system and the use of other BIONET
features such as electronic communications. The hfroduction to BIONET is given to each new lab
group when they sign up for BIONET. At 100 pages in length, the new "Intro" is greatly expanded
from its 70 page predecessor. A sample login session is included to assist the first time BIONET
user, and a trouble-shooting chapter covers many of the common problems that might be
encountered while using BIONET. Other new sections include information on contributed sofiware
and databases, utility programs, network electronic mail, and electronic submission of sequences to
the national databases using the XGENPUB program. A new version of the Intro&&on to BIONET
will be produced when BIONET ports over to the SUN computers which run the UNIX operating
system.

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3.1.1.6 Additional On-line EELP EXAMPLES

As part of the consultant's on-going effort to expand and improve the on-line documentation for
BIONET, several additions have been made to the HELP EXAMPLES system of on-line examples of
using various programs. The most important additions have been examples of the FASTA-MAIL
program, the QUEST program, and the use of the FIND and RETRIEVE commands to locate and
make personal copies of entries in the databases. The FASTA-MAIL example (on-line example no.
35) guides the user through submitting a FASTA-MAIL similarity search to the DNA or protein
databases, and then accessing the search results through the MM mail program. Much of the
explanation involves a discussion of the FASTA-MAIL scoring and alignment output and how to
interpret the results. The QUEST example (example no. 33) takes the user through using a
promoter `key" (consensus pattern) in the QUEST program to search the chimpanzee beta-globin
gene nucleic acid sequence for polymeraseI1 promoter sites.  Instructions are given for using the
GUIDE command to set up the search and open a file in which to collect matched sequences, loading
the promoter key pattern from IntelliGenetics' KeyBank database of consensus sequences, and
running the search. The FIND and RETRIEVE example (example no. 40) takes the user through a
typical sequence of locating a sequence of interest in the databases and then making a personal copy
of the sequence that can be freely modified by the user. Although most database retrieval tasks can
now be accomplished through the new Intelligenetics' FINDSEQ program (see below), the FIND
command is still an extremely fast method of doing keyword searches, in addition to being useful for
locating sequences in databases that are not yet handled by FINDSEQ such as KEYBANK and
VECTORBANK Constant revision and expansion of the on-line help system is needed as new
programs and databases are added to the BIONET system.

3.1.12 BIONET Newsletter

The BIONET staff began producing a hardcopy newsletter called BZONET News. Two issues have
been mailed to the user community (April and October 1988) and copies are included in Appendix III.

3.1.1.10 IDEAS programs

Five protein structure prediction programs from the IDEAS (Integrated Database and Extended
Analysis System) suite written by Dr. Minoru Kanehisa of the National Cancer Institute and Kyoto
University were released on BIONET in February. Since the IDEAS suite only runs on VAX/VMS
systems, BIONET provided remote access to a networked MicroVAX owned by IntelliGenetics on
which the suite is installed. To access the programs, BIONET users run a simple program which
logs them into the MicroVAX. Data files may be moved to the MicroVAX and output results files can
be transmitted back to the BIONET DEC-2065 for further analysis. Since its introduction, the
IDEAS suite has been used an average of 23 times per month. Further details are provided below
under PC and Minicomputer-based Software.

3.1.2 Collaborative Research
BIONET's collaborative community is made up of several components, encompassing efforts by
outside scientists working in conjunction with BIONET staff. In subsequent sections we discuss
each component in more detail:

o Collaborative Efforts by the BIONET research staff.  This covers research
performed by Drs. Jurka and Maulik together with outside investigators.
o DEC-2065 Software Contributors. This component includes those persons who have

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contributed software for use by the BIONET community on the central DEC-2065
computer;

o PC and Minicomputer-based Software.  This component includes our efforts to
gather and disseminate software of special utility to the community;
o Data Contributors. This component includes those persons who, together with the
BIONET staff, contribute data useful to the community;
o Liaison with Other Resources. Several accounts have been established to promote
sharing of information among molecular biology computing resources;
o Bulletin Boards and Leaders. This component includes those persons who have
agreed to maintain bulletin boards of special interest to scientists using BIONET.

3.121 Collaborative Efforts by the BIONET research staff
The BIONET research group which is headed by Dr. Jurka and includes Dr. Maulik and Mr. Liang
Jen Hong, our Applications Programmer, has engaged in collaborative research on the evolution of
Alu and protein sequences.  Other preliminary investigations in the area of protein structure
prediction have been pursued as well.

Dr. Jurka has developed collaborative research projects with Professor Roy Britten (CalTech,
abstract by Jurka and Britten at Cold Spring Harb. Symp. May, 1988), and Dr. Emile Zuckerkandl
(Linus Pauling Institute, abstract by Jerxy Jurka with Emile Zuckerkandl and Jerry Latter has been
presented on an International PBBS Meeting, held in Sept. 1988 in Corsica). Collaborative research
with both institutions is in progress and the abstracts describing this work are provided in Appendix
I. Dr. Jurka has also been appointed to the editorial board of the Journal of MoZecuZar Evolution.
He has also reviewed manuscripts for Genomics and Nucleic Acids Research.

In collaboration with Aleksandar Milosavljevic and Professor David Haussler from the University of
California at Santa Cruz, Dr. Jurka and Mr. Homg worked on applications of machine learning
algorithms to the classification of biological sequences. In particular, they investigated a class of
models of unsupervised learning, based on the principle of cognitive economy.  Using prototype
algorithms based on these models they were able to successfuly reproduce classification of aligned
human Alu sequences (Jurka and Smith, 1988). They intend to explore the applicability of these
models to the classification of unaligned sequences.

As part of an on-going research project designed to aid molecular biologists in predicting the
structural and functional properties of biological molecules from their sequences, BIONET has
created a "higher-order" database of protein structural characteristics.   Derived from the
Brookhaven database (PDB) of protein structures, the DSSP database is created using the algorithm
of Kabsch and Sander to create a dictionary of secondary structure of those proteins that are known
to be non-homologous at the level of sequence and structure. The dictionary describes the secondary
structural patterns in terms of helix, beta-bridge, beta-ladder, turn, bend, and coil and is also
annotated with comments taken directly from the principal literature citations describing the
structure. These include general features of the protein as well as specific annotations of residues
involved in active sites or regions, forming the core, etc. The annotations are in the format:
predicate-name/argumenW/terminator to make them easily readable by a suitable computer
program. BIONET has begun preliminary investigations with the Research Institute for Advanced
Computer Science (RIACS) at NASA/Ames to design a Lisp-based software package that will be able


P4lRR.Ol685-06

to infer structural features from this database.

3.1.2.2 DEC-2066 Software Contributors
The following are the major contributors of software for use by the community on the DEC-2065:

FA!STP, FASTN, and FASTA From Bill Pearson - BIONET users have largely migrated from the
older FASTP and FASTN programs this year to the FASTA-MAIL program which was described
above in the Service section. The user interface for FASTA-MAIL runs on the DEC 2065 but the
actual database searches are run remotely on BIONETs Sun 3/280 computer. The results are
returned to the user on the DEC by electronic mail. The FASTA-MAIL program is used about 950
times per month or about 32 times per day!!

BIONET users are currently using FASTP on the DEC at the rate of 266 times per month. FASTN
was removed from the DEC due to the advent of FASTA-MAIL and the wish to remove compute-
intensive searches from the DEC but the FASTP program (which consumes far fewer resources and
can be used to search user-created databases) was maintained.

MUT.,TAN - MULTAN is a program developed by Dr. Bill Bains for aligning multiple homologous
DNA sequences. While it is limited to being able to align sequences which are at least 60%
homologous, it is an extremely rapid program. Multiple sequence alignment is useful for BIONET
users studying evolution and for those trying to obtain a consensus from many sequences of similar
function. A new version of MULTAN has been received from Dr. Bains and will be made available
when BIONET users are transferred to the Sun computers.

BIOFOLD - Three years ago Dr. Michael Zuker, from the NRC laboratory in Ottawa Ontario made
BIOFOLD available as a program on the BIONET computer. This program predicts RNA secondary
structure and is used an average of 7 times per month.

ALIGN - Dr. Dan Davison, while a graduate student at Stony Brook at SUNY wrote and contributed
two versions of his ALIGN program. The first version runs directly on the BIONET computer and
can be used to align two very long DNA sequences including sequences which are not very
homologous to each other and contain large gaps. The alignments are significantly better than
similar heuristic alignments obtained from the SEQ SEARCH procedure in the BIONET core
programs. The alignments are not as good as obtained from the SEQ ALIGN procedure but Dr.
Davison's ALIGN program is significantly faster. The ALIGN program is used an average of 31 times
per month.

XPROF - Dr. Rose, at Pennsylvania State University, has contributed the DEC-VAX Fortran
version of his method for calculating hydropathicity profiles for proteins based on empirical
observations on the extent to which amino acid residues are found to be exposed or buried. This
program was released last year on the DEC and is used an average of 6 times per month.

XALIGN - As part of BIONEl"s effort last year to make available multiple sequence alignment
software, we prepared and released XALIGN, based on Bacon and Anderson's ALIGN program. This
program, as described in Bacon, D.J. and W.F. Anderson, J. Mol. Biol. 191: 153-161, 1986, finds
the best local alignments among up to five protein sequences. The program was developed to run on
most Fortran77-compatible computers and has been significantly modified to run on BIONET's DEC

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2665 computer. XALIGN uses a large amount of CPU time and we thus asked the BIONET
Community to use it during offpeak hours only, outside of 8 - 5 PST. The program has been used an
average of 6 times per month since its release.

3.1.2.3 PC and Minicomputer-based Software
PC and minicomputer-based software is available to BIONET users via the BIONET lending library.
This software runs on local computers instead of the DEC 2065 and, as such, helps reduce the
demands on our central resource. Other minicomputer software (IDEAS, see below) is available to
BIONET users on an IntelliGenetics-owned MicroVAX.

We began the lending library concept by making Kermit (a terminal emulator and file transfer
protocol from Columbia University) available to BIONET users. BIONET copies Kermit onto
diskettes provided by the users and returns the disks by regular mail. (Smaller lending library
programs are also available for downloading from the DEC 2065.) We currently make Kermit
available for Apple II, Macintosh, IBM PC and TRSSO model computers. We have extended our
lending library to include many BIONET user developed programs and a number of utility programs
that are useful for file transfer on IBM PC and Macintosh computers,  Software for VAX and Sun
minicomputers has also been added to the lending library. A catalog of lending library software is
available in Appendix lV. We report below the new contributed software during this past year.

MS-Kermit version 2.30

In May, version 2.30 of the public domain MS-Kermit communications package for IBM compatible
computers was added to the BIONET Lending Library for free distribution to users who send in a
blank diskette. This version of Kermit is significantly improved from its predecessor in that it is
able to emulate a Tektronix 4010 graphics terminal.  It also allows dialup procedures to be
automated in a command file. The ability to emulate a graphics terminal means that BIONET users
that have the Kermit package and an IBM PC or compatible can obtain the graphics output from
programs such as DDMATRIX, CLONER, HPLOT, and HCOMP over their telephone connection. If
they also have a graphics printing package, they can then print these graphic displays on their local
dot matrix or laser printer. BIONET took advantage of the new macro capability in MS-Kermit to
create login command files that allow users to dial a local Telenet or CompuServe number, and go
through the complete BIONET login procedure by simply typing a single command to the Kermit
program prompt. These login command files are given to users if they obtain Kermit from BIONET.
Instructions for using the package with BIONET, and the original Kermit manual are also
distributed to users. Since its release 74 requests for the new version of Kermit have been handled
by the BIONET administrator.

New version of MacDNA

MacDNA is a program written by Robert Schleif from the Dept. of Biochemistry, Brandeis
University. An updated version of MacDNA has been made available through the BIONET lending
library of software and for downloading from BIONKT. MacDNA is a small (65Kbyte>, self-contained
application that is fast and efficient at many straightforward DNA analysis tasks such as
translations, character stripping, restriction mapping, consensus searching, inverting, formatting,
etc. The program is not Mac-like, i.e., it uses no mouse or dialog boxes, but the current version will

30


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run under Multifinder. The current version has enhanced formatting and editing capabilities, and
does single letter translations, not included in the original version.

OligoMutantMaker

OligoMutantMaker from Kevin Beadle8 of the Univ. of Calif. at San Prancico has been made
available to BIONET users.  OligoMutantMaker simplifies the designing and screening of
oligonucleotide-directed single amino acid substitution experiments by searching for nucleotide
sequences which introduce a restriction endonuclease recognition sequence into the codon
substitution site of the mutant. The program utilizes the redundancy of the genetic code to generate
all possible nucleotide sequences for a given amino acid substitution (including nucleotide sequences
in which silent mutations are introduced into the 5' and/or 3' codons immediately adjacent to the
substitution site) and determines whether any restriction endonuclease recognition sequences are
present. Any nucleotide sequence containing a restriction site is displayed or printed along with
relevant information about the site such as its restriction enzyme(s), the random frequency of the
enzyme's recognition sequence(s), the prototype of the enzyme, isoschizomers of the enzyme, and the
unit cost of the enzyme from various biochemical suppliers.

OligoMutantMaker runs on the IBM PC and a version for the Macintosh is in preparation.

New Version of SEQAID II

SEQAID II is a multifunctional PC program for DNA and protein sequence analysis from Donald
Roufa and Douglas Rhoads of Kansas State University.  SEQAID II's functions include editing,
extracting sequences from a GenBank floppy disk release, dot matrix comparison, fragment sizer,
base composition, translations, protein structure and hydropathicity, restriction site search, and
locating potential exons by codon bias.

One of the new features in release 3.0 is a gateway to the GenBank database (floppy disk release)
and a user-friendly interface to search and extract genetic sequence data from the database.
Included is a utility, GBHEADERJXE, which permits users to modify the GenBank format file,
HDR550.GBK to accomodate future floppy disk releases.  As implemented, SEQAIDII accesses
floppy disk releases of GenBank either segmented on diskettes or combined in a subdirectory of the
user's harddisk.

SEQAIDII version 3.0 also contains a new module that scans anonymous nucleic acid sequences for
potential protein coding regions based upon codon usage frequencies. The module permits the user
to construct and amplify required codon bias tables from appropriate nucleic acid sequences (cDNAs
or spliced exon sequences) and to save the tables to disk.

IDEAS - As a sizeable minority of BIONET users had been requesting the availability of programs
for protein structure analysis and prediction, last year BIONET investigated the availability of
programs performing these types of analyses.  It was decided that the IDEAS suite (Integrated
Database and Sequence Analysis System) by Minoru Kanehisa of Kyoto University (and previously
the National Cancer Institute) would be an appropriate package to obtain. An older version of the
IDEAS package, ported to run on the BIONET DECSO computer, had already been made available
by Dr. Kanehisa but it lacked the newer structural analysis programs needed. Since the newer

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version ran only under the VMS operating system on VAX computers, in February 1988 BIONET, in
co-operation with IntelliGenetics, made available an IntelliGenetics VAX/VMS computer to BIONET
users (see Technological Research). Accordingly, BIONET set up an account on an IntelliGenetics
networked microVAX computer, and ported over the IDEAS package stripped of all programs save
those pertaining to structure analysis or prediction and that would ensure an added functionality to
BIONET. Complete on-line help files were written describing both the programs and the access
procedures, so that BIONET users could easily review the functionality of these new programs
before availing themselves of them.

The programs made available were:

o STRALI: The STRALI program performs a secondary structure prediction by homology
alignment (Kanehisa, unpublished). It searches for local regions of similarity between a
query protein sequence and one whose structure has been determined and deposited in
the Brookhaven Protein Databank. The Kabsch-Sander secondary structure
classification (see DSSP, below) is used to predict regions of secondary structure in the
query.

o CHOFAS: CHOFAS is the popular Chou-Fasman (Chou,P.Y. and Fasman,G.D.,
Biochem.(l978) 47:251-276) algorithm for secondary structure prediction complemented
by the Rose algorithm (Rose,G.D., Nuture(1978)272: 586-590) for beta-turn prediction.

o DELPHI: DELPHI performs protein secondary structure prediction by Robson's method
(Garnier et al.,J. Mol. Bio1.(1978)120:97-120). The program makes predictions for helix,
(extended) beta-sheet, reverse-turn, and coil. Decision constants, provided by the user,
can affect the predictions greatly.

o ALOM: This program attempts to identify membrane proteins based on a discriminant
analysis of hydrophobic amino acids (KIein,P. et al., in prep.). The program reports
whether the sequence is likely to be integral or peripheral, together with the estimated
likelihood.

o HCOMP: Displays on a graphics terminal the hyrophobicity profiles of two aligned
protein sequences. The profile is calculated by smoothing the Nozaki-Tanford
hydrophobicity scale. (Nozaki,Y. and Tanford, C. J. Biol. Chem. (1971) 246: 2211-2217).

o HPLOT: Plots the distribution of hydrophobic and charged amino acids using any of four
possible algorithms: Nozaki-Tanford (Nozaki,Y. and Tanford, C. J. Biol. Chem. (1971)
246: 2211-2217); Hopp-Woods (Hopp,T.P. and Woods,KR. Proc. Natl. Acad. Sci. USA
(1981) 70: 3024.3828); Eisenberg (Eisenberg et al., Proc. Natl. Acad. Sci. USA (1984) 81:
140-144); or Kyte-Doolittle (Kyte,J. and Doolittle, RF. J. Mol. Biol. (1982) 157: 105-132).

Since its introduction the IDEAS suite has been accessed an average of 23 times per month.

3.1.2.4 Data Contributors

BIONET has always provided rapid updates to all the major collections of sequence data including
GenBank and EMBL nucleotide sequence collections, and the NBRF/PIR Protein Data Bank. This
last year we have continued to expand the databases on-line that are related to molecular biology.
This has often involved establishing contacts with database managers and providing them with the
facilities on BIONET to maintain their data collections. The following summarizes our current and
projected activities in this area:

Restriction Enzyme Database. We continue to provide the community with the latest additions
to the Restriction Enzyme Database, through the cooperation of BIONET and Dr. Roberts at Cold

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Spring Harbor. Modifications are mailed electronically to BIONET after they are incorporated into
the on-line database at CSH. In addition, we provide the community with subsets of the list of
enzymes that are commercially available. These subsets have been revised using the data in the
July, 1988 version of the database. The lists are made available in IntelliGenetics format for use in
the SEQ and PEP programs.

The catalog sources for each of the files are listed below:

amersham.lst
brl.lst
anglian.lst
ibi. 1st
boehringer . 1st
neb.lst
phannacia.lst
promega.lst
usb.lst

arsham (8/87)
Bethesda Research Laboratories (6/881
Anglian Biotechnology Ltd. (l/88)
International Biotechnologies Inc (7/87)
Boehringer-Mannheim (5/88)
New England Biolabs (7/88)
Phannacia P-L Biochemicals (5/88)
Promega Biotec (g/87)
United States Biochemical Corporation (4/88)

These files are extremely useful since the full Roberts' database now consists of over 900 enzymes
and most users are only interested in enzymes which they can readily access. Users can also create
custom databases of enzymes using the information in the commercial enzyme lists to limit their
analyses to enzymes in their lab or from their favorite suppliers. Analyses run with these shorter
enzyme lists will, of course, also be correspondingly faster.

SWISS-PROT. Since June, 1987 the BIONET staff has made the SWISS-PROT protein sequence
database from Amos Bairoch available on BIONET. SWISS-PROT contains data obtained from the
NBRF/PIR database, data translated from the EMBL DNA sequence Data Library, as well as
sequences entered in-house. To maintain SWISS-PROT on BIONET requires conversion to both the
IntelliGenetics format and to the FASTA format for searching by the FASTA-MAIL program. In
addition, testing is done after the conversions are made to insure the integrity of the data.
Maintaining the original, IntelliGenetics, and FASTA formatted versions of SWISS-PROT currently
requires over 28 Mbytes of disk storage. When first released on BIONET, SWISS-PROT release 4.0
contained 1,036,010 residues in 4387 sequences.  Since then the databank has approximately
doubled; the current release 8.0 contains 2,224,465 residues in 7724 sequences.

Genetic Variations of Drosophila melanogaster - the `Red BoolF. BIONET has long had the
full text of Lindsley and Grell's classic work "Genetic Variations of Drosophila melanogaster"
available on line. This was made possible by help from the author, Dan Lindsley and with the
permission of the original publisher, Carnegie Institution of Washington. Dan Lindsley holds an
NLM grant to update this book and has also been forwarding chapters of the book to BIONET as
they are finished. Because of this, the most complete collection of Drosophila mutants are always
available on the BIONET computer. This book can be searched using one of several different text
searching programs (QUEST, FIND and XSEARCH) all of which allow searches for complex boolean
relationships between search terms. This also permits BIONET users to search for all genes by
name, by phenotype, by affected tissues, by genetic location and by cytological location. This size of
the text is still small enough to permit a complete serial search in a matter of seconds. This year
BIONET received and put on-line the revisions of the chapters describing loci and mutant alleles
whose names begin with the letters A through R.

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!W40 Mutant List. On July 15, BIONET made available a contributed database of the SV40 large
T antigen mutants compiled and maintained by Dr. J. M. Pipas of the University of Pittsburgh. The
database consists of five lists of mutants divided into the following groups: early region deletion
mutants including either sequence or map information; early region point mutants including either
base-pair change (and corresponding amino acid substitution) or map position; and the primary
sequence of truncated T antigen mutants. Each list is annotated and has a complete set of literature
references which describe the mutations listed in detail. Since its introduction on BIONET the
database has been distributed to five geographically distinct regions in the U.S. and in Europe using
BIONET's existing ARPANET and BITNET connections, as well as being used at the rate of 16
references per month on BIONET itself.

LiMB Database. In February, BIONET made available on-line the LiMB (Listing of Molecular
Biology databases) database, created by the staff at Los Alamos National Laboratories. LiMB
contains information about the contents of databases related to molecular biology as well as details
of how they are maintained.  It was created to facilitate the process of locating and accessing
databases that the research community depends on; it is also of use to those who are doing research
in designing and linking these databases. Information for each database includes the purpose of the
database, contact addresses including network addresses for the database staff, the history of the
database, source of the database data, literature references, cross-references to other databases,
details of the computer-readable form of the database, and the size of the database.

Removal of Brookhaven database from BIONET.  This year access to the Brookhaven
structural database had to be curtailed due the the imposition of an $8,500 per year fee for network
access by Brookhaven. Apparently a fee of this magnitude was necessary because the database
effort is largely funded through the sale of tapes. The database was continued only for in-house
research by the BIONET staff at the old fee of only about $260 per release. The low usage of the
database on BIONET due to the lack of graphics capabilities on the system precluded the
expenditure of $8,500. Only the DSSP program which extracted secondary structure information
was available for use on the system. Given different circumstances in the future the database may
be restored to BIONET. However the expenditure of such a large sum of money for this database
compared to the costs of other molecular biology databases (about 10x more expensive) could be
avoided if more funds were provided by the government to Brookhaven to finance its operation.

Protein Crystallography Directory. Dr. M. M. Teeter of Boston College maintains a list of the
electronic mail addresses of all protein crystallographers in the U.S., Canada, Europe, and
elsewhere. A copy of the latest version of the list is periodically sent electronically to BIONET over
BITnet, where it is made available to any BIONET users who wish to browse it using a free-form
text searching utility called FIND available on BIONET. All a user need do is attach the
appropriate network s&ix (e.g., .bitnet, .janet, or .eam) in order to send electronic mail to any of the
crystallographers listed in the database.

3.125 Liaison with Other Resources

Several accounts have been established on BIONET to promote interaction with other, related
Resources. The following is a summary of current sites with which we can exchange information.

BRTP Mailing List. Following the February 1988 meeting at the DRR BIONET established an

34


P41RBO1665-06

electronic mailing list for the Biomedical Research Technology Program. By sending a single mail
message to the address brtp@BIONET-2O.bio.net it is now possible for scientists at any of the BRTP-
funded resources to communicate with scientists at all other resources.

Molecular Biology Computer Research Resource. The MBCRR, at the Dana-Farber Cancer
Institute at Harvard, shares information through mail delivery via the GENE account and via the
bulletin board system.  An MBCRR bulletin board is available on the DEC 2065. Dr. Jurka at
BIONET has also continued his work on the MASE editor in collaboration with Mr. Donald Faulkner
at the MBCRR.

Molecular Biology Information Resource. The MBIR at Baylor formerly communicated with
BIONET through Dr. Lawrence's account on the BIONET computer. After having established an
Internet connection last year, the MBIR has been on our list of BIONET newsgroup recipients.

Pxwtein Identification Resource. BIONET has provided a bulletin board expressly for the PIR
which now allows members of that database staff to communicate easily with users around the
world. In addition the PIR along with GenBank and EMBL now receives automatic electronic data
submissions via BIONET's XGENPUB program using the common data submission form.

Genl3ank GenBank continues to utilize the GenBank bulletin board on BIONET which now has
worldwide distribution through the BIOSCI bulletin board network. GenBank also receives new
sequence data submissions from BIONET's XGENPUB program. XGENPUB was released in 1987
and has been used to submit a total of 202 new sequences to the databases; 166 during this reporting
period (12/87 - ll/88) for an average rate of 14 per month.

EMBL Databank The EMBL continues to utilize its bulletin board on BIONET to communicate
with the scientific community.  It also receives electronic data submissions from BIONETs
XGENPUB program as noted above.

Pittsburgh Supercomputer Center. From August 8 - August 12, Spencer Yeh, the BIONET
Applications Analyst, participated in the Biomedical Supercomputer Workshop at the Pittsburgh
Supercomputing Center @SC). The Pittsburgh Supercomputer Center utilizes a Cray Y-MP
supercomputer and will be the first supercomputer center to receive a Cray 3 computer when they
become available in 1990. In addition, the PSC has a three-year $2.2 million grant from the NH-I's
Division of Research Resources to provide the biomedical community with supercomputing
resources, training, and user support. Because of this, the PSC already has both the GenBank and
PIR databases on-line. The purpose of the visit to the PSC was to establish the feasibility of
providing BIONET users remote access to the PSC's Cray computer for computationally-intensive
tasks.

The workshop covered topics such as using the VAX frontends to the Cray, using existing software at
the PSC for biomedical research, and optimization/vectorization techniques for vectorizing
FORTRAN code on the Grays.  Discussions with Hugh Nicholas and David Heerfield, Scientific
Specialists at the PSC, addressed important issues such as the network connectivity of the PSC for
remote job submission, expected speed improvement on typical BIONET applications when ported to
the Cray, time allocation schemes for Cray CPU usuage, suitability of the Cray for languages other
than FORTRAN, especially C code, and the projected programmer time required for porting existing

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applications to the Cray environment. It was learned that the PSC is changing to a UNICOS (Gray
UNIX) operating system which will facilitate systems development efforts since the BIONET
systems programmers are already conversant with UNIX systems.

As a result of the discussions it was concluded that it would be feasible to allow BIONET users
remote access to the PSC's Cray. However, important issues still need to be addressed such as the
projected speed and reliability of the NSFnet over the next 5 years, network and Cray queuing
delays for remote jobs, and the overhead involved in submitting jobs to a supercomputer. While the
Cray can achieve speed increases of 10 - 100 times the performance of a SUN 3/280, the turnaround
time will probably be limited by the network response from California to Pittsburgh, and by the
queuing system at the PSC for batch jobs. Jobs which require less than 30 min. - 1 hr. of Sun CPU
time are probably too small to warrent the overhead of remote job submission to the PSC. The
workshop provided an excellent introduction to using Cray computers, and the information gained
will allow BIONET to evaluate its options for handling computationally intensive tasks.

Biological Matrix Workshop. BIONET has continued to support the Matrix project. Dr. Jurka,
the BIONET scientist, attended the last Matrix meeting in Washington in October and BIONET has
continued to maintain a newsgroup for the Matrix project.

BIOSCI bulletin board network distribution centers. BIONET initiated collaborations with
five other university sites to establish the BIOSCI bulletin board network. This is described in more
detail below.

Journal Editors on BIONICI'. Last year BIONET established accounts for the editorial board of
the Journal of Biological Chemistry, CABIOS, Cell, and the Washington office of Nature. This year
an account has been opened for the Journal of Bacteriology. These accounts serve several purposes.
First, they allow easy communication between the scientific community and the on-line editorial
staff. Second, they allow the editors access to sequence analysis software which they may use in
reviewing manuscripts. Third, the accounts increase awareness among editors of the advantages of
electronic networking, especially in regards to the problem of data submission to the nucleic acid and
protein sequence databanks. Although the usual reaction on the part of journal editors has been one
of reluctance to become actively involved in the data submission process (understandably so in light
of their heavy workloads), their involvement in BIONET alerts them to the availability of on-line
data submission software (the BIONET XGENPUB program described above) and they can then
pass this information on to other scientists.

This year Dr. Herb Tabor was trained in the use of the system by Dr. Kristofferson while in
Washington for the February DRR meeting.  Dr. Kristofferson also trained one of Dr. Tabor's
assistants at the J. Biol. Chem. offices in the use of electronic mail and file transfers. Joseph Palca
of the Washington office of Nature has actively used the system during the course of the year for
electronic communications. Recently the Jowxal of Bacteriology has been provided with an account
on BIONET for the purpose of advance publication of the table of contents of that journal. BIONET
will establish an electronic bulletin board for this purpose.

Training of the editorial staff in the use of BIONET can usually be accomplished by direct terminal
links with simultaneous verbal instruction over the telephone. In the course of about 45 minutes a
person can become proficient in using the electronic mail and bulletin board facility and also learn

36


P41RsRO1665-06

the use of the on-line help system. This allows them to explore other features of the system at their
leisure. The BIONET consultants are also available to assist the editors when called upon.

3.15.6 BulletinBoardsandLeadere
The following bulletin board topics are currently available on the system.

Bulletin Board Name
-------------------

AGEING
ASK-BIONXT
BIO-CONVERSION
BIO-MATRIX
BIONEWS (BIONET-NEWS)
BIOTECX
CONTRIBUTXD-SOBT
Et4BL-DATABANX
EwPLoYBfENT
GENBANK-BB
GENE-EXPBESSION
GXNWC-ORGANIZATION
INPO-1100
INFO-AILIST
INFO-AMIGA
INFO-ATARI16
INFO-IBM-PC
INFO-XCC
INFO-XXPMIT
INFO-LAW
IWO-WAC
INFO-XODEMS
INFO-NEURON
INFO-SUN-SPOTS
INFO-TELECOM
INFO-VAX

=THODS-AND-PEAGXNTS

Description
-----------
Scientific interest group
User queries and consultant responses
Scientific interest group
Application9 of computer9 to biological databases
General announcement9
Biotechnology issues
Information on program9 contributed by users
Coxmunications about EMBL databank concerns
Job openings
Cossauaications about GenBank database matters
Scientific interest group
Scientific interest group
Computer interest group
Computer interest group
Computer interest group
Computer interest group
CojPputer interest group
Cwuter interest group
Computer interest group
Assorted legal information
Computer interest group
Information aboout modems
Neural network computing
Computer interest group
Telecomaunications
Computer interest group
BBoard for I4BCRR announcements
For reagent exchanges and announcements about
lab methods
Scientific interest group
Scientific interest group
Information on cosxnun ications software
General PC software announcements
Nessages to and from the PIR database staff

mLECUL&R-EVOLUTION
ONCOGENES
PC-COA0RWICATIONS
PC-soPTwAPE
PIR
PLANT-MOLECULAR- BIOLOGY Scientific interest group
PROTEIN-ANALYSIS      Scientific interest group
RESEARCH-NEWS         General interest itams about science
SCIENCE-RESOURCES     Information about funding agency policy, etc.
SWISS-PROT          Nessages to and from the SWISS-PROT database staff
YEAST-GXNXTICS       Scientific interest group

The leaders of the indivdual boards are:

AGEING              Sydney Shall
ASK-BIONXT              David Xristofferson
BIO-CONVERSION         Eng-Leong FOO
BIO-MATRIX              Dan Davison
BIONXWS                David Xristofferson

37


P41ltRO1685-06

BIOTBCH                W Patnaik
CONTRIBUTED-SOFTWABX     BIONBT Staff
BMBL-DATABANK            Graham Cameron
                   & David Kristofferson
mlPLoYNENT              David Kristofferson
GENBANK-BB              Christian Burks
                    & David Benton
GENE-EXPRESSION          Bill Sofer
GBNOMIC-ORGANIZATION      Tom Marr
                        Susan RUSSO
MBTIiODS-AND--S     David Kristofferson
-LBCDLAR-EVOLUTION       Dan Davison
ONCOGENBS                David Steffen
PC-COMMUNICATIONS        David Kzistofferson
PC- SOFTNARE          Doug Brutlag
PIR                David George
PLANT-MOLECULAR-BIOLOGY   Robert Jones
PROTEIN-ANALYSIS         Amos Bairoch
REsmRm-NENS            Sunil Maulik
SCIENCE-RESOURCBX        l4ichele Cimbala
SWISS-PROT              Amos Bairoch
YEAST-GENETICS         John Thompson

Note that the INFO- bulletin board material is received from information sources outside of
BIONET.

This year was a particularly exciting time in the development ofthe BIONETbulletin board system
as it became the nucleus of the new international BIOSCI bulletin board system.

Because scientists work on a variety of computer networks around the world, BIONET recognized
the necessity of developing a mechanism to allow all of them to communicate without the necessity of
learning the peculiarities of accessing each network. We sought out computer sites on all major
international networks and arranged to have parallel copies of the original BIONET bulletin boards
accessible from these sites. Besides BIONET in the U.S., other major BIOSCI distribution sites are
now situated at the SERC laboratory in Daresbury, England; the University College, Dubulin,
Ireland; the University of Uppsala in Sweden, and the University of Helsinki in Finland. Recipients
of the bulletin boards from these sites are located around the world from New Zealand and
Australia, the Far East, and Israel, throughout Europe, and back to North America. The bulletin
boards are available to users on the ARPANET (from BIONET), BITNET (from BIONET), EARN
(from Dublin, Daresbury, Helsinki, and Uppsala), Usenet (from BIONET), NSFnet (from BIONET),
and JANET (from Daresbury). Users in any particular location need only post or receive messages
from their closest site. Any postings at any one site are automatically forwarded by the central
BIOSCI sites to all other participants on all of the above-listed networks.

A copy of the BIOSCI information sheet mailed electronically to people who request information is
provided in Appendix V.

The following list contains the number of messages posted to each BIOSCI board from 12/87 through
lY88. All told 956 messages were posted which was an increase of 74% over the previous year. As
many molecular biologists are only now discovering the use of electronic mail and bulletin boards we


expect that these high growth rates will continue into the foreseeable future. For the last five years
BIONET has looked on effort expended on electronic communications as a long-term investment.
This year it is clearly starting to pay off.

Bulletin Board      Messages Posted
--------------          ---------------

AGBING              0 (being established)
BIO-CONVERSION         6 (started end of 11/88)
BIO-MATRIX              46
BIONBWS                 187
BIOTBCH                118
CONTRIBUTED-SOE'T      21
EMBL-DATABANX            17
ENPLOYNENT              93
GENBANK-BB              33
GENE-EXPRESSION          14
GBNOMIC-ORGANIZATION       0
=THODS-AND-REAGENTS     105
MOLECULAR-BVOLUTION       44
ONCOGENES                12
PC-CO&QdUNICATIONS         12
PC-SOFTNARE              57
PIR                    18
PLANT-MOLECULAR-BIOLOGY    5
PROTEIN-ANALYSIS          33
RESEARCH-NEWS             72
SCIENCE-BESOURCBS         51
SWISS-PROT                 4
YEAST-GENETICS            8

3.1.3 Technological Research

3.1.3.1 Research Efforts by the BIONET Scientist
The activities during 1988 can be divided in the following three groups: (1) Studies on interspersed
repetitive elements with emphasis on Alu and Ll subfamilies; (2) Design and development of
software for sequence analysis (with emphasis on sequence extraction, multiple alignment and
classification); (3) Collaborative research on evolution of Alu and protein sequences (discussed above
under ColIaborative Research).

1. The biological findings on Alu classification have been published (Proc. N&l. Acad. Sci.
USA 85, 47754770, 1988 & Nucleic Acids Res. 16, 766, 1988 see Appendix I). In
addition to the work on Alu sequences, classes of KpnI sequences have been discovered.
A manuscript on this subject will be submitted by December 1988.

2. The following activities occurred: (a) Development of a sequence editor in collaboration
with Donald Faulkner of Harvard (Trends in Biochemical Sciences 13, 321-322, 1988).
After publication of the article, several new functions have been added to MASE upon
our suggestions: enhancements on COLUMN-CORRELATION, CREATE-LOCUS,
SIMILARITY-DISCARD-GAPS,    RENAME-LOCUS,    JUMP-TO-POSITION-
ABSOLUTE, modifications of PATTERN-HIGHLIGHT, MODE-ALIGNMENT, MODE-
DNA, MODE-PROTEIN. In addition, we assisted in testing of these and other
functions added to MASE during last year; 0.1) In-house work on a sensitive algorithm
for sequence classification with emphasis on Alu and KpnI repeats. Previous attempts
by other investigators to classify Alu repeats based on overall sequence similarities
proved unsuccessful (Bains, 1986). This was quite inevitable since the diagnostic

39


positions are only a small portion (l-6%) of the total number of bases in Alu sequences
and the distinction between them and the statistical noise could not easily be made
using standard tree analysis.

3.1.3.2 FASTA-MAIL

Because of the increasing demands on the DEC 2065 BIONET needed to find alternative computing

resources.  Towards the end of last year, Sun Microsystems generously donated a new central
computing facility to BIONET. Although some software development is necessary before we can
transfer users directly to the Sun system we were able to use it remotely fromj the DEC for database
searches through the use of our new FASTA-MAIL program. Much of the Dee's CPU resources were
being spent on genetic sequence library searches, which could take up to six hours of CPU time. In
an effort to ease CPU usage, we undertook to write an interface to FASTA, a sequence search
program written by William Pearson that runs under UNIX. The interface we devised is known as
FASTA-MAIL.

FASTA-MAIL will take submissions received via Internet mail, queue them for batch processing,
and mail the results back to the submitting address. On the DEC 2065 users run a simple program
and answer a few questions about the type of search that they wish to perform. This program
submits these instructions together with the query sequence data to the Sun 31280 computer at
BIONET. Currently, there are two queues on our Sun that run concurrently to handle protein and
nucleic acid searches, respectively. The batch processing program processes a limited grammar at
the beginning of each message so that it may pass command line arguments to FASTA.

On the Sun end a message is received by the mail processing agent known as sendmail, which
delivers the message to a program instead of a user. In this case, the program is one that checks
whether the user has requested a nucleic acid search or a protein search, and places the message in
the appropriate batch queue. It should be noted that as initially implemented, the batch queues
used were, in fact, printer queues because SunOS's batch facilities were too weak to support multiple
queues. Sequence data would enter through the print spooling system and be processed as printing
output until the output filter was called. At that point, we specified our own output filter, a program
known as fastaq. fastaq would then compare the sender of the database query with a list of
authorized users. Once validated, the message headers would be stripped (although the program
retained the return address), and a small set of parameters are read in - ktuple and database name.
The rest of the data is then passed to FASTA, whose output is sent directly to the UNIX mail
program.

Besides allowing access to BIONET users on the DEC 2065 computer, the FASTA-MAIL program
ushers in a new means of performing database searches. The program can be easily modified to
accept input from any electronic mail site in the world. Soon we expect to allow users at other
sites access, but outside jobs will be placed in a lower priority queue so as not to impact significantly
on our registered users. Expansion of the computer resources available at BIONET would allow us
to serve a far greater number of users very economically.

40


3.1.3.3 Development work on the new Sun central computing facility
In June of 1988, the six Sun 3/6OM computers and one 3/2808 computer donated by Sun
Microsystems arrived at BIONET. These computers are nearly operational and are providing some
service to the BIONET community, particularly via FASTA-MAIL as noted above. In addition to
many smaller projects (not listed), the following work on the new system has been accomplished:
o The BIONET/BIOSCI mailing lists that were administered on the DEC-20 are now being
   administered on the 3/28OS. This will take some of the load off of the DEC-20's already
   loaded mailer.

o The BIONET Sun has become the major distribution point for the BIONET/BIOSCI
mailing lists, passing them onto other major Internet sites.
o As mentioned previously, FASTA-MAIL was written and implemented in August so that
genetic sequence searches could be done on the Sun.
o A hierarchal help system has been implemented, and the BIONET documentation is
being moved into this system.

o newuser, a program developed by one of our staff is being implemented so that users will
configure their environments the first time they log into the Suns.

o MM, a mail management system has been installed. An almost identical mailer was run
on the DEC-20. This will eliminate relearning the electronic mail program when users
migrate to the Sun system.

o netnews, an electronic bulletin board system, has been installed.

We shall be begin moving users to the Suns from the DEC-20 as soon as accounting software is in
place.

3.1.3.4 BIONET Sated&e Software for VAXNMS systema

The BIONET Satellite software package provides the mechanisms for VAX/VMS computer systems
to exchange electronic messages with a variety of computer installations. Messages are sent using
telephone connections in a standard electronic mail format. Messages delivered are compliant with
the address conventions of RFC822 ( the standard for the format of Internet text messages).
Messages can be directed to individuals or sent to a bulletin board facility. The bulletin board
system implements the Standard for Interchange of USENET messages. This standard allows the
host system access into the USENET news network.  The software components consist of two
integrated subsystems which implement the functionality as described above. The Pascal Memo
Distribution Facility (PMDF) implements the MMDF protocol which provides the interface for
message transmission. NEWS, written by Geoff Huston is the system which provides general
conferencing in the form of a bulletin board service on the VAXAMS host system.

The BIONET satellite system was originally designed to connect to a DEC 2060 computer running
the TOPS-20 operating system. Message transmission to VAX/VMS systems was implemented using
a non-standard communcations protocol, CAFARD. This protocol would then interface with a mail
delivery system called Pony Express. Pony Express software is a proprietary product of SRI. The
acquisition of a SUN 3/280 as the primary host system for BIONET has necessitated changes in
system software. The SUN machine uses the UNIX operating system. To support the CAFAED and
Pony Express systems on the SUN machine would have required extensive development and
maintenance time. The alternative was to look for public domain software which would provide the
original functionality of the Satellite software. The selection of PMDF and NEWS were selected, in

41


P41R.RO1685-06

part, to realize these goals. The MMDF protocol has been used extensively on UNIX machines and
the PMDF system provides compatibilty on all current VA?WMS systems. The USENET network
has been used for years as a way to conference information between end users. The NEWS system
provides both connectivity and a user interface to USENET. These two subsystems are in the public
domain and have sauce code that is distributed. This new version of the Satellite software is now
undergoing local testing. Installation and documentation procedures for VAX/VMS sites are near
completion. It is anticipated that site testing will begin in early January 1989.

3.1.3.5 The RICH program

It is now known that the amino acid composition of a protein plays a major role in determining its
folded state (Sheridan RP et al. (1985) Biopolymers 24: 1995-2003). A fundamental pattem-
matching problem in protein sequence analysis is that of finding the largest subsequences given
certain density (composition) criteria only. For instance, one may wish to find the largest region in a
500 amino acid protein that contains ~20% proline and >30% cysteine residues. A related problem
may be finding all protein subsequences in a database that have greater than 60% hydrophobic
residues.

As a result of requests such as these from BIONET users, an algorithm has been developed that will
scan a sequence and heuristically discover the largest subsequence(s) that satisfy any given density
criteria.  Current algorithms for finding rich regions in sequences (such as RICH in the SEQ
program; Brutlag, D., Clayton J., Friedland, P., & Kedes, L.H. (1982) Nucl. Acids Res. 10: 2791, find
the first such subregion satisfying the density criteria and then expand in increments of one unit
(base or amino acid) until the combined density falls below the density threshold. Following this, the
region is reported, and the algorithm proceeds to find the next such region. The fundamental
problem with algorithms of this type is that they fail to look far enough ahead (or back) to see if
sufficiently dense regions could be incorporated with the current one in order to locate the largest
subsequence. Our algorithm uses a heuristic search procedure to find all seed regions satisfying the
density criteria and then attempts to link all such "seeds" together. By finding all seeds in the initial
pass, the algorithm circumvents the problems of the other implementations and produces the largest
subregion in the second (or following) iteration or optimizing pass.

The implementation of this algorithm, termed RICH, is near completion and will be available on
BIONET in 1989. The implementation has been optimized to allow RICH to scan entire databases
such as the NBRF-PIR protein sequence database and locate patterns describable by their density
characteristics.  Uses of the RICH program might include finding hinge structures in
immunoglobulin sequences (known to be rich in prolines and cysteines; Huber, R. and Bennett, W.S.
(1987) Nature 326: 334-335) or verifying that a protein sequence satisfies the PEST hypothesis
(Rogers, S., Wells, R., & Rechsteiner, M. (1986) Science 234: 364-3681, i.e., if its half-life is related to
the density of P (proline), E (glutamic acid), S (serine), and T (threonine) residues. A preliminary
search of the NBRF-PIR database using RICH has shown surprising results in the type of sequences
containing one or more dense PEST regions, and further studies to verify and quanti@ this effect are
progressing.  A manuscript describing RICH is in preparation, including examples and results
arising from using RICH to scan databases for densitydependent patterns.

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P41RRO1685-06

3.1.3.6 BioCard - a prototype menu-driven interface for BIONET
BIONET had previously investigated using application toolkits to develop graphics-based, menu-
driven interfaces to terminal emulators running locally on users' personal computers. The aim of
these investigations was to create an interface containing all the information about BIONET as
easily accessible, cross-referenced help text, and to allow users to activate software or database
search queries on BIONET using simple switches/buttons which would then run command-files on
the central BIONET computer. With the advent of HyperCard for the Apple Macintosh, an
information management system with an built-in applications generator, the creation on icons,

menus, "dialog" boxes, windows, and hypertext tailored specifically to BIONET users has become
straightforward. The first HyperCard application for BIONET, termed BioCard, consists of a set of
graphics screens containing icons for all of the information currently on BIONETs HELP ME
system, as well as buttons representing the most commonly performed tasks on BIONET. A user of
BioCard could have it automatically dial the nearest Telenet or CompuServe phone number, connect
to BIONET, and then run a sequence analysis program on the BIONET computer. The user can also
switch between remote (BIONET) mode, and local (BioCard) mode, thus allowing users to browse
through HELP information without having to quit their current application.

Currrently, BioCard consists of the complete HELP ME system of BIONET in an easily searchable
Hypertext format, as well as twelve buttons which, when activated, run the most commonly
automated sequence analysis functions on BIONET such as database similarity searches, restriction
mapping, sequencing gel assembly, etc. BioCard is also bundles with MacKermit, a public domain
terminal emulation package for the Macintosh from Columbia University. Under the Macintosh's
MultiFinder operating system, users can switch between local (BioCard) mode and remote (BIONET)
mode in a simple and intuitive manner.

One advantage of Hypercard over other applications generators is that it is easily customizable by
the user, even one with only a rudimentary familiarity with computers. Thus BIONET users will
have the opportunity to select those icons, windows, and buttons that they require and use most
frequently, while discarding others of no interest to them.  Further, new functionality may be
created by coupling or modifying the existing application icons. Finally, adventorous users may wish
to design their own interfaces using BioCard as a model. Thus BioCard serves as an opportunity for
BIONET to explore the many human factors engineering aspects of interface construction. Once
these factors are known (through feedback from the user community over the BIONET network),
BIONET plans to develop hardware-independent user interfaces using such windowing protocols as
X-Windows from MIT, a public domain windowing system that is finding acceptance with such
personal computer/workstation vendors as IBM, DEC, Sun, and Apple. A preliminary version of
BioCard is under testing, and a first release should be available in 1989.

3.1.3.7 XGENPUB

One of the original goals of BIONET was to aid in several of the database efforts including GenBank,
EMBL and the Protein Identification Resource.  Initially we felt that a major contribution that
BIONET could make would be to make these databases more readily available by providing software
tools for database searching and analysis. However it became clear that a great number of DNA
sequences were actually being determined using the IntelliGenetics GEL program on the BIONET
computer. We felt that BIONET could provide a further service to both the community and to the
database efforts by developing software that would allow the scientist to annotate his sequence

43


P41HRO1665-66

according to the standard GenBank format and mail the sequence and its annotation to GenBank
electronically. In 1987, BIONET completed work on and released the initial version of GENPUB,
currently named XGENPUB on the DEC-2065. The GENPUB program is a forms-oriented display
editor that allows a person to fill in a template based on the GenBank submission form (and which
can be readily changed if the GenBank form changes) giving all the requisite data about a sequence.
The program automatically inserts the sequence in the appropriate place in the form by copying the
sequence from a designated file on the BIONET computer. When the form is completed a single
keystroke forwards the information to both the GenBank computer at Los Alamos and the EMBL
computer in Heidelberg for inclusion in the next issue of the databases. At that point the entry is
verified by the GenBank and EMBL staff and if they have questions about the data they can query
the author by electronic mail at BIONET. To date, GENPUB has been used on BIONET to submit
202 sequences to the databases.

This year some new features were added to the XGENPUB program:
  o the ability to accept any sequence data file regardless of format;
  o the ability to preview the data submission form inside the XGENPUB program prior to
  going into the editor and,

o additional internal help documentation.

The PIR database was also added along with GenBank and EMBL as a recipient of data submissions
after a common data submission form was adopted by all three databases.

Direct electronic submission mediated by GENPUB eliminates many errors in transcription.
GENPUB takes sequence files directly from the software used to determine the sequence and
submits it to the database. If local PC software is used to determine a sequence, the data can be sent
to BIONET using an error-checking protocol such as Kermit or Modem and forwarded to GenBank
via GENPUB eliminating all transcription errors. More importantly, GENPUB recruits the scientist
determining a sequence to annotate it, eliminating the problems of reading and interpreting the
publication and further simplifying the job of sequence collection. This concept of recruiting the aid
of the scientists performing sequencing to help build the database was fundamental to the
IntelliGenetics application for the GenBank contract. Since that contract was awarded to
IntelliGenetics, GenBank has undertaken to make an even more sophisticated form of this program
which will include error checking on the data entered into the annotations and which will be
extremely portable, running on a number of microcomputers as well as mainframes. When this
program is distributed to scientists who are developing new sequences, it will markedly increase the
rate and quality of the data entering the GenBank database. BIONET is proud to have served the
community in this way and looks forward to even closer working relationships between itself and the
database efforts. We have taken an important role in not only making databases more accessible,
but have also taken an active role in helping accumulate the data as well.

3.1.4 BIONET Training Program
This year we have continued holding intensive training sesions in our in-house training facility and
have also given a number of demonstrations and lectures at outside sites.

We conducted two day, in-house training sessions in March, May, July, September, and November.

44


These bve courses served a total of 45 BIONET users. The courses continue to stress the integration
of the programs to solve problems. These problems include sequence entry and editing, sequencing
gel management, nucleic acid and peptide sequence analysis, database structure and sequence
retrieval, collecting sequences, pattern searching, and sequence similarity searches. In November
we expanded the training program to include an evening session on connecting to the BIONET
computer, transferring files, using a text editor, addressing electronic mail, and accessing other
BIONET programs. This additional session will be included in future in-house trainings, as well.
The schedules for each training are included in Appendix VI. The training material for these
sessions has also continued to be revised by the staif during the course of the year. A new training
manual for use in the courses has been produced and continually updated.

Outside seminars and demonstrations were held at Rutgers University, the NIH, the Protein Society
meeting, the FASEB meeting, the Annual Meeting of the Canadian Society of Microbiology in
Windsor, Ontario, and at Ohio State University at Wooster. A lecture is also planned next February
in San Francisco at the annual ASCB meeting. In addition to these events, Dr. Kristofferson helped
train the some of the journal staff at the of&es of the JournaZ of Biological Chemistry and the
Washington office of Nature as described above under Liasons with Other Resources.

Rutgers. BIONET presented a poster at the biannual meeting at Rutgers University's workshop
titled "Computers in Molecular Biology"  at the Waksman Institute/Center for Advanced
Biotechnology and Medicine, New Brunswick, New Jersey, April 13-15. Approximately 50 people
attended.  The poster described new developments at BIONET and included hands-on
demonstrations of using BIONET to solve molecular biology computing tasks. In addition, BIONET
participated in a panel discussion on the future of molecular biology computing. Fourteen BIONET
applications were obtained as a result of the meeting.

NIB Users' Group Meeting. BIONET, in association with IntelliGenetics, organized a User's
Group meeting at the National Institutes of Health, Bethesda, Maryland on August 25. The meeting
featured talks describing the BIONET resource and some of the software available on it. In depth
presentations were also given on sequence similarity searches and alignments, assembling and
managing sequencing fragments, and recent changes in the IntelliGenetics software package. A
total of 70 people attended the all-day session.

Protein Society Meeting. BIONET was invited to present a poster and demonstration at the
"Computer Workshop on Protein Analysis SoRware" at the Second Annual meeting of the Protein
Society in San Diego, August 13-17, 1988. Over 1000 attendees were expected to be present for this
meeting. Two posters were presented, titled " Protein Databases and Analysis Software on
BIONET" and "Locating Amino Acid Patterns by Composition" that described the current state of
research and technological development in protein analysis software at BIONET. In addition during
the 3 hour workshop over 30 applications were obtained and 50+ people were given hands-on
experience at logging into BIONET over the TelenetiCompuServe networks and making use of the
BIONET software.

FASEB Meeting. The BIONET Applications Analyst, Spencer Yeh, attended the annual meeting of
the Federation of American Societies for Experimental Biology (FASEB) in May. A portable
computer was used to demonstrate the BIONET system to meeting attendees. Use of the new

45


P41R.RO1665-66

Tektronix graphics display in the Kermit communications program was illustrated by modifying
circular plasmids within the CLONER program.  In addition to generating 39 requests for new
BIONET applications, the many current BIONET subscribers attending the meeting were able to
obtain personal assistance with analyzing their data on BIONET. Suggestions for improving the
Resource and general comments about the usefulness of the Resource to the laboratory biologist were
also conveyed to the BIONET staff.

Canadian Society for Microbiology. Dr. David Rristofferson was an invited speaker to address
the members during the opening symposium of the Canadian Society for Microbiology annual
meeting. The meeting occurred on June 20th in Windsor, Ontario, and the opening session, attended
by over 100 scientists, was entitled "Computers in Microbiology." The features of the BIONET
system were described and many stimulating questions and discussions followed the presentation.

Ohio State, Wooster. The Ohio State University at Wooster conducted a summer workshop on
DNA sequencing and cloning techniques attended by 27 scientists from throughout the Mid-West.
This workshop included sections on computer-aided sequence analysis. Dr. Kristofferson addressed
the group on June 21st. He also gave an on-line demonstration of BIONET following the lecture
during which several users acquired hands-on experience and had their questions answered about
the use of the Resource.

3.1.5 Resource Facilities

Previous reports have discussed the DEC-2065 and the various software and database libraries
provided by the BIONRT Resource. In this section we highlight significant changes and additions to
the suite of hardware and software that comprise BIONET.

3.1.5.1 BIONEJYSUN Agreement

Because the DEC-2065 is rapidly becoming obsolete and because equipment funds have been limited,
in the summer of 1987 we submitted a proposal to Sun Microsystems to obtain a new central
computing resource for BIONET. Sun agreed to donate a central Sun 31280 file server and six Sun
3/60 client workstations. This equipment arrived in spring and became operational in June. This
BIONET initiative has saved the NIH $150,000 in equipment costs! Development efforts on the new
system were described above under Technological Research.

3.1.5.2 Computer Hardware and Telecommunication Networks
HardWltlX!.

The current BIONET Central Resource Machine is a Digital Equipment Corporation 2065 computer
with an N120 ethernet network interface. The ethernet interface provides access to the ARPANET
and other IntelliGenetics' resources.

The hardware configuration is as follows:

KLlO-E Model R Procebdor:

2 MFZO/MG20 Memory controllers
3 Mw MG20 Manory
   bCA25 Cache Buffer Memory

46


P41RR01685-06

2 RX20 Massbw Channel8
NI20 Ethernet Interface

Console and Front End Processor:

POP-11/40 CPU, 32 XW 16 bit memory
RX02 Dual floppy disk drive8
8 DXill Terminal interface8       8 * 16 TTY line8 each = 128 line8
RHll MaSSbuS Channel

LP20 Line printer interface

Peripherals:

3 RP07 disk drives               1llMW each
RPO6 disk drive               39Mw
      372 Mw Total disk storage
TU78 1600/6250-BP1 tap8 drive
LP26 600 LPM Line printer

Disk space (data storage)

Public structure (PS:) disk space use on the 2065 is dynamic.   The
fOllOwing snapshot is representative of typical USage, and is taken
from December 1988.

Total disk space      433,000 (pages--222 million words)
Overhead/Common      <lSO,OOO> (Core, System and System Support Libraries)
Swapping Space        < 25,000,
Pile 8y8tw Overhead   < 88,000, (Directories and index page8)
                          ---s----m

170,000

BIONET Allocation
BIONET Usage 12/88

153,000   (90% of the available space)
<155,000>
---------

UnuSed space        < 2,000> (Available for BIONET growth)
We conclude from these figures that BIONET is currently using 8,000 pages more than its disk space
allocation.

SunComput43rs

In May of 1988, BIONET took delivery of a substantial donation of computing hardware from Sun
Microsystems. The BIONET staff, especially, Eliot Lear, one of the BIONET Systems Programmers,
has been working to configure the hardware and software on the new Suns, so that we may
gradually move users off of the Dee-20 and on to the new Sun computers.

The current Sun hardware configuration is as follows:

Sun Microsy8ttm8 3/28OS Data Center Server

1 Xylogics 753 3.0 MB/second S&ID disk controller
1 Xylogics 472 tape controller
1 Systech MT1 16 Channel Async Line Multiplexor
1 450A Sun Microsystem second Ethernet controller (first

47


P41RRO1685-06

controller included on CPU board)

Peripherals:

2 Sun 626A Fujitsu hf2361A Disk Drive8    550 MB each
CDC 9720-850 Disk Drive               675 MB each
     1775 ME (1.7 GB) Total disk storage
1 Sun 675A Fujitsu 6250/1600 BP1 l/2 inch tape drive

7 Sun Microsystems 3/60M Monochrome, diskless Workstations, each
     workstation configured with 12MB main mry

The 7 diskless Sun 3/60's and the Sun 3L280 are attached to a thin ethernet dedicated to providing
file service for the diskless 3/6O's. The second ethernet interface on the 31280 is connected to the
IntelliGenetics backbone ethernet. All network traffic, not destined for the diskless 3/60's, is routed
via this second ethernet interface, to leave the other network free for file service to the 3/6O's. A
Cisco Systems Gateway controls the routing of network traffic between the BIONET diskless Sun
network, two other local ethemets at Intelligenetics, the Arpanet, and the soon to be established
BarrNet connection. BIONET users of the Sun system have electronic mail, t&et remote login, and
fip file transfer access to the rest of the Internet. These services are also available between the Suns
and the Dee-20, until the phaseout of the Dee-20 is complete.

The Sun 3/280 and the 7 Sun 3/60's are accessible via the X.25 Public Data Networks, Telenet and
CompuServ (see description of X25 networks above). This connection is implemented by connecting
serial ports on our X25 Host Pads (described above) to ports on an existing Cisco Systems Terminal
Server. A terminal server is a device which allows users of serial terminal line ports to establish
login connections over the ethernet to hosts on the network. Using this mechanism, we are able to
distribute BIONET users across the 3/280 and 7 3/60's (plus any additional incremental resources
that might be added in the future) in a way which is transparent to the users. We are working on a
load balancing system, which will ensure that each time a user connects to BIONET, he/she will be
connected to the most lightly loaded computer resource available on our network.

A diagram of the entire BIONET computer system and network is included in Appendix VII.

Public Data Network Connection.

BIONET is accessed principally over the Telenetl and Compuserve Public Data Networks (PDN).
An X.25 PAD (packet assembler/disassembler) is located on-site for each PDN. This is known as the
Host PAD, or HPAD. It provides individual terminal ports which are cross-connected to those on the
DEC-2065. The Telenet trunk line operates at 9600 baud synchronously, and the PAD converts this
into up to 16 asynchronous ports whose speed is typically 1200 baud. A handshaking protocol is
employed to smooth over bursts of data during the multiplexing.

Connection to multiple Public Data Networks increases geographic accessibility, since areas which
are served poorly by one of the PDN's are often served well by the other. Reliability is also improved
by providing alternate access when service is poor or unavailable on one of the networks. Our
connection to both PDN's provided incentive to each PDN to offer us lower rates.

`The Telenet Public Data Network ie operated by U.S. Sprint.


P41RRO1685-06

ARPANET

BIONET maintains a connection to the Arpanet which is arranged through a DABPA-funded project
with IntelliCorp. In exchange for our assistance with the mechanics of the connection to ARPANET,
BIONET is able to make use of this connection for communications, especially electronic mail and
file transfer. Since there are mail gateways from the ARPANET to many other communications
networks, this connection greatly expands BIONETs reach-- including networks such as BITNET,
EARN and CSNET in addition to the DOD Internet.

The BIONET DEC-2065 is connected to a local ethernet at IntelliGenetics via its N120 ethernet
interface. A gateway connects the local ethernet to the Arpanet gateway at IntelliCorp via two
19.2KB leased lines.

NSFNET

In order to increase the reliability and speed of access to the DARPA/NSF Internet, BIONET has
plans to share a Tl (1.54 Megabits/Set) link to Barrnet (Bay Area Regional Research Network).
Barmet is the regional NSF sponsored network for the San Francisco Bay area. We expect our
Barmet connection to be operational by early January of 1989. This takes on additional importnce
because in recent months DABPA has taken some initial steps in the direction of scaling down the
Arpanet, and there has been talk about a complete phaseout at some unspecified time in the near
future.

3.1.55 Summary Statistics on Machine Use
Originally BIONET had access to 50% of the cpu of DEC 2965 computer and the rest was used by
IntelliGenetics and its parent company IntelliCorp.  With the availability of other computer
resources at IntelliGenetics this situation has now changed so that BIONET's share of the computer
has risen to 90%.

The cpu cycles of the DEC-2065 computer are allocated to the user community, including BIONET,
by the system's class scheduler. This scheduler is given the percentage of the machine to allocate to
each class of users. Any cycles not consumed by a given class ("windfall") are available to the rest of
the user community. This method was chosen so that cpu cycles not consumed by one segment of the
community could be used by other segments if needed, i.e., no cpu cycles are wasted if someone needs
them.

Because the class scheduler also adds to the system overhead, the number of user categories has
been reduced to two. This reduces system overhead and frees up computing resources. Currently
the batch queue and all users except IntelliGenetics commercial time sharing customers are in class
0 with a 90% allocation of the machine. Commercial time sharing users are in the second class and
have a 10% allocation of the machine.

The actual use of the machine by the BIONET community has been on average each month 88%,
substantially greater than the 50% of the total cpu cycles originally allocated for BIONET. As an
example, the percentage use of the machine for the month of November, 1988 is shown in Figure
III-l.

49


P41R.R01685-06

The data for BIONE'Fs percentage of system use are plotted in histogram form in Figure 11X-2. This
figure demonstrates that BIONET has utilized well over 50% of the total cpu cycles used on the
2065, and routinely consumes over 85% of the total cpu cycles used on the system. Last year this
statistic averaged about 80%.

In the following series of tables and figures, we provide further details on the actual use of the
system by the BIONET community. Looking first at use of the system in prime time (8 AM - 8 PM,
M-F, PST), data for cpu time and connect hours for the indicated segments of the community are
given in Tables III-3 and III-4 by month, and totals. The cpu data in Table HI-3 is also plotted in
histogram form in Figure 111-3. BIONET prime time cpu usage is up over 45% compared to the year
before!

The main conclusion derived from these date is the BIONET resource is being heavily utilized. This
clearly demonstrates the demand for the BIONET service and explains why we approached Sun
Microsystems to obtain additional equipment.

The total number of connect hours, prime time (Table 1114), for the category BIONET Users is up
over last year by 37%. This is due both to continued growth in the user community and because two
batch queues were opened during the year to run jobs around the clock on the DEC. Initially these
queues were used very heavily, but the advent of the FASTA-MAIL program which utilizes the Sun
3/280 computer for database searches has almost eliminated the use of the DEC batch facility for
routine database searches.

The data for non-prime time (weekends and 8 PM - 8 AM M-F) are shown in Tables III-5 and 111-6,
and the data on cpu time are plotted in histogram form in Figure 1114. Non-prime time cpu usage
by BIONET has increased by 82% over the same figure for last year! Non-prime time cpu usage
remains higher than prime time because of the courtesy of users in scheduling their DEC batch jobs
to run during the evening hours. In both the prime and non-prime time periods BIONET staff cpu
usage has declined compared to last year as the staff has moved their work to the Sun computers.
Accounting software is not yet available for these systems.

The data for total use of the Resource by BIONET are presented in Tables III-7 and III-8 and the
total cpu time is summarized in Figure 111-5. Overall cpu usage is up by 65% and total connect
hours have increased by 36% compared to last year.

There can be no doubt when viewing these statistics that the BIONET Resource is valued and
heavily utilized by the scientific community. Impressive growth in the user community (over 200
new labs this year) and the statistics on machine use indicate that BIONET may be the most heavily
utilized resource funded by the NIH.

Summary data for use of our telecommunications network are presented in Table III-9 and Figure
III-6 by month for the past 12 months' use of the Telenet and Compuserve networks. Total connect
hours increased by 85% over last year ! Note that network "connect hours" represent actual time
spent using Telenet or CompuServe while "connect hours" in previous figures include batch jobs, staff
usage, and system overhead. These latter factors are accounted for as time "connected" to the
computer but do not represent connections via Public Data Networks. Local use of the BIONET
direct-dial access lines are also not included in Table III-9 and Figure HIS.

50



                                 P41RR01685-06
Figure 3-1:  Actual use of the DEC-2065

for the Month of November, 1988

$
"
0

51


P41RR01685-06

Figure 3-2: MONET'S Percentage of Total System Use, 12/87 - 1 l/88

BIONET Percentage of
TOTAL System Use

100
90
80
70
60
50
40
30
20
10
0

Jan Feb Mar Apr May Jun Jul Aug Sep Ott Nov Dee

52


P41RR01685-06

Tabel W-3:   BIONET Prime Time CPU Minutes

Dee       24588.20        1076.90        782.60       26447.70
Jan        9840.70         414.00         169.80       10424.50
Feb       10605.80         372.90        335.25       11313.95
Mar       14010.30         649.90         251.45       14911.65
Apr       11521.40         331.10        298.70       12151.20
May       11980.10         391.50        153.45       12525.05
Jun       14747.70         338.70         229.30       15315.70
Jul       10960.00         312.50         258.00       11530.50
Aug       12371.60         288.00        167.40       12827.00
sep       12261.40         257.90         440.05       12959.35
act       10897.40         231.20        322.25       11450.85
Nov        9625.80         176.90        295.45       10098.15

BlONETUsers         BIONET
Except Staff          staff

BCRG Plus
System
Overhead

Total
BDNET
  Use

Total      153410.40        4841.50       3703.70      161955.60

Tabel M-4:   BIONET Prime Time Connect Hours

Dee
Jan
Feb
Mar
APT
May
Jun
Jul
Aug
z
Nov

BlONETUsers
Except Staff

BIONET
staff

BCRG Plus
System
Overhead

Total
BIONET
  USe

7798.00        2642.90        4134.35       14575.25
2718.80         952.80       1782.45        5454.05
3858.20         867.50        1728.60        6254.30
5642.74        1257.30       2112.30        9012.34
4654.20         978.80       1701.85        7334.85
4391.40         957.70        1652.45        7001.55
6065.20         992.00       2040.55        9097.75
5265.30         668.20        1569.30        7502.80
5629.10        955.40        1639.50       8224.00
5168.20         866.30        1928.90       7963.40
4101.40        773.80        1629.40        6504.60
3585.20        726.50        1624.30       5936.00

Total       58677.74       12639.20      23543.95       94860.89

53


P41RR01685-06

Figure 111-3: BlONET's Prime Time Use of the DEC-2065 12/87-l l/88

BIONET Usage during Prime Time
     in CPU Minutes

300001

25000
20000
15000
10000
5000
   0

Dee Jan  Feb Mar ADr Mav Jun  Jul Aua S

* BlONtr USERS     BIONET STAFF

SYSOVERHEAD O TOTAL BIONET

!p Ott Nov

54


                                      P41RRO1685-06
Tabel 111-5:   BIONET Non-Prime Time CPU Minutes

lac
Jan
Feb
Mar
Apr
May
Jut-i
Jul
5
Nov

39571.00         322.10        1287.75       41180.85
16832.60         117.00         278.75       17228.35
17766.40          90.70        1512.10       19369.20
24050.10         482.30        235.65       24768.05
19409.10         645.70        759.55       20814.35
20637.40          67.90        244.65       20949.95
25492.50          77.30         267.00       25836.80
18816.00         175.60         605.25       19596.85
21007.50          20.30         177.50       21205.30
17103.50          26.70        742.45       17872.65
14466.40          16.10        809.05       15291.55
14094.70          19.20        748.70       14862.60

Total      249247.20        2060.90        7668.40      258976.50

BlONETUsers         BIONET
Except Staff          staff

BCRG Plus          Total
System         BIONET
Overhead           USe

Tabel W-6:   BIONET Non-Prime Time Connect Hours

Feb
Mar
Apr
May
Jun
Jul
Aug
sep
act
Nov

BlONETUsers
Except Staff

BIONET
staff

BCRG Plus
System
Overhead

Total
BICNET
  Use

5439.10         450.70       5643.70       11533.50
1690.10         156.10       2640.40        4486.60
2557.80         117.60       2646.85        5322.25
4395.60         207.00       3146.75        7749.35
3170.90         199.70        2597.45        5968.05
3258.80         108.00       2579.00        5945.80
4045.40          87.60       3150.10        7283.10
4053.40          71.20       2248.00        6372.60
4743.40          67.70       2549.05        7360.15
3250.10         105.70       2887.10        6242.90
2553.00         33.40        2560.30        5146.70
2194.80          58.50       2524.55        4777.85

Total       41352.40        1663.20      35173.25       78188.85

55


P41RR01685-06

Figure 111-4: Bionet's Non-Prime Time Use of the Dee-2065, 12/87-l l/88

BIONET Usage During Non-Prime Time
       in CPU Minutes

40000

30000

20000

10000

   0

Dee Jan Feb Mar Apr May Jun Jut Aug Sep Ott Nov

OVERHEAD

56


                                     P41RR01685-06
Tabel M-7:   BIONET Total CPU Minutes

lhc
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
2
Nov

BIONET Users         BIONET
Except Staff           staff

BCRGPlus
System
Overhead

Total
BIONET
  lJsa

64159.20        1399.00        2070.35       67628.55
26673.30         531.00         448.55       27652.85
28372.20         463.60        1847.35       30683.15
38060.40        1132.20         487.10       39679.70
30930.50         976.80        1058.25       32965.55
32617.50         459.40         398.10       33475.00
40240.20         416.00         496.30       41152.50
29776.00         488.10         863.25       31127.35
33379.10         308.30         344.90       34032.30
29364.90        284.60        1182.50       30832.00
25363.80         247.30        1131.30       26742.40
23720.50         196.10        1044.15       24960.75

Total      402657.60        6902.40       11372.10      420932.10

Tabel Ill-&   BIONET Total Connect Hours

Dee
Jan
Feb
Mar
W
May
Jun
Jul
Aug
2
Nov

BlONETUsers
Except Staff

BIONET
staff

BCRG Plus
System
Overhead

Total
BKINET
  Use

13237.10        3093.60        9778.05       26108.75
4408.90        1108.90        4422.85        9940.65
6216.00         985.10        4375.45       11576.55
10038.34        1464.30        5259.05       16761.69
7825.10        1178.50        4299.30       13302.90
7650.20        1065.70        4231.45       12947.35
10110.60        1079.60        5190.65       16380.85
9318.70         739.40        3817.30       13875.40
10372.50        1023.10        4188.55       15584.15
8418.30         972.00        4816.00       14206.30
6654.40        807.20        4189.70       11651.30
5780.00         785.00        4148.85       10713.85

Total      100030.14       14302.40       58717.20      173049.74

57


P41RR01685-06

Figure 111-5: Bionet's Total Use of the Dee-2065 12/87-l l/88

Total BIONET Usage in CPU Minutes

70000

60000

50000

-10000

30000

$0000

10000

   0

Dee   Jan   Feb   Mar   AV  May   Jun Jul   Auti!  Sw   Ott   Nov

         * BIONET USERS O BIONET STAFF

I   SYS OVERHEAD O TOTAL BIONET  I

58


P41RR01685-06

Tabel 111-g: BIONET Network Usage Connect Hours

Dee
Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
2
Nov

Total       46652.77       18743.95       65396.72

Prime      Non-Prime          Total
6779.69        2717.94        9497.63
2246.27         857.43        3103.70
3047.92        1203.13        4251.05
4279.67        1774.58        6054.25
3620.61        1277.32        4897.93
3210.61        1259.72        4470.33
4421.23        1724.26        6145.49
3928.71        1860.19       5788.90
4573.06        2395.25        6968.31
4009.63        1543.09        5552.72
3516.30        1228.15        4744.45
3019.07         902.89        3921.96

59


P41RR01685-06

Figure 111-g: Total Telenet and CompuServe Network Use, 12/87-l l/88

BIONET Network Connect Hours

7000.00

6000.00

5000.00

4000.00

3000.00

2000.00

1000.00

  0.00

Dee Jan Feb Mar Apr May Jun  Jul Aug Sep Ott Nov

I W Prime    O Non-Prime I

60


P41R.R01685-06

3.1.5.4 Computer Software - Core Library

There was a new release (version 5.2) in August 1988 of the IntelliGenetics software suite that
makes up the Core Software Library. This software is made available to the BIONET community
immediately upon its formal release. Version 5.2 included the addition of new functionality to many
of the existing programs and also included a completely new program, FINDSEQ, that simplified the
retrieval of sequence data from the GenBank and PIR databases and also allowed for the
performance of Boolean searches. FINDSEQ was considerably more user-friendly and powerful than
the previously used FIND command, but was designed to handle searches that did not require the
power of the IntelliGenetic's QUEST database pattern searching program. The next release of the
IntelliGenetics software is scheduled for early 1989 and will be made available on BIONET. It will
feature significant new improvements in speed and sensitivity for database similarity searches.

3.1.5.5 Computer Software - System Library
During the course of the year most new systems software was added to the new Sun network. This
was documented above in the section entitled Development work on the new Sun central
computing facility.

3.1.5.6 Computer Software - Contributed Library
Software contributed to BIONET is placed in the <CONTRIBUTED> directory on the DEC-2065, to
which only the BIONET community has access.  Recently BIONET has also implemented an
"anonymous FI'P" capability on the new Sun 3/280 file server. This allows users on other Internet
computers to retrieve software from BIONET over the network through the use of the FI'P or File
Transfer Protocol. This method of retrieval is significantly faster than downloading files from
BIONET to a remote PC and does not run up communications charges.

Major software packages produced by BIONET collaborators and implemented on BIONET with the
aid of our staff have been summarized under Collaborative Research, above.  Refer also to the
software lending library catalog in Appendix IV.

3.1.5.7 Database Library

BIONET provides its users with a large number of different databases in support of molecular
biology and molecular genetic research, the most popular being the Roberts' restriction enzyme
database and the GenBank nucleic acid sequence database.  We provide database updates in a
timely manner to the community.  Our sequence database releases in IntelliGenetics' file format
have usually been 2-3 weeks after obtaining the tapes from NIH GenBank or the EMBL. The
original database format files are made available on the DEC-2065 usually the same evening of the
day on which the tapes arrive.

BIONET databases were discussed in detail in previous Annual Reports. New additions and
changes to the databases on BIONET during this year are described above under Data Contributors.
Here we document the use of the major databases on BIONET.

The DNA and protein sequence databases are used by BIONET scientists as a source of sequence
data and for searching. Two major types of searches are performed. The main usage occurs when
BIONET users search the database for similarities or homologies with sequences that they have
determined. The second type of search involves use of the QUEST program to find interesting


P41lXRO1685-06

consensus sequences that are known to have functional importance. This year our statistics on
database searching have been dramatically impacted by the introduction of the FASTA-MAIL
program.  Since accounting software is not yet available on the Sun system we cannot provide
accurate data for the number of individual GenBank, PIR, or SWISS-PROT database searches
performed by users. Statistics can, however, be provided for the overall total number of searches.
We know that the FASTA-MAIL interface on the DEC is utilized 950 times per month or 32 times
per day on average. FASTA-MAXL is used to search each of the three databases just mentioned.
BIONET has successfully promoted the use of FASTA-MAIL over the use of other database
searching software to reduce the load on the DEC 2065 computer. In addition the IntelliGenetics'
QUEST and IFIND programs are used 346 times per month for database searches.

Restriction Enzyme Database - Thanks to Dr. Richard Roberts, the chairman of the BIONET
National Advisory Committee, we have established one of the most up-to-date lists of restriction
enzymes available. Dr. Roberts maintains a restriction enzyme registry and distributes his updated
lists in an electronic message to BIONET. BIONET incorporates these new lists into its program
essentially immediately. These lists are used within the core program SEQ and PEP and are
referenced 459 times per month.

VectorBanktm . - Vectorbank is a collection of sequence data and restriction maps of important
cloning vectors, viruses and phages that is maintained by the IntelliGenetics staff. This database is
used by the CLONER program for manipulating restriction maps and simulating DNA cloning
experiments. Release 4.2 of VectorBank in May saw the revision of the restriction map data files for
all 146 vectors in the database. All single and double cutter sites using prototype enzymes from the
latest restriction enzyme database were included in the map data. Individual Vectorbank sequence
files are accessed up to 25 times per month.

KeyBanktm - KeyBank is a data bank of nucleic acid and protein consensus patterns collected from
the literature by IntelliGenetics, Inc. KeyBank is produced in a format suitable for use with the
QUEST pattern searching program of the IntelliGenetics Suite. Some of the patterns available
include binding sites, active sites, allosteric sites, phophorylation sites, cleavage sites, cap sites,
chromosome modification sites, polymerase binding sites, insertion sites, regulatory sites,
methylation sites, replication origins, satellite DNA, 2 DNA, and zinc finger regions. The latest
release (rel. 3.0, October 1988) has 1237 new patterns taken from more than 906 references. This
part of KeyBank occupies more than 690,000 bytes of memory. In addition, KeyBank also has files of
codon and restriction enzyme site patterns.  The contents of the key files in the main part of
KeyBank can be easily searched by using the many indices supplied with the database, such as the
organism, keyword, or citation indices. Individual database files in KeyBank are utilized up to 10
times per month.

3.2 Highlights

The sections above describe in detail our accomplishments in the several components of the BIONET
Resource. Considering that a very signficant fraction of staff time during our fifth year was involved
with planning, grant writing, and other obligations for our renewal, substantial progress was still
made on the Resource. Here, in brief, are some of the most notable:
o The new computer network donated by Sun Microsystems is being prepared for direct
  access by the user community by the BIONET systems staff (Mr. Rob Liebschutz and


P41RRO1685-06

Mr. Eliot Lear). As of August 1988 BIONET released the FASTA-MAIL program. This
provided our users on the DEC with electronic mail access to high-speed database
searches on our Sun 3/280 computer. Database search times were reduced from hours to
tens of minutes or less, and the average mid-day user load on the DEC 2965 dropped
by a factor of about three to five since it was no longer being used for these compute-
intensive tasks. FASTA-MAIL used the FASTA program obtained from Dr. William
Pearson, and the mail server portion was developed by Mr. Liebschutz, Mr. Lear, and
Mr. Spencer Yeh.

o The BIONET user community continued its vigorous growth, up 31% over last year for a
total of 867 laboratories on the system. On the DEC 2965 total cpu usage increased by
65% and total connect hours by 36% as compared to last year.

o Dr. Jeny Jurka, the BIONET Scientist, published important work in the area of
repetitive DNA sequence analysis. In conjunction with this research, new functionality
has been added to the Multiple Aligned Sequence Editor MASE) by the BIONET
applications programmer, Mr. Liang Jen Homg. This editor was originally developed by
Dr. Jurka and Donald Faulkner at Dana Farber's Molecular Biology Computer Research
Resource and then extended at BIONET over the past year. Work on the editor has also
involved collaborators from the machine learning group at the University of California at
Santa cruz.

o Dr. Sunil Maulik has continued work on the RICH program which performs database
searches for sequences of defined percent composition.  Dr. Maulik has obtained some
interesting preliminary results with the program.  He has also been involved in
developing a new HyperCard tm-based user interface for BIONET.
o The electronic communications network was significantly enhanced. The efforts of Dr.
David Kristofferson led to the formation of the international BIOSCI bulletin board
network. Besides BIONET in the U.S., other major BIOSCI distribution sites are
situated in England, Ireland, Sweden, and Finland. Recipients of the bulletin boards
from these sites are located in all parts of the globe. The bulletin boards are available to
users on the ARPANET, BITNET, EARN, Usenet, NSFnet, and JANET. Users in any
particular location need only post or receive messages from their closest site. Any
posting at any center is automatically forwarded by the central BIOSCI sites to all other
participants on the above-listed networks.

o Finally, the research conducted by BIONETs 867 laboratory groups was made
significantly easier by a total revision of the BIONET documentation and the production
of a new User Manual. This involved major efforts by BIONET staffer's Ms. Vickie
Johncox, Mr. Spencer Yeh, and Ms. Rathryn Berg. The documentation was sent free of
charge to all users on the system this past summer.

3.3 Administrative Changes

The following have been the administrative changes within BIONET during the past year. These
have come about for reasons ranging from personnel shifts to additions and resignations. None of
these changes has had a negative impact on the Resource itself, in particular, its "appearance" and
availability to the community.

o Mr. Liang Jen Homg was hired in May 1988 as the new BIONET Applications
Programmer to assist Dr. Jurka in his research efforts and to work on the BIONET
contributed software. Mr. Homg has a M.S. in Computer Science from San Jose State
University.

o Mr. Eliot Lear was hired in June by the Computer Facilities group at IntelliGenetics as
an additional system programmer.  Mr. Lear has a B.S. in Computer Science from
Rutgers and has run a state-wide network of Sun Microsystems file servers and


P41RRO1685-06

workstations. Since joining IntelliGenetics he has devoted a large fraction of his time to
bringing up the new Sun system for BIONET.

o Ms. Vickie Johncox, formerly a BIONET Scientific Consultant, left BIONET in June for
another job as head of IntelliGenetics' training group. BIONET hired Dr. Karen Davis,
formerly at the University of California, Santa Cruz, as her replacement.  Dr. Davis
started at the end of June. No disruption in the service occurred as Ms. Johncox's duties
were handled smoothly by Mr. Spencer Yeh and Drs. Maulik and Kristofferson.

o In November, Ms. Rathryn Berg resigned as BIONET Administrator to pursue work
elsewhere. Before her departure BIONET hired Ms. Cindy Eppard from IntelliGenetics
to replace Ms. Berg. Ms. Eppard learned her new responsibilities rapidly, and there was
no interuption in the establishment and processing of BIONET user accounts.

Despite the demands of their jobs, increased once again by a 31% growth in the size of the user
community this year, the BIONET stafF remains a highly dedicated group of individuals who are
interested in their work and in promoting the goals of the resource. BIONET has received many
testimonials to the quality of its staff and the support that they provide. Copies of a few of these
testimonials are included in Appendix VIII.

3.4 Resource Advisory Committee and Allocation of Resources
The membership of BIONETs National Advisory Committee has not changed this past year. The
NAC consists of:

o Dr. Richard J. Roberts, Ph.D., Chairman, Senior Staff Investigator, Molecular Biology,
Cold Spring Harbor Laboratory

o Professor John Abelson, Ph.D., Department of Biology, California Institute of Technology
o Professor Alan Maxam, Ph.D., Dana Farber Cancer Institute, Harvard Medical School,
Harvard University.

o Thomas Rindfleisch, M.S., Director, Knowledge Systems Laboratory, Department of
Computer Science, Stanford University.
o Professor Irwin Runts, Ph.D., Department of Pharmaceutical Chemistry, University of
California, San Francisco.

o Professor Charles Yanofsky, Ph.D., Department of Biological Sciences, Stanford
University.

o Professor Eric Lander, Ph.D., Harvard Business School
o Professor Joshua Lederberg, M.D., Ph.D., President, The Rockefeller University.

The last regularly scheduled meeting occurred on November 13, 1987 in Mountain View. Due to the
time occupied by preparations for the BIONET renewal the next NAC meeting has been postponed
until the first part of 1989.  When issues have arose during this last year we have consulted
members of the NAC by phone, especially our chairman, Dr. Roberts, and Mr. Thomas Rindfleisch
at Stanford.

Allocation of Resources. Tbe Committee agrees with our methods for allocating the Resource.
The DEC-2065 computer uses its windfall scheduler to allocate cpu time to the various categories of
users and overhead, as described under Resource Facilities. The cpu time is distributed on a first-
come, first-served basis. This method has been very successful. Considerably more than 50% of
CPU time (BIONET's original allocation) has been delivered to BIONET scientists (see Resource

64


Facilities, above). BIONET is now routinely using almost 90% of the DEC 2065 plus significant
resources on the new Sun computer system.

We continue to request that the community not have more than one person per PI group using
BIONET at the same time during prime time. The community continues to do an excellent job in
complying with this policy.

We continue to allocate additional disk space to PI groups involved in managing large sequencing
projects or extensive databases of sequences.  We do this on an ad hoc basis upon requests by
investigators. Our archive and retrieval system is working smoothly for archival storage and prompt
retrieval (one to two days) of important files.

3.5 Dissemination of Information of Resource's Capabilities
We discuss two areas related to dissemination of information about the Resource that we have
pursued this grant year.  The first is interactions with the scientific community through
participation at conferences and seminars.  The second is use of the electronic mail and bulletin
board facilities of the Resource itself to keep scientists worldwide aware of changes and
improvements.

3.5.1 Community Interactions and Awareness
We have used two methods this year to inform the community about BIONET and to solicit
applications for access to the Resource.  The first method has been the presentation of invited
seminars and participation at major conferences where we have presented lectures and/or have had
booths at exhibitions. These efforts are summarized above under BIONET Training Program. At
these conferences, we have distributed the standard applications packets to scientists, after
demonstrating to them the capabilities of the Resource.

The second method has been through the publication of journal articles which describe BIONET.
This year an article detailing new developments at BIONET appeared in vol. 16 no. 5 of Nucleic
Acids Research. A preprint of this article was in last year's Annual Report. An upcoming article will
appear in Protein Sequence and Data Analysis and is listed on the BRTP Training Form under
Scientifx Subprojects. A copy of this article is available in Appendix II.

3.5.2 Electronic Communications

The electronic communication facilities of BIONET provide another important way to disseminate
information about the Resource.  In addition, electronic mail and bulletin boards provide a
mechanism for scientific and technical interchanges among members of the community. With the
dissemination of BIONET bulletins to investigators outside of the Resource (via ARPANET, BITNET
and USENET), information about BIONET is being distributed electronically worldwide.
Information on the types of electronic mail communications with BIONET was summarized
previously in the discussion of the Collaborative Research component of the Resource.

65


P41RR01685-06

3.6 IMPORTANT Suggestions and Comments
For the BIONET staff the main `highlight" of this year was the renewal process of the BIONET
grant. We have not commented on this topic in this report because some issues are still under
negotiation with the NIH and are not yet for public disclosure. We do, however, have the following
general comments.

The regulations of the BRTP program at the Division of Research Resources require a Resource such
as BIONET to excel in several different areas: Technological Research, Collaborative Research,
Service, Training, and Dissemination.  Given the size of the BIONET user community and the
demands that it makes on our staff, it has always been a challenge to cover all of these areas
adequately. We have excelled in the areas of Service, Training, and Dissemination because, until the
addition of Dr. Jurka, that was where our talents lay and the sheer din of the user community would
have been deafening had we neglected the Service to devote more of our limited resources to
research. We feel that the real justification of this resource has been the quality of the research done
BY OUR HUNDREDS OF USERS and that any efforts on our behalf would always be dwarfed by
comparison.

Nonetheless, DRR regulations make it possible for a committee of ten people who are not even
users of the Resource to come in and possibly scuttle the entire operation because of deficiencies
in just one of the five mandated areas. Furthermore it is possible that such an event can occur
without ANY input from the almost 3000 users of the resource. 1 The fact that such an action could
lead to the disruption of research and electronic communications for almost 900 laboratories around
the world does not seem to be a perturbing factor in the renewal decision. This surely must be
one of the more amazing possibilities in the history of modern science!

If this year's report demonstrates anything, it shows that BIONET works and works
succeaafulIy, and that it has continued to make significant improvements over the last several
years. We have the largest user community of any similar resource in the world and it continues to
grow, even attracting users from locations that have competing facilities. Few, if any, other related
organizations can lay claim to the "resourcefulness" that BIONET has shown in successfully
pursuing its proposal to Sun Microsystems which resulted in the grant of $150,009 in new hardware.

We suggest that reason finally prevail in this process, and that the NIH recognize, preserve, and
expand this valuable resource.  DRR regulations should be reviewed and revised so that
organizations that are primarily devoted to Service need not excel in every single BRTP category in
order to survive.

To date the user community is only dimly aware of the forces impinging upon BIONET. Because of
the current negotiations with the NIH it has not been appropriate to involve them.  It would be
extremely interesting to see their reaction if events lead to the termination of the BIONET Resource,
but, if reason prevails, this disasterous event will not come to pass, and the users will continue to
invest their energies productively in their research.

66


P41RJ301685-06

I. BIONET Research Publications
Copies of six BIONET research publications and abstracts are included in this section.

6'7


Proc. Natl. Acad. Sci. USA
WWp. 4775-4778. July S88

A fundamental division in the Ah family of repeated sequences

  (evolutton/Alu subfatnilks/sewdary stllmre/cpG dimukottck)
JERZY Jum*t AND TEMPLE SMITH*

o Bione~, 700 East El Camino Real, Mountain View, CA 94040, and *Dana-Farber Cancer Institute, Harvard School of Public Health, 44 Binney Street.
Boston. MA 02115

Communicated by Roy J. &&en, March 10. 1988

ABSTRACT   The ALU family of repeated sequences from
the human genome contains two distinct subfamilies. This
divisiin is based on different base preferences in a number of
diagnostic sequence positiuns. One subfamily of the sequences,
referred to as the Alu-J subfamily, is very similar to 7SL DNA
in these positions. The other subfamily, Al&, can be divided
further into well-defii branches of sequences. These t'lnd@s
revisethepreviouspictureoftheAlufamilyandexposekir
complex evdutionary dynamics. They reveal 8equenee varla-
tions of potential importamx for the proliferatSon ofAlu repeats
andrelatethemtotheirstru&nd features. In addition, they
open the possibiity of using different types ofAlu sequences as
natural markers for studying genetic Memeats in the
genome.

A typical human Ah family member is a sequence 1300 base
pairs long and consists of two similar but not identical
subunits, Ah-left and Alu-right, connected by an adenine-
rich linker. Both halves ofAfu elements are related to the 7SL
RNA (1). Although Ah sequences are the most abundant
among middle repetitive elements in the human genome, their
biological role remains unclear (2). In this paper, we report on
the presence of at least four different types of Alu sequences,
which probably originated at different times in the history of
primates.

METHODS

A set of 125 complete or nearly complete human Ah
sequences were extracted from the GenBank DNA sequence
data base.0 The list of the GenBank loci used, positions of the
extracted sequences, and other specifications are given in the
legend of Table 1.7 The statistical analysis described below is
based exclusively on painvise comparisons of each Ah
sequence with the consensus sequence (see Fig. 1). using the
computer algorithm of Smith and Waterman (3). The overall
consensus sequence in Fig. 1 was derived from our data and
is slightly different from the one recently published (2). The
differences are exclusively within CpG doublets, which are
known to be variable in A/u repeats (4). Taking pairwise
comparisons as a starting point, the multiple alignment of the
analyzed set of sequences has been done by hand with a
specialized sequence editor (5). To detect sequence positions
with different base preferences (diagnostic positions), we
used "column-correlation" function incorporated in the
sequence editor (5). This function was originally designed to
perform automatic searches for compensatory mutations.

RESULT8

During a search for compensatory mutations in the multiply
aligned set of 125 Alu sequences, we noted an unusually high

lltc publication costs of this article were defrayed in part by page charge
payment. This article must thercforr hc hereby marked "ahcrriscmen/"
in accordance with 18 U.S.C. 01734 solely to indicate this fact.

proportion of correlated base occurrences in at least 15
sequence positions. These positions are referred to as diag-
nostic positions and are listed in column 1 of Table 1 (the
position numbers are the same as in Fig. 1). The observed
correlations in the diagnostic positions reflect different base
occurrences in different Ah subfamilies. It is shown below
that the most predominant bases in the 15 diagnostic posi-
tions belong to only one of the two basic types of Ah
sequences present in the analyzed set.

To segregate the most predominant type from the remain-
ing Ah sequences, we have used computer alignment (3) of
each Ah element with the Ah consensus from Fig. 1. The
average overall similarity between the 125 Ah sequences and
the Ah consensus is 83.88% with a SD of 5.63% (gaps
counted as single mismatches). These numbers are slightly
different if gaps are excluded from the analysis (see Table 3).
Given the overall similarity, we assume that the probability
of matching between any Ah sequence and its consensus in
a randomly chosen aligned position equals 0.83. Any Alu
sequence similar 40% or less to the consensus sequence in the
15 diagnostic positions has been defined as an Ah-J element.
The probability of only 6 matches or less in 15 randomly
chosen aligned positions can be calculated from the binomial
distribution and is <O.OOl. Following the statistical defini-
tion, we have found 31 Afu-J sequences in the analyzed set of
125 sequences. The remaining 94 sequences are referred to as
Ah-S sequences. The 3:l ratio of S/J Ah sequences explains
why the overall consensus sequences and Ah-S consensus
sequence overlap. We have found no sequences matching
seven or eight diagnostic consensus positions, which sug-
gests that the distinction between J and S sequence types is
quite sharp with few or no intermediate forms. As shown in
Table 1, in the diagnostic positions the J subfamily maintains
consistently different bases from those in the S subfamily.
The difference in base preferences between J and S subfam-
ilies is most evident at positions 94. 204, and 275 (Table 1).
For example, G-204 is present in 29 of 31 Afu-J sequences and
in only 1 of 94 Ah-S sequences. Similarly, G-94 and C-275 are
powerful diagnostic indicators that can be used for prelimi-
nary "by eye" identification of A/u-J elements.

As illustrated in Table 1 the most frequent bases in the J
subfamily are identical with those in 7SL RNA in 14 of 15
diagnostic positions. Furthermore, the differences between J
and S Ah elements correlate with differences in the adenine-
rich linker connecting the left and right halves of the Ah
dimer (positions 121-133 of the consensus sequence in Fig. 1;
data not shown). The triplet TAC in the middle of the linker
is present in 980% of Alu-S compared to only 20% of the
A/u-J sequences. It is not certain if the homologous TAC
triplet was ever present in many Alu-J sequences since their

"To whom reprint requests should be addressed.
tEMBL/GcnBank Genetic Seauencc Database (1987) GenBank
(IntelliGenetics, Mountain View, CA), Tape Rek&e 46i.O.
)The AI u sequences used in this study are available on the Bionct
computer in the file <jurka>human-aluseq. The data can also be
obtained by electronic mail from jurka@bionet-2O.a.

4775


4776   Evolution: Jurka and Smith                   Proc. Natl. Acad. Sci. USA 85 (1988)

Table 1. Dipgnostic base diiennces between major subfamilies of the Alu family

Consensus   Ah     Frequency of         Base in   Consensus

AIU

Frequency of

Base in

position   subfamily T C A  G (-) 7SLDNA   position   subfamily T C A  G (-) 7SLDNA

57 (C)      J      6   3200   2     A      163 (A)     J      12   325

  . .
        S     36 47 9 2 0
63 (A)      J      2 0 12 16 1     G
         S      1 287 4 0
65 0      J     30 1 0 0 0     T
         S     20 41 1 1 37
70 6-i)      J      2 21 0 7 1     T
        S      0 2 389 0
71 0-l      J      523 0 2 1     C
        S     90 4 0 0 0
94 cl      J      0 0 229 0     G
         S      487 11 1
101 (G)     J      2 0 19 10 0      A
        S      0 0 985 0
1% (A)     J      0 0 13 18 0     G
        S     0 093 10

         S      2   1 55 36
1~~4 (A)      J      1 0   6 22
         S      1   1 91 1
204 (A)     J      0 0   1 29
        S      0   0 89 1
208 (G)     J      0   0 20 11
         S      2   3 29 59
~0     J      6 22 1 1
        S     80 10 0 1
233 (A)     J     22 2 4 0
         S      0   090 3
275 (`l-l     J      128 10
        S     85 2 1 2

0      G
0
2      G
0
1      G
2
0      A
1
1      C
3
3      T
1
1      C
4

Consensus Positions are taken from Fii. 1. (-  Alignment gaps. Loci names and 5' + 3' positions of Alu-J and AI& sequences are listed
below as they appear in GenBank (release 46.0). l Alu reseats eomplcmcntary to tbc consensus sequence are listed in 3' + 5' order. Positions
Qnceded by b snd c indkate b and c btancbes of Ah-S sequences, respeetivcly, as defined in Table 2 and in the text. A&J:
HUMACHRA7(1580-1295); HUMADAG(4907-5201. 24773-24495, 31460-31747); HUMAPGCll(l982-2235); HUhlAPOE4(2562-2849);
HUMBLYM1(266-560); HUMERPA(1810-2100); HUMFJXG(2417224465); HUMFOL5(1577-1847); HUMlFNB3(2663-2405,13213-13489);
HUMlL2R8(1209-1486); HUMLDLR(4193-4485); HUMPOMC2(26-340); HUMPOMC6(26-303): HUMRSAOLD(498-790);
HUMRSKPA1(24-291); HUMTBB5(2922-2627, 2949-3239, 5611-5885); HUMTHBNB(3593-3883); HUMTPA(7512-7227, 8862-9165.
10801-10513. 16794-17114. 18878-19167. 20944-21250. 22262-22536. 26941-27228); M13121(1141-856). Ah-S: HUMAlATP(4932-5219);
HUMADAG(1672-1369.2357-2642; c: 56k-5893,8000-7720,8484-8193,13452-13741; 15386-15096.15806-16094,17224-16933.18414-187%.
19900-19613. 22527-22812, 25453-25163, 27269-26979; c: 28032-28320); HUMAGG(b: 1391-1106); HUMALBG(3287-3576, 6046-5759);
HUMANFA(c: 1340-1621; b: 1630-1919); HUMAPGAJ1(3291-3585, 6421-6709); HUMAPGAJl(2571-2860); HUMAPGCll(2254-2542);
HUMAPGEl(636-352,2427-2138.5049-4773); HUMClA21(347-60); HUMCL423(330-45); HUMClAJN1(992-1285); HUMFIXG(7298-7595;
c: 31537-31801. 35947-36248); HUMFOU(12S4-989); HUMGAST2(187~77); HUMGHV(2506-2248); HUMHBA4(2060-1773, 4297-4585,
8548-8836); HUMHBBRT(482-190, 1260-1548); HUMlFNB3(4648-4363, 7265-7545; c: 8975-8688); HUMlNS2(69-357);
HUMLDLlVS(291-8); HUMLDLR(L: 3715-4011); HUMMHDC3B(L: 3712-3424); HUMMHDRBYb: 2838-3124; c: 4063-4345);
HUMMYCRT(c: 3143-2876); HUMNGFB(c: 5259-5544); HUMPGMC(1392-1102, 7099-6603); HUMPGMCl(333-47); HUMRSAl(c:
508-803); HUMRSA27(1-251); HUMRSAlqb: 168-451); HUMRSABll(l-269); HUMRSAB13(11-295); HUMRSABlS(l-241);
HUMRSAB2(1-288); HUMRSAB6(1-256); HUMRSAB8(1-265); HUMRSAP3(b: 897-1186); HUMRSKA1(21-347); HUMSLJT1(568-280);
HUMTBB5(3289-3573. 4115-3849; b: 5241-4953; b: 6799-6516); HUMTBBM4O(c: 1828-2113); HUMTHBNB(1165-874, 3418-3110);
HUMTPA(5960-5671, 6746-6483. 739-1022. 10066-10355. 12986-12700; b: 17170-17455. 21279-21567. 21940-21651, 25619-25905.
26522-26811. 27879-28149; b: 28803-29090, 32922-33210, 34234-34503); HUMUG2PD(c: 546-260, 1685-1396); M1159l(b: 1404-1115);
M12036(637-362); M12929(592-302).

tinkers vary substantially in both their size and the primary
sequence.

Following the same approach, the S subfamily of the Ah
family has been found to contain other types of Alu se-
quences. Unlike the J/S division, the intra-S division is more
difficult to define statistically since the number of simulta-
neous differences between subsets of Ala-S sequences ap-
pears to be smaller and there is a number of intermediate
sequences virtually absent from the J-S junction. Therefore,
we first define the most distinct "b" branch of the S
subfamily as containing sequences that match 3 or fewer of
the 11 diagnostic positions listed in Table 2 and in Fig. 1.
There are 12 such elements in the analyzed set of W Afu-S
sequences. The average overall similarity between each
analyzed Ah-S sequence and the consensus sequence is
86.59 if every gap is counted as a single mismatch (Table 3).
Based on this number, we assume the probability of matching
the aligned consensus sequence at a randomly chosen posi-
tion to be 0.86. As calculated from the binomial distribution,
the probability of matching 3 or fewer of the randomly chosen
aligned positions is lo-`. The probability of matching exactly
4 and 5 positions equals 1.9 x 10" and 1.63 x 10e3,
respectively. We have found 5 sequences matching 4, and 6
matching 5 diagnostic positions in the analyzed set of 94
Ah-S sequences. These 11 sequences are arbitrarily defined
as a "c" branch of the S subfamily. AfIer segregation of the
b and c branches, the remainder of the A1u-S subfamily is
referred to as an "a-branch." Preliminary analysis of 71 Alu

elements from this branch revealed the presence of 16
sequences containing simultaneously thymine at position 244
and adenine at position 272, as opposed to C-244 and G-272
in the remaining 55 Ah sequences. In addition, 14 of the
above 16 sequences contain an extra adenine in position 244.
This suggests that the A/u-a branch may contain at least two
different types of Ah sequences and it can tentatively be
replaced by "d" and "e*' branches containing 16 and 55
sequences, respectively.

As illustrated in Table 2 and Fig. 1, the base preferences are
quite similar between Alu-b and Alu-c sequences up to
position 88. Further on, Ah-c remain similar to Afu-a with
the exception of guanine at position 163. Therefore, the c
branch can be viewed as an intermediate between the a and
b branches of the S subfamily. A unique feature of the c
sequences may be the presence of adenine at position 74. Gf
the 125 Alu sequences only 8 contain A-74 of which 7 belong
to the Alu-c branch defined above. The eighth Ah sequence
containing adenine at position 74 (HUMPOMCl) has all the
Alu-c features listed in Fig. 1: deletion at 64 and 65, A-78,
T-88, and G-163. Therefore, it can also be considered as an
Ah-c sequence.

Based on the analysis of phylogenetic trees, other authors
(6) have recently identified the Afu-b branch as a "subfamily
of the Ah family." The authors have pointed out differences
between the Alu consensus and the Alu-b sequences at
positions listed in Table 2 as well as in Fig. 1 and at other less
characteristic positions not included in our analysis.


Evolution: Jurka and Smith

Proc. Natl. Acad. Sci. USA 85 (1988)   4777

7SL
Alu-cons

7SL
Alu-J
Alu cons
Alu-c
Alu-b

7SL
Ah-J
Alu cons
Alu-c
Alu-b

7SL
Alu-J
Alu cons
Alu-c
Alu-b

7SL
Alu-J
Alu cons
Alu-c
Alu-b

7SL
Ah-3
Alu cons

7SL
Alu-J
Alu cons

1           15  16          30  31           45
-GCCGGGCGCGGTGG CGCGTGCCTGTAGTC CCAGCTACT-CGGGAG
GGCCGGGCGCGGTGG CTCACGCCTGTAATC CCAGC-ACTTTGGGAG

46          60  61          75  76         90
GCTGAGGCTGGAGGA TCGCTTGAGTCCAGG AGTTC....CCAGCC
            gt cc
GCCGAGGCGGGZGGA TCACCTGAGGTCAGG AGTTCGAGACCAGCC
                    --     (A) A         T
                    --            A         T

91         105  106        120  121        135
TGGGCAACATAGCGA GACCCCGTCTCT
  G       a     g
TGGCCAACATGGTGA AACCCCGTCTCTACT AAAAATACAAMATT

    T    C

136         150  151        165  166        180
-GCCGGGCGCGGTGG CGCGTGCCTGTAGTC CCAGCTACTCGGGAG
AGCCGGGCGTGGTGG CGCGCGCCTGTAifTC CCAGCTACTCGGGAG

                          G
                 G         G

181         195  196        210  211        225
GCTGAGGCTGGAGGA TCGCTTGAGTCCAGG AGTTCTGGGCTGTAG
            G          G               C
GCTGAGGCAGGAGAA TCGCTTGAACCC:GG AGGCGGAGGTTGCAG

                G R                  C

226         240  241        255  256        270
TGCGCCTGTGA... .G CCACTGCACTCCAGC CTGGGCAACATAGCG
       T
TGAGCC-GAGATCGCG CCACTGCACTCCAGC CTGGGCGACAGAGCG

271        285
AGACCCCGTCTCT
   C
AGACTCCGTCTCAAA AAAAA

Frcz.1.  Consensus sequence for 125 Ah sequences and the homologous regions of human 7SL DNA. Major and minor characteristic bases
for other types ofAIu sequences are printed in capital and lowercase letters, respectively, and correlate with the analysis in Table 1. Dots indicate
sequence regions absent from 7SL DNA but present in the Ah family. The remaining `ISL-spcciftc sequences are not shown. Dashes under
positions 64 and 65 indicate bases missing in Alu-b and Alec seauences. Additional characteristic positions not listed in Table 2 are put in
parentheses.

The diagnostic position 78 (Table 2) is in the middle of the
stretch 77-79, which can pair with base 87-89 containing
another diagnostic position 88. Bases 77-79 are within the
polymerase 111 promoter region (bases 74-86 in Fig. 1).
Correlation between occurrences of complementary bases at
positions 78 and 88 suggests the possibility of a weak
secondary interaction in this region. Another potential for
secondary interaction, already proposed for 7SL RNA (7,8),
exists between complementary bases 69-75 and 89-95. This
region includes 3 of the 15 positions distinguishing between
the J and S subfamilies and the complementarity is conserved
throughout the Alu family. The only AC mispairing has been
found in this region in the Aluc sequences. The role of the
above hypothetical structures is not clear, although their
location suggests involvement in Alu transcription. There is
also a possibility of a secondary interaction between bases
244 and 245 and bases 271 and 272 that includes bases at
positions diagnostic for putative d and e branches of the Ah
family discussed above.

Table 3 indicates that the average overall similarity be-
tween Alu-J and the Alu consensus sequence in nondiag-
nostic positions is lower than the average similarity between
Alu-S and the Alu consensus. This indicates that on average
Alu-J sequences are more diverse than Alu-S sequences. By

t test, one can find that differences between Alu-J/consensus
and Al&/consensus similarities are statistically significant
(P << 0.081). The conclusion holds true even if the general
Alu consensus is replaced by the Afu-J consensus (data not
shown). There is also a significant difference (P < 0.001)
between analogous numbers for a and b subdivisions of the
Alu sequences. The differences between Alu-b and Aluc
sequences are marginally significant (P < 0.05), and analo-
gous differences between Alu-a and Aluc are insignificant.

As pointed out before (4). CpG doublets undergo rapid
mutations in Alu sequences. This may result from a deami-
nation of methylated cytosine (for a review, see ref. 9).
Average CpG content is lowest in the J subfamily (3.84 r
2.01) as compared to analogous numbers for Alu-a (7.75 k
2.95), A/u-b (16.08 + 5.01). andA1u-c (9.54 2 3.75) branches
of the S subfamily. Significance levels for the differences in
the CpG content are virtually identical to those for the
similarity differences discussed in the preceding paragraph.

DISCUSSION

Given the similarity between Alu-J and 7SL RNA sequences
in the diagnostic positions, the large intra-subfamily diversity
and the low CpG content, we find the J sequences to be good


4778   Evolution: Jurka and Smith


Table 2.  Base preferences in Ihe S subfamily branches

Consensus   Branches      Frequency of
position    of Alu     T    C A G    (-)

78 0

88 G)

95 (C)

100 m

153 (C)

163 (A)

197 (C)

219 (G)

20    41     1     1     8
0     0     0     0    11
0     0     0     0    12
62     3     3     0     3
4     0     0     0     7
4     0     0     0     7
67     0     4     0     0
1     0     9     1     0
0     0    12     0     0
2     1     2    65     1
9     0     1     1     0
11     0     1     0     0
2    68     1     0     0
2     9     0     0     0
12     0     0     0     0
66     1     2     1     1
7     4     0     0     0
1    10     1     0     1
10    35     1    24     1
5     1     0     5     0
0     1     0    11     0
1     0    53    17     0
1     1     1     8     0
0     0     1    11     0
15    50     2     4     0
3     6     1     1     0
0     0     0    12     0
65     3     2     1     0
10     0     1     0     0
1     0     4     7     0
0     1     2    64     0
0     1     0    10     0
0    11     0     0     0

(-), Alignment gaps.

candidates for the early Afu elements derived from the 7SL
RNA (1). The base differences in the diagnostic positions and
the linker regions may be important for understanding how
this transformation occurred and are good targets for exper-
imental analysis. On the other hand, the least diverse Ah-b
sequences can be viewed as a relatively young branch of the
Ah family. There are three published examples of Ah
sequences that are believed to be inserted relatively recently
on the evolutionary time scale: in the a-satellite DNA of
African green monkey (lo), in the gorilla &globin gene
cluster (11). and at the Mlvi-2 locus of human cell lymphoma
(12). All these Ah sequences belong to the b branch defined
above.

While this paper was in review, other authors (13) reported
on a subdivision of the Ah family into three different
subfamilies corresponding to our J subfamily and two
branches (a and b) of the S subfamily. These two branches,
as well as the branch c, are virtually equally different from the
J subfamily of Ah sequences and similar to each other in the

Proc. Natl. Acad. Sci. USA 8S (1988)

Table 3.  Average overall similarities with the Alu
consensus sequence
         Gaps as
          mismatches     Gaps excluded
Ah type    Mean     SD     Mean     SD     Total

AU       83.88    5.63    86.39 4.38     125
J        79.20    4.35    82.83    2.27      31
s        86.59    3.39    88.75    1.98      94
a        86.52    3.26    88.59 1.79      71
c+b     89.20     4.14    91.15    3.08      23
E        87.04 4.25     89.45     2.87      11
        91.22 2.92    92.71     2.45      12
Sequence alignments have been made by using the computer
algorithm (2). The diagnostic positions have been excluded from
similarity calculations.

diagnostic positions from Table 1. Therefore, we consider
them as members of the S subfamily. The authors draw their
conclusions from analysis of pairwise difference distribution
among Alu sequences involving both the diagnostic differ-
ences discussed in this paper and a mutational noise. Our
analysis is based on multiple sequence comparisons, which
permits more rigorous distinction between diagnostic and
background differences. With this level of resolution we are
able to classify each Alu sequence individually. This, and the
analysis of the CpG content discussed in the accompanying
paper (14), opens a way to date the invasion of individual
genes by different types of Ah sequences and of genetic
rearrangements associated with this process.

We thank Donald Faulkner for professional computer assistance
and Roy B&en, Douglas Brutlag. Teny Friedemann, David Kristof-
ferson. and Randall Smith for critical and useful comments on the
manuscript.

1.
2.


3.

4.
5.

6.

7.

8.
9.
10.

11.

12.

13.

14.

Ullu, E. Bt Tschudi, C. (1984) Nature (London) 312, 171-172.
Kariya, Y., Kafo, K., Hayashizaki, Y., Himeno, S., Tarui, S.
Bt Mats&am, K. (1987) Gene 53, l-10.
$ith, T. F. 8r Waterman, M. S. (1981) J. Mol. Eiol. 145,195-
Bains, W. (1986) J. Mol. Evol. 23, 189-199.
Faulkner. D. V. 8t Jurka. J. (1988) Trends Eiochem. Sci., in
press.
Slagcl, V., Flemington. E.. Traina-Dorge, V., Bradshaw, H. &
Deininger, P. (1987) Mol. Mol. Evof. 4, 19-29.
Gundelfinger. E. D., Di Carlo, M., Zopf. D. & MeUi, M. (1984)
EMBO 1.3, 2325-2332.
Zwieb, K. (1985) Nucleic Acids Res. 13,6105-6124.
Bird, A. P. (1987) Trends Genet. 3, 342-347.
Grimaldi. G. & Singer, M. F. (1982) Proc. Nat/. Acad. Sci.
USA 79,1497-1500.
Trabuchct, G.. Chebloune, `Y., Savatier, P., Laucher. J.,
Faure, C.. Verdier, G. & Nigon, V. M. (1987) J. Mol. Eva/. 25,
288-291.
Economou-Pachnis, A. & Tsichlis, P. N. (1985) Nuchic Acids
Res. 13,837%8387.
Willard, C., Nguyen, H. T. & Schmid, C. W. (1987) J. Mol.
Evol. X,180-186.
Britten, R. J., Baron, W. F., Stout, D. & Davidson, E. H.
(1988) Proc. Nat/. Acad. Sci. USA 85, 4770-4774.


Reprintedfrom TrenQ in Biochemical Sciences- Augur, 1988   Vol. 13, No. 8

321

Multiple aligned sequence editor
     (USE)

Donald V. Faulkner and Jerzy Jurka

Cognitive capacities of the human
brain can not, so far, be matched by
computers. Even well optimized com-
puter programs have limited flexibility
in addressing the variety of problems
associated with sequence analysis.
Hence, we were motivated to design a
Multiple Aligned Sequence Editor
(MASE) which combines manual
sequence manipulations with standard
computer analysis. An earlier article in
TIBS described the adaptation of stan-
dard word processor software for a
similar purpose'.

MASE can be used for editing any
set of sequences. The total number and
size of sequences that can be edited
simultaneously depends on the com-
puter (typically. the total number of
characters should not exceed 1 x 106).
Sequences can be displayed on the
screen in two windows, each of which
can be moved either independently or
concurrently. An example of the
sequence display is shown in Fig. 1. The
window sizes depend upon the type of
terminal used. A standard VT100 ter-

D. V. Faulkner is or rhe Dono-Farber Cancer
lnstirure, 44 Binney Sweet. Boston, MA 02115.
USA. and 1. Jurko is OI Bioner. 700 Eosr El
Camino Real, Mounrain View, CA 94040, USA.

minal can display 21 sequences per
screen, with 25 characters in each
window. Any terminal size and type
which supports full screen editing can
be used by writing a UNIX termcap
entry. MASE can be run on computers
with the Berkeley UNIX operating
system.

Intrinsic functions

MASE provides over 80 intrinsic
functions, each of which can be selec-
ted with few key strokes from a menu
by calling the function COMMAND-
MODE(:). Individual functions can
also be fixed to different keys on the
keyboard using the BIND function,
and this assignment can be stored in a
separate file and loaded whenever the
sequence editor is used. A short defini-
tion of each intrinsic function can be
called up using question-mark key. Full
on-screen HELP and a tutorial are also
available. The intrinsic MASE func-
tions can be divided into the following
basic groups: (1) moving cursors and
searching for patterns; (2) modifying
the sequence data; (3) changing the
sequence display without affecting
input/output files; (4) window manipu-
lations; (5) sequence analysis; (6)
modifying MASE behaviour; (7) mis-

cellaneous, and (8) generation of for-
matted output. In this short article we
will outline only some of the capabili-
tiesof these functions.

Primary  modifications of  the
sequence data involve insertions/
deletions of alignment gaps in individ-
ual sequences or in the whole set. This
permits the alignment for maximum
similarity. One can begin with totally
unaligned sequences or use an output
from any sequence alignment program
as a starting point. However, the for-
mat of the pre-aligned sequences must
be as described below under the
`Sequence data files' and in the MASE
manual. The on-screen alignment can
be facilitated by changing the sequence
display, for example by highlighting
conserved sequence patterns, hydro-
phobic/hydrophilic residues, etc. (see
Fig. 2). One can also easily emphasize
differences between aligned sequences.
The sequences can be rearranged arbi-
trarily to facilitate direct by-eye com-
parisons. Furthermore, the window
display permits any two columns of
characters be placed next to each other
(e.g. columns45 and 71 in Fig. 1).

Sequence analysis involves on-
screen computation of consensus se-
quence, identity matrix, column com-

Vu&r Locus Name
  1 KlHlRG
  2 KlHUQU
  3 KlHUBI
  4 KlHWR
  5 KlHUDE
  6 KlHUEU
  7 KlHL&L
  8 KlHUHU
  9 KlHUKR
  10 KlHUKU
11 KlHULY
12 KlHIJW
13 KlHURE
14 KlHlRY
Dffset frua stst

   1                           45 71                          ii5
  -DIQMTQSPSSLSASVGDRVTIT~DINHYLtUYWW;PKKFIP D~#FTI~C~CPEDI~~DTLPRTFUW;TKLEIKR~
   DIQllTQSPSSLSASVGDRVTITCQASaDISDYLHSYOMBGWlPK FTFrIssLWEDIflTYYcwYDYLPuTFGcGn<vEIKw
   DIC!HTQSPSPLSFISVGDSVTITC(YISPDIRNSLIUY~ FWXiSLWEDFMYYCWWYTF~~IKRl
   DICPlTQSPSTLSRSVGDRVRITCRRSQNISSMLMWKFWXX FTLTISSLZPBBFA~GPGTKVDIKRl
   B-IZHTQSPSSL%SVGDRVTITCXiXQS~~ DFTLTISGLLfQFCITWCWSYTT YTFGPGTKvENTRl
   DIC!llTCEF'STLSRSVGDRVTITCf#SQSINllUWf~ FlLTISSLWDDFATYYCWNS -ii!iKKz
   DIOMClSPSSLSFlm;DRVTIICRFISQCIRNDLTUY~ FTLTISSLPEDFMYYCLP
   DIQtlTQSPSSLSRSVGIRVTITCRWlSISSYL%YQ@W#Q FTLTISSLW'EDFFlTYYCWNYITPTSFUXJRVEIKF!l
   DI-QHTQSPSTLSVSVGDRVTITCERSQTVLSYLrUYQQKPQW BFlFTISSVZF'ZBFClTYYCPlFGClGTKVRKRl
   DIC!~~T@PSTQPIWGDRVTITCR~~SQSINI~~ FlLTINSLLH'DDFFllYYCCQYSi?YPYTFGQGTKLDIKRl
   DICRlTQSPSSLSVSVGDRVTITCQfi~~ fTFTISSLQPEDIATYYCOQYMYPPTFCaCT
   DIWRSf'SSLSFlSVGBRVTITCRRSZTISSYLBWLa6a FTFTISSLzPzBFATYYrzSYSSPTTFCwIKRl
   DIWlTQSPSSLSITCQRSQDIIKYLHPIWTPU(FIP DYTRISSLPPEDIFlmCWWSLWTFGWKLC!IT
   DICWTQSPSSLSASVGDRVTITCCRSQDISIFLWCWPGw DFlFTIS%lPEDIb MYCWFaUTTFC
A, ignoring gaps 72


322

TIBS 13 -August I988

lumbr Locus Name
  1  KlHUAG
  2 KlHUAU
  3 KlHUBI
   4 KlHUAR
  5 KlHUDE
  6 KlHUEU
  7 KlHUGL
  8 KlHUHU
  9 KlHUKFl
  10 KlHUKU
  11  KlHULY
  12 KlHUOU
lffset from start

1, ignoring gaps     0

Fig. 2. A sequence display from MASE with T and S highlighted.

position and search for compensatory
mutations  using COLUMN-COR-
RELATION function. COLUMN-
COMPOSITION permits analysis of
base/amino acid frequencies at homol-
ogous sequence positions. COLUMN-
CORRELATION lists the total num-
ber of simultaneous and non-
simultaneous base differences between
the aligned sequences in any two
sequence positions. Each sequence can
be compared either to a reference
sequence (order independent), or to
its nearest neighbor (order depen-
dent).The probability of simultaneous
and non-simultaneous base variations
in the whole set is evaluated using the
binominal formula. The COLUMN-
CORRELATION   function   has

recently been used for secondary struc-
ture prediction and classification of
human Alu sequences2.

Sequence data files

Each input file contains one or more
sequences. A sequence can contain an
indefinite number of comment lines,
each having a semicolon in the first
column. The first line without semi-
colon contains the locus name which
can not exceed 20characters and this is
followed by the sequence information
lines each not exceeding 95 characters
in length. After insertion of alignment
gaps the modified sequence set is stored
in the same input format. Aligned out-
put file can be created by a separate

OUTPUT-ALIGNED function. Line
length, number of lines per page, pos-
itions of vertical and horizontal gaps,
all depend upon the chosen output
format.

Program availability

By the end of this year MASE will be
made available on-line for the Bionet
community. Other non-profit users can
obtain the source code for editor writ-
ten in C and MASE manuals from:
Susan Russo, MBCRR, Dana-Farber
Cancer Institute, 4-4 Binney Street,
Boston MA 02115, USA. Tel. (617)
732-3746.

Acknowledgements

This software was developed under
auspices of Molecular Biology Compu-
ter Research Resource which is suppor-
ted by NIH grant RR0227503. A
portion of the support came from the
NIH grant U41-RR-0168505 for the
Bionet resource. We thank Dr Temple
Smith for helpful suggestions and
strong encouragement for this project.

References

1 Boswell, R. B. (1987) Trends Biochem. Sci.
12.279-280

2 Jurka, J. and Smith, T. F. (1988) Proc. Narl
Acad. Sci. USA (in press)


Volume 16 Number 2 1988                       Nucleic Acids Research

Small cytoplasmic Ro RNA pseudogene and an Mu repeat in the human a-l globin gene

Jcrzy Jurka. Temple F.Smith' and Damian Labudd

Bionet. 700 East El Cammo Real. Mountain View, CA 94WO. `Dana-Farber Cancer Insntute, Harvard
University. 44 Binney Street. Boston. MA 021 IS, USA and zMedical Genetica. Hopttal Sainte-Justtne. 3175
Cotc SainteCattwme. Montreal. Quebec H3T ICY. Can&
Submttted November 6. 1987

The 5'-end of the previously studied Ah repeat from the al -globin gene (1) is flanked by a
sequence 80% similar to one of the full length human small cytoplasmic Ro RNAs (Fig.
la), denoted as HY3 (2). This is the first known example of a pseudogene for the Ro
scRNA. Only a few such pseudogenes are expected to exist in the human genome (2). The
pseudogene location next to the Ah sequence may suggest physical interactions between
HY3-like RNA and the Ah RNA prior to the reverse transcription. The 3'-flanking region
of the previously studied fuIl size Alu repeat is another unreported Alu sequence truncated
at the Eco RI restriction site (Fig. lb). In vitro transcription of the region analysed (1) gave
four RNA fragments. One of them, 86 nt long, is synthesized from the short class III
transcriptional unit located on the S-side of the Ah repeat (3). This location coincides with
the location of the HY3-like DNA sequence.

promoter?    GTGG-CNNAGTGG
HY3        GGCTGGTCCGAGTGCAGTGGTGTTTACAACTAATTGATCACAACCAGTTA    50
         ???????? ??????????? o o ????????????????? ?????????
????o     GGCTGGTTGGAGTGCAGCGCTTTTTACAATTAATTGATCAGMCCAGTTA 52
                                                            (a)
HY3       CAGATTTCTTTGTTC~TT~T~~ACT~CCA~T~TTCACTTGA~T-AGCCTTT   101
        ,t/**t* I,   *tt******t*ttt*,,******** tt**** ***r*
3'-al    TAAATTTATCiiTTTCCTTCTCCACTCCTGCTGCTTCAGTTGACTAAGCCTAA 104

promoter     GTGGCNNAGTGG
AlU       GGEGGGCG'XGTGG-CTCACGCCTGTAATCCCAGCACTTTGGGAGGCCG     49
        o ?????????????? ??????????????????????????????????
????o     GGTTGGGCACAGTGGCCTCACGCCTGTAATCCCAGCACTTTGGGAAGCCA    471
                                                           (b)
promoter                        GGGTTCGANNCC
Ah       AGGCGGGCGGATCACCTGAGGTCAGGAGTTC                      80
        o ??????????????   ??????????????
????o     AGGTGGGCAGATCAC--AAGGTCAGGA%TTC                     ???

Fig. 1. (a) Sequence alignment between HY3 (2) and the comsponding 3'-al-globin region (1). Putative
p~lyt~ter~e II1 pmmoter is indii (I?) Genomic Alu consensus (4). aligned 10 the 3'-al-giobin sequence at
positions 423-500. Promoter boxes (5) 81c indicated. Sequences and numbering of the 3'-al-globi region
are identical lo those in (1). Exact matches (*). purine-putinJpyrimidine-pyrimidine replacements (I). and
gaps (-) ate indicated in both alignments.

(1) Shen, C.-K.J. and Maniatis, T. (1982) J. Mol. Appl. Gen. 1, 343-360. (2) Wolin,
S.L. and Steitz, J.A. (1983) Cell 32, 735-744. (3) Hess, J., et al. (1985) J. Mol. Biol.
184, 7-21. (4) Schmid. C.W. and Shen, C.-K.J. (1985) in Molecular Evolutionary
Genetics (McIntyre, R.J. ed.). pp. 323-358. Plenum Publishing. New York. (5) Fowlkes,
D.M. and Shenk. T. (1980) Cell 22.405-413.

766

9 I R L Prasl Limitad, Oxford, England.


Genome Mapping

J. Jurka

EVOLUTION OF HUMAN ALU REPEATS: IMPLICATIONS FOR
GENOME  S UDIES
J. Jurka T  and R.J. 2Britten, 21 Bionet,  Mountain
View, California;   California Institute of Tech-
nology,  Pasadena,  California.

     The human Alu family of repeated sequen-
ces contains at least five different subfamilies
referred to as Alu-b,c,d,e and j which are arran-
ged according to their position on the evolutio-
nary time scale from the most recent, Alu-b, to
the oldest, Alu-j, subfamily. This is identified
by computer analysis of the correlated, subfamily
-specific nucleotide occurrences in a number of
the diagnostic Alu sequence positions. A sPecia-
lised sequence editor has been developed to pur-
sue these studies.

     Members of each subfamily show different
nucleotide preferences in the diagnostic Alu
sequence positions.which can easily be identified
Another characteristic feature of different sub-
families is a systematic difference in the CpG
content from an average 16.08 per sequence in
Alu-b to an average 3.84 per sequence in the
Alu-j subfamily. It is proposed that subsequent
generations of Alu subfamilies have been trans-
cribed from different CpG-rich source genes. The
genes presumably have replaced each other during
the evolutionary history of primates. Once inte-
grated into the genome, a copy of the source gene
is no longer under selective pressure to maintain
the original CpG content of the source gene.
Therefore,
mination   one can observe a time-dependent eli-
      of CpG from Alu sequences.
    The classification of Alu repeats, based

on the diagnostic base differences and the cpG
"decay) provide a method to date both the invasion
of individual genes by Alu elements and the
genetic rearrangements associated with this
process.


STUDIES ON RAT LIVER CYTGCHROMES P450 USING
COMPARATIVE SEQUENCE ANALYSIS

           1            1          2
Elzbieta Holsztynska , David J. Waxman and Jerzy Jurka
(1) Department of Biological Chemistry and Molecular Pharmacology,
Dana-Farber Cancer Institute, Harvard Medical School; (2) Bionet,
National Computer Resource for Molecular Biology.

Currently, about 70 full-length sequences for cytochrome P-450 from
9 eukaryotic species and one prokaryote are available. The
crystallographic model of bacterial cytochrome has been reported
(Poulos et al., 1987, J. Mol. Biol. 195,687) and has been used as a
reference to evaluate our structural predictions using a variety of
theoretical and computer methods. Postulated regions of potential
structural-functional importance in P450 PB4(IIBl) include: (1)
combined membrane insert halt-transfer signal residues; (2) sites of
interaction with cytochrome b5 and CAMP-dependent kinase; (3)
internal halt-transfer sequence; (4) large hypervariable region; (5)
predicted dioxygen binding site; (6) NADPH-P450 reductase
interaction site; (7) conserved region and (8) axial cysteine heme-
binding region. We used Multiple Aligned Sequence Editor (Faulkner
and Jurka, 1988, TIBS, in press), to elaborate a synthetic model
correlating sequence variations in homologous positions of the
aligned set with the functional map as well as with predicted and/ or
reported structural features. Results of this study will be presented.

(Protein Sot. Meeting, San Diego, August 1988)


Locating Amino Acid Patterns in Proteins by Composition
            Sunil Maulik

BIONET c/o IntelliGenetics, Inc., 700 E. El Camino Real, Mountain View, CA.,
                    94040.

It is known that the amino acid composition of a protein plays a major role in
determining its folded state [l]. A fundamental pattern-matching problem in
protein analysis is that of finding sequences (or sub-sequences) given certain
compositional criteria only. For instance, one may wish to find all regions of
unspecified length >20% proline and >30% glycine residues in a (say) 500
amino acid peptide sequence.  A related problem may be to find all
hydrophobic regions (~50%) in a protein. An algorithm has been developed that
will scan a sequence and find sub-sequences (of any length) satisfying given
compositional criteria. The implementation of this algorithm, termed RICH, is
near completion and will be available soon on BIONET. Uses of the RICH
program might include finding hinge structures in immunoglobulin sequences
(known to be rich in proline and cysteine residues [2] ); or verifying if a protein
satisfies the PEST hypothesis [3] i.e.  if its half-life is related to the
compositional content of P (proline) E (glutamic acid) S (serine) and T
(threonine) residues.

References:
1. Sheridan RP, et al.,(l985) Biopolymers 24: 1995-2003
2. Huber R and Bennett WS (1987) Nature 326: 334-335
3. Rogers S Wells R and Rechsteiner M (1986) Science 234: 364-368


P41RRO1665-06

II. BIONET Training Publications
A copy of one BIONET informational publication is included in this section.

66


Protein Seq Dana Anal (1988) 2:11~1

`iEm
0 Springtr-Vcrlag 1988

Protein databases and software on BIONET

Sunil Maulik

BIONET c/o IntclliG~netics incorporated, 700 East El Camino Real, Mountain, View, CA, 94040 USA

Abstract. BIONET provides databases, software, and ne-
tworking/communications tools to over 2500 molecular bi-
ologists worldwide. Software for the analysis of nucleic acid
and protein sequence data is provided by both IntelliGenet-
its. and academic contributors. BIONET is currently imple-
menting dedicated high speed servers for searching protein
databases, as well as providing more flexible tools for pro-
tein structure recognition and prediction. In this review,
protein databases and analysis software available on the
BIONET resource are described, and progress in providing
new tools for structure prediction, comparative sequence
analysis, and pattern recognition using Artificial Intelli-
gence (AI) techniques are summarized.

file-transfer capabilities to any host computer on the AR-
PAnet and to a large number of Internet hosts on Bitnet,
Usenet, CSnet. etc. This extremely high level of connectivity
has facilitated both communication and collaboration be-
tween scientists worldwide. As an example, Dr. M.M.
Teeter of Boston College (Teeter@ bcchem.Bitnet) main-
tains a database of the electronic mail addresses of protein
crystallographers throughout the U.S. and Europe. This
database is accessible on BIONET for use with the MM
mail software.

What is BIOSET?

BIONET is a national computer network for molecular
biologists and biochemists. It is a non-profit resource
funded by a co-operative agreement between the NIH. Divi-
sion of Research Resources and IntelliGenetics. of Moun-
tain View. California (No. 5 UJl RR01865-06). BIONET
provides access to biological databases. software for analyz-
ing the data. and communication and networking tools for
the distribution of data. software. and computing resources.
BIONET maintains databases and software for both nucleic
acid and protein analysis. This review covers the protein
analysis component of BIONET only.

An annual subscription fee of S400 allows academic or
non-profit users unlimited access fo the BIONET computer
(a DEC-2065 time-shared mainframe, with additional net-
work access to a Sun 31280 database server and a micro-
VAX) including payment of telecommunication costs. Ac-
cess limited only to the communications facilities is also
available. International users are waived the subscription
fee. but are required to pay their own telecommunication
costs. Over 750 laboratories are currently subscribed to
BIONET from the U.S., Europe, and Japan, corresponding
to over 2500 researchers.

BIONET is using its extensive networking and commu-
nications facilities fo maintain a number of electronic bulle-
tin-boards (BBoards) dealing with different aspects of mo-
lecular biology. These BBoards are distributed throughout
the world. Messages are exchanged with the SEQNET
BBoard in Europe (SEQNET is BBoard service run by Drs.
Michael Ashbumer and Martin Bishop of Cambridge Uni-
versity), and are received as far away as Israel. Korea,
Taiwan, Australia, and even the U.S. research station in
Antarctica! Recently, BIONET helped initiate and partici-
pated in the formation of the worldwide BIOSCI BBoard
network. Of particular interest to scientists uorking with
proteins are the PROTEIN-ANALYSIS and METHODS-
AND-REAGENTS BBoards. The former is moderated by
Amos Bairoch of the University of Geneva (Bairoch@ cgec-
mu51.Bitnet) and deals with such topics as protein chemis-
try, sequence analysis. and structure prediction. The latter
is an all-purpose BBoard for nucleic acids and proteins.
and contains requests and data on topics such as codon
usage tables, peptide synthesizers, cDNA libraries, anti-
bodies, and protein engineering/site-directed mutagenesis.

Protein databases and software

BIONET is directly connected to Telenet and Compu-
Serve which provide 24-h access to the BIONET computer
via a local telephone call from most cities in the United
States. These networks allow scientists in Europe and Japan
to access BIONET using international carriers such as Eur-
onet, Datax-P. Transpac, and Venus. BIONET is also di-
rectly connected to the ARPAnet which provides mail and

BIONET provides the Protein Identification Resource
(PIR) protein sequence database [l], the SWISS-PROT pro-
tein sequence database distributed by the European Molec-
ular Biology Laboratory (EMBL)(Z], and the KeyBank'"
database of protein (and nucleic acid) sequence patterns
(provided by IntelliGenetics). KeyBank'" contains protein
and DNA consensus sequences (motifs) described using the
QUEST programs pattern language (31. Thus. at a single
resource information at several levels of biological function
are immediately accessible to the researcher. IntelliGenetics
provides a suite of programs that readily access information
in these databases. The IFIND program will rapidly search
a query sequence against any sequence databank utilizing

Ms. No. 0 35 Author hiw       Ms. A - As  Pages A- 4
Springer-Verlag. Heidelberg / H. Stiirtz AG. Wl?rzburg
Rovisorischc Scitcnzahlcn / Provisional page numbers    I. Kerr.    Date 2?~!?!:8.8


the algorithm of Wilbur and Lipman [4]. report similarity
scores. and display optimized alignments between the query
and similar database sequences. The QUEST program can
also search any sequence databank with a given sequence
pattern and locate exact matches of that pattern to se-
quences or subsequences in the database. QUEST may also
be used in conjunction with the predefined patterns in Key-
Bank'" to locate particular subsequences (for instance, sig-
nal sequences) within a given sequence of interest.

Sequences located by either IFIND or QUEST may be
simultaneously aligned by GENALIGN. a multiple se-
quence comparison program utilizing the regions method
of Martinez [q. GENALIGN will align up to 49 protein
or nucleic acid sequences simultaneously. The program al-
lows the user to choose between several different amino
acid "alphabets" when comparing protein sequences, in-
cluding the Jimenez-Montano and Zamora-Cortina alpha-
bet of evolutionary similarity [6], the Miyata alphabet of
physico-chemical similarity [7l, a hydrophobic-hydrophilic
alphabet, or a hydrophobic-neutral-hydrophilic alphabet.
In addition. users haveil flexibility to create and use their
own amino acid alphabets.

IntelliGenetics' PEP program allows the user to perform
various analyses on peptide sequences. These include sec-
ondary structure predictions by the Chou-Fasman algo-
rithm [El, and hydropathicity calcuations using the Hopp-
Woods [9] or Kyte-Doolittle [lo] procedures. In addition,
PEP can simulate chemical cleavage by a variety of pro-
teases and chemical treatments (a database of eight com-
mon proteases and live forms of chemical cleavage exist
within the program'. and users can add or create their own
database), determine amino acid composition. molecular
weight. and p!. The program also performs rapid and rigor-
ous similarity comparisons between peptide sequences. (The
former using a modified [ll] version of the Kom-Queen-
Wegman algorithm [12], and the latter using the Needle-
man-Wunsch algorithm [13] as modified by Smith-Water-
man [ 141.) PEP determines reverse translations using codon
preference tables followed by restriction site mapping and
splicing functions. Finally, PEP also contains a generalized
"window" algorithm. that allows users to define any char-
acteristic pattern in a sequence that can be determined using
a moving weighted average (either arithmetic or geometric)
over the sequence. For example, the window function in
PEP may be customized to predict the existence of mem-
brane-associated alpha-helices using the algorithm of Rao
and Argos [15].

In contrast to the other programs in the IntelliGenetics
suite, the SIZER program does not deal directly with K-
quence data, but instead may be used to calibrate gel elec-
trophoresis bands against given standards, SIZER can use
either the Duggleby [16]. Southern [17], or spline [18] meth-
ods of curve fitting. SIZER may be used with either protein
or nucleic acid gel fragment data.

In addition to the IntelliGenetics suite of programs for
nucleic acid and protein sequence analysis, BIONET pro-
vides selected software from academic researchers. Contrib-
uted programs that deal with various aspects of protein
analysis currently on BIONET include:

' The protcaxs arc: trypsin. chymotrypsin. pepsin. thermolysin.
clostnpain. Staphylococcal proteax, Myxobacter protease, and
proendopeptidax. The chemical treatments are: cyanogen. bro-
mide. hydroxjamine. iodosobenzoare, pH 2.5. and iodoacetamide

(i) FASTP. which utilizes the algorithm of Lipman and
Pearson 1191 to implement rapid and sensitive similarity
searches of the PIR or SWISS-PROT protein databanks
(ii) The PROT3 [20], ALP3 [21] and XALIGN 1221 pro-
grams for multiple sequence alignment of protein sequences
(iii) The XPROF program for the prediction of protein
hydropathicity using Rose's algorithm [23]
(iv) The IDEAS suite of structural programs [24] including
DELPHI (secondary structure prediction by Robson's
method [ZS], HPLOT (distribution of hydrophobic and
charged residues using the Nozaki-Tanford [26] or Eisen-
berg et al. [271 methods), HCOMP (comparison of hydro-
phobicity profiles), and ALOM (prediction of membrane-
spanning regions by disctiminant analysis [28]).
(v) The DSSP program [29] for the determination of protein
secondary structure from the Brookhaven Protein databank
atomic coordinates.

BIONET also creates software which promotes efficient
use of the resource. Recent software additions include the
BIFIND and BFASTP database interactive command-file
generators. BIFIND and BFASTP serve to insulate naive
users from the intricacies of performing database similarity
searches. They prompt the user for sufficient information
to perform the database search, display menus of the differ-
ent databases. and use intelligent defaults for all other pa-
rameters. They produce as their output command (batch)
files, which, when submitted. instruct the appropriate data-
base searching program (IFIND in the case of BIFIND.
FASTP in the case of BFASTP) to run the searches as
batch (remote) jobs.

Sew directions

An extremely rapid version of FASTP implemented to run
on Sun workstations has been provided to BIONET by
Dr. Warren Gish of U.C. Berkeley. Named FASTP-mail.
it is accessible by sending an electronic mail message con-
taining the query sequence to an IntelliGenetics Sun data-
base-server. The program automatically reads the message
and then proceeds to search the entire PIR database in
as little as 30 seconds. It remails the search output (top
20 scoring database sequences as well as optimized align-
ments) back to the user. The implementation allows the
user to proceed with other tasks on BIONET, or even log-
off, while the search is taking place.

BIONET is implementing a FASTA-mail program (that
searches both the GenBank. EMBL nucleotide libraries and
the PIR,`SWISS-PROT peptide libraries). Thanks to a gen-
erous equipment donation from Sun Microsystems,
BIONET will be moving to a network of Sun workstations
and file-servers during 1988. The new network will result
in a greater than tenfold increase in the amount of total
compute power available on BIONET. The implementation
of dedicated database-servers on this network will become
increasingly important as both the resource and the data-
bases continue to expand.

BIONET has initiated a collaboration with RIACS (Re-
search Institute for Advanced Computer Science) to use
the Connection Machine II (301 for database searches. The
Connection Machine II is an example of a massively paral-
lel architecture. BIONET intends to implement software


`3

for rapid database similarity searches and large-scale se-
quence alignments within this parallel environment.

In the last fifteen years protein structure prediction has
become an increasingly important research topic. There is
a growing demand from the BIONET community for more
flexible and accurate tools for protein structure prediction
and analysis. BIONET intends to build upon its expertise
in comparative sequence analysis [31] and to collaborate
with outside experts on proteinstructure recognition, analy-
sis. and prediction to rexarch and develop new tools in
this field. Protein structure prediction by knowledge-based
analysis requires as its starting point a database of structur-
al knowledge [32] obtained from the analysis of known pro-
tein structures. Rules inferred froni such a database are
then combined with either pattern-matching [33] or multiple
sequence alignment algorithms [34] to predict secondary
structural elemenrs. and, optimally, a single tertiary struc-
ture. If the protein sequence shares a substantial primary
sequence similarity (> 50%) to a sequence of known struc-
ture. multiple sequence alignment, followed by molecular
modeling. generally allows a plausible structure to be built
[35. 361. Even in the absence of known structure, predicted
secondary structural elements, hydropathicity. chain flexi-
bility, and evolutionary information combined in a multiple
sequence alignment can provide increased accuracy in pre-
dicting the fold of a protein [36-381. As obtaining optimal
multiple sequence alignments algorithmically remains an
unsolved problem. many researchers prefer the interactive
alignment and analysis of sequences. A prominent example
of software capable of such a task is the Multiple Aligned
Sequence Editor (MASE) developed by Faulkner and Jurka
[39], which combines interactive sequence manipulations to-
gether with standard sequence analysis functionality.
MASE runs on computers under the UNIX operating sys-
tem. It will be available on-line later this year when
BIONET moves to the SUN network.

A complementary approach to protein structure predic-
tion by comparative sequence analysis is one of pattem-
matching. The QUEST program described earlier already
provides one means of fmding sophisticated patterns in se-
quences. Houever. to recognize structural features of pro-
teins, more complex pattern searching tools are needed.
A pattern language for protein structure (termed PLANS)
has already been implemented [40]. An artificial-intelligence
based program called MATCH has been written by R.
Abarbanel. MATCH can tind elements of secondary struc-
ture such as turns with better than 90% accuracy [33. 411.
Tertiary structure predictions using both pattern-matching
and combinatorial approaches are now being attempted [42,
431. BIONET intends to make MATCH and other pro-
grams developed for combinatorial prediction of protein
tertiary structure available on the resource. Many of these
p&grams result from the research of F.E. Cohen and I.D.
Kuntz at UCSF [33. 40, 411 with whom BIONET intends
to collaborate on disseminating research software tools. The
programs as well as the knowledge-bases upon which they
depend will be expanded to improve the accuracy of predic-
tive techniques.

A related pattern-matching problem is that of finding
sequences (or sub-sequences) given certain compositional
criteria only. For instance, one may wish to find all regions
(of unspecified length) >20% proline and >30% glycine
residues in a 500 amino acid peptide sequence. A related
problem may be to find all hydrophobic regions (>50%)

in a protein. An algorithm has been developed that will
scan a sequence and find sub-sequences (of any length) sat-
isfying given compositional criteria. The implementation.
termed RICH. is in progress. (Details of the algorithm and
its implementation will be described elsewhere.) Uses of
the RICH program might include finding hinge regions in
immunoglobulin sequences (known to be rich in proline
and cysteine residues 1441); or verifying if a protein satisfies
the PEST hypothesis [45], i.e., if its half-life is related to
the compositional content of P (proline) E (glutamic acid)
S (serine) and T (threonine) residues.

Summary

With efforts underway to sequence complete genomes, the
number of actual and deduced protein sequences will in-
crease rapidly. Sequence databases and software must be-
come integrated with "higher order" databases (e.g., of
protein structural characteristics). Suitable software must
also be developed to link them together. BIONET already
acts as a central repository for sequence databases, sequence
and pattern searching software, and electronic media for
the acquisition and dissemination of new biological infor-
mation. Additionally, BIONET is actively pursuing re-
search to create new algorithms to allow the functional
characteristics of protein molecules to be deduced from
their sequences.

References

1. George DG. Baker WC, Hum LT (1986) h'AR 13: 11-16
2. Hamm GH. Cameron GN (1986) NAR 15-10      i +YF.
3. Abarbanel RA (1984) NAT 12: 263-280
4. Wilbur WJ. Lipman DJ (1983) Proc Nat1 Acad Sci USA
80: 7X-730

5. Sokl F. Martinez HM (1986) NAR 14:363-374
6. Jimenez-Montano M. Zamora-Cortina L (1981) Proceedings.
VII International Biophysics Congress, Mexico City
7. Miyata T. Miyazawa S, Yasunaga T (1979) J Mol Evol
12:219-236

8. Chou PY. Fasman GD (1974) Biochemistry 13:211-245
9. Hopp TP. Woods KR (1981) Proc Nat1 Acad Sci USA (1981)
78: 38'13828

10. Kyte J. Doolittle RF (1982) J Mel Biol 157: 105-t 19
11. Brutlag D. Clayton J, Friedland P, Kedes LH (1982) NAR
  10: `79-294

12. Kom LJ, Queen CL, Wegman MN (1977) Proc Nat1 Acad
Sci USA 74:4401-4405

13. Needleman SB, Wunsch CD (1970) J Mol Biol48:443-453
14. Smith TF, Waterman MS (1981) J Mol Biol 147: 195-197
15. Rao MJK, Argos P (1986) Biochem Biophys Acta 869: 197-214
16. Duggelby R, Kinns H. Rood JI (1981) Anal Biochem
  110:49-55

17. Southern EM (1979) Anal Biochcm 100:31%323
18. Vandergmft JS (1983) Spline interpolation.-!n: 111 111 (eds)
Introduction to numerical computations, 2Jd cd. Academic
Press. San Francisco. pp 126-l 38
19. Lipman D, Pearson W (1985) Science 227: 1435-1441
20. Murata ,U. ~~~~~~ ~~~(1985) PNAS 82:3073-3077
21. Goloh 0 (1986) J Theor Biol 121:327-337
22. Bacon DJ, Anderson WJ (1986) J Mel Biol 191: 153-161
23. Rose GD. ~~~~~~ 111 Science (1985) 229: 834-838
24. Kanehiw M (1984) IDEAS. Integrated database and extended
analysis system for nucleic acids and proteins. User manual.
Laboratory of Mathematical Biology, NIH, Bethesda, MD
25. Gamier J. Osgulhorpc DJ. Robson B (1978) J Mol Biol
  120:97-120

26. Nozaki Y. Tanford C (1971) J Biol Chem 246:X11-2217


27. Eiscnberg D. Weiss RM. Tcrwilliger TC (1984) Proc Nat1 Acad
I     Sci USA 81: I@144
28. Klein P, Kanehisa M, DeLisi C (1985) Biochem Biophys Acta

. is

  815:468476
29. Kabsch W, Sander C (1983) Biopolymers 22:2577-2637
30. Hilli D (1985) The connection machine. MIT Press, Cambridge.
  MA

31. Jurka J, Smith TF (1988) Proc Nail Acad Sci USA
  85:4775-4778

32. Blundcll TL (1987) Nature 326: 347-352
33. Cohen FE. 111111111(1983) Biochemistry 22:4894-4904
34. Webster TA, Lathrop RH, Smith TF (1987) Biochemistry
26:695(M957

35. Lcsk AM. Chothia CH (1986) Phil Trans R Sot London Scr
  A 317:345

36. Brown JH. ~~#~~~~~~(1988) Nature 332:845-850
37. Crawford 1P. Niermann T. Kirschner K (1987) Proleins

118-129

38. Pearl LH. Taylor WR (1987) Nature 329:351-354
39. Faulkner DV. Jurka J (1988) Trends Biochem Sci [l:II-ll
40. Abarbanel RA (1984) Ph.D. thesis, University of California.

San Francisco

41. Cohen FE. ~~~~~~ llfl(l986) Biochemistry 25 : 266271
42. Cohen FE. 111 llIll[ (1986) Science 234:349-352
43. Webster TA, Lathrop RH, Smith TF (1987) Biochemistry

26: 6950-6957

44. Huber R, Bennett WS (1987) Nature 326:334-335
45. Rogers S, Wells R, Rechstcincr M (1986) Science 234: 364-368

Received May 31, 1988 / Accepted June 27, 1988


P41R.RO1685-06

III. BIONET Newsletters
Copies of the two BIONET Newsletters are included in this section.

69


BIONET NEWS,

vol. 1 no. I

Automatic Dab Submission to Gen-

Bank, EMBL, and NBRF-PIR

BIONlZ users can conveniently submit
sequence data using the XGENPUB pro-
gram. XCENPUB helps to annotate and
electronically submits sequence data to
GenBank, the National Institutes of HeaIth
DNA sequence libmry, EMBL, the nucle&
tide sequena database from the European
Molecular Biology Laboratory, and NBRF-
PIR. the National Biomedical Reseaxh
Foundation's protein sequence databaw

Sequence authors are encouraged to
submit their sequence data once it has
been derived. Direct computer-mdable
author submissions decrease the time
delay with which the data is incozponti
into the databases and can improve the
completeness and accuracy of the annota-
tion and sequence data.

XGENPUB is located on-line and can be
accessed by simply typing "XGENPUB" at
the system Q prompt. The program
accepts sequence data in files from the
authofs BIONET' dimory and prompts
the user for the name of the sequence tile
and the sequence name. XCENPUB
initiates an editor for completing a
submission form and insats the sequence
data at the end of the form. When the w
exits from the session, the completed entry
is automatically mailed to Genepnlc
EMBL, and NBRF-PIR using the AR-
PANFI' computer network A copy of the
submission will also be sent to the
author's mail file. Sequence data will be
present in the databases within four
months of submission.

The vast rate of growth that datati are
experiencing moves sequence submission
responsibilities to the authors. Many
journals are now requesting that their
contributing authors submit the data One
journal, Nucleic Acids Rcpatrcfi,  nsquln6
evidence that data have been submitted
before it will consider a manuscript for
review. The XCENKJB program providee
BlONET users with a convenient method
with which to fulfill their sequence

submI6hK!splmallFuxtha
information repniing XGENPUB may be
Obhhdby viewingthe on4ine help topic
HELPXGENPUB. 0

Bfficient Database Searching

Inmeff&toinc6ase&cientutiKzation
OfthWSOurce,BiONEThuproduesd
"GmmandFrlccena;rtorr" These
progr8Inecrrotcafonnatted~of
Pmgnm-for-OV
ofthese4perlcadahbaaes6rchlng
roftwamon BIONEI'. BIFIND, BFASIP,
andBFASlNservetoinsulateurarl+om
theintriuderoftheIFIND,FAsrpand
FA!ZNdatabasesimkityreafihing
prognma~chpromptsthewformlfK-
dent infi3xmuion to perform the simikity
~disptaysmaIusoftheWeraltda-
tah6chok6,andw&lteuigmt
defaultsforallother F----Y-
produaasthelroutputoommuld(ktch)
fK6,whi&whensubmltted,iIutnKtthe
appropde database seaxhing prognm
tonmthesearchasabatchorremotejob.
The w may then safely log-off the
BlONET system, knowing that the output
ofthewseafihwillbestoredina
filefors~tenalyslsoncethelYatch
jLJbhrM.On-linchelpIZldShfor~Iltha
pgXUtlSbytypingHELPfOUCtWdbythC
Irrov-

lheGIMUndF~Cenmtol%undago
continusd development. As an example,
BIFINDwilIsoonbeabletosaveBUTH
mh and alignment msults. With the ad-
vent of BIONEI"s new FA!Z'P-maiI data-
baseserv6(thats6xhfstheentirePIR
pt&l&hbWlkhUlitthS3OSSiOXdS-
3Gtim6fasterthananyotherimpkmen-
tationofFASTP),anewBFASIPisbeing
implemalted,thatmakesuseofthisnew
rava~WatchtiforaFASIN-
nuKpmgranlapableof#udrirythe
GmBank nudeicaddequencedaerbr#
fiumorerapIdlythanprduslyapabk
and a comspondIng new BFASIN pn+
w--o

Revised Introduction to BIONIC

InMay,arevisedversionoftheintru-
duciontoBIONET willbesenttoalIofthe
principal hestlgatom subsaii to

April 1988

BIONET. The Introductfon to BIONET
irthehandbooksenttonewsubsuii
which provides basic information for
accessing and using BIONEK

The new version includes two new
sectionswhichshouldbenefitusers
greatly. A Troubleshooting chapter
helpsuse!rsdlagnoseand solvecommonly
ermxmmd tech&al problems. A
"Progmn I&amptcr" appendix provides
anmtated examplesof thirteen common
biologial tasks and how the BIONET
pplIUlMykUS?dtOSOlolvethUlL
Manymorepnqmmexampleswillbe
available In printed form when the new
InteKiGenetics User Manual, described in
a separate atick+ is distriiuted to
BIONET usws this spring.

. Revisions to the lnfmdlcction to BIONET
include the addition of a step-bytep
`example of a BIONET us&s first login
8ession Itcovaahowtoamnect,loginto
the BIOm UIKIunt, read login messages,
and get help. The chapter desuibing the
BIONET programs now includes a com-
plete list of the contributed molecular
biology soHware that is available on-line
or thmugh the BIONET Lending Library
of diskettes. The chapter on databases has
bS6leXplhdtOXfdleettheMWOrguri-
Wlon of the databases as well as recently
added databasee such as SWISS-F+RUT.
Thlschapteralsod6aiithenew
XGENPUB pmgrim which idlows eke
tronic submiasion of sequena data and
annotations to all of the principal daht-
bases. Mivheffotihasbeengiventiin-
C0rp0ratlng w suggestions fur changes
into * new Intro. We hope that it allows
userstospendlessti.meleaming,and
mans time using the BIONET mso\~lcc o


BIONET Communications Reach
      Worldwide

The BIONET National Computer Resource
for Molecular Biology is far more than
merely a facility to analyze DNA sequent.
In addition to its analysis software and
molecular biology databases, the Resource
offers aace8.s to powerful electronic commu-
nications networks with worldwide scope
Use of this facility is completely free to
BIONFI account holders.

BlONET maintains the most extensive
network available of molecular biology
ekctronic bulletin boards fbboardsl. Nine
teen bulletin boards are maintained on the
BIONET mainframe computer and copies of
posted messages are read around the world.
The BIONET bboards provide scientists with
the means of contacting a large community
of colleagues by simply sending a single
electronic mail message. A detailed list of
bboards can be seen on BlONlX by typing
HELP BBLIST after the Q prompt.

BlONET bulletins are distributed via the
ARPANET, BITNET, and USENEkomputer
networks and can be received by scientists
without BIONET accounts who have elec-
tronic mail addresses on any of these
nctworks.There is no charge for this service.
Users on any of these networks can also post
messages directly to any of these bboards
without editorial intervention. One simply
uses the address format
BBOARDNAMEBBIONET-20ARPA.
BIONET users can post messages to the
bboards by simply entering the bboardname
in response to the To: prompt when sending
a message in the MM mail program.

Copies of each message are automatically
relayed around the world via our direct
network contacts and through our message
exchange agreement with the SEQNET
bboard service in Cambridge, England.
Messages are read by scientists in places as
far away as Japan, Korea, Taiwan, Israel, and
Australia.

If there are many people working on a local
campus computer at your institution who
may be interested in partldpadng in the
bboards, please have the person in charge of
your computer facility contact
kristofferson@bionet-2O.arpa. We can then
arrange an efficient redistribution scheme at
your site

Electronic bulletin boards are just beginning
to be discovered by the molecular biology

community. Bboanis truly have the
potential to revo1uti0niz.e the way that
dence is done In this field! o

Addressing Electronic Mail Outside
     bf BIONET

Many BIONET users have colleagues with
BlTNETor EARN (European Academic
Rexarch Network) electronic mail ad-
dressesandwonderhowtosendmailto
them. The procedure is extremely simple,
andtherearr,nochargesforsendingmail
messages. One sends a message using the
normal BIONET mail routine; only the
address for the message is slightly
different- Suppose that a colleague's
BITNET address is SMI'IHBYALEVM.
Enter SMTH0YALEVM.B~ in
reqmue to the To: prompt in the MM mail
)xogram:

MM>send

To: smith@yalevm.bitnet

TheprocedumisthesameforEARN
addresses. SMRH@EMBLbecomes
SMITHOEMBLEARN. Actually
SMIlH@Eh4BLBITNET will also work
for EARN addresses, since the two
networks are eswntially the same. EARN
is BITNET In continental Europ

srne users have trouble addmssing mall
to English addresses. The network used in
England is called JANET (Joint Academic
NETwork). Unfortunately, JANET ad-
dressesarearrangedinreverse order of
that used in many other countries, inchld-
ingtheUS. To circumvent this problem,
BIONET has implemented the .JANET
address extension (properly termed a
"pseudodomain"). To send to a JANET
address mad30uk.ac.caabio, add
JANET to the end of the address:

  mad3Ouk.ac.cambio.janet
Note that case does not matter in mail ad-
dresses.

Fmlly, sometimes one encounters trouble+
some addresses that contain !`r
foop!roplnet@pri.com. what e"tt!k&J
these addresses at the To: prompt, enclose
the part of the address to the left of the
einquotes. Enter.

   `foopIropfnetVprLcom
AU of the l's should be enclosed between
the two quotation marks for the address to
be properly interpmted by the BIONET
mail software.

This short article should provide the

means to contact users almost anywhere in
the network& world provided one has
theirektmnicaddress. Ofcourse,aswith
regular mail, one somehow has to find that
address by other means. International
usm dirtxtories 1~ still in their infancy,
but there are means of determining
addresses if the telephone or regular mail
can not be used to find this. The BIONET
consultants can help with this and any
other communications questions. please
do not hesitate to send them an e-mail
message to the BIONET address or call
them at (415l324-4363. O

User Manual Replaces Short Course

A User Manual, which replaces the current
Short Course, will be made available to
BIONET Principal Investigators this
Spring. Each Kncipal Investigator wiLl
receive one copy of the manual.

The User Manual is organized into the
fobving sequence analysis topics:

- sequence entry and editing
-sequence location

- sequence and map display
- sequendng project management
. - squence translation
- seqmmce aompositlon
-sequencestlucture
- restriction analysis
-cloning simulation
- sequence comparision and alignment

Each topic contains annotated program
examples illustrating the step-by-step
procedures involved in performing
SpeciBc operations. The User Manual also
includes sections on tile structure, data-
bases, and program commands. Addi-
tional information may be obtained from
Kathryn Berg at (4151962-7337. o

Subscription Fees

Each year BIONET account holders are
assessedaflatS4OO.OOfeeusedtocover
telecommunications charges. New ac-
counts are billed when the BIONET
account is activated. For renewing
subscrilbers, a reminder notice is sent out,
foliowed by an invoice a few weeks later.
It would be appreciated if the invoices for
both new accounts as well as renewing
subsuii would be paid within a
reasonable length of time.

We have instituted a policy of freezing
delinquent aaounts if the yearly subscrip
tion fee is not forthcoming. If your
account ha5 been frozen, please call
(415x362-7337. 0


aosendusyour.writtcn&tsaIJout
BIONET (signed Iutdcopy,`plase!l~ ;;::.:? .,
Plea5edetaBboththe~alld -: -+ :
negative aspects of the w   .,.
toenableusto~thesevke thaw?:
provide ourmailingaddres?ir:
                 :
                   : ;i-
  Dr.DavidKristofdaroa 
        mm ., .:>;>f ;" ;. .
     c/o in-    .-
  7ooEElCiminolbrl -,:
   MomaInview,cA 94040 :
             .

Wewouldalsoappresiatethreecopisoof
anypublicationsfromyourlabomtory
sinceDeamber1,l9s7thathaveentaBed
theuseofBIONET. Thankyouin
advance for your coopaaioh

AJ~w~~MT4-~ Progrsm
Speeds Similarity Searches.

A5thl?fil%t5teptowardsutil&ingthenew
BIONEl' Sun computers, the FASTA-
MAIL pqram, developed by BIONET's
SySb3lt5progrpmmcn(EliOtIauudROb
Liebsdtutzl, was ndeased August 17.
SIncethf&overtloo~PfRand
SWISSPRUI' seprchc) have k run by
BIONEl=uaek TheFASI'A-MAIL
programallowsauaertomtdamaIl
message containing a nudek add or
pmteinquayseqmmcetoaBIONETSun
computer. A sequence similarity mh is
thenp&ormedagainstanucleicacidor
protein databank using the FASTA
program developed by William Pearson

,m ds I& file on the BIONEI' DEC-
206!5amputer.TheF~A~isan
rbprond. version of the older FASIF and
FASTNpro%rrma Byincorporating
dktinctsaningmatricee,bothproteinand
DNAman?execukdbythesame
pmgmu~ FASA inchrdes an additional
8kpInthecalculationoftheinitial
pebwI5e5ImBarity5unethatauo~ ,_

Gfurthw co+i&ion and optimkd
aP=nL.

To IPY FASTA-MAIL on BIONET, the
qtmrysequauemustadheretostandard
InteUadc5 file format axwention5.
BsauseFAST'A-MAiLisamailserver,
truootherstipuUonsalsoapply:(1)no
I&&thsfik~kI~gerthUtBO~
actemandQ)tkeGinonlybeone
sequmceintheflk TostuttheFASTA-
MAIL pugram, type FAZTA-MAIL at the
XoNEr-4rpmnptThepqmmi5
quita simple and user&mdIy, but we do
urgeusersto~dtheon4ineHEL.P
FASA-MAIL help topic at the "(D"
prompt. ThisdocumentatIonexplainsthe
FASI'Aaawingpmdure,givesp~~&cd
advia about submit&q jobs, and contains
biwogrrphic- forti-
Asaondhclptoplc,HELPEf35,- -.. -
pmvkleaacompIekste+y+tepaumple
oftlmprogramfromsubmissionofa
FASTA-MAILjobthroughareviewofthe
dts. Panun~thattheuserdloo5ea
arewhichdatabasetosesrchandthe
-valuedTKV. TheKWPvaluei5
aImlopstothethcoRDs~parameter
intheiFiNDpnpm. Itdetetminashow
nunyautsecdvelaid~mustmatdtin
tkfIntpasskfactkregionkconsId-
eredbytbeprogram. AlowwKTUP
vaIlaeIlKmUasthe!sensitlvityofthe
5eaebutk?ngthensthereprrhtime
arponatklly. The tumamundtimeon
seamhesdependsprimar@onfrow
mUrpthIBjObSprcilltheqwUetUKlthc
lciuPvahas. Currently,job55c?emtok

October 1988

running in 10 to 20 minutes for protein
searchesandinlto3hoursforfuIl
GenBankwb.

Many users have asked questiona regard-
ing the interpretation of search results.
Bear in mind that a high similarity score
cannot prove biological homoIogy, it can
only provide evidence FASIA-MAIL
provides three scores for each sequence
displayed, INITN, INm, and OPT. INlTl
isthesameastheoldFASIPorFASIN
"INIrscoreandispknariIyincIudedin
FASI'A-MAIL for comparison with zsults
from these older programs. The INITN
score is the score used on the first pass to
rank sequences or discard those below a
cutoff score. While the INITN score does
allow gaps within matching regions, the
gap-penalty does not vary with distance at
this point, nor does the program chadc to
saeifthereisabetteralignmentofthetwo
s4quence5 yielding a better scorn These
are induded when the final OPT score is
calculated by a Needleman-Wunsch/
Smith-Watterman-type of alignment on
the region surrounding the highest scoring
initial region. The alignment displayed is
based on this final alignment procedure.
Thus, while the INlTN score determines
whichsequencesarckeptandtheorderin
which they are displayed, the OET score
gives a more precise gauge of the similar-
ity of sequences. However, since the OPI
ttcore is not calculated for each equence in
the database, there is no mean and
standard deviation generated for the OPT
5am5. TheINlTNsoDrestlllmustbeused
when one compares the high saxing
sequences agahst the fuIl population.

Two method6 ue useful for deteeminlng
stat&ticsl significe~ One can simply
c~mpate the INITN score of the sequence
in question with the moan and standard
deviation of al1 INITN scores to e how
many standard deviations above the mean
thescorelies. Ho wevex,onemustkeepin
mind that in a sufficiently large
database! search there usually wiIl be
soma random matches that stllI acore even
thRe or four standard deviations above


the mean. To see if a high mring *
quence is truly similar to the target quetY,
Lipman and Pearson suggest randomizing
one of the two sequences and then scoring
it with the same INTIN or OPT scoring
pmceduR IftheorigInaIun.randomi7Jsd
score is much higIwr than random&d
runs, this indicates that the high score is
related to the sequence order and not just
to the seque~e composition. William
Pearson has developed a program, EDa
which perfwms this Monte Carlo simula-
tion. This program wiII be avai&bIe on
BIONEI' when users are shifted to the Sun
system. Contact Dr. Pearson directly if
youwishtoobtainEDF2forPCsor
mainframe Unix computers.

The original paper by Wa Pearson and
David Lipman descriig the FASTA
program, "Improved toob for bioIogkaI
sequence compark4oq" appeared In the
April 1988 issue of PNA5 (VoI. 85, pp.
2444-2448). WiIliam Pearson may be
contacted by e-mail at *wrp&&ginia.edu"
or at the Department of Biochemistry; Box
440, Jordan I&II; Univ. of Virginia;
Charlottesville, VA 22908. BIONEI' would
like to thank Williun Pearson and David
Lipman for aBowIng BIONET users access
to their programs and for pmvIdIng details
regarding the program'5 xoms. We look
forward to receiving comments and
suggestions about the FABTA-MAIL
P*f3=m.

               . .  A..-'
           .
Multiple Aligned Sequence
   Editor (MASE)

:

A*yrisof*,&&&&g
number of sequmcwcanbequite~~
fmstrating and, above al& tinuxonsum-
ing. Most aIgorithms can only handIe a+
quences of limited sizl Outputs fnnn
available multiple sequence alignment
algorithms often need hyther refinement
to make bioIogIcaI saw. These and &ted
problems with sequemzanaIysIs 5timu-
lated my joii, effort with Don FauIkner to
design and dcvclop a gener&purpose
multiple sequence editor now aIled
MA5E.OurworkbeganbackIn1986when
I was a rawch kllow at Harvard. It has
been continued a5 a adkborative pmjxt
since 1 joined BIONET as a scientist In the
middle of 1987. An introductory artide on

MA~canbefoundintheAugustissue
of Trends in BIochemIaI sdences, (Fat&
ner, D.V. 6r lurks, J; voI. 13, pp. 321322).
A limited number of reprints of this
article are still avaiIabIe from'either a
author.

MASEcankin5tded~wmpuhr5
nmningtheBer3&yUNlXoperating
system. Many non-pm8t meardt gmups
wIthaae5storBerkel5yUtisy5te!m
have!aIraadyiKquimd&ee!&orfreeof .
dwgefmmHarvad. lbrthosenon-
pmfitretedm3whodonothavexN55
totheappmpriatehanIwaru,BIONETb
auknizedtodis&ukMASEon-lin.
Currently, BIONET Is moving from our
DEC-20 to a network Ot do~td BUN
computa8.Afhrthi5tmNitton,MAsE
wIBbemadeavGlabkeu-IIneinIate1988~
aeuIy1989.FmmthershnicalpoIntof
ViWitWOUkikVUp5dUltOknOW
howmanypotentiaIBlauEr5ub5aii
needthesequa~editrx. Wetherefore
a5kpotentjainewusa5tomailm
ektmnk message tojurka@bionet-
2O.bioaet. Thank you for your help.

JazyJ-

November Training

A.reyouanewuserofBIONED Orare
youanarpaienaedu.5wwantingto
becomemorepmBcientwIthyouruseof
BioNlm PlantopartIdpteinan
upcoming BIONEI' training xssion.

Then&tckssisschduidbrNovember
17-l% 1988. Topics covued wiII indude
5aquenNdataentryandalitin&5e!qu~
inggdmuugrmcnt,nudekacidand
psptidesaqlKnNd*dahbasestruc-
tumaMi5expmcerdli#al,colkcting5e-
qumar=fi-gfotprttans,
aqencedmitrity-, and ekc-
tmnicmail.

Thetrdning565IonwiBnmfmm8~30
until5M)pmandfmm63Ountil9M)pm
oaNovemIJer17th. TheevanIng&SsIon
willcQvertheTOPSZOopntIng5y5tem,
COOUtt&tiON SOhVUe, th OldiN hdp
fadlitiee#a5wdlasan owmkw of contrii
lltd5oftwmmIhe seasian on November
l&hbe@nsatgM)amwIthaqtmstionand
answerseUion.EnmaIinunutionbegins
at9zOOamandcondudesa5100..Lunch is
provided each day. Evening me& are not

provided, but there are reasonably priced
restaurants within wdking distance.

BIONET is located in the InteIIiCenetics
Building, 700 East EI Camino EeaI (at the
inters&ion of Highway 85) In Mountain
View, CA. This is within easy driving
clhance from either the San Francisco or
San Jose airports.

Thecostofthetrainingiskeptlowto
encourageuserstoattend. ThereguIar
priceIs$lOOforthetwodaypmgmm. To
help offset the additIonsI cost of tanspor-
tation, outof&ate subscribers pay $50.00.

ThetraIningisBmitedto12pwpIeona
&t-come, first-serve basis. To reserve a
pIace, caII Kathryn Berg at (415) 962-7337.
We aaept purchase orders, MasterCard,
VISA, or pasonaI check Payment should
beswtto:

    BIONEI'
c/o IntelliceNtic5
7wEa5tElcaminoRNl
Mountain View, CA 94040

We are enthusiastic about the BIONEf
resource and look forward to facilitating
~youruseofit.

1989 Training Schedule

Listed below are the dates for BIONIC
mining in 1989. Note that the course has
ban expanded to cover additional topics,
5udt a5 contriited software, file transfers
and tekcommunications.

Febmaxy6,7'
Mardi 15,16,17
  May 17,lS. 19
  July 19,20,21
September 20` 21.22
' November 15,16,17
o wening session on 2/7

BIONET I'kws is pvblishcd by the BIONFT
GnnpnkrJz?soura s-q contributors to t/is
is5wppn KlAy?l&g,KamDaoir,~nzy
lurka, muid Kristoh, and spcnccr Yeh.
Yonr conwunts 0ld sqgestfons on this
pubriatfonnngn?attyoppnciPtcd.


P41lXEtO1685-06

IV. BIONET Software Lending Library Catalog
A copy of the catalog is provided in this appendix.

70


Version 1.5/KB/ll-88

PC-Lending Library Software

Please send one disk for each program requested. Include a self-addressed, stamped mailer along with
the disks and request form. This will greatly streamline the ordering procedure. Send your request to
BIONET, 700 E. El Camino Real suite 300, Mountain View, CA 94040. Substantive questions about the
programs, or any difficulties with program functions should be addressed to bionet@BIONET-20.arpa.

I. Communications/Terminal Emulation
Multipurpose public-domain software for logging on to BIONET, VT100 emulation and file transfer.
Various formats are available:

o Kermit--for IBM PC compatible computers
o MacllKermit and Kermit Shareware--for Apple Macintosh, Macintosh II, and Macintosh SE
computers (send 2 disks for these programs)

II. Editor

o Micro-emacs-text editor available for BIONET subscribers.  Either high or low density
versions may be requested.

Ill. Contributed Software
All programs come on 5 l/4 " diskettes

SEQAIDII
D. J. Roufa and D. D. Rhoads
Division of Biology
Kansas State University
Manhattan, KS 66506

Droufa@BIONET-20.arpa

SEQAIDII is a multifunctional program for DNA sequence analysis.

System requirements:  IBM PC compatible computer. Working files require 200 000 bytes of disk
storage space.

Flies Included:

o INSTALL.DOC--Documentation file
o SEQAIDFD.EXE--Self-extracting arechive of SEQAIDII files
o SEQAIDII.NEW--Release notes for version 3.0

o README.DOC--Installation instructions


PCZUCKER
Michael Zuker
National Research Council
Biological Sciences
Room 3115
100 Sussex Dr.
Ottawa, ON Kl A OR6 CANADA

ZukerQBIONET-20.arpa

PCZucker is a program for global prediction of RNA secondary structure.

System requirements: IBM PC compatible computer

The programs require a minimum of 512 kbytes of RAM, version 2.0 (or later) of DOS, and a floppy
containing 360 kbytes, or 1.2 mbytes.

Flies Included:

o Two versions of PCFOLD

1. PCFOLD.EXE--packs the arrays, to extend the length of the longest fragment it can
fold (425 bases maximum).

2. PCFOLD2.EXE--does not contain packing, can handle only fragments of 345 bases
maximum.

  o README.DOC--Documentation and explanation of program use
  o MENUDAT,MENUDAT2-Data files needed by the program
  . FOLD.ENR-Energy file
  o FOLD.BAT--Batch file to run PCFold program
  o FOLD2.BAT--Batch file to run PCFold2 program
  o PSTV-Default sequence file
The source code is available in:
  o SOURCES.1 --Source programs for PCFOLD.EXE
  o SOURCES.2~-Source programs for PCFOLD2.EXE


MOLECULE
John Thompson
Carnegie-Mellon University
Biological Sciences
616 Mellon Institute
4400 Fifth Ave
Pittsburgh, PA 15213

Woolford.Thompson@BIONET-20.arpa
This disk contains compressed files for John Thompson's MOLECULE program for display of secondary
structure prediction. The programs read .CT files produced by Zuker's PCFOLD program, and display a
2D graphic representation of the structure. Three versions of MOLECULE are available, but only one
need be accessed, depending on the monitor-type being used.

System requirements: IBM PC compatible computer

Flies included:

o CMOLECULE.ARC--IBM colorgraphics monitor
o EMOLECULE.ARC--EGA monitor (Enhanced Graphics Adaptor)

o HMOLECULE.ARC--Hercules Monochrome Card

o o .DOC--instructions on program use
o `.PAS--source code, in Turbo Pascal

ALIGN
Dan Davison
Dept of Biochemical and Biophysical Sciences
University of Houston
University Park
4600 Calhoun
Houston, TX 77064

Goad.Davison@BIONET-20.arpa

Keith Thompson
Biology Dept.
Brookhaven Nat'1 Lab
Long Island, NY

The ALIGN.DOC file provides a detailed description of how the program compares sequences the user
has submitted. The documentation includes a step-by-step explanation of the procedures involved, and
clarifies program parameters. Several types of output are available. The program has the capacity to
print any or all of the following:

1. imput data (amino acid or nucleotide sequences typed in by user)
2. table of matches--which show actual start and stop positions of each match found
3. a listing of matched and unmatched areas

System requirements: IBM PC compatible computer

Files Included:

  o ALIGN.EXE--executable version of ALIGN
  o ALlGN.DOC--documentation and explanation of program use
There are many other accessory files also on this disk.


ALP3tALN3
Osamu Gotoh
Dept. of Biochemistry
Saitama Cancer Center Research Institute
Ina-machi Saitama 362
JAPAN

This diskette contains some implementations of the algorithm for aligning three protein or DNA sequences
described in Gotoh, 0. (1966) J. Theor. Biol. 121,327-337.

System requlrements: IBM PC compatible computer

Files included:

o ALN3.EXE--program for aligning three DNA sequences
o ALPS.EXE--program for aligning three protein sequences
o MDM-1 .DAT--program data file
o MAKMDM.EXE--accessory file

Teat data files:
  . Sl .SEQ    DNA

  . S2.SEQ "

  . S3.SEQ "

   Pl .PEP   Protein

  . P2.PEP "

  . P3.PEP "
Also included:
  o SEQFORM.DOC --describes the sequence file format to use when running ALN3/ALP3.

PLASMID PAINT
Joe Liisick
Dept. of Pathology, M-01 2
U C San Diego
La Jolla, CA 92013

Jlipsick@BlONET-20.arpa

PLASMID PAINT, a program written in Microsoft QuickBASIC 2.0, allows one to draw plasmids.

System requirements: IBM PC compatible computer with a CGA-compatible adapter and screen

Files included:

o PLASMIDC.EXE--executable file
o PAINT.BAT--short batch file which loads the DOS GRAPHICS command and then runs
PLASMIDC.EXE.

o README.DOC--explanation of program use


OLIGO MUTANT MAKER
Kevin Beadles
1044 l/2 Shrader St.
San Francisco, CA 94117

(415)759-0148 (Kevin will call you back collect if he must return your call.
OligoMutantMaker simplifies the designing and screening of oligonucleotide-directed single amino acid
substitution experiments by searching for nucleotide sequences which introduce a restriction
endonuclease recognition sequence into the codon substitution site of the mutant.

System requirements: IBM PC compatible computer.

Files Included:

o README.DOC--Documentation file

o BWMUTANT.COM--Executable file for monochrome monitors.

o CMUTANT.COM--executable file for color monitors.
o CUTTERS.DAT--Binary database of enzyme cleavage and availability data.
o CODONID.DAT--Standard genetic code.
o [ENZYME.TXT]--This file is created every time the program is run. It contains a copy of the
results of the last analysis.


P4lR.R01685-06

V. BIOSCI Bulletin Board Network Information
A copy of the information sheet which is mailed electronically to interested parties is included in this
section.

71


BIOSCI BULLETINS
----------------

The following is a list of bboards available for distribution to sites
on the ARPANET/Internet, BITNET, EARN,  NETNORTH,  and JANET.   For each
of  these bboards  a list of   BITNET name abbreviations and analogous
USENET newsgroup names  are listed below.   Finally,  we provide a list
of  the various sites  (nodes) that distribute the bboards and the
address format for posting messages.  Note that messages posted at any
node are automatically redistributed to all other nodes on  the BIOSCI
network and subsequently to their readers.

BBOARD NAME
_----------
AGEING
BIONEWS
BIOTECH
BIO-CONVERSION
BIO-MATRIX
CONTRIBUTED-SOFTWARE

EMBL-DATABANK
EMPLOYMENT
GENBANK-BB
GENE-EXPRESSION
GENOMIC-ORGANIZATION
METHODS-AND-REAGENTS
MOLECULAR-EVOLUTION
ONCOGENES
'PC-COMMUNICATIONS
PC-SOFTWARE
PIR
PLANT-MOLECULAR-BIOLOGY
PROTEIN-ANALYSIS
RESEARCH-NEWS
SCIENCE-RESOURCES
SWISS-PROT
YEAST-GENETICS

BITNET abbreviations
established:

BBOARD NAME               BITNET/EARN Name
----e---e--                       a-----F-----m---
AGEING                      AGEING
BIONEWS                     BIONEWS
BIOTECH                     BIOTECH
BIO-CONVERSION              BIO-CONV
BIO-MATRIX                  BIOMATRX
CONTRIBUTED-SOFTWARE         SOFT-CON
EMBL-DATABANK                EMBL-DB
EMPLOYMENT                  BIOJOBS
GENBANK-BB                  GENBANKB
GENE-EXPRESSION              GENE-EXP
GENOMIC-ORGANIZATION         GENE-ORG
METHODS-AND-REAGENTS         METHODS
MOLECULAR-EVOLUTION          MOL-EVOL
ONCOGENES                   ONCOGENE
PC-COMMUNICATIONS            SOFT-COM
PC-SOFTWARE                 SOFT-PC
PIR                          PIR-BB

PLANT-MOLECULAR-BIOLOGY

PLANT

TOPIC
--e-s

Scientific Interest Group
General announcements
Biotechnology issues
Scientific Interest Group
Applications of computers to biological databases
Information on molecular biology programs

contributed to the public domain
Messages to and from the EMBL database staff
Job opportunities
Messages to and from the GenBank database staff
Scientific Interest Group
Scientific Interest Group
Requests for information and lab reagents
Scientific Interest Group
Scientific Interest Group
Information on communications software
Information on PC-software for scientists
Messages to and from the PIR database staff
Scientific Interest Group
Scientific Interest Group
Research news of interest to the community
Information about funding agencies, etc.
Messages to and from the SWISS-PROT database staff
Scientific Interest Group

(<= 8 characters)   for  each bboard have been


PROTEIN-ANALYSIS             PROTEINS
RESEARCH-NEWS                RESEARCH
SCIENCE-RESOURCES            SCI-RES
SWISS-PROT                  SWISSPRT
YEAST-GENETICS               YEAST

Note: For the database bboards addresses such as PIR, EMBL, etc., were
avoided since these may conflict with other BITNET addresses at the
EMBL, etc.,  if they join the distribution scheme.

Equivalences of the Unix USENET newsgroup names to the ARPANET mailing
list names follow:

bionet.general   ................... ..BIONEW S
bionet.jobs   ...................... ..EMPLOYMEN T
bionet.technology.general   ........ ..BIOTEC H
bionet.technology.conversion   ..... ..BIO-CONVERSIO N
bionet.molbio.news   ............... ..RESEARCH-NEW S
bionet.molbio.ageing   ............. ..AGEIN G
bionet.molbio.bio-matrix   ......... ..BIO-MATRI X
bionet.molbio.methds-reagnts   ..... ..METHODS-AND-REAGANT S
bionet.molbio.genbank   ............ ..GENBANK-B B
bionet.molbio.embldatabank   ....... ..EMBL-DATABAN K
bionet.molbio.pir   ................ ..PI R
bionet.molbio.evolution   .......... ..MOLECULAR-EVOLUTIO N
bionet.molbio.gene-express   ....... ..GENE-EXPRESS10 N
bionet.molbio.gene-org   ............ .GENOMIC-ORGANIZATION
bionet.molbio.oncogenes   .......... ..ONCOGENE S
bionet.molbio.plant   .............. ..PLANT-GENETIC S
bionet.molbio.proteins   ........... ..PROTEIN-ANALYSI S
bionet.molbio.swiss-prot   ......... ..SWISS-PRO T
bionet.molbio.yeast   .............. ..YEAST-GENETIC S
bionet.sci-resources   ............. ..SCIENCE-RESOURCE S
bionet.software.pc   ............... ..PC-SOFTWA RE
bionet.software.pc.comm   .......... ..PC-COMMUNICATIO N
bionet.software.contrib   .......... ..C~NTRIBUTED-SOFTWAR E

BIOSCI Nodes
-------a----

Information about the BIOSCI bboard network can be obtained by mailing
to the address
                      biosci@xxxx

where   xxxx  can be any of the following node addresses, e.g.,
biosci@uk.ac.daresbury in  the United Kingdom.
outside of Europe and North                 Interested parties
                    America should contact whichever node is
most convenient.  Messages can be posted directly to bboards at any of
these nodes by using the address format

bboard@xxxx

where bboard is a name from the lists above and xxxx is from the   node
list below,   e.g.,  bionews@umdc.bitnet Note that   nodes  listed as
(Internet) sites utilize  the long bboard names as indicated in the
first list above and  nodes listed as BITNET,  EARN, or JANET use   the
BITNET abbreviated bboard names.

Europe                              North America
-------------------------------------      ----------------------------------
Sweden     bmc.uu.se      (Internet)    net.bio.net (Internet)
UK      uk.ac.daresbury (JANET)
Ireland    irlearn.bitnet           bionet-20.bio.net (Internet)
                   (BITNET/EARN) umdc.bitnet
Ireland    irlearn.ucd.ie                    (coming soon, BITNET)
                   (Internet,
      but uses BITNET bboard names)


P41REO1685-06

VI. BIONET Training Schedules
The schedules for the five Mountain View Trainings are included.

72


Training Schedule

March 17 & 18,1988

Thursday, March 17

Time      Topic
9:00- 9:45    Introduction
9:45-10:15   Sequence Entry
10:15-10:30   Break
10:30-l 2:00  Sequencing Project Management
12:00- 1 :oo   Lunch
1 :oo- 1:20    Cloner Demonstration
1:30- 2:oo   Sequence Alignment
2:00- 3:15   DNA Sequence Analysis
3:15- 3:30    Break
3:30- 4:45   Peptide Sequence Analysis

Friday, March 18

Time
9:00- 10:30

10:30-l 0:45
10:30-l 2:oo

12:00- 1 :oo
1 :oo- 3:oo
3:00- 3115
3:15- 4:oo

instructor
Nancy Bigham




Vicki Johncox


Constance Gensch
David Kristoff erson



Spencer Yeh

Instructor
Constance Gertsch

Topic
Database Structure
and Simple Searches
Break
Finding Sequences
in the Databases
Lunch
Sequence Alignment
Break
Electronic mail and bulletin boards


An Introduction to the IntelliGenetics Suite
       Course Schedule
     May 19 and 20,1988

Thursday, May 19

    8:30 9:00   Introduction
          Overview of the IntelliGenetics Programs

     9:oo   9:45  Sequence Data Entry and Editing

   9:45 lo:30 System commands

    lo:30 lo:45 Break

Nancy Bigham

    10:45 l2:OO

    12:oo l:oo

    1 :oo   2:15

    2:15   2:30

     2:30   3145

     3:45   5:oo


Friday, May 20

    8:00 9:00

     9:oo 1030

10:30 10:45

10:45 12:oo



12:oo l:oo

1:OO 3:15

3:15 3:30

3:30 4:30

Sequencing Gel Management

Lunch

Nucleic Acid Sequence Analysis

Break

Peptide Sequence Analysis

Electronic Mail and Bulletin Boards
File Transfer

Open Classroom

Database Structure
and Sequence Retrieval

Break

Collecting related sequences,
searching for patterns

Lunch

Sequence Similarity Searches

Break

Review and Questions

Vicki Johncox

Trish Benton

Spencer Yeh

Dave Kristofferson

Consultants

Beth Swank

Sunil Maulik

Nancy Bigham


An Introduction to the IntelIiGenetics Suite
           Course Schedule

July 14 and 14 1988

Thursday, July 19

8:30

8:45

9:oo

9:45

10:30

10:45

12:oo

1 :oo

2:oo

3:15

3:30

8:45

9:oo

9:45

10:30

10:45

12:oo

l:oo

2:oo

3:15

3:30

5:oo

Introduction
Overview of IG Programs

Vickie Johncox

BIONET Resource          Dave Kristofferson

Operating System.
File Structure

Vickie Johncox

Electronic Mail

Kathy Berg

Break

Sequence Entry

Vickie Johncox

Lunch

Sequencing Gel
Management

Spencer Yeh

NA and Peptide Sequence Analysis

Break

Problems


Friday, July 20

8:00   9:oo

Open Classroom

Consultants

9:oo

10:30

Database Structure
and Sequence Retrieval

Vickie Johncox

10:30 10:45

Break

10:45 12:oo

Collecting Sequences.
Pattern Searching

12:oo l:oo

l:oo   3:15

Lunch

Sequence Similarity
Searches

Sunil Maulik

3:15   3:30     Break

3:30

4:30

Review and Questions

Vickie Johncox


An Introduction to the IntelIiGenetics Suite
       Course Schedule
   September 15 and 16 1988

Thursday, September 15

8:30  8:45



8:45  9:oo



9:oo  9:30



9:30  lo:oo


lo:oo  10:15



10:15  10:30


10:30  11:15



11:15  12:oo



12:oo l:oo


l:oo  3:15



3:15  3:30


3:30 5:oo

Introduction

Introduction to
BIONET

Operating System,
File  S true ture

Electronic Mail

Organization of
IntelliGenetics

Break

Sequence Data Entry
and Editing

Sequencing Gel
Management

Lunch

NA and Peptide
Sequence Analysis

Break

Problems

Vickie Johncox

Dave Kristofferson

Trish Benton

Kathy Berg

Murray Summers

Karen Davis

Trish Benton

Karen Davis


Friday, September

   8:OO 9:oo


   9:oo 10:30

10:30 10:45


10:45 12:oo

12:oo l:oo


l:oo 3:15

3:15 3:30


3:30 4:oo


4:oo 4:15

16

Open Classroom

Consultants

Database Structure
and Sequence Retrieval

Trish Benton

Break

Collecting Sequences,
Pattern Searching

Lunch

Sequence Similarity
Searches

Break

Problems

Review and Questions         Vickie Johncox

Sunil Maulik


An Introduction to the IntelliGenetics Suite and BIONET
           Course Schedule
       November 17 and 18,1988

Thursday, November 17

8:30

9:oo

9:15



9%

10:15


10:30



11:15



12:oo

l:oo

1:30



3:30

3:45






5:oo

9:oo

935

9:45

Introduction

Introduction to BIONET

Operating System,
File Structure

10:15

10:30

11:15

Electronic Mail

BREAK

12:oo

1:oo

1:30

3:30

Sequence Data Entry
and Editing

Sequencing Gel
Management ,

LUNCH

3:45

500

Problems

NA and Peptide
Sequence Analysis

BREAK

Problems

6:30     BREAK

6:30     9:oo    BIONET-specifii topics            BIONET staff

Vickie Johncox

Dave Kristofferson

Vickie Johncox

Karen Davis


An Introduction to the IntelliGenetics Suite and BIONET
           Course Schedule
       November 17 and 18,1988

Friday, November 18
    8:00     9:oo

    9:oo     10:30

10:30     10:45

10:45    12:oo

12:oo     1 :oo


l:oo     3:oo

3:oo     3:15

3:15     3145

3:45     4:oo

Open Classroom

Database Structure
and Sequence Retrieval

BREAK

Collecting Sequences,
Pattern Searching

LUNCH

Sequence Similarity
Searches

BREAK

Problems

Review and Questions

4:oo

coo    BIONET-specific topics

Consultants

Karen Davis

Karen Davis

Vickie Johncox

Vickie Johncox

BIONET staff


Introduction to BIONET
Thursday Evening Schedule
  November 17,1988

630

7:15

Connecting to BIONET,
File Transfers

David Kristoff erson

7:15

7:45

TOPS-20 Operating System,
EMACS Text Editor

Karen Davis

7:45     8:00     BREAK

8:00

8:30

Bulletin Boards, E-mail
Addresses, Help Me

8:30

9:oo

Other BIONET Software          Spencer Yeh

Introduction to BIONET
Friday Afternoon Schedule
  November 18,1988

4:oo     5:oo     FASTA-MAIL

Karen Davis

SpencerYeh


P41R.R01685-06

VII. BIONET Computer Facilities
A diagram of the BIONET computer facilities follows on the next page.

73


I W-B-.

I  z :
:z;
'6,
: G'
:c;:
Iti'
I    'I

I
I z :

I E :
:c
----I


P41R.R.O1685-06

VIII. Testimonials

Copies of two recently received testimonial letters are included in this section.

74


HARVARD MEDICAL SCHOOL

DEPARTMENT OF BIOLOGICAL CHEMISTRY
   AND MOLECULAR PHARMACOLOGY

Tel. (617) 732- 2046
    Fax: 7384516
   Internet: Hinh@BIONET-20.bio.net

25 Shattuck Street
Boston. Massachusetts 02115

Jay Hirsh
Assoc. Professor
260 Longwood Ave.

October 21,1988

Re: Statement in support of NIH grant to the BIONET computer facility.

To whom it may concern:

We have been members of the BIONET computer facility for approximately 3 years. We have found
this facility to be of great importance to our research. We make extensive use of the homology
searching and sequence analysis routines, and are just beginnning to take full advantage of the database
searching routines. These analyses have uncovered a number of promoter elements of the Drosophila
dopa decarboxylase gene (D&z) that am conserved through evolution and are functionally important
((Scholnick et al (1986) Science 234,998-1002; Bray & Hirsh (1986) EMBO J., 5,2305-2312;
Bray et al (1988) EMBO J.,7,177-188.). We enclose a copy of this last reference. Even though it
did not utilize directly the Bionet resource, this manuscript shows directly that one of the initially
identified conserved elements is a CNS-specific regulatory element.

We have recently extended this analysis to a cell-specfic enhancer of Ddc (Johnson, McCormick, Bray
& Hirsh, in preparation), an -800 bp segment that contains a number of functional elements. This
approach is also proving to be valid in this region, in that a number of conserved elements are showing
interesting in vivo functions in regulating different aspects of the neuronal pattern of expression of Ddc,

Within the past year, we have also begun to make extensive use of the Bionet E-mail facility. We now
routinely communicate with European and American colleagues using this system Given the cost of
international phone calls, our use of this facility alone would pretty much justify the yearly bionet fee.

1


We continue to use this facility, even though there is presently a local computer facility claiming to offer
comparable services at a comparable cost. This decision is due to the superb level of support that we
have derived from the Bionet staffi Questions regarding operation of the system are answered expertly
and promptly. The programs and program manuals have been continually evolving, such that the
programs are now accessible to even novice users in the lab. When I have made queries-such as
wondering whether database searches couldn't be done without hanging on the phone for hours- I have
been surprised and amazed that the staff have gone to what appear to be major efforts to implement
such searching programs. Our experiences with the aformentioned local computer facility, where the
level of staff support was almost nill, have shown the value of such services.

In summary, I hope that NIH will continue to support the Bionet resource. This program has attracted
a dedicated and skilled staff that provides services and support that we have not been able to find
elsewhere. An interruption in the funding of this program would certainly cause disruption to a large
number of users who are highly dependent on these people and this system

Sincerely,

2


THE JOHNS HOPKINS UNIVERSITY

      BALTIMORE, MARYLAND 21218

DEPARTMENT OF BIOLOGY

Noverrber 28, 1988

Dr. David Kristofferson
BIONET
c/o Intelligenetics
700 E. El Camino Road
Mountain View, CA 94040

Dear Dave,

   I am addressing this letter to you personally because I
dislike the "To Whom It May Concern" format - it makes me feel
as though I am writing to an answering machine.  Please feel free
however to show the contents to anyone.

   There are several statements that I would like to make about
BIONET, its services, its staff and the Intelligenetics suite of
DNA and protein sequence analysis software that they offer.

1. BIONET offers a level of support for the researcher that
cannot be duplicated by many universities or research centers.

   While DNA/protein sequence analysis software can be
put on the university or medical school mainframe computer
or run on personal computers, the problem has always been
w: training people in fundamentals (try to find
understandable documentation on some of these programs) and
then teaching them the significance of the variables involved
and the limitations of the algorithms used. BIONET has made
a considerable effort to address these problems.  They offer
affordable training at Mountain View, California and are
willing to come to you if they have staff and time.  They
have made a real attempt to write complete and understandable
and useful documentation for the system and the programs.   Most
important is their online help and on-call systems experts who
answer immediate questions about programs and data.  It is
difficult for a systems operator at a university computer
center to be well-versed on all the software running on the
machine.  Even if they are, they need the time to explain
the programs.  These overworked systems operatqrs must rely
on local experts or bright students to help people with questions
about individual programs.  Molecular biologists need easily
accessible, experienced professionals to answer their questions.
BIONET offers those professionals.

2. The BIONET staff are highly competent and extremely
cooperative. I have nothing but praise for the personnel at
BIONET and the service and support they offer. Over the last
year, I have received advice (good advice, by the way), had
problems solved, and talked about on-site training.  Everyone
who dealt with me was knowledgeable, professional and very
helpful. I really appreciate having these people on call to
answer my questions
and skill.      , which they do with remarkable rapidity


BIONET, page 2

3. The systems analysts and programmers at BIONET listen to
the users.  Several times I have had questions about the
running of a program or suggestions about format or documentation
and the people at BIONET always listened and corrected the
program's problem or explained to me my problem.  They are
always improving the system and it is wonderful never to be
stuck with a "quirk" in the program.

4. Access to BIONET is very easy for the computer novice.
As the computer "expert" in Dr. Roseman's laboratory, I find
that people are very cautious about accessing the mainframe
computer on campus.  Mistakes cost money and since the
system is not dedicated to one suite of software, the interface
has to be more complicated.  Most people don't want to learn
DOS for the PC let alone a whole new operating system for
the Vax or the IBM.  BIONET allows the novice easy access
while retaining all the system commands for the more expert
or more daring among us.  People in my laboratory are connecting
to the BIONET system with ease and learning as they work. This
is an immense time-saver for me since the online help keeps
them from calling my name every five minutes and distracting
me from my own research.

5. BIONET provides communication lines between scientists
that are easily used and therefore frequently used.   For a
while BIONET was the only large scale bulletin board system
for molecular biologists.  Now more and more communications
lines are open, but the problem is again getting people to
use them.  Some of them require Bitnet or Arpanet, etc. and
that requires people to access their mainframe computers and
learn how to run electronic mail systems that are not as
user-friendly as the BIONET system.  BIONET is now acting as
a gateway to a number of these systems and I for one, am
grateful that I can hear the news, or send the news without
running one more program.

  I am very pleased with EIONET and the Intelligenetics programs.
They have enabled my colleagues and me to further our work in
molecular biology by providing the sequence analysis software in
a form that we could learn and understand. I believe that BIONET
provides necessary and unique services for the research scientist.

Very truly yours,
,.iG y7I-Y 7c 4 -7 I /'. J$-
Donna K. Fox, Ph.D.'
Associate Research Scientist