silent mutagenesis

Duke Groebe drg at prophet.pharm.pitt.edu
Fri Nov 12 08:42:58 EST 1993


In article <9311101526.AA06935 at net.bio.net>,
RGB12955%USA.decnet at USAV01.GLAXO.COM ("USA::RGB12955") wrote:
> 
> In a recently received NEB Transcript, Raj Shankarappa refered to an in press
> method to use PCR to introduce silent mutations.  Raj, if you are still out thercould you email the protocol to me as a postdoc in my lab is about to embark
> on a project to synthesize a cassetable gene?  I am not on methods and reagents
> list anymore, so could you send the copy to me at RGB12955 at usav01.glaxo.com ?
> 
> Thanks,
> Rich Buckholz



It sounds like you're looking for a computer program called SILMUT.  This
program is referred to in two articles published in BioTechniqes
(Shankarappa et al., 1992, vol. 12, #3, p. 382 and Shankarappa et al.,
1992, vol 12, #6, p. 882).  You can get SILMUT by anonymous ftp to
ftp.bio.indiana.edu in the molbio/ibmpc directory.  A Gopher server can do
essentially the same thing.

Here's the READ.ME file for SILMUT:

""""
SILMUT and TABLE programs in this package help you to identify
regions in a sequence which can be altered to introduce restriction
enzyme sites and other sequences by silent mutations.  The work is
based on our following publications.

1.  B. Shankarappa, D.A. Sirko, and G.D. Ehrlich.  A General Method
for the Identification of Regions Suitable for Site-Directed Silent
Mutagenesis.  BioTechniques 12, No. (3): 382-384

2.  B. Shankarappa, K. Vijayananda, and G.D. Ehrlich.  SILMUT: A
Computer program for the Identification of Regions Suitable for Silent
Mutagenesis to Introduce Restriction Enzyme Recognition Sequences.
BioTechniques 12, No. (6): 882-884.

If you have any questions, please contact one of us at the
following address.

1.  Dr. Raj Shankarappa,  bsh at med.pitt.edu
Research Associate,
University of Pittsburgh,
Department of Pathology,
Pittsburgh, PA 15261
email:  bsh at med.pitt.edu
phone: (412) 648-1986 or 648-9763 or 648-9026
fax (412) 648-1916

OR

2.  Mr. Vijayananda, vijay at litsun.epfl.ch
Office: EPFL - LIT
EL - ECUBLENS
CH-1015  lAUSANNE    
SWITZERLAND
+41-21-693.47.03

Residence:
K. Vijayananda
16 Ch. du martinet
ch-1007 Lausanne
Switzerland
+41-21-24.03.00

We would like to wish you good luck in your research efforts and
hope these programs have been helpful to you.

====================================================================

Please read the following if you have questions about using the
program SILMUT and TABLE

The disk contains the following files.

1.  Silmut.c:  documented program of silmut.exe
2.  Silmut.exe:executable file compiled by C compiler
3.  Table.c:   documented program of table.exe
4.  Table.exe: executable file compiled by C compiler
5.  Dbase1:    file containing single letter amino acid codes and
               the corresponding codons.
6.  Dbase2:    file containing the common 30 recognition sites for
               restriction enzymes.
7.  gpgr.in:   Input file containing the nucleic acid sequence of
               the conserved GPGR amino acid motif of HIV-1 virus.
8.  gpgr.out : Actual output file generated by silmut.exe for the
               input in gpgr.in file.


If you intend to change any files to suit your particular use, we
suggest you retain a copy of the above files as a precaution.

Silmut program identifies the potential for silent mutagenesis to
introduce any 6-base sequence such as restriction enzyme sites.

Table program provides the sets of amino acid motifs obtained by
translation of any 6-base sequence in three reading frames"

The Silmut and Table programs have been written in C language and
can be used in any IBM compatible computers.  The programs can also
be run in UNIX or other systems that support the C compiler.


Silmut program identifies the silent mutation potential for the
introduction of 6-base restriction enzyme sequences by translating
the RE recognition sequence in three reading frames to obtain a set
of amino acid motifs and finding a match for the amino acid motif
with the amino acid sequence provided as the input.  Thirty
selected restriction enzymes which do not contain degeneracy in
their recognition sites have been provided in file dbase2.  This
file can be edited in a DOS text editor to remove any restriction
enzyme sequences from being recognized or add new 6-base sequences.

The maximum number of restriction enzyme sites that can be included
in this file is 100. 


The program is invoked by typing SILMUT whereby you will have the
option to chose the analysis of a nucleic acid sequence; or the
amino acid sequence; or exiting the program.  The default modes for
input and output are screen.


If you choose to analyze a sequence stored as a file, that file
must be in a DOS format.  The file should contain 1 (for AA) or 2
(for NA) in the first line, sequence of nucleic/amino acid in the
second line and 3 (for exiting) in the third line [for an example
please see the file gpgr.in].  We recommend that you use a DOS
editor like Q.EXE which can handle lines lengths of 500 or more
characters.  If you use other programs like WordPerfectTM, which
usually handle about 52 characters in a line, you may have to split
the sequence into separate overalpping lines.  Also make sure you
save the file in a DOS format.  The proper syntax for evaluating a
sequence in a file is as follows.

silmut -i file1 -o file2

Here the input from file1 is analyzed and the output is directed to
file2.

Different combinations of input and output from or to the screen
can be used by omitting appropriate parameters following the
command silmut.  eg., Silmut -i file1 will obtain the information
from file1 and output is directed to the screen.  Similarly, 
silmut -o file2 will take the input from screen and direct the
output to a file called file2.  


Table program provides a table/listing of amino acid motifs
obtained by translating a 6-base sequence in three reading frames.
Translation in the first reading frame yields a sequence of two
amino acids, while translation in second and third reading frames
will yield degeneracies in the first and third position.  Each of
these amino acid motifs is separated in the output for clarity. 
Due to longer lines, the end of the line might be wrapped onto the
next line.  You may correct for this by editing and printing the
file using a word processing program after setting a wider margins
or setting smaller sized letters.


The default inputs are the files dbase1 and dbase2, for translation
of recognition sequences of 30 commonly used restriction enzymes.
If you want to change the codon specificities, you have to edit the
file dbase1.  If you want to add or delete any restriction enzyme
sites, or any other 6-base sites like splice sites etc., you need
to edit the file dbase2.  This editing can be done in a DOS
environment using any of the softwares like Q.exe.  If you use
other softwares like WordPerfect, make sure the file is saved as a
DOS text file.  Also, make sure that the spacing and other format
of the files dbase1 and dbase2 are retained as before.


The default output is the screen.  If you chose the output to be
directed to a file the proper syntax is 

table output.tab

where output tab is the file you want the output to be directed.
If you have any questions, please contact either of us at the
following address.

1.  Dr. Raj Shankarappa,
Research Associate,
University of Pittsburgh,
Department of Pathology,
Pittsburgh, PA 15261
email:  bsh at med.pitt.edu
phone: (412) 648-1986 or 648-9763 or 648-9026
fax (412) 648-1916

OR

2.  Mr. Vijayananda, vijay at litsun.epfl.ch
Office: EPFL - LIT
EL - ECUBLENS
CH-1015  lAUSANNE    
SWITZERLAND
+41-21-693.47.03

Residence:
K. Vijayananda
16 Ch. du martinet
ch-1007 Lausanne
Switzerland
+41-21-24.03.00

We would like to wish you good luck in your research efforts and
hope these programs have been helpful to you.

If you find any bugs with this program, we will be thankful if you
can inform us.  We have an intention of improving the program in
the near future. 

Following is the output from TABLE program for 30 selected RE
sites.  Please adujust the margins so that all the data is in one
line.  The order of the contents of the following table is RE,
Recognition site, 1st and 2nd AA obtained in the first reading
frame, 1,2,3rd AA obtained from 2nd reading frame, and 1,2,3rd AA
obtained from the third reading frame.  


(PLEASE SET THE MARGINS WIDE ENOUGH TO ACCOMODATE THE WRAPPED LINE)


HindIII                                      AAGCTT  K L  XQKE  A
FLSYXCW LSXPQRITKVAEG   S FL
MluI                                         ACGCGT  T R  YHND  A
FLSYXCW LSXPQRITKVAEG   R V
SpeI                                         ACTAGT  T S  YHND  X
FLSYXCW LSXPQRITKVAEG   L V
BglII                                        AGATCT  R S  XQKE  I
FLSYXCW LSXPQRITKVAEG   D L
StuI                                         AGGCCT  R P  XQKE  A
FLSYXCW LSXPQRITKVAEG   G L
BanIII/BspXI/ClaI                            ATCGAT  I D  YHND  R
FLSYXCW LSXPQRITKVAEG   S IM
Cfr6I/PvuII                                  CAGCTG  Q L  SPTA  A
VADEG   FSYCLPHRITNVADG S CXW
NdeI                                         CATATG  H M  SPTA  Y
VADEG   FSYCLPHRITNVADG I CXW
NcoI                                         CCATGG  P W  SPTA  M
VADEG   FSYCLPHRITNVADG H G
Cfr9I/SmaI/XmaI                              CCCGGG  P G  SPTA  R
VADEG   FSYCLPHRITNVADG P G
SacII                                        CCGCGG  P R  SPTA  A
VADEG   FSYCLPHRITNVADG R G
PvuI/RshI/XorII                              CGATCG  R S  SPTA  I
VADEG   FSYCLPHRITNVADG D R
EagI/XmaIII                                  CGGCCG  R P  SPTA  A
VADEG   FSYCLPHRITNVADG G R
BsuMI/PaeR7I/XhoI                            CTCGAG  L E  SPTA  R
VADEG   FSYCLPHRITNVADG S SR
PstI/SflI                                    CTGCAG  L Q  SPTA  A
VADEG   FSYCLPHRITNVADG C SR
EcoRI/RsrI/Sso47I                            GAATTC  E F  XRG   I
LPHQR   LSXWPQRMTKVAEG  N S
SacI/SstI                                    GAGCTC  E L  XRG   A
LPHQR   LSXWPQRMTKVAEG  S S
EcoRV                                        GATATC  D I  XRG   Y
LPHQR   LSXWPQRMTKVAEG  I S
SphI                                         GCATGC  A C  CRSG  M
LPHQR   LSXWPQRMTKVAEG  H A
NaeI                                         GCCGGC  A G  CRSG  R
LPHQR   LSXWPQRMTKVAEG  P A
NheI                                         GCTAGC  A S  CRSG  X
LPHQR   LSXWPQRMTKVAEG  L A
BamFI/BamHI/BamKI/BamNI/BstI/Bst1503I        GGATCC  G S  WRG   I
LPHQR   LSXWPQRMTKVAEG  D P
NarI                                         GGCGCC  G A  WRG   R
LPHQR   LSXWPQRMTKVAEG  A P
ApaI                                         GGGCCC  G P  WRG   A
LPHQR   LSXWPQRMTKVAEG  G P
Asp78I/KpnI                                  GGTACC  G T  WRG   Y
LPHQR   LSXWPQRMTKVAEG  V P
SalI                                         GTCGAC  V D  CRSG  R
LPHQR   LSXWPQRMTKVAEG  S T
ApaLI                                        GTGCAC  V H  CRSG  A
LPHQR   LSXWPQRMTKVAEG  C T
HpaI                                         GTTAAC  V N  CRSG  X
LPHQR   LSXWPQRMTKVAEG  L T
AccIII/BspMII                                TCCGGA  S G  FLIV  R
IMTNKSR FSYCLPHRITNVADG P DE
NruI/Sbo13I                                  TCGCGA  S R  FLIV  A
IMTNKSR FSYCLPHRITNVADG R DE
XbaI                                         TCTAGA  S R  FLIV  X
IMTNKSR FSYCLPHRITNVADG L DE
AtuCI/BclI/BstGI/CpeI                        TGATCA  X S  LMV   I
IMTNKSR FSYCLPHRITNVADG D HQ
BalI                                         TGGCCA  W P  LMV   A
IMTNKSR FSYCLPHRITNVADG G HQ
.

""""


Good luck,

Duke



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