I have just sent the IBM-PC program RAMHA to the following archives:
EMBL: embl-heidelberg.de (uuencoded self-extracting LHARC file)
Indiana: ftp.bio.indiana.edu (self-extracting LHARC file + readme file)
U.Houston: evolution.bchs.uh.edu (uuencoded self-extracting LHARC file)
RAMHA: Monte Carlo simulation of the random mutagenesis of synthetic cDNAs
David P. Siderovski
Department of Medical Biophysics, University of Toronto,
Toronto, Ontario M4X 1K9 CANADA
INTERNET: siderovski at galen.oci.utoronto.ca
RAMHA version 1.0 is provided as a self-extracting LHARC archive
(RAMHA1.EXE) containing the simulation program, all associated support files,
and a user's manual. To extract the files, just type RAMHA1 at the DOS prompt.
Random mutagenesis is a powerful tool in protein structure-function analysis.
One approach is the synthesis of cDNA sequences using doped nucleoside
phosphoramidites. A Turbo PASCAL program, RAMHA, was written for
modelling this method of random cDNA mutagenesis on PC-compatible computers.
Editing facilities allow the target cDNA to be entered directly as nucleotide
sequence or by back-translation of the encoded polypeptide, as entered using
one-letter amino acid abbreviations; back-translation is based on pre-defined
codon frequency tables supplied for several common research organisms or on
user-defined tables as created within RAMHA. Sequences can also be
imported from and exported to GCG sequence file format.
After a target cDNA has been entered or imported into RAMHA, the user can
directly simulate the random mutagenesis of this sequence.
Several parameters controlling the mutagenesis simulation can
be specified, including nucleotide doping level, number of iterations of the
simulation algorithm, and sequence(s) of enzyme site(s) to be detected within
"synthesized" oligos. The user can also test a range of nucleotide doping
levels using batch execution.
RAMHA will then simulate the synthesis of a user-specified number of
oligos, translate each into polypeptides, and accumulate various statistics,
including average percent similarity of mutant proteins to wildtype sequence,
frequency of resultant proteins retaining wildtype sequence, and percent of
premature open-reading frame terminations. In addition, RAMHA can scan
"synthesized" genes for new internal restriction sites arising from nucleotide
substitution and report on "hotspots" within the wildtype cDNA sequence.
By accumulating data on the number and position of stop codons (TAA, TAG, TGA)
arising in "synthesized" oligonucleotides, RAMHA can report on the
frequency of premature open-reading frame terminations & the number of mutant
proteins truncated before the end of a critical domain. Tests on a cDNA
containing all possible codons have revealed the requirement
for two distinct "anti-termination" strategies, schemes circumventing the
random introduction of stop codons (TAG, TGA, TAA), which have been added to