The spontaneous mutation frequency was 1.0 X 10^-9 mutants per viable cell for StrR, < 4.66 X 10^-10 mutants/viable cell for StrD, and < 4.66 X 10^-10 mutants/viable cell for StrI as well. The StrR mutant phenotype had the highest frequency, which means that most of the mutations that occurred conferred resistance to streptomycin. Equal mutation frequencies for StrD and StrI were obtained. The fact that the mutation frequencies for StrD and StrI were less than 4.66 X 10^-10 mutants/viable cell means that while some StrD or StrI mutations may have occurred, such mutants exist in too few numbers to be visible to the naked eye.
Comparing the nucleotide sequence of the mutants to that of the wild type, it can be inferred that there is a missense base pair substitution because there is simply a replacement of adenine with cytosine at base pair position 129. This cannot be a silent mutation because silent mutations result in no change in phenotype, whereas a phenotypic difference is present in the mutants. Additionally, there is no insertion or deletion of nucleotides. This alone is sufficient to reject frameshift mutations, which are caused by insertions or deletions, as a hypothetical cause.
However, frameshift and nonsense mutations can also be eliminated based on the amino acid sequence as well as the mere presence of colonies. Because frameshift mutations change the downstream reading frame and nonsense mutations involve a premature stop codon, both types of mutations result in truncated or, in the case of frameshift mutations, altered proteins. This would be evident in the amino acid data in the form of a radical alteration in the sequence. This is not the case because only a single amino acid changed: lysine to asparagine. Even this distortion is far from dramatic: both amino acids are polar and hydrophilic. However, there is a key difference in that lysine is basic whereas asparagine is neutral. Furthermore, because both types of mutations alter amino acid sequences radically, they would thus radically alter the expression of the rpsL gene, resulting in a dysfunctional or absent 30S subunit of the bacterial ribosome. Since cells require functional ribosomes to exist at all, the presence of viable cells or colonies eliminates any possibility of frameshift or nonsense mutation.
The PCR product and DNA sequence were accurate because the absorption reading was 19.4 micrograms per millileter and the DNA was able to be successfully sequenced, with the identity of each nucleotide being clearly distinguishable. The amino acid sequence and conclusions drawn from it can be confirmed in the literature. Table 2: Selection of streptomycin-resistant rpsL mutations, which lists all amino acid changes that result in the StrR phenotype and confirms the change from lysine to asparagine as one such possibility (J Biol Chem, 2009). Furthermore, the StrD mutants result from a mutation in the 90th amino acid, from proline to arginine or lysine. Additionally, StrI (StrP in the table, for "streptomycin pseudo-dependent") results from a mutation in the same amino acid, but to glutamine. The numbering of amino acid positions in the sequence differs in the table due to counting the amino acids post-translationally, since bacteria remove methionine residues after alanine, glycine, serine, proline, valine, threonine or cysteine residues (Hayes, written communication).