Although the insertion does not interrupt the reading frame, the remainder of the sequence that is alignable with oleander is highly divergent.
Introns may interrupt the reading frame of a gene between two consecutive codons (phase 0 introns), between the first and second nucleotide of a codon (phase 1 introns), or between the second and the third nucleotide (phase 2 introns) [ 40].
Among the 52 mutations, 31 (61%) were missense or inframe deletions, and the remaining 21 (39%) were predicted to interrupt the reading frame (nine stop mutations, six splice junction mutations, and six frameshift deletion or insertion mutations).
In contrast to SNPs, the effects of 1 and 2 bp indels are more predictable, as the 241 indels found in CDS interrupt the reading frame and protein sequence while the 35 indels identified in splice sites may lead to intron retention and the 4 indels found to eliminate termination codons potentially produce a longer protein.
The second line of evidence relates to intron position: (1) in all cases, the introns are in phase 0. They therefore interrupt the reading frame but not the last codon of the first exon at the donor splice site; (2) the intron sequences interrupt the coding sequences at an almost fixed position, close to codon 55, within the first internal loop defined by the seven transmembrane domains.
A total of 1,063 spliceosomal introns were initially predicted to interrupt the reading frames of 947 protein-coding genes (847 genes have a single intron).
The changed nucleotides could interrupt the original reading frame if they are not multiples of 3 base pairs.
Pseudogenes are often detected during manual curation of these gene predictions, because the gene model cannot be modeled consistently with homologous protein alignments due to sequence degeneracy that results in stop codons that interrupt the open reading frame.
However, the deletion of exon 13 does not interrupt the open reading frame and there are no indels or point mutations present elsewhere in the coding region, that would lead to frameshifts or premature stop codons.
However, deletions interrupting the reading frame and leaving an open-reading frame of only 540 bp in V. album strongly suggest that the gene is nonfunctional.
