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Summary: Smart Race cDNA amplification kit (Clontech). Expressed sequence tags were amplified
by PCR with the universal adapter primer provided with the kit and the various, specific
internal primers.
Complete macronuclear gene-sized chromosomes
Telomere-specific primers in combination with internal gene sequences allow a
straightforward recovery of the complete gene30
. The specific (internal) primers were
based on the DNA sequences of internal fragments of the various genes, which were
recovered previously by PCR with degenerated primers for conserved parts of the various
genes.
Phylogenetic analysis
Protein sequences were aligned with ClustalW and Muscle; unequivocally aligned
positions were selected with Gblocks or manually. Phylogenies were inferred with
maximum likelihood by using a discrete gamma-distribution model with four rate
categories plus invariant positions and the Poisson amino acid similarity matrix, and
neighbour joining as implemented in ClustalW, correcting for multiple substitutions with
the Gonnet amino acids identity matrix, and bootstrapping with 100 samples.
ORFs with a lower size limit of 100 nucleotides were identified with ORF Finder
(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). tRNAs were identified with tRNAscan-SE
(http://www.genetics.wustl.edu/eddy/tRNAscan-SE). Potential mitochondrial import
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