To determine the relationship of PCN to other Class III HD ZIPs, a phylogenetic analysis of Class III HD ZIP sequences from whole-genome sequencing projects was undertaken (Materials and Methods).
In particular, exon/intron arrangements of ZIP gene sequences encoding for protein sequences upstream of the PL domain are most diverse and may be encoded by a single exon (e.g. mouse ZIP10) or multiple exons with non-conserved exon/intron boundaries across ortholog ZIP sequences.
To firmly establish which model, if either, is correct, additional Prnp and/or Sprn ortholog sequences from early chordates will need to be uncovered and compared to ZIP sequences.
A phylogeny with eight species, including published HD-Zip sequences from maize, poplar and cucumber, was largely congruent with the phylogeny generated using five species.
The probable HD-Zip sequences were re-aligned to the profile HMM using hmmalign, available in the tool HMMER, and were viewed in SeaView.
When cells traveling in the bloodstream find their ZIP code sequence is recognized by the cells lining a particular region of the blood vessel system, they can probably squeeze through the blood vessel wall and into the surrounding tissue.
The downstream LDR primer contains a fluorescent tag that becomes covalently joined to the oligonucleotide containing the zip code sequence if ligation is successful.
Four different upstream primers, each containing a unique zip code sequence and a different base at their 3' ends are used to screen for substitution mutations at each nucleotide position.
This deletion corresponds to 84 amino acids of ZIPCO including the entire 4th trans-membrane domain, the ZIP signature sequence and conserved histidine and glycine residues, and part of 5th trans-membrane domain.
A 540 bp-long internal fragment of ZIPCO encoding the fourth TM region, including the ZIP signature sequence and part of the fifth TM region of the protein, was deleted and replaced by the hDHFR selectable cassette (Fig 2A).
