For this analysis, pathway position was defined as the number of steps required to transduce the signal from the most upstream element (Grb2, position 0), to each of the pathway components (with proteins RSK1 3 sharing position 6, the most downstream position of the core pathway; Figure 2A; Additional file 1: Table S5).
The CDS boundaries were determined as follows: the upstream CDS boundary was selected by choosing the most upstream start codon position from the set of isoforms; the downstream CDS boundary was selected by choosing the most upstream stop codon position from the set of isoforms that map to the gene (if more than one authentic stop codon exists, the most upstream stop codon position is chosen.
The region between the most upstream TSS (corresponding to position -221) and the first ATG, and the region between the first and the second in-frame ATGs, both, alone and even stronger in combination, are able to drive the expression of a reporter gene in promoter-less vectors.
The most surprising result was the upstream position of Hh signaling in the head because this differs from the Drosophila situation, in which ocular wg expression is unaffected in hh mutants, while the hh stripe is slightly altered in wg mutants (Gallitano-Mendel and Finkelstein, 1997).
All genes share 3 identical intron positions within the 5' portion of the gene and, as already observed [ 8, 39], the positions of the two most upstream introns are also shared by the NF genes.
The altered patterns of starts observed in most upstream or downstream shifting start site mutants appear to be stereotypical to each class, meaning the positions of starts that are more likely used at ADH1 in mutants are the same within each mutant class as defined by upstream or downstream shifting, not by whether they are in GTF subunits or the Pol II active site.
Although the correlation was not absolute and most likely depended on each specific mRNA sequence (e.g. internal polyA-stretches), there was a clear correlation between the upstream position of the probe and the length-distribution of the cDNA.
So what is the most upstream regulator?
Pathway position was measured following Ramsay et al.'s (2009) pathway pleiotropic index, in which groups of enzymes are numbered relative to pathway branch points from most upstream to most downstream.
