Supernatants were centrifuged at 14,000× g for 15 min to clear cellular debris, and then were concentrated to ∼30 μl with Amicon 30 kDa filters (UFC503024, Millipore), then mixed with an equal volume of 2× sample buffer and heated at 95°C for 5 min. Duplicate 4 15% gradient mini-Protean TGX gels (456-1084, Biorad, Hercules, CA) were used to run samples.
The optimal primer annealing temperature (TM) was determined on BCBL-1 DNA extracts in duplicate with a temperature gradient from 55.0°C to 63.0°C on an iQ5 Real-Time PCR Detection System (Bio-Rad, Australia) using the QuantiFast SYBR Green PCR Kit (Qiagen, Australia).
Under the radial gradient, click Duplicate.
Furthermore, human gene duplicates exhibit a skewed gradient of distribution along the chromosomal length with significant clustering in pericentromeric regions.
As a result of manual annotation, approximately 50% of the predicted human and axolotl paralogs were discarded and 4 of the 1 1 orthologs were re-classified as duplicated axolotl loci (anterior gradient 2 homolog, integral membrane protein 2b, stress-associated endoplasmic reticulum protein 1, and parvalbumin).
32 Briefly, each gel band was analyzed in technical duplicate and separated over a gradient from 2%to3535% acetonitrile (0.1% formic acid) on a 100-μm × 100-mm C18BEH column (Waters, Milford, MA) running at 600 nL/min for 1 hour.
This nonrandom, pericentromeric gradient of duplications in the human genome has been noted by preceding studies of rodent paralogs [ 54], human gene duplicates on Chromosome 22 [ 55] as well as at a genome-wide scale [ 18, 49, 50, 56, 57].
Semi-quantitative RT-PCR experiments for validation of regulated mRNAs from polysome gradient fractions were performed in duplicate.
Because the strength of purifying selection depends on the number of duplicates (Lynch and Conery 2000; Jordan et al. 2004), the polarity of selective constraints along the IT pathway in vertebrates may result from a gradient in the number of duplicates.
