SAE Cell Culture.
Cultures were initiated from S. acanthias early embryos in a basal nutrient medium developed originally for salmonid and zebrafish cells and applicable to cells of
marine origin as well (35, 36). This medium was supplemented with purified peptide growth factors, additional nutritional supplements, and low concentrations
of serum, as described (5). Cultures were grown at 18°C on collagen-precoated vessels in ambient carbon dioxide. The cells have been cultured in a
continuously proliferative state for 3 years.
EST-Based Identification of Genes Expressed in the SAE Cell Line.
Total RNA was extracted from 5 × 108
SAE cells at passage 25 by using TRIzol reagent. After mRNA isolation,
the primary cDNA library was directionally cloned into CMV Sport 6.1
(Invitrogen, Carlsbad, CA) and normalized by using subtractive
hybridization at two different Cot values, resulting in a 280-fold
reduction in the abundance of β-actin sequences. Average insert size
was 2.0 kb. More than 5,000 inserts were sequenced from the 5′ end.
Among these, a portion was also sequenced from the 3′ end. Of the ESTs
resulting from the 3′ sequencing, 611 were of base-identification
quality and length to be acceptable to GenBank. The average length of
the high-quality base sequence for these 3′-sequenced clones was 422
nt. Total data, prepared as described above and accepted by GenBank,
were 5,213 5′ EST and 611 3′ EST sequences and gene-identification
blast search. These data are publicly available through National Center
for Biotechnology Information and include all sequences to which this
work refers.
Identification of 3′ UTR Phylogenetic Footprints.
Inserts
sequenced from the 3′ end were screened against the National Center for
Biotechnology Information (NCBI) nucleotide database (nonredundant)
using MEGAblast (NCBI). Forty-eight of these returned significant
matches. Of these, 33 showed no BLASTx match and were analyzed further
as potential 3′ UTRs. Each of these sequences was compared by BLASTn to
all sequences in the NCBI database by using default parameters (37). To screen for potentially conserved sequences, we set threshold values of 80% nucleotide sequence identity, S scores of ≥60, and Expect (E) values <10−4.
Eight ESTs from identified genes showed regions of 20–203 nt that were
conserved in the homologous genes of a range of vertebrates. At this
point, additional related sequences within the 3′ UTRs of the eight
genes were further examined by inspection if E < 5 × 10−2.
CHAOS+Dialign (38)
was used for multiple alignment and program-based identification of
phylogenetic footprints; sequences of at least 20 bp met the threshold
values of the BLAST search given above and shared at least 80% identity
in three or more species. Percent identity of footprints in each gene
was determined from ClustalW pairwise alignment scores (39). Within these footprints, motifs were identified by comparisons between CHAOS+Dialign and FootPrinter (40).
Default parameters were used for all programs except FootPrinter, which
was set to detect 10-nt motifs with zero parsimony. EditSeq software
(DNAStar, Madison, WI) was used to determine percentage A+T. All
sequences were analyzed with RepeatMasker to detect possible repetitive
DNA elements (RepeatMasker Open 3.0. 1996–2004, www.repeatmasker.org).
PCR Methods.
Primers were designed to amplify highly conserved regions and interregion areas identified in the 3′ UTR of the fbxw7 (F-Box and WD-40 domain) gene. Genomic DNA was extracted from livers of S. acanthias and L. erinacea [whole zebrafish (Danio rerio)], the XM swordtail/platyfish (Xiphophorus) cell line (41), and kidney of the elephant shark (chimera) C. milii.
The sequence of forward primer fbxw7-F-1 was:
5′-TTGCTGGTCAGTCTTAGTG-3′. The sequence of forward primer fbxw7-F-2
was: 5′-ACAACTCAACATGAACTGTG-3′. The sequence of reverse primer fbxw7-R
was: 5′-GAATCAAACCATAGACACAATCC-3′. For PCR, the initial denaturation
step (94°C, 2 min) was followed by 10 cycles of 94°C (1 min), annealing
(47°C, 1 min), and elongation (74°C, 1 min). This was followed by 20
cycles at 94°C, 30 sec; 47°C, 30 sec; and 72°C, 45 sec. The final
elongation step was 72°C, 5 min, and products were then held at 4°C.
Product sizes were analyzed by electrophoresis on 1.2% agarose gels and
imaged with the Kodak (Rochester, NY) Gel Logic 100 system.
Answers to all your Biology Questions
