Alignment of Predicted Amino Acid Sequences for Three Feline <i>FLJ36031Y</i> Genes and Human Autosomal <i>FLJ36031</i>

<p>Above the alignments, asterisks indicate sites that are conserved. Below the alignments are histograms displaying level of amino acid conservation across the alignment. Note that the human FLJ36031 protein contains a long stretch of mostly unique amino acids from position 40 to 111, whereas the carboxyl terminus of the feline FLJ36031Y proteins (positions 232–318) is unique.</p

Retention Frequency of Different Classes of Cat Y Chromosome Genes in the Feline RH Panel

<p>The retention frequency of single copy X-degenerate genes (boxed in green) is similar to the genome-wide average of 0.39 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020043#pgen-0020043-b042" target="_blank">42</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020043#pgen-0020043-b044" target="_blank">44</a>]. Single copy X-degenerate genes close to the centromere (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020043#pgen-0020043-g005" target="_blank">Figure 5</a> for <i>SRY</i> FISH results) and potentially low–copy number genes (e.g., <i>HSFY</i>), are slightly elevated (boxed in blue). The five multicopy genes (boxed in red) have considerably higher retention frequencies than the single-copy genes, confirmed by the broad Yq chromosomal distributions determined by FISH (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020043#pgen-0020043-g005" target="_blank">Figure 5</a>).</p

Multicopy Distribution of Novel Y Chromosome cDNAs Confirmed by FISH

<p>FISH of feline cDNA probes on male domestic cat metaphase preparations. For each novel gene, the signal is detected only on the Y chromosome and is generally restricted to the long arm. Note that no hybridization signals were found on the distal short arm, which is heterochromatic [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020043#pgen-0020043-b030" target="_blank">30</a>]. For multicopy genes we also show the hybridization results on interphase nuclei. An <i>SRY</i>-containing BAC clone hybridized to the short arm of the Y chromosome near the centromere. A magnified view of the reverse-DAPI banded Y chromosome is shown in the upper left corner of each image, both with (right) and without (left) hybridization signals.</p

Male-Specific Amplification of <i>TETY2</i> and <i>CUL4BY</i> STS Primers in Cat and Dog

<p>A fragment of both genes has been PCR amplified with Y chromosome STS markers in matched cat and dog male and female genomic DNA samples. A 1-kilobase (kb) DNA ladder is shown to the left and right. PCR products are of the expected size in male DNAs, whereas no or nonspecific amplification is observed in female DNAs. The third lane in each set of primers (−) is a no-DNA control reaction. Dog ESTCO610012 is orthologous to cat <i>TETY2.</i></p

Establishment, Characterization, and Toxicological Application of Loggerhead Sea Turtle (Caretta caretta) Primary Skin Fibroblast Cell Cultures

Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 μM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1–10 μM benzo­[<i>a</i>]­pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells

Additional file 1: of Long-term expansion of primary equine keratinocytes that maintain the ability to differentiate into stratified epidermis

Figure S1. Equine keratinocytes (EK-100) were cultured in various culture conditions. Representative phase contrast images of primary equine keratinocytes (EK-100) cultured in (a) co-culture with irradiated fibroblasts+ 10 uM Y-27632, (b) F + 10 uM Y-27632 (c,d) CNT with or without 10 uM Y-27632 and (e)KSFM+ 10 uM. All images were taken on and day7 following initial culture without passage (×10 magnification. Size bars = 400 μm). Top right images show enlarged magnification (×40 magnification, size bars = 100 μm). Figure S2. Fluorescence-activated cell sorting (FACS) analysis of human keratinocytes (HFK) and mouse fibroblasts (j2) using pan-cytokeratin antibody. HFK cells were incubated without (a) pan-CK antibody or (b) with pan-CK antibody, (c) J2 fibroblasts without pan-CK antibody, or (d) with pan-CK antibody. Figure S3. Validation of antibodies for equine tissues. Specificity and reactivity of CK-14 was tested by using diluted concentration of CK14 1:600, 1:5000, and no antibody respectively in (a) breast cancer tissue and (b) equine skin tissue. All images (×40 magnification, scale bar = 100 μm) are representative of three experimental repeats. (PPTX 5023 kb

Summary data for the scarlet macaw first-generation draft <i>de novo</i> genome assembly with comparison to the initial turkey and chicken genome assemblies.

<p>Summary data for the scarlet macaw first-generation draft <i>de novo</i> genome assembly with comparison to the initial turkey and chicken genome assemblies.</p

Summary of Roche 454 Titanium and Illumina sequence data used for <i>de novo</i> assembly of the scarlet macaw genome.

<p>Summary of Roche 454 Titanium and Illumina sequence data used for <i>de novo</i> assembly of the scarlet macaw genome.</p

Relationship Between Total Contig Length (Kbp) and Total Contig Number for the Scaffolded Scarlet Macaw (<i>Ara macao</i>) Genome (SMACv1.1).

<p>The y-axis represents total contig length, expressed in kilobase pairs (Kbp), whereas the x-axis represents the total number of scaffolded contigs. Based on the estimated size of the scarlet macaw genome (1.11–1.16 Gbp), ≥90% of the assembled genome was captured within approximately 95,000 contigs.</p

Chicken-Scarlet Macaw (<i>Ara macao</i>) Comparative Chromosome Painting (ZooFISH).

<p>Using chicken flow sorted macrochromosomes (GGA1-GGA9) as well as GGAZ and GGAW, the homologous chromosome segments of the scarlet macaw were established via fluorescent <i>in situ</i> hybridization. All flow sorted probes were validated via hybridization to chicken metaphase spreads (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062415#pone.0062415.s001" target="_blank">Figure S1</a>).</p