Primer sequences used for real time PCR experiments and FISH probe preparation.

a<p>PCR product size in base pairs,</p>b<p>primer used in combination with HSFY-E1F,</p>c<p>with HSFY-E2R.</p><p>The table shows the primer name, primer sequences, the predicted PCR product size (base pairs, bp), GenBank accession number of the sequence that was used as the target for primer design, gene name and symbol, and the unique annealing temperature (°C) that was used for the PCR amplification.</p

<i>HSFY</i> copy number in the calibrator sample measured by real time PCR with three different primers.

<p>There is no significant difference in mean <i>HSFY</i> copy number as measured by three different primer sets (HSFY8: HSFY8F/HSFY8R; HSFY10: HSFY10F/HSFY10R, and HSFY16: HSFY16F/HSFY16R) which target different regions of the gene. <i>HSFY</i> copy numbers for primer sets HSFY8, HSFY10 and HSFY16 were based on 20, 4 and 3 separate real time PCR runs, respectively.</p

Gel electrophoresis of hybridization probes (A) and flourescence <i>in situ</i> hybridization (FISH) analysis (B–G).

<p>A) The largest intense band in lane 1 shows 2072 bp fragment of the 100 bp DNA ladder (Invitrogen Canada Inc.). PCR products amplified by primer pairs HSFY-E1F/HSFY-E2R, HSFY-E1F/HSFY-E1R, HSFY-E2F/HSFY-E2R were loaded into lanes 2, 3, 4, respectively. FISH images show the hybridization of the same products in the same order, thus using the whole gene (E), exon 1 (F) or exon 2 (G) specific probes. Images in the upper row (B, C, D) show the metaphase chromosomes with only 4′,6-diamidino-2-phenylindole (DAPI) counterstaining. Bar represents 10 µm.</p

Sequence alignment of the conserved DNA-binding domain in various species.

<p>The conserved heat shock factor (HSF) type DNA binding-domain shows a high degree of sequence homology among different species. Amino acids conserved between at least 5 proteins are marked with shaded boxes. The accession numbers for the proteins deposited in the sequence database on the NCBI website are as follows: hHSFY1 NP_149099.2; hHSFY2 NP_714927.1; mHSFYL NP_081937.1; rat NP_001012132.1; rhesus macaque ACL51668.1; cat NP_001035212.1; bovine (Hereford) NP_001070474.1.</p

Comparison of the percent homologies (%) of the complete <i>HSFY</i> protein (A) and HSF-type DNA-binding domain (B), for all species analyzed.

<p>In all cases, the percent homology was highest at the conserved HSF-type DNA-binding domain with the exception of an analysis of the two human HSFY copies which showed that both the complete protein sequence and conserved region were identical (100% homology). Bovine showed 51% and 72% homology with human HSFY for the complete protein and conserved domain, respectively. Bovine HSFY was most closely related to cat HSFY with a complete protein homology and conserved domain homology of 58% and 81%, respectively. The accession numbers for the proteins found in the NCBI database are as follows: human hHSFY1: NP_149099.2; hHSFY2: NP_714927.1, mouse NP_081937.1, rat NP_001012132.1, rhesus macaque ACL51668.1, cat NP_001035212.1, bovine NP_001070474.1.</p

<i>HSFY</i> copy number (n = 24), <i>HSFY</i> mRNA levels (n = 22) and percent 56-day non-return rate (NRR) for all bulls.

<p>N/A = testis sample not available.</p><p>There were no significant differences in mean HSFY copy number among bulls and the average was 73.3±0.8 copies. HSFY mRNA levels did vary significantly among bulls (p<0.0001) with an average of 0.489±0.038 relative to GAPDH. The 56-day non-return rates, a measure of field fertility, ranged from 49.6%–77.3%, with an overall average of 65.9%.</p

Gel electrophoresis of the real time PCR product of both male and female DNA samples for <i>HSFY</i> and <i>ZAR1</i>.

<p><i>HSFY</i> shows male-specific amplification indicating that the target genes are on the Y chromosome. Zygote arrest 1 (<i>ZAR1</i>) is the autosomal control gene which shows amplification in both the male and female sample. N) Negative control M) 100 bp ladder – the top and bottom band represent 200 and 100 bp, respectively (Invitrogen Canada Inc.).</p

Comparison of the predicted amino acid sequences of <i>HSFY</i> in Hereford and Holstein cattle.

<p>The mRNA sequences are 99% homologous and differ by only one base pair (c.1192A>C). This missense substitution mutation (p.<i>Tyr266Ser</i>) results in an amino acid change from tyrosine (Y) in Hereford to serine (S) in Holstein in the predicted protein sequence (shown by the box). This mutation is not found in the conserved HSF-type DNA-binding domain which is shown by the underlined sequence.</p