Figure 1.
A calf with perinatal weak calf syndrome.
The birth weight was 16 kg (normal average; 28.4±4.24 kg). The gestation period was 296 days (normal average; 288.9 days). The affected calf was weak and had difficulty nursing. The calf died at 2 days old.
Figure 2.
Genome-wide homozygosity mapping of perinatal weak calf syndrome.
(A) The results of the genome-wide homozygosity mapping of perinatal weak calf syndrome with a paternal half-sib family composed of 13 affected and 30 normal animals sired from Bull A using ASSHOM (•) and ASSIST (X). The black horizontal bars mark the limits between the 29 autosomes. (B) The results of the homozygosity mapping on BTA 8. (C) The LRT-statistic profile for the disease on BTA 8. The horizontal line indicates the threshold for the 1% chromosome-wise significance level. The dashed line indicates information content (right y-axis). Microsatellite positions are indicated as filled triangles under the x-axis, respectively. The filled boxes on the x-axis represent the 95% confidence interval. The 95% confidence interval was flanked by IDVGA-52 (79.3 Mb) and MNB-38 (102.6 Mb), corresponding to the results of homozygosity mapping.
Figure 3.
Genotypes of affected and normal animals for SNPs on BTA8.
Overlapping blocks of extended homozygosity in the affected animals are shown in grey. The interval flanked by ARS-BFGL-NGS-108358 and ARS-BFGL-NGS-28685 (4.04 Mb) with shared homozygosity in 11/13 affected animals is determined as the critical region. In normal animals, grey blocks indicate the same homozygous haplotype shown in the affected animals with at least 6 SNPs.
Table 1.
Confirmation of 3 SNPs in CENPP, IARS and LOC786526.
Table 2.
Genotyping with the Bull A family used for mapping.
Figure 4.
Alignment of the region flanking Val79Leu, and IARS aminoacylation activity.
(A) Val79 is extremely conserved, and the flanking regions are also conserved in mammals. Top: cattle mutant and wild-type sequences. Middle: other mammalian sequences. Below: non-mammalian sequences. (B) NIH 3T3 cells were transfected with wild-type (Val-79) and mutant (Leu-79) V5-His-IARS cDNA plasmids. Whole-cell lysates were treated with His-tagged protein-binding magnetic beads (Dynabeads TALON). Immunoblot analysis was performed with anti-V5 antibody (Invitrogen) and anti-IARS antibody (H-219, Santa Cruz). 1. Purified wild-type V5-His-IARS. 2. Wild-type V5-His-IARS-transfected whole-cell lysate treated with TALON beads. 3. Purified mutant V5-His-IARS. 4. Mutant V5-His-IARS-transfected whole-cell lysate treated with TALON beads. Most of the V5-His-tagged protein was absorbed by TALON beads. The molecular weights of recombinant V5-His-IARS proteins (r-IARS) were higher than the endogenous murine IARS protein (e-IARS). The r-IARS fractions did not contain detectable e-IARS. (C) The aminoacylation activity of purified r-IARS (n = 3, in triplicate) shown with the standard deviation; 100% corresponds to 1.22 pmol/µg-protein, 30 min. Revelomycin A, 1 µg/ml. A t-test was conducted to obtain the p values.
Table 3.
Further genotyping of IARS mutations.
Table 4.
IARS mutations in random dead calves and growth-retarded calves.