Methods 150

4.4.2.1 Expression of AFF1 in Lenses

A sheep CDS sequence for AFF1 was assembled using BLAST matches between the cattle CDS (accession number NM_001191525, from http://www.ncbi.nlm.nih.gov/nuccore) and the sheep sequences available at

http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid=9940. The sheep CDS was used to design primers using the online Repeatmasker (Tarailo-Graovac et al, 2009) and Primer3 (Rozen and Skaletsky, 2000) programs. Primer sequences were designed only to match areas of known sheep sequence. Seven pairs of primers were designed, and each primer was named with the name of the gene, the letters A-F, and an F for “forward” or an R for “reverse”. The primers designed to amplify the entire gene were named AFF1F and AFF1R (Table 28).

The lens RNA used in the following procedures came from normal cultured lenses UN, VN, and TN. As described in Section 4.3.4.1, the letter N means that the lens were not exposed to opacity-inducing substances and the other letters refer to the animal the lens came from. These lenses were cultured and RNA was extracted as described in Section 4.3.3.1. , Leukocyte RNA from the affected sheep 47/07 which was used in NUDT9 sequencing was also used.

10.5 μL of the RNA extract and oligo dT primers (Invitrogen) were used to prepare cDNA with Superscript III reverse transcriptase (Invitrogen). PCR was carried out using the cDNA

at high fidelity. The PCR recipe and cycle was as described in section 4.3.3.2, except that an annealing temperature of 59°C was used. A 2μL sample of each PCR product was run on 2% agarose gel, 90V 1 h.

The PCR product was used to prepare two sequencing mixtures, one with the forward primer and one with the reverse primer. Each sequencing mixture contained 3.2 μL of a 1μM solution of the primer, the equivalent of 6.1ng of PCR product DNA, and sterile deionised water to 15 μL. The amount of DNA needed was calculated by allowing 2ng per 100 bases of the sequence. The sequencing mixtures were submitted to the Allan Wilson Centre Genome Service at Massey University, and sequenced as described in section 3.3.2.3. A BLAST search was performed to confirm that the sequence was from AFF1 cDNA and therefore that AFF1 is expressed in the cultured lens.

4.4.2.2 Sequencing of AFF1

The other primer pairs (Table 28) were used in PCRs to amplify regions of the AFF1 CDS. PCRs and agarose gels were carried out as in section 4.3.3.2. If there was no amplification or if the band was too faint, the PCR was repeated at an annealing temperature of 55°C. PCR products were purified with an AxyPrep PCR Cleanup Kit (Axygen).

The PCR products were sequenced as described in section 3.3.2.3. Each sequence was used in a BLAST search of the bovine genome to confirm that it matched only the AFF1 sequence. The DNAMAN (Lynnon) program was used to align each sequence with the bovine AFF1 CDS, and also its counterpart sequence generated from the other primer in the pair. The bovine sequence was used because the sheep sequence was too incomplete to align with most of the sequences. Sequences were cropped to the region of highest homology with the bovine sequence and each alignment was repeated. Sequences were assembled together using the Genedoc program (Nicholas et al, 1997), placing normal and affected sequences adjacent.

Only half of the AFF1 CDS was successfully sequenced using the original set of primers, so additional primers were designed and used in more PCRs. These were AFF1DNAF and AFF1DNAR, designed from bovine DNA sequence to amplify a 921-base region from genomic DNA, AFF1TOPF and AFF1TOPR, designed from bovine cDNA sequence to

amplify the start of the CDS from cDNA, and AFF1B2F, designed from bovine cDNA sequence to be used with AFF1DR to amplify a 1500-base section of the CDS from cDNA (Table 28). Two additional primers were designed from new sequence generated from the AFF1B primers and normal cDNA. These were named AFF12GAPF and AFF12GAPR (Table 28). The generation of this sequence is described in section 4.4.3.2.

Table 28: AFF1 Primers. The sizes predicted from bovine sequence (accession number

NM_001191525) are given in bp. There are two forward primers designed for AFF1BR, and sizes for both sets are given. The positions of these primers are shown in Figure.

Name Sequence Size (bp) Tm (°C)

AFF1F AGCAGGTAGTCCCGTCACC 60.1 AFF1R AAACCATCCTGTCCAACCAA 3986 60.2 AFF1AF AGTCTCCACGCCAAAAGCTA 60.0 AFF1AR GCCATGAGTGGGTCATTTTC 691 60.3 AFF1BF GGCAGCAGACCTTTGAGAAA 661 60.5 AFF1B2F GCTCCTAGCGAGTCTCCTGA 59.9 AFF1BR TGACTTTGGTCAGCCAGTTG 615 59.9 AFF1CF CCTCAGCACATTCCAGCAG 60.6 AFF1CR CTTTGGAGGGCTGGTCCTT 561 61.5 AFF1DF CTGCCTGCAGGTGGAGAG 60.7 AFF1DR CTGGCGTTTTCTGAGCTTCT 546 59.8 AFF1EF CAGAAGACAAACAGCCACCA 59.9 AFF1ER ACGCATGCATAAAACAGCAA 800 60.3 AFF1FF GGAGACCGTGGACCTCATTA 59.9 AFF1FR CTTGACTGGGACCCTATGGA 303 59.9 AFF1DNAF ACGGCATTATGTGACGTGGT 61.3 AFF1DNAR ATCTTCCTTCCCGCAGAAAT 1057 60.0 AFF1TOPF ATGGACGGCAGACCTTGA 60.2 AFF1TOPR TTCCCCAGCCTATTTTCAGA 301 59.6 AFF12GAPF AGTTCTGCAGTCCCAAACCA 60.7 AFF12GAPR GAAAGGTGGCTTCTCTGAGG 153 59.0

4.4.2.3 Mutation Analysis of AFF1

Affected and normal sequences were compared to find candidate OHC mutations. The 47/07 affected sheep is assumed to be heterozygous for the cataract locus. Therefore if the AFF1 sequence contains the OHC mutation, the 47/07 cDNA wil be a mixture of the normal and affected alleles while the normal cDNA will only have the normal allele. The sequencing results will show a two-base code for the cataract sequence and a one-base code for the same position in the normal sequence. The electropherogram should indicate that the two-base code is a true heterozygote with roughly equal peaks. It is also possible that 47/07 is a homozygote, in which case the cataract sequence will have one base and the same position in the normal

4.4.2.4 Comparison of AFF1 Exon Boundaries

The human AFF1 gene has 20 exons with 19 exon boundaries other than the start and stop codons. In order to compare the exon boundaries between human and sheep sequences, the human AFF1 CDS was aligned with the sheep cDNA sequence generated in this chapter.