Studies on the Ovine CLN5 gene : a dissertation submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours at Lincoln University

Abstract

Neuronal ceroid lipofuscinoses (NCLs, Batten disease) are group of inherited childhood neurodegenerative diseases. Protein filled storage bodies accumulate in neurons as well as in most other cells. This protein storage is specific. Subunit c of mitochondrial ATP synthase accumulates in nearly all forms of the disease. Each form is caused by mutations in a separate gene and there are at least 8 of these. It has been proposed that these genes encode components of a specific pathway or oligomeric complex for subunit c turnover. NCLs also occur in animals, the best described being a form in New Zealand sheep that are orthologous to the human CLN 6 late infantile variant disease. This investigation was aimed at determining the sequence of the ovine gene for another human form, CLN5, to allow studies of co-operativity and cross regulation between these gene products. Sequence of ovine CLN5 was found but it is most likely that the gene is differently organised in sheep than in humans. PCR primers designed from the human gene sequence amplified products from exons I and IV of human genomic DNA but only from the region of the equivalent ex on N in sheep. A 125 bp oville fragment had 97% homology to part of human ex on IV. Primers degenerate at the 3' end were tested to ensure that lack of ovine ex on I amplification was not due to specific nucleotide differences at the polymerase start site. Different annealing temperatures and magnesium concentrations were also tested. Primers to cDNA spaning exons 1-3 gave several PCR products, but the band of the predicted size contained cDNA of which 55 bp were 88% homologous with a human type III inositol triphosphate receptor, reading in the reverse direction on the complimentary strand. The region for human CLN5, at 13q21, is gene rich and CLN5 is overlapped by at lest 2 other genes reading in the opposite direction, coding in both exons and introns of CLN5. cDNA arising from splicing variants of the human CLN5 have also been detected, but not fully characterised. The exon-intron boundary at the 3' end of ex on I is read through by at least one of these. Full genomic sequencing of the region is required to work out the structure of ovine CLN5.