**2.2.1 Analysis of the** *IGF2/H19* **locus**

8 Bacterial Artificial Chromosomes

content has shown a bias towards the insertion of young SINE and LINE elements and segmental duplications (Kortschak et al., 2009). As some differences in gene content between platypus and echidna X chromosomes have been identified, a comparison of the gene and repeat content of their Y chromosomes could provide important insight into the evolution of this complicatied sex chromosome system. Undoubtedly, a BAC-based approach will

The unexpected finding of no homology between monotreme and therian sex chromosomes begged the question as to how monotremes achieved dosage compensation. BAC clones were instrumental in determining the expression status of platypus X-borne genes in RNA-FISH experiments. Genes on platypus X chromosomes were monoallelically expressed in approximately 50% of cells and were biallelically expressed in the remainder, and so it appeared that the platypus employs a very leaky form of X inactivation for dosage compensation (Deakin et al., 2008a). This stochastic transcriptional regulation resembled the leaky inactivation of X-borne genes in the wallaby (Al Nadaf et al., 2010), suggesting that despite different origins of the X chromosome in monotremes and marsupials, their X inactivation mechanisms may have evolved from an ancient stochastic monoallelic expression mechanism that has subsequently independently evolved in the three major

In an attempt to further characterize features of the platypus X inactivation system, BAC clones were used to examine replication timing and X chromosome condensation, two features common to X inactivation in therian mammals. Replication timing of X-borne genes was determined by hybridizing fluorescently labeled BACs to interphase nuclei and counting the number of nuclei with asynchronous replication represented by double dots over one homologue of the gene of interest and a single dot over the other. These dot assays revealed asynchronous replication of some regions on the X chromosomes, namely those not shared on the Y (Ho et al., 2009). Condensation status of three platypus X chromosomes was determined by hybridizing two BACs mapped to opposite ends of the chromosome and measuring the distance between the two signals on the two X chromosome homologues. Only one X chromosome (X3) displayed signs of differences in chromosome condensation. Consequently, chromosome condensation may not play a significant role in platypus dosage compensation (Ho et al., 2009). It would be interesting to perform these same experiments in echidna for comparative purposes. Since an echidna BAC library is available, it is hoped that

Most autosomal genes in diploid organisms are expressed from both the maternal and paternal copies at equal levels. However, there are roughly 80 exceptional genes in eutherian mammals that are monoallelically expressed in a parent of origin fashion. The silent allele is marked (imprinted) by epigenetic features, such as CpG methylation and histone modifications. The evolution of a genomic imprinting mechanism appears counterintuitive since surely it would be more advantageous to have two expressed copies of a gene to protect the individual against deleterious mutations occurring in one copy. Consequently, genomic imprinting raises many questions regarding the how and why genomic imprinting evolved, although there appears to be some link between the evolution

continue to be the best strategy for obtaining Y-specific sequence.

this data will be obtained in the future and such a comparison made.

of viviparity and genomic imprinting (Hore et al., 2007).

mammalian lineages (Deakin et al., 2008a, 2009).

**2.2 Evolution of genomic imprinting** 

The *IGF2* imprinted locus has been extensively characterized in humans and mice, and was the first gene reported to be subject to genomic imprinting in marsupials (but not monotremes) (O'Neill et al., 2000). Elucidating the mechanism by which this is achieved was the subject of a number of subsequent studies. Sequence comparisons between the nonimprinted *IGF2* locus of platypus and the imprinted locus of marsupial and eutherian mammals were made in an attempt to identify potential sequence elements required for imprinting of this locus. A platypus BAC clone containing the *IGF2* gene was fully sequenced and compared to opossum, mouse and human. This study failed to identify any sites of differential methylation in intragenic regions but did uncover strong association of imprinting with both a lack of short interspersed transposable elements (SINEs) and an intragenic conserved inverted repeat (Weidman et al., 2004). Isolation of an opossum BAC clone (Lawton et al., 2007) and more extensive interrogation of the locus, identified a differentially methylated region (Lawton et al., 2008). This BAC clone was used in RNA-FISH experiments to show that demethylation of this differentially methylated region results in biallelic expression of *IGF2* (Lawton et al., 2008). Therefore, differential DNA methylation does indeed play a role in *IGF2* imprinting in marsupials.

In humans, *H19* is a maternally expressed long non-coding RNA located near the *IGF2*  locus*.* While protein coding genes in this region were easily identified from genome sequence, the low level of sequence conservation typical of non-coding RNAs made the identification of *H19* more challenging. Three wallaby BACs spanning the the *IGF2/H19*  locus were isolated by screening the library with probes designed from all available vertebrate sequences for genes within the region (Smits et al., 2008). Sensitive sequence similarity searches of the sequence obtained from these BAC clones identified a putative *H19* transcript with 51% identity to human *H19.* This sequence was found to be absent from the opossum genome assembly and hence, a BAC clone containing the opossum *H19* orthologue was isolated and sequenced. Like eutherians, *H19* is maternally expressed in marsupials (Smits et al., 2008).
