**2.1.1 The marsupial X chromosome**

4 Bacterial Artificial Chromosomes


**Species Genes or Region Purpose Reference** 

Platypus *SOX 3* Mapping (Wallis et al., 2007b)

(*M.domestica*) Immunoglobulins Mapping (Deakin et al., 2006a)

(*M.domestica*) T cell receptors Mapping (Deakin et al., 2006b)

Platypus *DMRT* cluster Sequencing (El-Mogharbel et al.,

Platypus Defensins Mapping (Whittington et al.,

Platypus *SOX9* and *SOX10* Mapping (Wallis et al., 2007a)

Dunnart *LYL1* Sequencing (Chapman et al., 2003) Tammar wallaby Prion protein gene Sequencing (Premzl et al., 2005)

Tammar wallaby Mucins & Lysozyme Mapping (Edwards et al., 2011) Tammar wallaby *SLC16A2* Sequencing (Koina et al., 2005) Tammar wallaby *BRCA1* Mapping (Wakefield & Alsop,

Table 3. Studies in marsupial and monotreme comparative genomics that relied on BAC

Determining the evolutionary origins of marsupial and monotreme sex chromosomes was the driving force behind much of the gene mapping conducted in these species. The earliest gene mapping work showed that at least some genes found on the human X chromosome were also on the X in marsupials, resulting in the hypothesis that the X chromosome of these two mammalian groups had a common origin. Gene mapping using heterologous probes and radioactive in situ hybridization (RISH) supported the extension of this hypothesis to include monotremes. However, it was only when BAC clones became

**2.1 Origins of marsupial and monotreme sex chromosomes** 

pathway genes Mapping (Grafodatskaya et al.,

cell receptors Mapping (Sanderson et al., 2009)

pigments Sequencing (Wakefield et al., 2008)

2007)

2008)

2007)

2006)

BACs have played a role.

Platypus Sex determination

Tammar wallaby Immunologulins & T

Tammar wallaby Cone visual

Echidna and

Opossum

Opossum

clones.

Like humans, marsupial females have two X chromosomes whereas their male counterparts have a X and a small Y chromosome, meaning that they require a mechanism to compensate for the difference in dosage of X-borne genes between females and males. Several decades ago, it was shown that several X-linked genes in human were also located on the X in marsupials and one X chromosome was inactivated in somatic cells to achieve dosage compensation. However, even in these early studies, striking differences in the characteristics of X inactivation in eutherians and marsupials were evident. Marsupials were found to preferentially silence the paternally derived X chromosome rather than subscribing to the random X inactivation mechanism characteristic of eutherian mammals. This inactivation was found to be incomplete, with some expression observed in some tissues from the inactive X and thus, appeared to be leakier than the stable inactivation observed in their eutherian counterparts (reviewed in Cooper et al., 1993). Therefore, there was a great interest in investigating the marsupial X chromosome and X inactivation in greater detail, a task in which marsupial BAC libraries have been indispensable.

The first step towards gaining a deeper understanding of X inactivation in marsupials was determining the gene content of the marsupial X chromosome. Early gene mapping studies showed that not all genes located on the human X chromosome were present on the X in marsupials. This was supported by cross-species chromosome painting which showed that the human X chromosome could be divided into two regions; one being a region conserved on the X chromosome both in marsupials and human, referred to as the X conserved region (XCR), and a region added to the X chromosome in the eutherian lineage - the X added region (XAR) (Glas et al., 1999; Wilcox et al., 1996). This added region corresponded to most of the short arm of the human X chromosome.

Progress in determining the boundaries of the XCR and XAR was slow until the release of the opossum genome assembly, which revealed this boundary in this species and pathed the way for detailed gene mapping in a second species, the tammar wallaby. Wallaby specific overgos were designed for human X-borne genes from sequence generated by the genome sequencing project and used to screen the wallaby BAC library in large pools. BACs for these genes were mapped to wallaby chromosomes using FISH. Genes from the XAR mapped to chromosome 5 (52 genes) and the XCR genes mapped to the X chromosome (47 genes). This mapping data enabled comparisons in gene order to be made between wallaby, opossum and human, revealing a surprising level of rearrangement on the X chromosome between these species (Deakin et al., 2008b).

One region that was of particular interest for comparative gene mapping in marsupials, given the differences in X inactivation between marsupials and eutherians, was the X inactivation center (XIC) located within the XCR on the human X chromosome. This region contains the *XIST* (X inactive specific transcript) gene, a master regulatory non-coding RNA transcribed from the inactive X, and a number of other non-coding RNAs that play an important role in X inactivation (reviewed in Avner & Heard, 2001). The *XIST* gene is poorly conserved between eutherian species (Chureau et al., 2002; Duret et al., 2006; Hendrich et al., 1993; Nesterova et al., 2001). Sequence similarity searches failed to identify any sequence with homology to *XIST.* As a consequence, a BAC-based approach was taken to determine whether *XIST* was present in marsupials.

Three independent research teams used similar BAC-based approaches to determine the location of genes flanking the eutherian XIC locus on marsupial chromosomes. Shevchenko et al (2007) isolated BACs containing *XIST-*flanking genes as well as other genes from the XCR in two opossum species (*M.domestica* and *D.virginiana*). FISH-mapping of these BACs in both species revealed an evolutionary breakpoint between *XIST-*flanking genes. Likewise, Davidow et al (2007) and Hore et al (2007) mapped BACs identified to contain *XIST*flanking genes from BAC-end sequence data generated as part of the opossum genome project and mapped them to different regions of the *M.domestica* X chromosome. Further sequence searches around these flanking genes failed to identify an orthologue of *XIST*  (Davidow et al., 2007; Duret et al., 2006) and it was concluded that the *XIST* gene is absent in marsupials (Davidow et al., 2007; Hore et al., 2007)*.* This conclusion was further supported by mapping of *XIST*-flanking genes to opposite ends of the tammar wallaby X chromosome (Deakin et al., 2008b). Hence, marsupial X inactivation is not under the control of *XIST* but then this raised more questions regarding marsupial X inactivation. Is there is a marsupial specific X inactivation centre? To answer this question, a more detailed investigation of the status of inactivation of marsupial X-borne genes was required.

Fortunately, the BACs isolated for mapping genes to the tammar wallaby X chromosome could be used construct an 'activity map' of the tammar wallaby X chromosome, where the inactivation status of X-borne genes at different locations along the X was determined. By using RNA-FISH, a technique that detects the nascent transcript, it was possible to determine the inactivation status of an X-borne gene within individual nuclei. The large insert size of BAC clones makes them ideal for hybridization and detection of the nascent transcript. Al Nadaf et al (2010) determined the inactivation status of 32 X-borne genes. As was suggested by earlier studies using isozymes, X inactivation in marsupials is incomplete. Every gene tested showed a percentage (5 – 68%) of cells with expression from both X chromosomes. This activity map of the wallaby X chromosome demonstrated no relationship between location on the X chromosome and extent of inactivation, suggesting that there is no polar spread of inactivation from a marsupial-specific inactivation center (Al Nadaf et al., 2010).

Although there are still many questions to be answered concerning marsupial X chromosome inactivation, BAC clones have proven to be extremely valuable resources for these studies and have resulted in the rapid advance of knowledge in this field. Further work is already underway to construct activity maps of genes in other species, using BACs from the opossum and the devil. Including a further species, the bandicoot (*I. macrourus*) would be particularly interesting as this species has an extreme version of X inactivation where they eliminate one sex chromosome (either a X in females or the Y in males) from somatic cells. The availability of a BAC library for this species makes it possible that this research could be carried out in the future.

### **2.1.2 Gene content of the marsupial Y chromosome**

Although gene poor, the Y chromosome has an exceptionally important function, being responsible for sex determination and other functions in male sex and reproduction. A comparison of the chimpanzee and human Y chromosomes demonstrates the rapid evolution of the Y chromosome (Hughes et al., 2010). Extending this comparison to include marsupials would provide even further insight into the evolution of this remarkable chromosome. Orthologues of several eutherian Y-borne genes were mapped to the Y chromosome of marsupials but it was of more interest to see if there were novel genes found on the marsupial Y, which could be revealed by sequencing a marsupial Y chromosome.

Sequencing of the highly repetitive Y chromosome is extremely difficult by shot-gun sequencing. A BAC-based approach is seen as the best option to obtain well-assembled sequence. A novel method has been used to obtain Y specific BAC clones in the wallaby, in which the Y chromosome was isolated by flow sorting or manual microdissection and used to probe a wallaby BAC library and create a sub-library enriched with Y-specific BAC clones (Sankovic et al., 2006). Sequencing of two of these clones resulted in the identification of novel genes on the Y chromosome, *HUWE1Y* and *PHF6Y* (Sankovic et al., 2005)*.* These genes are not on the Y chromosome of eutherians but do have a homologue on the X chromosome. It is hoped that more of these Y-specific BACs will be sequenced in the future to enable the evolutionary history of the therian Y chromosome to be unraveled.

### **2.1.3 Gene content of the platypus sex chromosomes**

6 Bacterial Artificial Chromosomes

1993; Nesterova et al., 2001). Sequence similarity searches failed to identify any sequence with homology to *XIST.* As a consequence, a BAC-based approach was taken to determine

Three independent research teams used similar BAC-based approaches to determine the location of genes flanking the eutherian XIC locus on marsupial chromosomes. Shevchenko et al (2007) isolated BACs containing *XIST-*flanking genes as well as other genes from the XCR in two opossum species (*M.domestica* and *D.virginiana*). FISH-mapping of these BACs in both species revealed an evolutionary breakpoint between *XIST-*flanking genes. Likewise, Davidow et al (2007) and Hore et al (2007) mapped BACs identified to contain *XIST*flanking genes from BAC-end sequence data generated as part of the opossum genome project and mapped them to different regions of the *M.domestica* X chromosome. Further sequence searches around these flanking genes failed to identify an orthologue of *XIST*  (Davidow et al., 2007; Duret et al., 2006) and it was concluded that the *XIST* gene is absent in marsupials (Davidow et al., 2007; Hore et al., 2007)*.* This conclusion was further supported by mapping of *XIST*-flanking genes to opposite ends of the tammar wallaby X chromosome (Deakin et al., 2008b). Hence, marsupial X inactivation is not under the control of *XIST* but then this raised more questions regarding marsupial X inactivation. Is there is a marsupial specific X inactivation centre? To answer this question, a more detailed investigation of the

Fortunately, the BACs isolated for mapping genes to the tammar wallaby X chromosome could be used construct an 'activity map' of the tammar wallaby X chromosome, where the inactivation status of X-borne genes at different locations along the X was determined. By using RNA-FISH, a technique that detects the nascent transcript, it was possible to determine the inactivation status of an X-borne gene within individual nuclei. The large insert size of BAC clones makes them ideal for hybridization and detection of the nascent transcript. Al Nadaf et al (2010) determined the inactivation status of 32 X-borne genes. As was suggested by earlier studies using isozymes, X inactivation in marsupials is incomplete. Every gene tested showed a percentage (5 – 68%) of cells with expression from both X chromosomes. This activity map of the wallaby X chromosome demonstrated no relationship between location on the X chromosome and extent of inactivation, suggesting that there is no polar spread of

Although there are still many questions to be answered concerning marsupial X chromosome inactivation, BAC clones have proven to be extremely valuable resources for these studies and have resulted in the rapid advance of knowledge in this field. Further work is already underway to construct activity maps of genes in other species, using BACs from the opossum and the devil. Including a further species, the bandicoot (*I. macrourus*) would be particularly interesting as this species has an extreme version of X inactivation where they eliminate one sex chromosome (either a X in females or the Y in males) from somatic cells. The availability of a BAC library for this species makes it possible that this

Although gene poor, the Y chromosome has an exceptionally important function, being responsible for sex determination and other functions in male sex and reproduction. A comparison of the chimpanzee and human Y chromosomes demonstrates the rapid

inactivation from a marsupial-specific inactivation center (Al Nadaf et al., 2010).

whether *XIST* was present in marsupials.

research could be carried out in the future.

**2.1.2 Gene content of the marsupial Y chromosome** 

status of inactivation of marsupial X-borne genes was required.

Monotremes, like other mammals, have male heteromorphic sex chromosomes, but their sex chromosome system is somewhat complex. Female platypuses have five different pairs of X chromosomes and their male counterparts have five X and five Y chromosomes that form a multivalent translocation chain during male meiosis (Grutzner et al., 2004). Similarly, the echidna (*T. aculeatus*) has five X chromosomes in females, and five X and four Y chromosomes in males (Rens et al., 2007). Early gene mapping studies using RISH with several heterologous probes suggested that at least one monotreme X chromosome shared homology with the therian X (Spencer et al., 1991; Watson et al., 1992; Watson et al., 1990). Subsequent mapping of BAC clones containing *XIST-*flanking genes indicated that at least some therian X-borne genes had an autosomal location in the platypus (Hore et al., 2007). The sequencing of the platypus genome made it possible to more thoroughly investigate the gene content of all platypus X chromosomes. By FISH-mapping BACs end-sequenced as part of the genome project, it became evident that, in contrast to the original gene mapping data, the platypus X chromosomes share no homology the therian X. Instead, at least some of the X chromosomes share homology with the chicken Z. Genes from the XCR were located on platypus chromosome 6 (Veyrunes et al., 2008). Furthermore, mapping of platypus X chromosome BACs onto male chromosomes identified the pseudoautosomal regions on the platypus Y chromosomes, providing the first glimpse into the gene content of the platypus Ys. Finding a lack of homology between monotreme and therian X chromosomes had a major impact on our understanding of the timing of therian sex chromosome evolution and provided surprising insight into the ancestral amniote sex determination system, which may have resembled the ZW system observed in birds (Waters & Marshall Graves, 2009).

The complicated sex chromosome system of monotremes makes determining the sequence of platypus Y chromosomes especially interesting. Since only a female platypus was sequenced as part of the genome project, no Y-specific sequence was obtained (Warren et al., 2008). Kortschak et al (2009) isolated and sequenced six Y-specific platypus BAC clones. The gene content of these BACs has not been reported but a detailed analysis of the repeat 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 continue to be the best strategy for obtaining Y-specific sequence.

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

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