PMC

Metaphase spreads from male zebra finches. Z chromosome components are: a Zsm(submetacentric)/Zsm, b Zm(metacentric)/Zm, and c Zsm/Zm. Inverted image of DAPI staining. Bar, 10 μm

Testis-specific expression of PAK3 related gene located on Z chromosome (PAK3Z). RT-PCR analysis for brain, lung, and testis/ovary cDNA samples showed that PAK3Z expression is specific to testis, suggesting the involvement of PAK3Z in male-specific functions

Z chromosome karyotypic polymorphism in UCLA zebra finch population. Based on the location of centromere, Z chromosomes are categorized as submetacentric Z (Zsm) and metacentric Z (Zm). Inverted image of DAPI staining

Identification of Z chromosome inversion type by PCR. The 3-kb fragment was amplified from metacentric Z (Zm) and 1 kb from submetacentric Z (Zsm). Animals a, d, f have metacentric Z and b, c have submetacentric Z. e Male and has both types of Z chromosome

Partial alignment of PAK3 protein sequences. Predicted genes 1 and 2 (Fig. ) showed high homology to PAK3 from the other species. Asterisks indicate conserved amino acids in all nine sequences

Distribution of Zsm and Zm chromosomes in Australia. Bar graph shows the percentage of each chromosomes (gray: Zsm, black: Zm; Electronic supplementary material Table ). Map was made based on the web site (http://www.aquarius.geomar.de/). The bar for Lombok/West Timor is from Timor zebra finch (T. guttata guttata) samples

Diagram of relative position of FISH signals and late replication site on Zsm and Zm chromosomes. On the Zm chromosome, the 033L23 BAC signal (HINT1 gene) is larger at the major site and smaller at the minor site. We have previously reported the gene locations for the Zsm chromosome (Itoh et al. )

Location of DMRT1 (028P16, green), HINT1 (033L23, green), and late replication site (red) on Zsm and Zm chromosomes. The signal of 033L23 was split into two spots on Zm chromosome: stronger signal on long arm and weaker signal on short arm. Thus, the origin of 033L23 BAC clone is the Zsm chromosome since there is only one signal. Counter staining: DAPI

Alignment of 033L23 sequence (Zsm) to the corresponding zebra finch genome (Zm; from Ensembl: www.ensembl.org). The region between 0 and 50,000 bp of the 033L23 BAC clone should contain the inversion point since there are many repetitive units as an indicator of rearrangement. Asterisk shows the location of DNA fragment where ZINVG-F1 and ZINVG-R1 primers amplify

Genescan (http://genes.mit.edu/GENSCAN.html) predicted four putative genes on 033L23 BAC sequence. Genes 1 and 2 have a homology to PAK3 and gene 3 has a homology to HINT1. Regions highly homologous to PAK3 and HINT1 are shown above the line. Vertical bars indicate putative exons identified by Genescan, but some of these may not be related to PAK3 and HINT1. The breakpoint of the Z chromosome inversion is probably somewhere within genes 1 or 2

Comparison of zebra finch PAK3 protein sequences to chicken. The protein sequence of zebra finch PAK3 located on chromosome 4 has high homology to that of the chicken (99% identity). Predicted genes 1 and 2 from BAC clone 033L23 (Fig. ; Zsm) and predicted gene 5 from the published zebra finch genome sequence (Zm; Ensembl: www.ensembl.org) have regions homologous to chicken, but lack several amino acid sequences. The percentage of identities to chicken is shown only for the alignment with the highest score

Dynamic inversion model of the zebra finch Z chromosome. The physical map of Zm chromosome is more similar to the gene location of chicken Z chromosome. Considering the positions of FISH signals and late replication site on Zsm and Zm, one possible explanation for this karyotypic polymorphism is an inversion of the large middle region of Z chromosome. 033L23 BAC probe contains HINT1 gene and shows two signal locations in Zm (major and minor). The heterochromatic region on the long arm of chicken Z chromosome was reported by Hori et al. (1996). A similar region showed a pattern of late replication based on the BrdU incorporation assay (Itoh et al., unpublished data)