Isolation and sequencing of doublesex / male abnormal 3 ( DM ) related transcription factor ( Dmrt ) genes from the Asian toad Bufo gargarizans ( Cantor , 1842 )

The doublesex/male abnormal 3 (dsx/mab-3 or DM) domain gene family involved in sexual development encodes putative transcription factors including a DNA-binding homology motif, the DM domain. We used highly degenerate primers to clone and sequence seven distinct DM related transcription factor (Dmrt) genes from the Asian toad (Bufo gargarizans Cantor, 1842). A database search for the cloned sequences revealed the following percentage identity with the homologous Dmrt genes of the human: BgDmrt1 = 97%, BgDmrt2 = 97%, three isoforms of BgDmrt3 (BgDmrt3a = 93%, BgDmrt3b = 95%, BgDmrt3c = 100%) and two isoforms of BgDmrt5 (BgDmrt5 = 97%, BgDmrt5 = 91%). Based on DM domain amino acid sequence similarities we constructed a phylogenetic tree which grouped vertebrate and invertebrate Dmrt genes into seven distinct subfamilies. The DM domains of both human and the newly-discovered Bufo gargarizans genes contained two conserved zinc-chelating sites (CCHC and HCCC), except BgDmrt3b, which contained the CCRC and HCCC sites.

The DM domain gene family has some members with a conserved DNA-binding DM domain encoding putative transcription factors related to the sexual regulators Doublesex (DSX) from Drosophila melanogaster and Male abnormal 3 (MAB-3) from Caenorhabditis elegans (Erdman and Burtis, 1993;Raymond et al., 1998).The DM domain has a highly intertwined structure that chelates two zinc atoms, and makes specific DNA contacts predominantly in the minor groove (Zhu et al., 2000).Doublesex and MAB-3 related transcription factor (Dmrt) genes have recently been cloned from a wide range of vertebrates, including fish, amphibians, reptiles, birds and mammals (Mastuda et al., 2002;Smith et al., 2002;Kettlewell et al., 2000;Shibata et al., 2002).These genes have been found to play essential roles in a variety of sex developmental processes, and at least some of these functions have been conserved.For example, Dmrt1 gene knock-out mice have severe defects in testis differentiation and DMY mutant medaka fish (Oryzias latipes) show sex reversal during which XY fish carrying a mutant DM-domain gene on the Y chromosome (the DMY gene) develop not as males but as females (Raymond et al., 2000;Mastuda et al., 2002).The frog (Rana rugosa) Dmrt1 gene is expressed in the differentiating testis but is not detectable during ovarian differentiation (Shibata et al., 2002).The zebrafish (Danio rerio) Terra/Dmrt2 gene is an early left-sided expressed gene that links left-right patterning with bilateral synchronization of the segmentation clock (Saude et al., 2005).The Dmrt3 gene is primarily expressed in the forebrain, neural tube and nasal placode of both mice and chickens (Smith et al., 2002), while the expression of Dmrt5 in adult zebrafish is restricted to brain cells and developing germ cells (Guo et al., 2004).
The widely distributed Asian toad (Bufo gargarizans Cantor, 1842), also called the Chusan Island toad, plays an important ecological role in the environments where it lives.Although Shang (1983) showed that Bufo gargarizans has a ZZ/ZW type chromosomal sex determination system, the molecular mechanisms of sex determination and differentiation remain unclear for this species.As a prelude to understanding the involvement of Dmrt genes in the sexual development of this toad, we cloned the DM domain gene family of B. gargarizans using degenerate primers from genomic DNA.In the present paper we report the cloning and nucleotide sequencing of seven different Dmrt genes from B. gargarizans.The phylogenetic relationships between the DM domain genes of verte-brates and invertebrates were also examined based on DM domain amino acid sequences.
We obtained three female and two male B. gargarizans from Wuhu city (Anhui province, China) and humanly sacrificed them.Total genomic DNA was isolated from muscle tissues by routine protocols (Sambrook et al., 1989).To amplify the conserved DM domains we used a pair of degenerate primers D1 (5'-TGCG(AGC)(AC)G(A G)TGC(AC)G(AG)AA(CT)CACGG-3') and D2 (5'-C (GT)(GC)AG(GC)GC(GC)ACCTG(GC)GC(AGCT)GCC AT-3') (Ren et al., 2001), the expected amplification fragment length being 140 bp with these primers.The polymerase chain reaction (PCR) was carried out in a 25 μL reaction mixture containing 50 ng of genomic DNA, 1.5 mM Mg 2+ , 200 μM of each dNTP, 0.2 μM of each primer and 1 unit of Taq DNA polymerase (Promega, Peking, China).The cycling conditions were as follows: 32 cycles of 30 s at 94 °C, 40 s at 62 °C and 40 s at 72 °C , followed by 7 min elongation at 72 °C.The PCR products were cloned using the pMD 18-T Vector (TaKaRa, Dalian, China) and transformed into Escherichia coli strain DH5α (Sangon, Shanghai, China) and 115 white clones were transferred to a plate of clones from an initial culture plate of lysogeny broth (LB) media containing 5-bromo-4-chloro-3-indolyl-β-Dgalactopyranoside (X-gal) and isopropyl-β-D-thiogalactoside (IPTG) and 75 positive clones with insert PCR fragment were confirmed using colony PCR (Shen et al., 2000).The distinctive positive clones were screened using single-strand conformation polymorphism (SSCP) (Nie et al., 1999) and sequenced using the universal sequencing primer on an ABI377 auto-sequencer (Applied Biosystems).All DNA sequences were analyzed using the basic local alignment search tool (BLAST) and CLUSTAL X1.8 programs and the phylogenetic tree was constructed using the molecular evolutionary genetic analysis (MEGA) program V2.1 (Kumra et al., 2001).
Using the B. gargarizans genomic DNA as template we obtained a 140 bp PCR fragment and we obtained seven distinct positive clones from the DNA of the male and female toads, although there was no difference between the clones in respect to the sex of the toads from which the clones originated (Figure 1).
Database searches and phylogenetic analysis of the clones uncovered seven unreported Dmrt sequences, representing distinct genes, which we named by adding the prefix Bg (Bufo gargarizans) and submitted to GenBank as Bufo gargarizans doublesex and mab -3 related transcription factor (BgDmrt) genes.The genes and their GenBank accession numbers and sequence identity to homologous human DMRT genes (in parentheses) are: BgDmrt1 (DQ217562, 97%), BgDmrt2 (DQ217563, 97%), BgDmrt3a (DQ217564, 93%), BgDmrt3b (DQ217565, 95%), BgDmrt3c (DQ217566, 100%), BgDmrt5a (DQ217569, 97%) and BgDmrt5b (DQ217571, 91%).Note that BgDmrt3 has three isoforms and Dmrt5 has two iso-forms.The putative amino acid sequences of the BgDmrt genes and the alignment between corresponding human DMRT sequences are shown in Figure 2. The DM domains of both human and the newly-discovered B. gargarizans genes contained two conserved zinc-chelating sites (CCHC and HCCC), except BgDmrt3b, which contained the CCRC and HCCC sites.It is probable that the DM domain genes act as a transcription regulator in sex determination and other developmental processes, mediated by the DNAbinding zinc motif (Zhu et al., 2000).
The putative amino acid sequences of the seven clones were compared with 34 published Dmrt gene sequences accessible at the NCBI BLAST server (Table 1), the DMdomain neighbor-joining phylogenetic tree being shown in 1190 Chen et al.  Figure 3. Phylogenetic analysis showed that all these Dmrt genes were clustered into seven different subfamilies (Dmrt1-7), and confirmed the identity of the BgDmrt genes identified by us.However, the BgDmrt5b was not included in the Dmrt5 group, implying more accumulation of amino acid substitutions in BgDmrt5b than in other members of the Dmrt5 cluster.We also compared four different invertebrate DM domain proteins with other vertebrate DM domain proteins using Mab-3 from the nematode Caenorhabditis elegans as the outgroup and found that Dmrt2 was related to Dmrt11E from the fly Drosophila melanogaster, Dmrt4 was related to Drosophila melanogaster Dmrt99B and Dmrt5 was related to Caenorhabditis elegans F10C1.5, suggesting that both the Dmrt2 and Dmrt4/Dmrt5 groups are ancient because they both contain invertebrate sequences.Ottolenghi et al. (2002) used structural analysis to show that the human genes DMRTA1, DMRTA2 and DMRTA3, also called DMRT4, DMRT5 and DMRT3 respectively (the alternative nomenclature for human DMRT genes follows Volff et al. (2003), likewise for DMRTB1 and DMRTC2, which was called DMRT6 and Dmrt7 respectively), all have a DM domain and a DMRTA domain, phylogenetic analysis showing a relationship between DMRTA1 and DMRTA2 while DMRTA3 was more distant.Our neighbor-joining analysis supported a relationship between vertebrate Dmrt4 and Doublesex/male abnormal 3 (DM) related transcription factor (Dmrt) genes from the Asian toad 1191 Dmrt5 but, in contrast, no closer phylogenetic relationship could be detected between Dmrt3 and Dmrt4/Dmrt5, although this does not preclude a close relationship.Volff et al. (2003) have pointed out that different Dmrt genes can be under very different evolutionary constraints.In our analysis the short branches in the Dmrt3 group suggests very strong sequence conservation, while the fact that the branches in the Dmrt1 group appear to be longer indicates much more relaxed evolutionary constraints for these sequences.This is the first report describing Dmrt genes from B. gargarizans.Several of the clones obtained in this study indicate that Dmrt genes that are duplicated in B. gargarizans are present in mammals as single copies.For example, the human genome contains single copy of DMRT3 (DMRTA3), whereas this gene is present in at least three copies in B. gargarizans.Intriguingly, the BgDmrt3a gene is divergent from other Dmrt3 genes in the phylogenetic tree and the zinc-chelating site I of BgDmrt3b is cystine, cystine, arginine, cystine (CCRC) not cystine, cystine, histidine, cystine (CCHC).According to Prince and Pickett (2002), gene duplication is a mechanism by which new gene functions may be acquired.The very recent duplication of the Dmrt1 has apparently led to the formation of the master male-determining DMY gene in the medaka fish, and similar scenarios might have generated new paralogs of other Dmrt genes in different taxa (Mastuda et al., 2002;Volff et al., 2003).The Dmrt gene duplication in B. gargarizans may have been the result of such a process, unlike the situation in teleost fish, which may have been the result of the ancient duplication of the whole genome.However, how these Dmrt genes function in the sexual development B. gargarizans still needs further experimental exploration.

Figure 3 -
Figure3-Neighbor-joining phylogenetic tree (1000 replicates) of some vertebrate and invertebrate doublesex/male abnormal 3 (dsx/mab-3 or DM) related transcription factor (DMRT) gene family members based on amino acid sequence data.Nematode Mab-3 sequences were the outgroup.Branches with less than 50% support have been collapsed.The toad DM domain sequences in the tree were the Bufo gargarizans sequences obtained during the research described in this paper.