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Clone and Sequence Analysis of Sox Genes in Rana tientaiensis
YAN ZHANG1, LIU WANG NIE1,*, LIANG YAN1, PING PING ZHENG1 AND WEN CHENG1
1Life Science College, Anhui Normal University; The Key Laboratory of Biotic Environment
and Ecological Safety in Anhui Province, Wuhu 241000, Anhui, China.
*Corresponding author E-mail: lwnie@mail.ahnu.edu.cn

Abstract.- The Sox genes of Rana tientaiensis were amplified and cloned using highly degenerate primers designed
from the conservative motif (HMG-box) of the human SRY gene. The SSCP technique was used to detect different
clones. Seven distinct Sox gene fragments were obtained from both male and female R. tientaiensis; no sexual differences were observed. Seven of these fragments (named RtSox3a, RtSox3b, RtSox3c, RtSox4, RtSox11, RtSox12, andRtSox14) exhibited 95%, 95%, 95%, 97%, 98%, 97%, and 97% similarity (respectively) to the corresponding homologous human SOX genes. The eighth fragment showed 79% and 77% similarity to the human SOX21 and SOX14
genes, as well as varying levels of similarity to other group B Sox genes. The eighth gene, provisionally named
RtSoxB14, may be a new member of the Sox gene family or a derivative of an existing Sox gene. Phylogenetic analysis suggests that the RtSox genes are highly conserved members of the SoxB, SoxC and SoxD gene groups. Sequence
analysis further illustrates that the gene Sox3 found in R. tientaiensis are duplicates of those seen in the mammalian
Sox gene family. Amino acid positions 15–19 are characteristic of each group in the Sox family.
Keywords.- Sox genes, SSCP, Rana tientaiensis, subgroup diagnosis.

Introduction
The Y chromosome-linked gene SRY is a dominant
inducer of testis development in mammals (Sinclair etal., 1990) and a founding member of a gene family withsequence homology to the High Mobility Group (HMG)
domain (Fawcett and Klymkowsky, 2004). Since discovery of the SRY gene, many members of the SOX/Sox(SRY-related HMG box) gene family have been foundthroughout the vertebrates, showing at least 60% protein
similarity to the SRY HMG domain. Genes in each subgroup show over 80% similarity. These SOX/Sox genes
have also been found to be involved in physiologicalprocesses such as sex determination and the development of the CNS, neural crest and endoderm (Bowles etal., 2000). Sox1, Sox2, Sox3 and Sox11, for instance, areexpressed mainly in the developing nervous system(Collignon et al., 1996; Pevny et al., 1998), and Sox4 isessential for heart and lymphocyte development(Schilham et al., 1996). The SOX/Sox genes have been
divided between ten subgroups, named A to J (Table 1),
not all of which occur in the same taxa (Bowles et al.,2000); groups I and J (containing sox31, sox32 andsox33), for instance, are only found in Zebrafish (Girardet al., 2001; Lunde et al., 2004).
Some amphibians have ZZ/ZW or XX/XY modesof sex determination, but most species do not have heteromorphic chromosomes, making the study of evolution and the mechanism of sex determination in theseorganisms interesting. Rana tientaiensis (2n = 26) is oneof these species without identifiable sex chromosomes© 2008 by Asiatic Herpetological Research(Guo et al., 1991). In this paper, we describe the cloningand sequencing of the eight Sox genes in Rana tientaiensis with the aim of researching the diversity and evolution of this gene family. Sequence analysis indicates thatsome of these genes in R. tientaiensis are duplicated.

Materials and Methods
Two male and two female Rana tientaiensis were captured from Taolin and Tingxi Anhui Provinces, China.
Genomic DNA was isolated from muscle tissues using
routine protocols. A pair of degenerate primers (sn1:ATGAAYGCNTTYATGGTNTGG; sn2: GGNCGRTAYTTRTARTCNGG) were designed using multiplealignments of the HGM-box sequence of SRY, corresponding to the MNAFMVW and PDYKYRP motifs
found in the HMG boxes of a wide range of Sox proteins.
PCR reactions were 30 μl in volume, including
18.75 μl ddH2O and approximately 100 ng of genomic
DNA, 1.5mM Mg2+, 120 μM dNTP, 0.3 μM/primer and1.25 μl Taq polymerase. PCR cycling conditions were 1cycle for 5 minutes at 97°C, followed by 35 cycles with
40 sec. at 94°C, 40 sec. at 53°C, 1 min. at 72°C, andfinally 72°C for 10 minutes to complete the final reaction.
Clones were genetically sequenced to detect thepositive clones with Sox DNA insertions. PCR products
were detected by 1.5% agarose gels and cloned by pMD
18-T Vector (purchased from TAKARA). Positiveclones were screened by SSCP (single-strand conforma-

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Table 1. Classification of the Sox gene family.
AB

C

DE

F

GH

I

J

SRY

Sox1
Sox2
Sox3
Sox14
Sox21
Sox25

Sox4
Sox11
Sox24
Sox22

Sox5
Sox6
Sox12
Sox13
Sox23

Sox8
Sox9
Sox10

Sox7
Sox17
Sox18

Sox15
Sox16
Sox20Sox30

Sox31

Sox32
Sox33

Figure 1. Amplified Sox gene fragments from 1: male
Rana tientaiensis, 2: female R. tientaiensis,3: Human; 4:negative control; M: DL2000 marker (TaKaRa).

tion polymorphism) analysis and sequenced with theuniversal sequencing primers on an ABI377 autosequencer. DNA sequences were analyzed using theBLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and
CLUSTALX programs. Molecular Evolutionary Genetic
Analysis (MEGA) software was used to construct thephylogenetic tree.

Results and Discussion
A 203 bp fragment of Rana tientaiensis genomic DNAwas obtained using the degenerate PCR primers listedabove. The fragment was identical to that found in thehuman genome (Fig. 1), indicating that these fragments
belong to homologous genes.
Of the 113 white clones (i.e., those with insertions),
64 were positive for the Sox insertion, as confirmed
through nucleotide analysis following genomic amplification. Eight distinct positive clones were found in bothmale and female Rana tientaiensis; there were no sexual
differences.
Seven of the eight distinct genes were named as follows: RtSox3a, RtSox3b, RtSox3c, RtSox4, RtSox11,RtSox12, and RtSox14. The amino acid sequences fromthese genes had 95%, 95%, 95%, 97%, 98%, 97%, 97%
and 97% similarity (respectively) to homologous SOX
genes in Human. These seven genes belonged to theSoxB, SoxC and SoxD subgroups, all of which lackintrons (Bowles et al., 2000). The 9th Sox gene was pro-visionally named RtSoxB14; the amino acid sequencesfrom this gene had 79% similarity to the Human SOX21
gene, 77% similarity to the Human Sox14 gene, as wellas varying levels of similarity to other group B Sox
genes. Nucleotide and putative amino acid sequences forthe eight Sox genes are listed (Fig. 2)
The amino acid sequences from the eight clones
were compared to 44 published Sox gene sequences in
GenBank, including sequences from Human (HomoSRY,
SOX1, 2, 3, 4, 7, 9, 11, 12, 14, 15, 21, 30), Mouse(MusSox1, 2, 3, 4, 7, 9, 11, 12, 14, 15, 21, 30), Gallus
gallus (GallSox2, 3, 9, 11 , 14, 21), Danio rerio(DaniSox1, 2, 4, 11), Xenopus laevis (XenopusSox2, 4,11), Takifugu rubripes (TakifuguSox1, 14b) and Eremias
breuchleyi (EbSox2, 4, 11, 12, 14 ,21) (Table 2). All
sequences were analyzed using neighbor-joining (NJ)
methods by MEGA 2.0 (Fig. 3).
Amino acid sequences between the RtSox genes
were highly conserved. Representatives of the Sox genein other species were also highly conserved with muchsimilarity between sequences. Gene duplication has
likely caused most of the diversity seen in the HMG box
superfamily, for which the Sox genes show the highest
mutation rate (Laudet et al., 1993). The high similarity
seen between the Rana and human genes in this study
are certainly indicative of gene duplication.
In the case of Sox12, amino acid sequences werenearly identical, although the 10th amino acid was N
instead of H in Human and Mouse (Fig. 2). In Sox 4, the48th amino acid was R instead of Q in Mouse, and inSox11, the 48th amino acid was D instead of N in Mouse,
Human and several other species. The high degree ofsimilarity amongst these genes suggests that they belong
to the same gene family, and may perform similar roles
amongst taxa. For example, the three highly conserved
genes in group C display overlapping expression patterns, and Sox4 and Sox11 display overlapping expression patterns in the mouse embryonic pancreas(Lioubinski et al., 2003).
According to Laudet et al. (1993), Sox4 is considered to be an early offshoot of the SRY gene in the Sox
family phylogeny. The conservative nature of Sox4
homologues in non-mammalian amniotes is interesting
because in mammals, the SRY gene exhibits rapid evolu-2008

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Table 2. Sox genes sequence in different species
Sequence

Accession number

Human sapiens

Sequence

Accession number

Mouse musculus

HomoSOX1

NP_005977

MusSox1

BAC75667

HomoSOX2

CAA83435

MusSox2

NP_035573

HomoSOX3

CAA50465

MusSox3

AAH52024

HomoSOX4

NP_003098

MusSox4

NP_ 033264

HomoSOX7

NP_113627

MusSox7

NP_035576

HomoSOX9

CAA86598

MusSox9

AAH04064

HomoSOX11

BAA88122

MusSox11

NP_033260

HomoSOX12

CAB81632

MusSox12

NP_035568

HomoSOX14

AAI06731

MusSox14

XP 284529

HomoSOX15

NP_008873

MusSox15

NP_033261

HomoSOX21

NP_009015

MusSox21

NP_808421

HomoSOX30

NP_848511

MusSox30

AAF99391

HomoSRY

AAT37462

Danio rerio

Gallus gallus
GallSox2

NP_990519

GallSox3

NP_989526

GallSox9

DaniSox1

NP_001032751

DaniSox2

NP_001002483

DaniSox4

BC065354

DaniSox11

CAB87379

GallSox11

NP_990518

Xenopus laevis

GallSox14

NP_990092

XenopusSox2

AAB62821

GallSox21

BAA77266

XenopusSox3

P55863

XenopusSox11

Q91731

EbSox2

DQ067423

Takifugu rubripes

EbSox4

DQ067426

TakifuguSox1

AAQ18494

EbSox11

DQ067427

TakifuguSox14b

AAQ18499

EbSox12

DQ067428

EbSox14

DQ067430

EbSox21

DQ067433

Eremias breuchleyition, possibly caused by Y-linked inheritance (Tuckerand Lundrigan, 1993). The limited diversity of this genein non-mammalian taxa may be due to the retention ofan ancient conserved function.
It is likely that Sox3 is the closest homologue to theSry gene based on nucleotide sequence data.
Furthermore, Sox3 is located on the mammalian X chromosome, and is highly similar to SRY (Sinclair et al.,1990), suggesting that they arose through duplication of
their common ancestral during differentiation of the sex
chromosomes (Collignon et al., 1996; Foster andGraves, 1994; Stevanovic et al., 1993). This is significant because the X and the Y chromosomes are thought
to have arisen from a common “autosome” ancestor in
the lineage that gave rise to mammals (Wright et al.,1993). Further examination of Sox genes in lower vertebrates, Prototheria (monotremes) and Metatheria (mar-supials) will be necessary to establish the evolutionary
origins of Sry. The three conservative Sox3 genes (sometimes found within the same species or individual) canbe identified by the following variations in amino acidsequence: RtSox3a has an F at position 46 and an H atposition 58; RtSox3b has an F at position 46 and an M at
position 58; RtSox3c has an I on position 46 and an M
on position 58.
The RtSoxB14 is unique among the Sox genes inhaving the amino acid sequence VITEH at positions 15–19, a K at position 44 and an S at position 50. In the phylogenetic tree (Fig. 3), although RtSoxB14 was moreclosely related to subgroup B than other groups, the origin and classification of this gene is ambiguous.
Previously, genes encoding proteins with more than
60% similarity to the SRY HMG domain have been
named Sox (SRY box) genes, and Sox genes with at least

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Figure 2. (A) Alignment of nucleotide sequences (above), (B) Alignment of amino acid sequences (Opposite page, top),(C) Percentage amino acid similarity between Rana tientaiensis Sox clones as determined by the sequence identity
matrix function in Bioedit (Opposite page, bottom).

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157

Figure 2 (continued).

80% similarity have been placed in the same subgroup.
However, as more and more Sox genes are identified, theability to accurately classify these genes decreases. For
example, the genes sox30 and Ce-soxj have only 46%
and 48% similarity to Human SRY HMG. Bowles (2000)
attempted to alternatively diagnose the gene family bypossession of the amino acid sequence “RPMNAF”,
which is highly conserved across genes, however, thissequence was also found in the taxonomically ubiquitous gene cic, so the sequence “RPMNAFMVW” wasprovided as a replacement (Bowles et al., 2000).
Now that a sequence identifying the Sox gene family has been identified, can sequences be found to characterize the Sox subgroups? Following analysis of theavailable sequences (Figure 4), it appears that positions
15–19 may have be useful for this purpose. The
sequence “MAQE(D)N” may work for group B (exceptfor HomoSOX3 and MusSox3), “IMEQS” for group C,“IMEQW” (for Sox12) or “ILQAF” (for Sox5, Sox6 andSox13) for group D, “LADQY” for group E, “LAVQN”(for Sox7) or “LAQQN” (for Sox17 and Sox18) forgroup F, “MAQQN” for group G and “LAKAN” forgroup H. RtSoxB14 can be separated from the remaining
Sox genes by the unique sequence VITEH, although inmammals (HomoSOX3 and MusSox3) it is changed to“MALEN”, like the sequence for SRY. This further supports a close relationship between the SOX3 and SRYgenes.

Several Sox genes appear to have been duplicatedin Rana tientaiensis: RTSox3a, RTSox3b and RTSox3c.
Similar duplications in amphibians are uncommon, butthey are more frequently encountered in teleosts: Sox1,4, 9 and 14 has been duplicated in the sea bass (Malykaet al., 2003) and Sox21 has been duplicated in theZebrafish (Argenton et al., 2004). The “duplicationdegeneration-complementation” model developed byForce et al. (1999) suggests that the partition of ancestral
subfunctions is an important mechanism leading to thepreservation of multiple gene copies; this model predicts
that the probability of gene conservation will be higher
in more complex genes with a larger number of subfunctions (Force et al. 1999). Most duplicate genes in Ranatientaiensis have silent mutations (except RtSox3a,which has an encoded amino acid mutation), but itwould appear that the sequences are under selective
pressure and may indeed perform separate subfunctions.
Future studies investigating Sox genes in Rana tientaiensis will likely provide much insight into duplicate genes.

Acknowledgments
We are grateful to the reviewers for numerous valuablesuggestions on the manuscript. This research was supported by National Natural Science Foundation of China(No. 30640048, No. 30770296), the Natural Science
Foundation of Anhui Education Department(KJ2007A022).

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Figure 3. Phylogenetic analysis of Sox/SOX gene family

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Submitted: 25 October 2006
Accepted: 23 September 20072008