Isolation and sequence analysis of <Emphasis Type="Italic">Sox</Emphasis> genes from lizard <Emphasis Type="Italic">Eremias</Emphasis> multiocellata

The Sox (SRY-related high-mobility-group box) family of genes shares a conserved HMG box and is involved in a diverse range of developmental processes and sex determination in vertebrates. Twenty Sox genes are present in the genomes of humans and mice, but far less is known about the Sox gene family in reptiles. Using two pairs of highly degenerate primers designed from a multiple alignment of Sox amino acid sequences in several species, different positive clones were obtained from male and female Eremias multiocellata, a viviparous lizard which is subject to TSD (temperature-dependent sex determination). These clones were sequenced and identified. They are members of the SoxB (Sox2, Sox14), SoxC (Sox11, Sox12) and SoxE (Sox9a, Sox9b, Sox10) groups. No sex-specific differences were observed. Based on the amino acid sequence similarities, the phylogenetic analysis was carried out and these genes clustered with their orthologues. In addition, we found the gene duplication in E. multiocellata, it may be a mechanism to produce new functional genes.     

鲤鱼中Sox9b基因的克隆和表达

Sox9基因是一个重要的转录调控因子,参与性别决定及软骨等多种组织和器官的发育过程。本研究利用简并引物扩增鲤鱼基因组DNA,首次发现在鲤鱼中存在两种形式的Sox9基因。二者在保守盒区编码的氨基酸序列相同,并都存在一个内含子,但内含子序列差异很大,分别长704bp和616bp。在此基础上采用RACE技术克隆了鲤鱼Sox9b基因的5’端和3’端,通过拼接获得了2447bp的全长cDNA序列。编码428个氨基酸。其中96—174位共79个氨基酸为HMG保守盒。将鲤鱼Sox9b基因与三刺鱼等九种动物的氨基酸序列相比较发现。它们的同源性高达75％以上,显示soz9基因在进化中较保守。应用半定量RT—PCR技术对成体鲤鱼不同组织中Sox9b基因的表达进行了分析。结果表明该基因广泛表达,尤以脑及精巢中表达最为丰富。     

鲤鱼中Sox9b基因的克隆和表达

Sox9基因是一个重要的转录调控因子,参与性别决定及软骨等多种组织和器官的发育过程。本研究利用简并引物扩增鲤鱼基因组DNA,首次发现在鲤鱼中存在两种形式的Sox9基因。二者在保守盒区编码的氨基酸序列相同,并都存在一个内含子,但内含子序列差异很大,分别长704bp和616bp。在此基础上采用RACE技术克隆了鲤鱼Sox9b基因的5’端和3’端,通过拼接获得了2447bp 的全长cDNA序列,编码428个氨基酸。其中96-174位共79个氨基酸为HMG保守盒。将鲤鱼Sox9b基因与三刺鱼等九种动物的氨基酸序列相比较发现,它们的同源性高达75%以上,显示Sox9 基因在进化中较保守。应用半定量RT-PCR技术对成体鲤鱼不同组织中Sox9b基因的表达进行了分析,结果表明该基因广泛表达,尤以脑及精巢中表达最为丰富。     

The SOX gene family: function and regulation in testis determination and male fertility maintenance

The Sox (Sry-type HMG box) genes encode a group of proteins characterized by the existence of an SRY (sex-determining region on Y chromosome) box, a 79 amino acid motif that encodes an HMG (high mobility group) domain which can bind and bend DNA, which is the only part in SRY that is conserved between species. The Sox gene family functions in many aspects in embryogenesis, including testis development, CNS neurogenesis, oligodendrocyte development, chondrogenesis, neural crest cell development and other respects. The Sox gene family was originally identified through homology with Sry. The Sry gene is the mammalian testis-determining gene. It functions to open the testis determination pathway directly and close the ovary pathway indirectly. Sry and Sox9 are the most important two genes expressed during testis determination. Besides, researchers have found that Sox8 and Sox9 have functions in the male fertility maintenance after birth. In this review, information was evaluated from mouse or from human if not mentioned otherwise.     

大绿蛙6个Sox基因的克隆及进化讨论

Sox基因家族编码一类转录调控因子,他们参与到个体发育的许多过程,如中枢神经系统的形成、性别分化、骨和淋巴细胞的发育等.许多动物体内都检测到Sox基因,基于序列及其结构特点,Bowles等将其划分为10个族.本文采用简并PCR技术,扩增了大绿蛙Sox基因的HMG-box保守区,经SSCP及序列分析,获得6个基因并分别命名为:RlSox3a、RlSox3b、RlSox3c、RlSox11、RlSox14 和RlSox21.所获序列无性别差异,经系统发生分析发现分属于大绿蛙Sox家族的B 和C 亚族.其中RlSox3基因出现多拷贝,为Sox基因家族进化的DDC(duplication-degeneration-complementation)模式提供了一定的分子证据.结合GenBank中已登录的31个Sox基因氨基酸序列,使用MEGA 3.0软件构建NJ (neighbor-joining) 系统发生树,讨论了Sox基因的系统发生历程.     

Cloning and functional analysis of the Sry-related HMG box gene, Sox18

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii) an intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor -2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue.     

人类性别决定和性别分化研究进展

SRY基因在人类性别分化中起着关键作用,目前研究认为SRY仅是涉及性别决定过程的基因之一,其他基因和SRY相关基因SOX9,抗副中肾激素基因AMH,编码缁类因子的基因SF1,X－连锁的DAX基因,wilm‘s肿瘤抑制基因WT1等基因都参与了人类性腺分化和发育,本文拟就人类性别决定基因的研究进展及其与人类性别分化的关系作一综述。     

Characterisation and expression of Sox9 in the Leopard gecko, Eublepharis macularius

Since the discovery of the sex-determining gene, Sry, a number of genes have been identified which are involved in sex determination and gonadogenesis in mammals. Although Sry is known to be the testis-determining factor in mammals, this is not the case in non-mammalian vertebrates. Sox9 is another gene that has been shown to have a male-specific role in sex determination, but, unlike Sry, Sox9 has been shown to be involved in sex determination in mammals, birds, and reptiles. This is the first gene to be described that has a conserved role in sex determination in species with either chromosomal or environmental sex-determining mechanisms. Many reptiles do not have sex chromosomes but exhibit temperature-dependent sex determination (TSD). Sox9 has been shown to be expressed in both turtle and alligator during gonadogenesis. To determine if Sox9 also has a role in a gecko species with TSD, we studied gonadal expression of Sox9 during embryonic development of the Leopard gecko (Eublepharis macularius). Gecko Sox9 was found to be highly conserved at the nucleotide level when compared to other vertebrate species including human, chick, alligator, and turtle. Sox9 was found to be expressed in embryos incubated at the male-producing temperature (32.5 degrees C) as well as in embryos incubated at the female-producing temperatures (26 and 34 degrees C), Northern blot analysis showed that Sox9 was expressed at both temperatures from morphological stages 31 to 37. mRNA in situ hybridisation on isolated urogenital systems showed expression at both female- and male-producing temperatures up to stage 36. After this stage, no expression was seen in the female gonads but expression remained in the male. These data provide further evidence that Sox9 is an essential component of a testis-determining pathway that is conserved in species with differing sex-determining mechanisms.