Isolation and sequence analysis of sox genes from lizard Eremias 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.     

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.     

Related function of mouse SOX3, SOX9, and SRY HMG domains assayed by male sex determination

Sox genes encode proteins related to each other, and to the sex determining gene Sry, by the presence of a DNA binding motif known as the HMG domain. Although HMG domains can bind to related DNA sequences, Sox gene products may achieve target gene specificity by binding to preferred target sequences or by interacting with specific partner proteins. To assess their functional similarities, we replaced the HMG box of Sry with the HMG box of Sox3 or Sox9 and tested whether these constructs caused sex reversal in XX mice. Our results indicate that such chimeric transgenes can functionally replace Sry and elicit development of testis cords, male patterns of gene expression, and elaboration of male secondary sexual characteristics. This implies that chimeric SRY proteins with SOX HMG domains can bind to and regulate SRY target genes and that potential SRY partner factor interactions are not disrupted by HMG domain substitutions. genesis 28:111-124, 2000.     

The Sry-related HMG box-containing gene Sox6 is expressed in the adult testis and developing nervous system of the mouse.

We have cloned and sequenced a full-length cDNA for the HMG box-containing, SRY-related gene Sox6 from mouse. The deduced protein sequence of Sox6 has considerable homology with that of the previously determined Sox5 sequence. It seems likely that these genes have diverged more recently than other members of the SOX gene family, although the two genes map to different chromosomes in the mouse. In common with Sox5, Sox6 is highly expressed in the adult mouse testis and the HMG domains of both proteins bind to the sequence 5'-AACAAT-3'. This suggests that the two genes may have overlapping functions in the regulation of gene expression during spermatogenesis in the adult mouse. However, Sox6 may have an additional role in the mouse embryo, where it is specifically expressed in the developing nervous system.     

池蝶蚌Sox2 基因在不同组织及不同月龄精巢中的表达

Sox 基因家族在胚胎发育过程和性别分化中起重要作用, 为研究池蝶蚌中Sox 基因的功能, 以人SRY基因HMG-box 保守区的序列设计简并引物, 以雌、雄池蝶蚌基因组DNA 和精巢cDNA 为模板进行扩增, 获得了2 个不完全相同的序列, 分别为DNA-HMG1、DNA-HMG2 和cDNA-HMG, 长度均为220 bp, 编码73个氨基酸。与人等物种Sox1、Sox2、Sox3 及Sox14 有很高的同源性, 雌雄个体之间没有序列差异性。采用RACE-PCR 扩增获得了池蝶蚌性腺Sox2 部分cDNA 片段, 长度为1774 bp, 该序列核苷酸与欧洲帽贝的SoxB和人类的Sox2 的同源性最高; 在部分开放阅读框249 个氨基酸残基中, 具有Sox 家族典型的HMG-box 结构域, 与人类、小鼠、原鸡和斑马鱼等Sox2 的HMG-box 同源性为98%。为了解该基因在各组织中的表达情况,采用实时荧光定量PCR 方法分析了外套膜、闭壳肌、鳃、肠、肝、肾、精巢和卵巢在内的8 种组织hs-Sox2的表达情况, 结果显示, hs-Sox2 基因在8 种组织中均有表达, 其中在肾脏中的表达量最高, 其次是肠与闭壳肌, 在雄性性腺中的表达量明显高于雌性性腺, 在肝脏中的表达量最低; 为了解hs-Sox2 在不同性腺发育时期的表达情况, 采用实时荧光定量PCR 方法分析了5 个不同月龄的精巢组织中hs-Sox2 的表达情况, 结果显示在39 月龄性腺的表达量最高, 其次是16 月龄性腺, 63 月龄蚌中的表达量最少。以上结果表明, hs-Sox2 基因可能参与了池蝶蚌精巢的发育及功能的维持。       

野生中国大鲵Sox基因的克隆与序列分析

Sox基因家族是一类编码转录因子的基因家族,其产物具有一个HMG-box基序保守区,调节动物的性别决定与分化过程,并参与多种器官的发育。参照人SRY基因HMG-box保守区序列及有关文献,设计一对简并引物,PCR扩增了野生雌性中国大鲵（Andrias davidianus）基因组DNA,结果得到了一条长约220 bp的产物片段;菌落PCR扩增结果表明,克隆后的白色菌落中90%以上都是阳性克隆;通过SSCP技术筛选到3种有差异的阳性克隆,进行测序,获得了3个Sox基因：Sox4a、Sox4b与Sox14。对所得序列进行序列比对和聚类分析,结果显示该基因在分子进化上具有高度的保守性。对中国大鲵Sox基因的研究,目前国内外尚未见报道。为研究中国大鲵性别决定机制以及Sox基因进化提供了分子遗传学资料。     

两种淡水鱼类的Sox基因

ＴＨＥＳＯＸＧＥＮＥＳＩＮＴＷＯＳＰＥＣＩＥＳＯＦＦＲＥＳＨＷＡＴＥＲＦＩＳＨＥＳ两种淡水鱼类的Ｓｏｘ基因ＫｅｙｗｏｒｄｓＳｅｘ,Ｉｎｔｒｏｎ,Ｆｒｅｓｈｗａｔｅｒｆｉｓｈ关键词性别内含子淡水鱼类ＴｈｅＳｒｙｉｓｔｈｅｓｅｘｄｅｔｅｒｍｉｎｉ...     

Sox15 is up regulated in the embryonic mouse testis

The mammalian sex determining region on the Y chromosome, SRY, is the founding member of the SOX gene family. SOX genes share a common DNA-binding motif termed the HMG box and have diverse roles in vertebrate embryonic development and tissue differentiation. Sox15 expression was analysed during mouse embryogenesis by whole-mount in situ hybridisation and Real Time RT-PCR. Sox15 was found to be expressed in developing mouse gonads from 11.5 dpc to 13.5 dpc with a peak of expression at 12.5 dpc. Expression was approximately twice as high in the male gonad as in the female gonad.     

Sox genes in grass carp (Ctenopharyngodon idella) with their implications for genome duplication and evolution

The Sox gene family is found in a broad range of animal taxa and encodes important gene regulatory proteins involved in a variety of developmental processes. We have obtained clones representing the HMG boxes of twelve Sox genes from grass carp (Ctenopharyngodon idella), one of the four major domestic carps in China. The cloned Sox genes belong to group B1, B2 and C. Our analyses show that whereas the human genome contains a single copy of Sox4, Sox11 and Sox14, each of these genes has two co-orthologs in grass carp, and the duplication of Sox4 and Sox11 occurred before the divergence of grass carp and zebrafish, which support the "fish-specific whole-genome duplication" theory. An estimation for the origin of grass carp based on the molecular clock using Sox1, Sox3 and Sox11 genes as markers indicates that grass carp (subfamily Leuciscinae) and zebrafish (subfamily Danioninae) diverged approximately 60 million years ago. The potential uses of Sox genes as markers in revealing the evolutionary history of grass carp are discussed.     

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.     

Sox8基因HMG盒区内含子剪接位点分析

Sox基因参与广泛的发育调控过程.为了确定Sox8基因HMG盒区内含子的大小及剪接位点,通过计算机分析本室克隆得到的泥鳅Sox8基因包括HMG盒区在内的一段基因组序列,推测在HMG盒区可能存在一个内含子,进一步通过RT-PCR方法,克隆泥鳅Sox8基因HMG盒区cDNA片段,与基因组序列比较分析,确认在泥鳅Sox8基因的HMG盒区存在一个内含子,并确定了该内含子的序列及剪切位点.同时,比较分析了人和泥鳅Sox8在HMG盒区的内含子的剪切位点.结果显示,Sox8基因HMG盒区内含子剪切位点在进化上是保守的。 Abstract:Sox genes have diverse roles in developing processes,it was inferred there may be one intron in the HMG box by analyzing the genomic DNA sequence of the paramisgurnus dabryanus Sox8 gene.RT-PCR was used to verify the intron and its splicing site.First strand of the cDNA reverse-transcribed from liver RNA was amplified using the primers flanking the HMG box.RT-PCR products were cloned into the pUC18 plasmid and sequenced.The eomparison between the cDNA and genomic DNA sequence revealed the splicing site of the intron existing in the HMG-box of the paramisgurnus dabryanus Sox8 gene.We also compared the splicing site of the intron in the HMG-box of Sox8 gene between paramisgurnus dabryanus and human.These results suggest that the splicing site in the HMG-box of Sox8 gene was conserved.