猪bmp15基因报告载体的构建及其特异性

为了研究骨形态发生蛋白15(bmp15)基因的表达和调控特性,通过克隆猪bmp15基因2.2 kb启动子片段,构建pBMP15-EGFP报告载体,实现监测干细胞向类卵母细胞分化的过程。以猪卵巢组织和中国仓鼠卵巢细胞(CHO)、成肌细胞(C2C12)、猪羊水干细胞(pAFSC)为材料,通过RT-PCR、免疫荧光、细胞转染、显微注射检测bmp15组织特异性表达,并且通过单层细胞诱导检测该基因体外示踪类卵母细胞获得过程的能力。RT-PCR结果显示bmp15在猪的卵巢组织中特异表达,在CHO中表达,而在C2C12和pAFSC中不表达。卵巢组织切片免疫荧光检测结果显示bmp15表达于卵泡发育的各个阶段。瞬时转染不同细胞发现启动子只在CHO中有活性,而在C2C12和pAFSC中均无活性。显微注射重组质粒片段结果显示增强绿色荧光蛋白(Enhanced Green Fluorecence Protein,EGFP)在卵母细胞体外成熟18 h启动表达,并能够持续至4-细胞期胚胎。单层细胞诱导结果显示诱导12 d的pAFSC出现携带EGFP的圆形细胞团。说明bmp15具有表达特异性和示踪干细胞诱导分化为类卵母细胞的潜能。     

Structure and expression of the mouse prealbumin gene

We cloned a genomic DNA fragment which covers the entire sequence of the mouse prealbumin gene and then studied the structure. The coding regions are separated into four exons by three introns, and these numbers, the sizes of the exons and the relative sites of the exon-intron junctions are all in complete agreement with those determined for the human gene. The sequences of four exons can be aligned perfectly with that of the previously determined mouse prealbumin cDNA. In addition to the exon regions, we found two highly conserved DNA regions between the mouse and human prealbumin genes, one in the 5'-flanking region of the gene and the other in the 3' end region of the first intron. These DNA regions contain several consensus glucocorticoid receptor-binding site sequences, and the latter also contains an enhancer sequence present in the immunoglobulin kappa-chain joining-constant kappa intron. RNA hybridizing to the mouse prealbumin cDNA was detected in the extracts from liver, brain, and kidney, but was not detected in testes, spleen, or heart. Little change was caused in the level of prealbumin mRNA in the liver by administration of dexamethasone to mice.     

Structure and sequence of the Drosophila zeste gene.

The zeste gene of Drosophila affects the expression of other genes in a manner that depends on the homologous pairing of the chromosomes bearing the target gene. Zeste mediates transvection effects, the ability of one gene to control the expression of its homologous copy on another chromosome. We have determined the structure of the zeste gene and several mutants bearing partial deletions and the sequence of the z+, z1, zop6 and z11G3 alleles. The predicted zeste protein has an unusual structure including runs of Gln, Ala and alternating Gln Ala. Contrary to expectations the z1, zop6 and z11G3 mutations can each be attributed to single amino acid changes. The analysis of the mutants suggests that the zeste gene product is required for normal expression of at least some genes and we argue that za mutants may have residual function.     

Structure and sequence of the human alpha-L-iduronidase gene.

In humans, a deficiency of the lysosomal hydrolase alpha-L-iduronidase (IDUA;EC 3.2.1.76) results in the lysosomal storage of the glycosaminoglycans heparan sulfate and dermatan sulfate, thereby causing the lysosomal storage disorder mucopolysaccharidosis type I. The gene for IDUA is split into 14 exons spanning approximately 19 kb. We report the sequence of two non-contiguous segments of the IDUA gene, one 1.8-kb segment containing exons 1 and 2 and surrounding sequences and a second segment of 4.5 kb containing the last 12 exons. The potential promoter for IDUA has only GC box type consensus sequences consistent with a housekeeping promoter and is bounded by an Alu repeat sequence. The first two exons of IDUA are separated by an intron of 566 bp, then there is a large intron of approximately 13 kb, and the last 12 exons are clustered within 4.5 kb. No consensus polyadenylation signal was found in the 3' untranslated region, although two variant polyadenylation signals are proposed.     

Coding sequence and flanking regions of the mouse vimentin gene

Summary Using a polyclonal antibody, a cDNA clone coding for part of mouse vimentin was identified in a gt11 expression library. DNA from this clone was used to screen a genomic library from Ehrlich Ascites Tumor cells for the mouse vimentin gene. A clone was found which contained the whole coding sequence and a large part of the 5- and 3-untranslated sequences. It was used to prepare a construct equivalent to a full-length cDNA clone. Extensive homologies to the vimentin sequence from other species were found for the coding and 3-untranslated sequences and the promoter region.     

Structure of the mouse C-reactive protein gene

A genomic DNA clone corresponding to the mouse C-reactive protein (CRP) has been isolated and characterized. The mouse CRP gene is 1.9-kilobase pairs in length and contains a single intron of 213-base pairs which interrupts the codon for the 2nd amino acid residue of the mature CRP protein. We compared nucleotide sequences of the mouse and human CRP genes and discussed structures of possible regulatory sequences. With this characterization, the isolation and sequence analyses of a set of mouse and human pentraxin genes, i.e. CRP and serum amyloid P component genes is not complete.     

The nucleotide sequence of the mouse immunoglobulin epsilon gene: comparison with the human epsilon gene sequence

We have determined the nucleotide sequence of the immunoglobulin epsilon gene cloned from newborn mouse DNA. The epsilon gene sequence allows prediction of the amino acid sequence of the constant region of the epsilon chain and comparison of it with sequences of the human epsilon and other mouse immunoglobulin genes. The epsilon gene was shown to be under the weakest selection pressure at the protein level among the immunoglobulin genes although the divergence at the synonymous position is similar. Our results suggest that the epsilon gene may be dispensable, which is in accord with the fact that IgE has only obscure roles in the immune defense system but has an undesirable role as a mediator of hypersensitivity. The sequence data suggest that the human and murine epsilon genes were derived from different ancestors duplicated a long time ago. The amino acid sequence of the epsilon chain is more homologous to those of the gamma chains than the other mouse heavy chains. Two membrane exons, separated by an 80-base intron, were identified 1.7 kb 3' to the CH4 domain of the epsilon gene and shown to conserve a hydrophobic portion similar to those of other heavy chain genes. RNA blot hybridization showed that the epsilon membrane exons are transcribed into two species of mRNA in an IgE hybridoma.