X-连锁迟发性脊椎骨骺发育不良基因剪接受体突变对mRNA加工的影响

X-连锁迟发性脊椎骨骺发育不良（spondyloepiphyseal dysplasia tarda, SEDL）是一种少见的由SEDL基因突变引起的骨软骨发育障碍性疾病,病变主要累及腰椎和近端承重大关节。为研究SEDL基因剪接受体突变(IVS2 -2A→C)对mRNA加工的影响,从该突变所致SEDL患者,以及健康对照者外周血中提取总RNA,逆转录合成cDNA, 以此为模板进行聚合酶链式反应（polymerase chain reaction, PCR）,对PCR扩增产物采用双向直接测序和非变性聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis, PAGE)方法进行分析。测序结果发现IVS2-2A→C突变患者的一种cDNA外显子2与外显子4直接拼接,显示外显子3全部丢失;另一种cDNA外显子1与外显子4拼接,显示外显子2和外显子3均缺失;在健康对照者也发现了外显子2缺失的cDNA。PAGE发现患者和对照者都存在两种RT-PCR产物,长度分别为567bp、425bp以及679bp、537bp,证实了测序结果。这说明SEDL基因第二内含子剪接受体突变（IVS2-2A→C）导致其外显子3在mRNA加工过程中全部丢失,由于SEDL基因的翻译起始位点位于外显子3,它的缺失可能使生成的mRNA不能被翻译,从而引起SEDL发生;外显子2位于5′ UTR,它的缺失提示SEDL基因存在选择性剪接,正常人也存在缺失外显子2的cDNA,说明这种选择性剪接对临床表型的影响似乎并不大,它对基因表达水平和表达调控是否有影响还需要进一步研究。     

PC-1增强受体酪氨酸激酶家族成员EphA3在前列腺癌细胞中的转录

为了研究前列腺癌相关基因(prostate and colon gene 1, PC-1）对受体酪氨酸激酶家族分子EphA3表达的影响,用RT-PCR、实时PCR和Western印迹检测表达不同水平PC-1的前列腺癌细胞系LNCaP和C4-2中EphA3的表达情况. 发现PC-1可诱导EphA3基因表达上调. 采用荧光素酶实验检测PC-1对于EphA3启动子转录活性的影响,结果显示,PC-1对转录起始位点上游916 bp的启动子活性没有影响,而可增强转录起始位点上游2011 bp启动子的活性.对EphA3启动子－916 bp～－2　011 bp区域进行生物信息学分析,结果显示,此区域包含HSF、NF-1、Nkx-2、SP1和GATA-1等多种转录因子结合位点.实验结果表明,PC-1可通过影响EphA3启动子诱导EphA3基因高表达,其调控区域位于转录起始位点上游－2　011　bp至－916 bp之间,提示PC-1可能通过影响一些结合于此区域的转录因子来影响EphA3启动子的转录活性.     

Phospholipase D is required in the signaling pathway leading to p38 MAPK activation in neutrophil-like HL-60 cells, stimulated by N-formyl-methionyl-leucyl-phenylalanine.

Human acute myelogenous leukemia cells (HL-60 cells) can be induced to differentiate to neutrophils by exposure to dibutyryl-cyclic AMP. The differentiation of HL-60 cells allowed the mitogen-activated protein kinases p38 and p44/p42 to be rapidly and transiently activated upon stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Western blot analysis using phosphospecific p38 and p44/p42 mitogen-activated protein kinase antibodies showed that increasing concentrations of ethanol or 1-butanol but not 2-butanol (0.05-0.5%) inhibited fMLP-induced p38 activation but did not inhibit p44/p42 activation. These data indicated that activation of phospholipase D (PLD) was required for activation of p38 but not p44/p42. We compared the effect of fMLP with those of tumor necrosis factor alpha (TNF alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We found that ethanol did not inhibit p38 phosphorylation upon stimulation with either GM-CSF or TNF alpha. These results suggested that in cells stimulated with fMLP, PLD was upstream of p38. To further test the involvement of PLD, we used antisense inhibition of human PLD1 expression. Treatment with antisense oligonucleotides inhibited p38 but not p44/p42 phosphorylation. These data supported a role for human PLD1 in fMLP-induced p38 activation in neutrophil-like HL-60 cells. In addition, the results obtained with TNF alpha and GM-CSF demonstrated that p38 activation occurred independently of PLD activation.     

水稻OsGSTL1启动子的分离和表达分析

从水稻基因组文库中筛选得到一个水稻GST基因,命名为OsGSTL1.半定量RT-PCR分析表明OsGSTL1基因的表达不受绿磺隆、乙烯利、脱落酸、水杨酸和茉莉酸甲酯的诱导,因此该基因可能与植物抗逆性无关.为了研究OsGSTL1启动子在植物体内的表达特性,将OsGSTL1起始位点5＇端上游不同长度的调控序列与报告基因GUS融合,并在洋葱表皮瞬间表达和拟南芥中稳定表达.研究表明：在洋葱表皮细胞中,160bp及更长的上游调控序列均能启动GUS基因的表达;而在转基因拟南芥中,含有2155 bp的上游序列的PGZ2.1：：GUS具有时空表达的特性,在转基因的早期幼苗中GUS基因在子叶中特异性表达,但在根中没有表达;而在幼苗生长的后期,根、茎、叶中都有少量的表达.但包含1 224 bp的上游序列的PGZ1.2：：GUS却表现为组成型表达的特性.由此推测,OsGSTL1启动子启动的基因表达可能与幼苗的营养代谢相关;而OsGSTL1启动子的时空表达相关元件可能位于OsGSTL1翻译起始位点5＇端上游-2155 bp至-1224 bp范围内.     

A conditional U5 snRNA mutation affecting pre-mRNA splicing and nuclear pre-mRNA retention identifies SSD1/SRK1 as a general splicing mutant suppressor.

A combination of point mutations disrupting both stem 1 and stem 2 of U5 snRNA (U5AI) was found to confer a thermosensitive phenotype in vivo. In a strain expressing U5AI, pre-mRNA splicing was blocked before the first step through an inability of the mutant U5 snRNA to efficiently associate with the U4/U6 di-snRNP. Formation of early splicing complexes was not affected in extracts prepared from U5 snRNA mutant cells, while the capacity of these extracts to splice a pre-mRNA in vitro was greatly diminished. In addition, significant levels of a translation product derived from intron containing pre-mRNAs could be detected in vivo. The SSD1/SRK1 gene was identified as a multi-copy suppressor of the U5AI snRNA mutant. Single copy expression of SSD1/SRK1 was sufficient to suppress the thermosensitive phenotype, and high copy expression partially suppressed the splicing and U4/U6.U5 tri-snRNP assembly pheno-types. SSD1/SRK1 also suppressed thermosensitive mutations in the Prp18p and U1-70K proteins, while inhibiting growth of the cold sensitive U1-4U snRNA mutant at 30 degrees C. Thus we have identified SSD1/SRK1 as a general suppressor of splicing mutants.     

ACYP1 gene possesses two alternative splicing forms that induce apoptosis

In human cell lines two products of the ACYP1 gene were detected by RT-PCR. In addition to the expected amplicon corresponding to the CT form of acylphosphatase (320 bp) a second unexpected one (400 bp) was characterized as the result of an alternative splicing in which an extra 79 bp long exon is inserted between the two known exons. This new product, indicated as CTsv, was cloned and expressed. We performed the ectopic expression of the two alternative splicing forms. Both CT and CTsv products were able to induce a proapoptotic effect when expressed in HeLa cell line, despite the fact that the CTsv protein did not show any acylphosphatase activity.