Immunological cross-reactivity between H-2Dk product and DTIC-treated H-2d lymphoma

The experiments reported here concern the characterization by techniques of in vitro cell-mediated immunity of the antigens induced by 5-(3,3' dimethyl-1-triazine)-imidazole-4-carboxamide (DTIC) on L1210, a chemically-induced lymphoma of DBA/2 mice (H-2d). This series of experiments with the DTIC-treated L1210 tumour show the presence of an 'H-2D'-like antigen which resembles the Dk gene product/s of the H-2k haplotype.     

IMMUNOLOGICAL CROSS-REACTIVITY BETWEEN H-2DK PRODUCT AND DTIC - TREATED H - 2d LYMPHOMA

The experiments reported here concern the charcterization by techniques of in vitro cell-mediated immunity of the antigens induced by 5-(3,3'dimethyl-1-triazine)-imidazole-4-carboxamide (DTIC) on L1210, a chemically-induced lymphoma of DBA/2 mice (H-2^d). This series of experiments with the DTIC-treated L1210 tumour show the presence of an H-2D'-like antigen which resembles the D^k gene product/s of the H-2^k haplotype.     

Uterine expression of alternatively spliced mRNAs of mouse splicing factor SC35 during early pregnancy

RNA differential display was applied to identify genes critical for the establishment of pregnancy in the mouse. One of the gene fragments identified was homologous to human SC35 splicing factor; the mouse counterpart had not then been cloned. To obtain the full cDNA sequence of the mouse gene, a cDNA library was screened and four positive clones were fully analysed. Sequencing analysis indicated that we had cloned alternatively spliced mRNA species of mouse SC35 splicing factor. A map of splicing structure for this gene's pre-mRNA was then proposed and region-specific mRNA species were tested on Northern blots. This analysis indicated that the overall expression level of SC35 mRNA was much higher in implantation sites than in inter-implantation sites in the mouse uterus during early pregnancy. The expression of alternatively spliced mRNAs for SC35 was differently regulated both during early pregnancy and by steroid hormones. Embryo-derived factors were also implicated in the up-regulation of SC35 mRNA at implantation sites. These results demonstrate, for the first time, that an essential splicing factor is regulated in a complex manner during implantation in the mouse uterus. Hence, its correct regulation could be important for the success of pregnancy.     

Unscheduled expression of capsid protein IIIa results in defects in adenovirus major late mRNA and protein expression

Adenovirus gene expression is to a large extent regulated at the level of alternative RNA splicing. For example, in the major late region 1 (L1) unit, a common 5' splice site can be joined to two alternative 3' splice sites, resulting in the formation of the so-called 52,55K (proximal 3' splice site) or the IIIa (distal 3' splice site) mRNAs. Whereas, the 52,55K mRNA is expressed both early and late during infection, the IIIa mRNA is strictly confined to the late phase of the infectious cycle. We have previously shown that IIIa mRNA splicing is subjected to a tight viral control of IIIa 3 splice site usage. In an attempt to determine why adenovirus uses elaborate mechanisms to confine IIIa mRNA production to the late phase of infection, we characterized the phenotype of a recombinant adenovirus expressing the IIIa protein from an inducible tetracycline regulated gene cassette. The results show that expression of the IIIa protein during the early phase of infection results in a significant reduction in late viral protein synthesis and a moderate block to viral DNA replication. Interestingly, unscheduled IIIa protein expression resulted in a perturbation of the accumulation of alternatively spliced L1 mRNAs. Thus, 52,55K mRNA accumulation was inhibited while no effects on endogenous IIIa mRNA expression was detected.     

An alternatively-spliced mRNA in the carboxy terminus of the neurofibromatosis type 1 (NF1) gene is expressed in muscle

The gene for neurofibromatosis type 1 (NF1) was identified bypositional cloning and found to contain two alternatively splicedexons. The first described alternatively spliced exon (exon23a) is located within the GAP-related domain of the gene andinserts an additional 63 nucleotides into the NF1 mRNA. Thesecond alternatively spliced exon (exon 48a) is located nearthe extreme carboxy terminus of the gene and inserts an additional54 nucleotides into the mRNA. This second isoform, termed 3'ALT,was originally detected while screening a fetal brain cDNA library.Examination of its expression by reverse-transcribed RNA PCRdemonstrates high level of expression in cardiac muscle, skeletalmuscle and smooth muscle. Trace levels of expression are detectedin brain and nerve. The 3'ALT isoform is expressed in fetalcardiac muscle, adult left ventricle and cardiac Purkinje cells.Further confirmation of the existence of this isoform was obtainedby blotting the PCR products with a radiolabeled oligonucleotideentirely derived from sequences contained within exon 48a andby direct sequencing of the PCR products. Additionally, thisisoform is expressed in muscle tissues from other vertebratespecies. The expression of this isoform in muscle suggests thatthe NF1 gene may play additional tissue-specific roles in muscledevelopment and signal transduction.     

神经母细胞瘤SK-N-SH细胞内DHRS4L2基因一种新选择性剪接亚型S4的克隆和亚细胞定位

目的 神经母细胞瘤SK-N-SH细胞系脱氢酶/还原酶(SDR家族)成员4类2[dehydrogenase/reductase(SDR family)member 4 like 2,DHRS4L2]基因的一种新的选择性剪接亚型克隆、生物信息学分析及其亚细胞定位.方法 以SK-N-SH细胞cDNA为模板,PCR扩增DHRS4基因簇Ea1转录本.将PCR产物A-T克隆至pGEMT-Easy质粒,对质粒进行DNA Sanger测序.将测序所得序列用NCBI ORF finder分析其编码区,用Motif Scan分析预测蛋白氨基酸序列.用Clustal Omega进行蛋白序列比对分析.将新亚型完整编码框cDNA以及删除偶核定位信号的编码框分别插入pEGFP-C1质粒,所得质粒和空质粒分别转染SK-N-SH细胞,在荧光显微镜下观察转染表达蛋白亚细胞定位.结果 用RT-PCR和Sanger测序方法发现,SK-N-SH表达DHRS4L2 Ea1转录本,未检测到其表达脱氢酶/还原酶(SDR家族)成员4类1[dehydrogenase/reductase(SDR family)member 4 like 1,DHRS4L1]的Ea2转录本.DHRS4L2 Ea1表达一个新的选择性剪接亚型DHRS4L2-S4(KU141377),由AY616183基础上在Ea1与E2外显子之间插入新外显子Ej形成,外显子Ej含有新亚型翻译起始密码子ATG.转录本KU141377预测蛋白羧基端具有偶核定位信号(bipartite nuclear localization signal,NLS),提示其可能定位于细胞核.绿色荧光蛋白融合蛋白实验显示,在SK-N-SH细胞该蛋白定位于细胞核.该蛋白还含有一个甘氨酸密集区(glycine-rich region)和阿片样生长因子受体重复(opioid growth factor receptor repeat)序列.结论 研究发现SK-N-SH细胞表达的一种DHRS4L2新选择性剪接亚型KU141377,其预测编码蛋白含有细胞偶核定位信号,融合荧光蛋白实验显示该新亚型定位于细胞核,这为后续研究DHRS4L2在神经母细胞瘤中的潜在功能奠定基础.