Increment of hFIX expression with endogenous intron 1 in Vitro

This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous intron 1 sequence.hFIX minigene was obtained with middle sequence truncated intron 1 inserted into the relative site of hFIX cDNA,and plasmid vector pKG5i‘IX,retroviral vector G1NaCi‘IX were constructed.These vectors were transduced into target cells of PA317,C2C12,primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF).The expression level of mixed colonies are PA317/pKG5i‘IX,151 ng/10^6 cells/24h;PA317/G1NaCi‘IX,308 ng/10^6 cells/24 h;C2C12/G1NaCi‘IX,186 ng/10^6 cells/24 h;RSF/G1NaCi‘IX,1929 ng/10^6 cells/24 h;HSF/G1NaCi‘IX,1646 ng/10^6 cells/24 h.These results indicated that hFIX minigene with intron l is able to increase the expression level to about 3 times of that of hFIX cDNA.Meanwhile,in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from intron splicing during viral production,a retroviral vector G1NaCi‘IXR with reversely inserted hFIX minigene expression cassette was constructed.The expression level of reverse constructor in PA317 cells was 390 ng/10^6 cells/24 h with 79% of bioactivity.PCR detection of HT/G1NaCi‘IXR cells infected with PA317/G1NaCi‘IXR supernatant confirmed the existence of intron 1 sequence.These results suggested that expression vector with forward-inserted intronl-carrying hFIX expression cassette can be used in directed gene transfer,but when using the retroviral-mediated gene transfer system,reversely-inserted intronl-carrying hFIX expression cassette should be considered.     

内含子二级结构与剪接位点

对６７个内含子的二级结构进行分析后,我们发现内含子两末端的碱基Ｇ绝大多数是游离的,剪接过程中形成“马套”结构的分枝点Ａ有８０％以上位于环区或游离的单链区,内含子５’端的Ｇ与分枝点Ａ在空间位置上彼此靠近。     

Characterization and organization of gene families at the Gli-1 loci of bread and durum wheats by restriction fragment analysis

Summary The polymerase chain reaction (PCR) can be used to detect polymorphisms in the length of amplified sequences between the annealing sites of two synthetic DNA primers. When the distance varies between two individuals then the banding pattern generated by the PCR reaction is essentially a genetic polymorphism and can be mapped in the same way as other genetic markers. This procedure has been used in a number of eukaryotes. Here we report the use of PCR to detect genetic polymorphisms in cereals. Known gene sequences can be used to design primers and detect polymorphic PCR products. This is demonstrated with primers to the -amylase gene family. A second approach is to use semi-random primers to target diverse regions of the genome. For this purpose the consensus sequences at the intron-exon splice junctions were used. The targeting of the intronexon splice junctions in conjunction with primers of random and defined sequences, such as -amylase, provides a source of extensive variation in PCR products. These polymorphisms can be mapped as standard genetic markers.     

An 8-bp deletion in mNOTCH4 intron 10 leads to its retention in mRNA and to synthesis of a truncated protein

Notch signaling participates in the development of multicellular organisms by maintaining self-renewal potential or inducing differentiation of numerous tissues. In this study, we characterized Notch4, the evolutionary most distant and least studied Notch family member. We identified a Notch4 inter-strain polymorphism with a previously undescribed mRNA variant. This longer Notch4 mRNA, which represented up to one-third of total Notch4 mRNA, resulted from intron 10 retention. Analysis of Notch4 intron 10 revealed that an 8-bp deletion, reducing its length from 68 to 60 bp, strictly correlated with its retention. Further experiments demonstrated that intron length was the only cause of the mis-splicing. Moreover, this mRNA variant resulted in a truncated protein containing half the extracellular domain of Notch4, including the ligand-binding domain.     

A soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) polyubiquitin promoter gives strong constitutive expression in transgenic soybean

The success of plant genetic transformation relies greatly on the strength and specificity of the promoters used to drive genes of interest. In this study, we analyzed gfp gene expression mediated by a polyubiquitin promoter (Gmubi) from soybean (Glycine max) in stably transformed soybean tissues. Strong GFP expression was observed in stably transformed proliferative embryogenic tissues. In whole transgenic plants, GFP expression was observed in root tips, main and lateral roots, cotyledons and plumules in young plants as well as in leaf veins, petioles, flower petals, pollen, pods and developing seeds in mature plants. GFP expression was localized mainly in epidermal cells, leaf mesophyll, procambium and vascular tissues. Introduction of an intron-less version of the Gmubi promoter (Gmupri) displayed almost the same GFP expression pattern albeit at lower intensities. The Gmubi promoter showed high levels of constitutive expression and represents an alternative to viral promoters for driving gene expression in soybean.