Prospects for nuclear gene phylogeography

In phylogeography, an empirical focus on gene lineages enables the history of population processes to be inferred from the simultaneous analysis of temporal and spatial patterns. Rapidly evolving cytoplasmic DNA has been the empirical workhorse propelling the success of this nascent field. Now, as more sophisticated historical models are being tested, there is a growing need for phylogeography to expand from a largely marker-specific discipline to a more general analytical approach that can be applied across independent loci. Recent results using nuclear haplotypes to study phylogeography indicate that the anticipated technical and biological hurdles can be overcome in many taxa to achieve phylogeographical comparisons across unlinked loci. Although many challenges remain, a more complete understanding of the historical, demographic and selective processes shaping phylogeographical patterns is emerging.     

The modified castor bean catalase intron is incompletely spliced in tobacco and Arabidopsis

In an attempt to insert the modified castor bean catalase intron (mCBC intron) into the coding sequence of the Cre recombinase gene, we found that the mCBC intron was not completely spliced from the resulting iCre gene in tobacco and Arabidopsis. Sequencing and allele-specific PCR analyses indicated that six nucleotides (UUACAG) at the 3′ terminus of the mCBC intron were retained in the mature mRNA of the iCre gene. Moreover, the mCBC intron was incompletely spliced from the Gus gene in pCAMBIA vectors. A mutational analysis of the mCBC intron demonstrated that the incomplete splicing was due to an artificial 3′ splice site introduced by the insertion of an adenine, which created a TAG (stop) codon near the 3′ splice site of the original CBC intron. Deletion of the inserted adenine or the six nucleotides that were retained from the mCBC intron led to the complete removal of the intron from the resulting iCre2 and iCre3 genes. Thus, in this study, we not only characterized the incomplete splicing event of the mCBC intron in tobacco and Arabidopsis, but also reported the construction of two intron-containing Cre recombinase genes that are useful for plant biotechnology applications.     

Tactics targeting circular mRNA biosynthesis

Faced with the development of mRNA technology in the field of medicine and vaccine, circular mRNA (circmRNA) becomes a strong alternative to mRNA for its circular secondary structure and higher stability. At present, the synthesis of circmRNAs has been realized by ligating linear mRNA precursors and is limited by poor efficiency. To solve this challenge, this study started with ribozyme catalysis and enzymatic reaction to explore different circmRNA biosynthesis strategies. In terms of ribozyme method, by screening different group I intron self-splicing system sequences, the sequence from thymidylate synthase (Td) gene of phage T4 showed the highest ligation efficiency. In terms of enzyme method, with the help of 20-bp homologous arm, T4 Rnl 2 was determined as the ligation method with the highest ligation efficiency. By comparing the two ligation methods, the expression level of circmRNA ligated by T4 Rnl 2 was 86% higher than that ligated by Td ribozyme. Based on these ligation methods, the screening results of internal ribosome entry site (IRES) sequences showed that mud crab dicistrovirus IRES was an IRES sequence with high ribosome binding ability and could be widely used in circmRNAs for efficient and stable translation in mammalian cells. These results should provide positive guidance for the industrial production of circmRNAs and the development of mRNA vaccines. Eventually, circmRNAs could widely function in the field of biomedicine.     

Profiling lariat intermediates reveals genetic determinants of early and late co-transcriptional splicing

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The biology of yeast mitochondrial introns

Molecular Biology Reports -     

The 5′-UTR intron of the midgut-specific BmAPN4 gene affects the level and location of expression in transgenic silkworms

Introns are important for regulating gene expression. BmAPN4, which has a 5′-UTR upstream intron (5UI), is specifically expressed in the entire silkworm midgut. In our previous study, the promoter region upstream of the 5UI of BmAPN4 was cloned and identified as the P3 promoter (P3P) with activity only in the anterior midgut. In this study, the sequence consisting of the P3P and the 5UI was cloned and named as P3P+5UI. A transgenic vector was constructed in which EGFP was controlled by P3P+5UI. Transgenic P3+5UI silkworms were generated by embryo microinjection. RT-PCR showed P3P+5UI activity throughout the larval stage. Intense green fluorescence was seen only in the entire midgut of P3+5UI silkworms and expression was confirmed by RT-PCR. qPCR revealed that expression of EGFP in the anterior midgut of P3+5UI silkworms was 64% higher than in P3 silkworms, indicating the 5UI sustained intron-mediated enhancement of gene expression. These results suggested that the BmAPN4 5UI affected the level and site of expression. The 5UI was cloned and added behind P2P, another specific promoter with activity only in the anterior midgut of silkworm, to construct the P2P+5UI and transgenic P2+5UI silkworms. Expression patterns were the same for P2P+5UI and P2P, suggesting that the 5UI of BmAPN4 did not affect P2P. This study found that the BmAPN4 5UI affected the amount and location of gene expression. Its influence appeared to be dependent on a specific promoter.