PCR primers for fish G6PD gene intron and characterization of intron length variation in the albacore Thunnus alalunga

Polymerase chain reaction (PCR) primers to amplify the fourth intron of glucose‐6‐phosphate dehydrogenase (G6PD) gene were designed. A large length variation of amplified fragment was observed in the Atlantic albacore sample with a moderate level of heterozygosity (H_E = 0.488). Nucleotide sequence analysis revealed deletion or insertion of a large nucleotide block (110 base pairs) to be responsible for the length difference. Successful amplification of single or two fragments was confirmed in the northern bluefin tuna and Pacific saury, indicating the wide cross‐species applicability.     

A 51 bp indel polymorphism within the PTH1R gene is significantly associated with chicken growth and carcass traits

Parathyroid hormone (PTH) is a crucial regulator of calcium homeostasis and bone remodeling, and the parathyroid hormone 1 receptor (PTH1R) belongs to a class II G-protein-coupled receptor. PTH activates PTH1R, which mediates catabolic and anabolic processes in the skeleton. However, the functional mechanism of PTH1R has not been thoroughly elucidated in organisms. This study identified a 51 bp indel mutation in the first intron of the PTH1R gene and elucidated the effect of this gene mutation on the growth and carcass traits in chickens. The results indicated that the 51 bp indel was significantly associated with subcutaneous fat thickness, abdominal fat weight, body weight and daily gain over 4–8 weeks. Furthermore, we found that PTH1R gene expression was highest in the kidney and liver tissues, and it showed a trend of decreasing in leg and breast muscle tissues at different embryonic stages. In addition, we examined the expression of the three genotypes of the PTH1R gene in the liver, breast muscle and abdominal fat and found that the II genotype was significantly higher than the DD and ID genotypes. In summary, these findings suggest that the PTH1R gene can serve as a potential molecular marker for chicken breeding.     

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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