Location of ribosome-binding sites in the nin5 region of bacteriophage lambda

Seven ribosome-binding sites on DNA have been located within the region defined by the nin5 deletion as well as several ribosome-binding sites on each side of the nin5 region. These were mapped by electron microscopy relative to the end points of the nin5 deletion and two Tn903 transposons, one inserted into gene Rz and another inserted near gene Q. These ribosomes binding sites within the nin5 region may correspond to polypeptide initiation sites for up to seven new dispensible lambda genes.     

Functional analysis of RPS27 mutations and expression in melanoma

Next‐generation sequencing has enabled genetic and genomic characterization of melanoma to an unprecedent depth. However, the high mutational background plus the limited depth of coverage of whole‐genome sequencing performed on cutaneous melanoma samples make the identification of novel driver mutations difficult. We sought to explore the somatic mutation portfolio in exonic and gene regulatory regions in human melanoma samples, for which we performed targeted sequencing of tumors and matched germline DNA samples from 89 melanoma patients, identifying known and novel recurrent mutations. Two recurrent mutations found in the RPS27 promoter associated with decreased RPS27 mRNA levels in vitro. Data mining and IHC analyses revealed a bimodal pattern of RPS27 expression in melanoma, with RPS27‐low patients displaying worse prognosis. In vitro characterization of RPS27‐high and RPS27‐low melanoma cell lines, as well as loss‐of‐function experiments, demonstrated that high RPS27 status provides increased proliferative and invasive capacities, while low RPS27 confers survival advantage in low attachment and resistance to therapy. Additionally, we demonstrate that 10 other cancer types harbor bimodal RPS27 expression, and in those, similarly to melanoma, RPS27‐low expression associates with worse clinical outcomes. RPS27 promoter mutation could thus represent a mechanism of gene expression modulation in melanoma patients, which may have prognostic and predictive implications.     

染色质调节元件与不同启动子的相互作用对基因表达调控的影响

为探究DNA序列元件对不同启动子调节转基因稳定表达的影响,利用遍在染色质开放元件 (Ubiquitous chromatin opening elements,UCOE) 和基质黏附序列 (Scaffold/matrix-attachment regions,MAR) 分别与含增强子的oct4基因启动子、含CpG岛的sox2基因启动子和不含调控元件的nanog基因启动子以及同时包含增强子和CpG岛的CMV启动子组合构建pOCT4-MAR、pOCT4-UCOE、pSOX2-MAR、pSOX2-UCOE、pNANOG-MAR、pNANOG-UCOE、pCMV-UCOE、pCMV-MAR等质粒,分析这些质粒稳定转染后的表达量和嵌合表达差异。结果发现,UCOE与含增强子元件的oct4启动子组合能较稳定高效表达,而MAR与含CpG岛的sox2启动子组合能较稳定高效表达。利用排除位置效应原因的嵌合表达对染色质高级结构调控基因表达的稳定性分析表明：(1) 通常情况下UCOE比MAR调节的表达载体的表达更高效和更稳定;UCOE连接含CpG岛的启动子形成开放染色质调节的高表达更稳定;(2) MAR与启动子上TATA盒或增强子可能通过染色质环产生高表达,但相对不稳定。结论：染色质调节元件UCOE和MAR与启动子调控元件之间能通过染色质开放状态或染色质环调控基因稳定表达。     

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era. Streptomyces phage-derived phiC31 integrase, which mediates an irreversible site-specific cassette exchange between the phage attachment site (attP) and the bacterial attachment site (attB), provides a promising option for the construction of a controllable gene-expression system. Here, we report a phiC31 integrase-mediated promoter flip system (FLIP) for the inducible expression of target genes in silkworm (Bombyx mori). First, we constructed a FLIP reporter system, in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene. The coexpression of a C-terminal modified phiC31-NLS integrase carrying a simian virus 40 (SV40) nuclear localization signal (NLS) effectively flipped the BmAct4 promoter through an attB/attP exchange, thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line, BmE. Subsequently, the FLIP system, together with a system continuously expressing the phiC31-NLS integrase, was used to construct binary transgenic silkworm lines. Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter, with an approximately 39% heritable transformation efficiency in silkworm offspring, leading to the constitutive and high-level expression of DsRed in silkworms, which accounted for approximately 0.81% of the silkworm pupal weight. Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.