结核分枝杆菌H37Ra FadD3基因克隆与表达研究

为探索FadD3在结核分枝杆菌胆固醇分解代谢中的作用,从结核分枝杆菌H37Ra的全基因组中克隆了FadD3基因,并在大肠杆菌BL21中表达.根据NCBI公布的结核分枝杆菌全基因组序列设计一对引物,PCR扩增FadD3基因.PCR扩增产物与克隆载体pGEM3Zf(+)进行拼接,得到重组基因FadD3-pGEM3Zf(+),再转化到大肠杆菌DH5ɑ得到克隆.PCR检测阳性克隆,再与表达载体pYUB28b拼接,得到重组质粒FadD3-pYUB28b.阳性重组质粒亚克隆到大肠杆菌BL21宿主菌进行自体诱导表达.经过表型筛选及鉴定分析,已成功构建了重组表达质粒FadD3-pYUB28b.SDS-PAGE和Western blotting证实,重组基因FadD3-pYUB28b在大肠杆菌BL21中有表达产物.实验结果表明,以pYUB28b为表达载体,FadD3重组基因在大肠杆菌表达体系于温度分别为18,28 ℃有包涵体形式的表达蛋白,在37 ℃无表达产物.     

Deregulated MYC expression induces dependence upon AMPK-related kinase 5

Deregulated expression of the MYC oncoprotein contributes to the genesis of many human tumours, yet strategies to exploit this for a rational tumour therapy are scarce. MYC promotes cell growth and proliferation, and alters cellular metabolism to enhance the provision of precursors for phospholipids and cellular macromolecules. Here we show in human and murine cell lines that oncogenic levels of MYC establish a dependence on AMPK-related kinase 5 (ARK5; also known as NUAK1) for maintaining metabolic homeostasis and for cell survival. ARK5 is an upstream regulator of AMPK and limits protein synthesis via inhibition of the mammalian target of rapamycin 1 (mTORC1) signalling pathway. ARK5 also maintains expression of mitochondrial respiratory chain complexes and respiratory capacity, which is required for efficient glutamine metabolism. Inhibition of ARK5 leads to a collapse of cellular ATP levels in cells expressing deregulated MYC, inducing multiple pro-apoptotic responses as a secondary consequence. Depletion of ARK5 prolongs survival in MYC-driven mouse models of hepatocellular carcinoma, demonstrating that targeting cellular energy homeostasis is a valid therapeutic strategy to eliminate tumour cells that express deregulated MYC.     

Taxonomy: Sus bucculentus revisited

In 1997, the rediscovery of Sus bucculentus in Laos was announced by Groves et al.--this wild pig species had gone unrecorded since first being described in 1892. Although the identification of the new specimen was based initially on morphology, the authors also used a 7% sequence divergence from the common Eurasian pig S. scrofa (based on their analysis of 327 base pairs of the gene encoding mitochondrial 12S ribosomal RNA) as support for the species status of S. bucculentus. Concerned about the large divergence reported for a relatively conserved gene, and the absence of the sequence in any public database, we analysed an additional tissue sample from the specimen and found only 0.6% divergence from S. scrofa. Our more extensive analysis places the sample within the S. scrofa clade, calling into question the species status of S. bucculentus and demonstrating the need for both phylogenetic and morphological evidence in defining species.     

Constituants amers deBrucea amarissima structures des brucéines A,B et C

Summary Evidence is presented for the structures (II a), (II b) and (II c) for the bruceines A, B and C, the bitter principles isolated from the seeds ofBrucea amarissima.

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18ème Communication sur les constituants amers des Simarubacées; 17ème Communication, voir².     

A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans

Germline stem cells are defined by their unique ability to generate more of themselves as well as differentiated gametes. The molecular mechanisms controlling the decision between self-renewal and differentiation are central unsolved problems in developmental biology with potentially broad medical implications. In Caenorhabditis elegans, germline stem cells are controlled by the somatic distal tip cell. FBF-1 and FBF-2, two nearly identical proteins, which together are called FBF ('fem-3 mRNA binding factor'), were originally discovered as regulators of germline sex determination. Here we report that FBF also controls germline stem cells: in an fbf-1 fbf-2 double mutant, germline proliferation is initially normal, but stem cells are not maintained. We suggest that FBF controls germline stem cells, at least in part, by repressing gld-1, which itself promotes commitment to the meiotic cell cycle. FBF belongs to the PUF family ('Pumilio and FBF') of RNA-binding proteins. Pumilio controls germline stem cells in Drosophila females, and, in lower eukaryotes, PUF proteins promote continued mitoses. We suggest that regulation by PUF proteins may be an ancient and widespread mechanism for control of stem cells.     

A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans

Messenger RNA regulation is a critical mode of controlling gene expression. Regulation of mRNA stability and translation is linked to controls of poly(A) tail length. Poly(A) lengthening can stabilize and translationally activate mRNAs, whereas poly(A) removal can trigger degradation and translational repression. Germline granules (for example, polar granules in flies, P granules in worms) are ribonucleoprotein particles implicated in translational control. Here we report that the Caenorhabditis elegans gene gld-2, a regulator of mitosis/meiosis decision and other germline events, encodes the catalytic moiety of a cytoplasmic poly(A) polymerase (PAP) that is associated with P granules in early embryos. Importantly, the GLD-2 protein sequence has diverged substantially from that of conventional eukaryotic PAPs, and lacks a recognizable RRM (RNA recognition motif)-like domain. GLD-2 has little PAP activity on its own, but is stimulated in vitro by GLD-3. GLD-3 is also a developmental regulator, and belongs to the Bicaudal-C family of RNA binding proteins. We suggest that GLD-2 is the prototype for a class of regulatory cytoplasmic PAPs that are recruited to specific mRNAs by a binding partner, thereby targeting those mRNAs for polyadenylation and increased expression.     

Olfaction: scent-triggered navigation in honeybees

The honeybee, Apis mellifera, navigates rapidly and accurately to food sources that are often kilometres away. They achieve this by learning visual cues, such as the location and colour of nectar-bearing flowers, and chemical cues, such as the scent and the taste of the nectar. Here we train bees to visit differently scented sugar feeders placed at specific outdoor locations and find that they can be induced to visit the same locations simply by having the corresponding scent blown into the hive, even when the destinations no longer have the food or carry the scent. A familiar nectar scent can trigger specific memories of a route and therefore expedite navigation to the food source.     

Molecular breeding of viruses

Genetic recombination is a major force driving the evolution of many viruses. Recombination between two copackaged retroviral genomes may occur at rates as high as 40% per replication cycle. This enables genetic information to be shuffled rapidly, leading to recombinants with new patterns of mutations and phenotypes. The in vitro process of DNA shuffling (molecular breeding) mimics this mechanism on a vastly parallel and accelerated scale. Multiple homologous parental sequences are recombined in parallel, leading to a diverse library of complex recombinants from which desired improvements can be selected. Different proteins and enzymes have been improved using DNA shuffling. We report here the first application of molecular breeding to viruses. A single round of shuffling envelope sequences from six murine leukaemia viruses (MLV) followed by selection yielded a chimaeric clone with a completely new tropism for Chinese Hamster Ovary (CHOK1) cells. The composition and properties of the selected clone indicated that this particular permutation of parental sequences cannot be readily attained by natural retroviral recombination. This example demonstrates that molecular breeding can enhance the inherently high evolutionary potential of retroviruses to obtain desired phenotypes. It can be an effective tool, when information is limited, to optimize viruses for gene therapy and vaccine applications when multiple complex functions must be simultaneously balanced.