Specific expression of a β-tubulin gene (GhTub1) in developing cotton fibers Specific expression of a b-tubulin gene (GhTub1) in developing cotton fibers

Eukaryotic microtubular cytoskeleton plays crucial roles in various cellular processes, in-cluding cell division, polar cell expansion and cell differentiation[1, 2]. The basic filament-forming unit of microtubules is a heterodimer protein composed of a-tubulin and b-tubulin polypeptides. In the higher plants studied, both a- and b-tubulin genes form multigene families. While some of these tubulin genes are expressed constitutively, many others exhibit tissue-, organ- or cell type-specific exp…     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Heterologous expression of a gene for thermostable xylanase from <Emphasis Type="Italic">Chaetomium</Emphasis> <Emphasis Type="Italic">thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia</Emphasis> <Emphasis Type="Italic">pastoris</Emphasis> GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml⁻¹ while that of recombinant without intron (xyn669) was 1.26 U ml⁻¹ after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.     

Overexpression of an optimized <Emphasis Type="Italic">Aspergillus sulphureus β</Emphasis>-mannanase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

β-Mannanase (EC 3.2.1.78) is a key enzyme to hydrolyze the β-mannosidic linkages in mannan and heteromannan. The expression of a wild type β-mannanase (manWT) of Aspergillus sulphureus in Pichia pastoris is not high enough for its application in feed supplement. To earn a high expression level, the manWT gene was firstly optimized to manM according to the code bias of P. pastoris, which was then inserted into pPICzαA and transformed into P. pastoris strain X-33. In the induction by methanol, β-mannanase was expressed in high level with 32% increase in comparison with the manWT gene expressed in P. pastoris in shaken flask. In a 10-L fermenter, the manM was expressed in 9-fold higher level than that in shaken flask, which yielded the enzyme activity of 1100 U/mL. This is the first study on codon bias effect on the β-mannanase gene expression level, which helps to achieve high β-mannanase yield and enzymatic activity in P. pastoris.     

Enhancing functional expression of heterologous lipase in the periplasm of <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Bold"> </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Functional expression of heterologous Pseudozyma antarctica lipase B (PalB) in the periplasm of Escherichia coli was explored using four fusion tags, i.e. DsbC, DsbA, maltose-binding protein (MBP), and FLAG in the sequence of increasing expression efficacy. Amongst these fusion tags, FLAG and MBP appear to be the most effective ones in terms of boosting enzyme activity and enhancing solubility of PalB, respectively. Overexpression of these PalB fusions often resulted in concomitant formation of insoluble inclusion bodies. Coexpression of a selection of periplasmic folding factors, including DegP (and its mutant variant of DegP_S210A), FkpA, DsbA, DsbC, and a cocktail of SurA, FkpA, DsbA, and DsbC, could improve the expression performance. Coexpression of DsbA appeared to be the most effective in reducing the formation of inclusion bodies for all the four PalB fusions, implying that functional expression of PalB could be limited by initial bridging of disulfide bonds. Culture performance was optimized by overexpressing FLAG-PalB with DsbA coexpression, resulting in a high volumetric PalB activity of 360 U/L.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Physical mapping of the <Emphasis Type="Italic">Rf</Emphasis><Subscript><Emphasis Type="Italic">1</Emphasis></Subscript> fertility-restoring gene to a 100 kb region in cotton

Cytoplasmic male sterility (CMS) plays an important role in crop heterosis exploitation. Determining one or more nuclear genes that can restore male fertility to CMS is essential for developing hybrid cultivars. Genetic and physical mapping is the standard technique required for isolating these restoration genes. By screening 2,250 simple sequence repeat (SSR) primer pairs in cotton (Gossypium hirsutum L.), we identified five new SSR markers that are closely linked to the Rf ₁ gene, a fertility restorer gene of cotton for CMS-D2. Based on our previous fine mapping of the Rf ₁ gene and assemblage of three published STS markers, we constructed a high-resolution genetic map of Rf ₁ containing 13 markers in a genetic distance of 0.9 cM. The 13 molecular markers were used to screen a bacterial artificial chromosome (BAC) library from a restorer line 0-613-2R containing Rf ₁ gene, which yielded 50 single positive clones. There was an average of 3.8 clones ranging from 1 to 12 BAC clones per PCR marker. These 50 clones produced an average insert size of 120 kb (ranging between 80 and 225 kb). Thirty-five primer pairs were designed based on 38 sequences of BAC ends, and two new STS markers tightly linked to Rf ₁ gene have been tagged and integrated into this map. The physical map for the Rf ₁ gene was constructed by fingerprinting the positive clones digested with the HindIII enzyme. We were able to delimit the possible location of the Rf ₁ gene to a minimum of two BAC clones spanning an interval of approximately 100 kb between two clones designated 081-05K and 052-01N. Further work using these two BAC clones will lead to isolation of the Rf ₁ gene in cotton.     

Expression of β-carotene hydroxylase gene (<Emphasis Type="Italic">crtR-B</Emphasis>) from the green alga <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis> in chloroplasts of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

A carotenoid gene (crtR-B) from the green alga Haematococcus pluvialis, encoding β-carotene hydroxylase that was able to catalyze the conversion of β-carotene to zeaxanthin and canthaxanthin to astaxanthin, was cloned into Chlamydomonas reinhardtii chloroplast expression vector p64D to yield plasmid p64DcrtR-B. The vector p64DcrtR-B was transferred to the chloroplast genome of C. reinhardtii using micro-particle bombardment. PCR and Southern blot analyses indicated that crtR-B was integrated into the chloroplast genome of the transformants. RT-PCR assays showed that the H. pluvialis crt R-B gene was expressed in C. reinhardtii transformants. The transformants rapidly synthesized carotenoids in larger quantities than the wild-type upon being transferred from moderate to high-intensity white light. This research provides a foundation for further study to elucidate the possible mechanism of photo-protection by xanthophylls and other carotenoids in high light conditions or through exposure to UV radiation.     

Genome-wide analysis and identification of the <Emphasis Type="Italic">SMXL</Emphasis> gene family in apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis>)

Strigolactones (SLs) are a recently discovered type of plant hormone that controls various developmental processes. The DWARF53 (D53) protein in rice and the SMAX1-LIKE (SMXL) family in Arabidopsis repress SL signaling. In this study, bioinformatics analyses were performed, and 236 SMXL proteins were identified in 28 sequenced plants. A phylogenetic analysis indicated that all potential SMXL proteins could be divided into three groups and that the SMXL proteins may have originated in Bryophytes. An analysis of the SMXL chromosomal locations suggested that gene duplication events at different times led to expansion of the SMXL family members in Angiospermae. Subsequently, the gene structure and protein modeling of MdSMXLs showed that they are highly conserved. The expression patterns of MdSMXLs indicated that they were expressed in different organs of apple (stems, roots, leaves, flowers, and fruits) at varying levels and that MdSMXLs may participate in the SL signaling pathway and the response to abiotic stress. This study provides a valuable foundation for additional investigations into the function of the SMXL gene family in plants.     

Influence of <Emphasis Type="Italic">RAR</Emphasis> α gene on <Emphasis Type="Italic">MDR1</Emphasis> expression and P-glycoprotein function in human leukemic cells

Background  

Multidrug resistance (MDR) phenotype of malignant cells is the major problem in the chemotherapy of neoplasia. The treatment of leukemia with retinoids is aimed on the induction of leukemic cells differentiation. However the interconnections between retinoid regulated differentiation of leukemic cells and regulation of MDR remains unclear.