Purification of Lac repressor protein using polymer displacement and immobilization of the protein

Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K₁₂BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl--D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.     

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Summary Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that theEscherichia coli lac operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two syntheticlac operators.Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-β-d-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of theE. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.     

Design and expression of oligomeric fibronectin fusion protein: a strategy for enhancing cell adhesion activity

Cell adhesion to extracellular matrices, including fibronectin, results in clustering of integrins in focal adhesions. To promote the clustering of fibronectin and thus enhance its activity at the sites of focal adhesion formation, we have engineered a fusion protein containing recombinant fibronectin fragment (hFN) connected to the tetramerization helix domain of lac repressor for oligomeric assembly. Purified Lac-hFN fusion protein exhibited significant increase of cell adhesion and proliferation of GF cells compared with hFN alone (p < 0.05).     

Error estimation and global fitting in transverse-relaxation dispersion experiments to determine chemical-exchange parameters

Off-resonance effects can introduce significant systematic errors in R₂ measurements in constant-time Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation dispersion experiments. For an off-resonance chemical shift of 500 Hz, ¹⁵N relaxation dispersion profiles obtained from experiment and computer simulation indicated a systematic error of ca. 3%. This error is three- to five-fold larger than the random error in R₂ caused by noise. Good estimates of total R₂ uncertainty are critical in order to obtain accurate estimates in optimized chemical exchange parameters and their uncertainties derived from χ² minimization of a target function. Here, we present a simple empirical approach that provides a good estimate of the total error (systematic + random) in ¹⁵N R₂ values measured for the HIV protease. The advantage of this empirical error estimate is that it is applicable even when some of the factors that contribute to the off-resonance error are not known. These errors are incorporated into a χ² minimization protocol, in which the Carver–Richards equation is used fit the observed R₂ dispersion profiles, that yields optimized chemical exchange parameters and their confidence limits. Optimized parameters are also derived, using the same protein sample and data-fitting protocol, from ¹H R₂ measurements in which systematic errors are negligible. Although ¹H and ¹⁵N relaxation profiles of individual residues were well fit, the optimized exchange parameters had large uncertainties (confidence limits). In contrast, when a single pair of exchange parameters (the exchange lifetime, τ_ex, and the fractional population, p_a), were constrained to globally fit all R₂ profiles for residues in the dimer interface of the protein, confidence limits were less than 8% for all optimized exchange parameters. In addition, F-tests showed that quality of the fits obtained using τ_ex, p_a as global parameters were not improved when these parameters were free to fit the R₂ profiles of individual residues. Finally, nearly the same optimized global τ_ex, p_a values were obtained, when the ¹H and ¹⁵N data sets for residues in the dimer interface, were fit independently; the difference in optimized global parameters, ca. 10%, was of marginal significance according to the F-test.     

HMQC and HSQC experiments with water flip-back optimized for large proteins

An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a ¹⁵N, ¹H-HMQC spectrum of uniformly ¹⁵N/²H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different ¹⁵N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-¹⁵N-HMQC and 3D¹⁵ N-HMQC-NOESY-¹⁵N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly ¹⁵N/¹³C/²H-labelled S. aureus DNA gyrase B.     

Improved magnetization alignment schemes for spin-lock relaxation experiments

A pair of pulse schemes that spin-lock magnetization efficiently are presented. The design of the sequences benefited from a particularly simple relation that is derived describing to first order the evolution of any magnetization component due to the application of an off-resonance 90° pulse. The sequences are shown theoretically and experimentally to significantly outperform the 90°-delay-90° element that is often used in current applications. It is shown that alignment of magnetization to within 1° of the effective field can be obtained over a bandwidth extending between [−ω_SL, ω_SL], where ω_SL is the strength of the spin-lock field using a simple scheme that is an order of magnitude shorter than an adiabatic pulse that might also be used for a similar purpose. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of -galactosidase with symmetric variants of - and -centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the -centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the -centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for -centered trp operator variants with exchanges in positions 3, 4 and 5.     

Mutants in position 69 of the Trp repressor ofEscherichia coli K12 with altered DNA-binding specificity

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turn-helix (HTH) motif of the Trp repressor, and guanine in position 9 of the -centred consensustrp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical -or -centredtrp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the -centredtrp operator. There are also interactions with other bases in positions 8 and 9 of the -centredtrp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus -centredtrp operator and a similartrp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the -centredtrp operator shows a change in the specificity of binding to a -centred symmetricaltrp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the -centredtrp operator.     

Extending the range of amide proton relaxation dispersion experiments in proteins using a constant-time relaxation-compensated CPMG approach

Relaxation compensated constant-time Carr–Purcell–Meiboom–Gill relaxation dispersion experiments for amide protons are presented that detect s-ms time-scale dynamics of protein backbone amide sites. Because of their ten-fold larger magnetogyric ratio, much shorter 180° pulses can be applied to ¹H than to ¹⁵N spins; therefore, off-resonance effects are reduced and a wider range of effective rf fields can often be used in the case of ¹H experiments. Applications to [¹H-¹⁵N]-ubiquitin and [¹H-¹⁵N]-perdeuterated HIV-1 protease are discussed. In the case of ubiquitin, we present a pulse sequence that reduces artifacts that arise from homonuclear ³J(H_N-H) coupling. In the case of the protease, we show that relaxation dispersion of both ¹H and ¹⁵N spins provides a more comprehensive picture of slow backbone dynamics than does the relaxation dispersion of either spin alone. We also compare the relative merits of ¹H versus ¹⁵N transverse relaxation measurements and note the benefits of using a perdeuterated protein to measure the relaxation dispersion of both spin types.     

An allelic exchange system for compliant genetic manipulation of the select agents Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei and B. mallei are Gram-negative bacterial pathogens that cause melioidosis in humans and glanders in horses, respectively. Both bacteria are classified as category B select agents in the United States. Due to strict select-agent regulations, the number of antibiotic selection markers approved for use in these bacteria is greatly limited. Approved markers for B. pseudomallei include genes encoding resistance to kanamycin (Km), gentamicin (Gm), and zeocin (Zeo); however, wild type B. pseudomallei is intrinsically resistant to these antibiotics. Selection markers for B. mallei are limited to Km and Zeo resistance genes. Additionally, there are few well developed counter-selection markers for use in Burkholderia. The use of SacB as a counter-selection method has been of limited success due to the presence of endogenous sacBC genes in the genomes of B. pseudomallei and B. mallei. These impediments have greatly hampered the genetic manipulation of B. pseudomallei and B. mallei and currently few reliable tools for the genetic manipulation of Burkholderia exist. To expand the repertoire of genetic tools for use in Burkholderia, we developed the suicide plasmid pMo130, which allows for the compliant genetic manipulation of the select agents B. pseudomallei and B. mallei using allelic exchange. pMo130 harbors an aphA gene which allows for Km selection, the reporter gene xylE, which allows for reliable visual detection of Burkholderia transformants, and carries a modified sacB gene that allows for the resolution of co-integrants. We employed this system to generate multiple unmarked and in-frame mutants in B. pseudomallei, and one mutant in B. mallei. This vector significantly expands the number of available tools that are select-agent compliant for the genetic manipulation of B. pseudomallei and B. mallei.     

Controlled gene expression utilising Lambda phage regulatory signals in a cyanobacterium host

Summary This study presents plasmid systems that utilize regulatory signals of bacteriophage Lambda to accomplish regulated expression of cloned genes in an A. nidulans R2 derivative strain. An operator-promoter region and the temperature-sensitive repressor gene cI857 of bacteriophage Lambda were employed. Linked to a cyanobacterial replicon, the plasmid vectors efficiently transformed Anacystis and were stably maintained within this host. The cat structural gene, encoding chloramphenicol acetyltransferase, was used to demonstrate that expression can be regulated by temperature shift. We have identified in extracts from the vector bearing Anacystis, a protein similar in size and immunology to the Lambda repressor. The systems described should allow controlled expression of adventitious genes in the cyanobacterial host.Abbreviations AP^r ampicillin resistance - Cm^r chloramphenicol resistance - CmActase chloramphenicol acetyltransferase - Km^r Kanamycine resistance - [ ] indicates plasmid carrier state     

Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis

Two plasmid-based expression vectors have been constructed where one allows intracellular production of recombinant proteins while the second directs the proteins into the culture medium. Both vectors use the strong promoter preceding the groESL operon (codes for the essential heat shock proteins GroES and GroEL) of Bacillus subtilis fused to the lac operator allowing their induction by addition of ITPG. While the background level of expression of these expression cassettes is very low in the absence of the inducer, an induction factor of about 1300 was measured. When the genes htpG and pbpE (coding for a heat shock protein and a penicillin-binding protein, respectively) were fused to the groE promoter, the amount of recombinant protein produced after addition of IPTG represented 10 and 13%, respectively, of the total cellular protein. To obtain secretion of recombinant proteins, the coding region for the signal peptide of the amyQ gene encoding an alpha-amylase from Bacillus amyloliquefasciens was fused to the groE promoter. High-level secretion of amyQ alpha-amylase and cellulase A and B of Clostridium thermocellum was demonstrated.     

Use of a promoter-specific probe to identify two loci from the Rhizobium meliloti nodulation regulon

The nodulation regulon of Rhizobium meliloti AK631 includes several operons (nodABC, hsnABC, hsnD, efn locus) which have in common a consensus promoter sequence called the nod box. A synthetic nod box probe was used to identify two additional nod boxes, n4 and n5, which were subcloned for study. By constructing lac fusions, we show that n4 and n5 sponsor induction of downstream regions as previously shown for n1-nodABC and n2-hsnABC. Using site-directed Tn5 mutagenesis, we find that the n5 locus plays a significant role in nodulation of alfalfa and sweetclover, whereas the n4 locus is important for alfalfa, but not for sweetclover. Hybridization data suggest that the n5 locus is conserved among Rhizobium species. In contrast, the n4 locus seems to be unique to Rhizobium meliloti strains, in agreement with the host-specific phenotype of n4 locus mutants. Thus, the use of a promoter probe allows us to identify nodulation genes which may be overlooked by standard methods such as random Tn5 mutagenesis.     

Magnesium deficiency in expanding leaves of Phaseolus vulgaris-gas exchange and nutrient concentrations

Phaseolus vulgaris was cultured either with or without magnesium in an aerated nutrient solution in growth chambers from 21 days after germination. Five days after transfer to Mg-deficient nutrient solution, terminal leaflets of first trifoliate leaves stopped expansion. From the fifth day after transfer, the net assimilation rate, the transpiration rate and the leaf water vapour conductance of first trifoliate leaves of the deficient plants declined. Following resupply of Mg on the seventh day after transfer to the Mg-deficient solution, the assimilation rate increased to 93% by the 12th day, the transpiration rate to 76% and the leaf water vapour conductance to 50% of the control plants.     

半红树植物黄槿的气体交换特性

半红树植物是一类既可在海岸潮间带又可在陆地生长和繁殖的海陆两栖植物。夏季强光和高温是海岸带生境突出的特点,半红树植物如何在这样的自然生境中生存和繁衍是值得探讨的问题。该研究以具有较高应用价值的锦葵科(Malvaceae)木槿属(Hibiscus)的常绿半红树植物黄槿(H.tiliaceus)作为研究对象,采用LI-6400便携式光合测定系统(Li-Cor Inc.,USA)对南亚热带气候条件下的广东省珠海淇澳岛红树林自然保护区自然生长的黄槿在夏季高温季节的叶片气体交换特性进行测定,探讨了其在自然生境下的光合行为和相关的形态解剖特征,揭示其生境适应性的光合生理生态机制。结果表明:(1)黄槿净光合速率、气孔导度、胞间二氧化碳浓度和蒸腾速率的日变化均表现为单峰型曲线,中午未出现明显光抑制现象。(2)黄槿较高的净光合速率与其较高的气孔导度相关,而后者则可能与其较大的气孔密度和具有表皮毛等叶片解剖结构特征有关。(3)黄槿光饱和点、光补偿点、最大净光合速率和日均净光合速率均符合阳生植物特征,因而是一种阳生性树种。以上结果说明黄槿在自然环境中具有的快速生长特性可能与其较强和较稳定的光合能力有关,该研究结果为深入揭示黄槿以及其他半红树植物适应海岸带高温强光生境的生理生态机制提供了基础数据,为黄槿的合理栽培及造林实践提供了理论参考。     

地衣芽孢杆菌对南美白对虾零水交换养殖系统细菌群落的影响

【目的】地衣芽孢杆菌（Bacillus licheniformis）对南美白对虾（Penaeus vannamei）免疫反应、抗病性和营养的影响已被广泛研究,但零水交换养殖系统下地衣芽孢杆菌对对虾肠道和养殖水环境微生物群落的影响尚不清楚。【方法】通过收集添加地衣芽孢杆菌在饲料或水中后,对虾肠道、池水和池低沉积物样品,通过16S rRNA基因测序和线性判别分析（linear discriminant analysis effect size,LEfSe）进行微生物分析。【结果】结果表明,添加地衣芽孢杆菌对对虾的生长影响较小。此外,添加方式的不同对对虾肠道菌群的影响较小。但添加地衣芽孢杆菌可以有效地改变对虾肠道微生物群落,并改善对虾免疫力。【结论】这些结果有助于全面了解在零水交换养殖系统中,通过饲料和水添加地衣芽孢杆菌后对虾肠道和环境的变化,从而为选择正确的益生菌以及如何添加益生菌维持对虾健康提供基础信息。     

Gas exchange of ears of cereals in response to carbon dioxide and light

One cultivar each of spring wheat (Triticum aestivum L. cv. Arkas), oat (Avena sativa L. cv. Lorenz), and barley (Hordeum vulgare L. cv. Aramir) was chosen in order to study the relative contributions of individual bracts to the gas exchange of whole ears. The distribution and frequency of the stomata on the bracts were examined. Gas exchange was measured at normal atmospheric CO₂ (330 bar) and at high CO₂ (2000 bar) on intact ears and on ears from which glumes or lemmas and pleae (wheat and oat) or awns (barley) had been removed.The relative contribution to the gas exchange of the whole organ is highest for the awns of barley ears. In wheat, the contribution of the glumes is slightly higher than that of the inner bracts before anthesis. Two weeks after anthesis the inner bracts contribute more than the glumes. This tendency of increasing importance of the inner bracts is also found in oat ears, but the relative amount of CO₂ uptake by the glumes is higher than in wheat. These changes during ontogeny result from the better supply of light to the inner bracts caused by opening of the ears' structures during grain filling, which in part compensates for the decreasing photosynthetic capacity.The ratio of the photosynthesis rate at high CO₂ to that at normal CO₂ is lower for the glumes of oat and for the awns of barley than for the other bracts.Abbreviations A³³⁰, A²⁰⁰⁰ net photosynthesis rate, A³³⁰ at normal atmospheric CO₂ (330 bar), A²⁰⁰⁰ at high CO₂ (2000 bar) - PPFD photosynthetic photon flux density - p^c intercellular partial pressure of CO₂     

The response of CO2 exchange rate to photosynthetic photon flux density for several Populus clones under laboratory conditions

No significant differences were found between four mathematical equations describing the response of CO₂ exchange rate to photosynthetic photon flux density in seven poplar clones under laboratory conditions. Choice of an optimal equation for poplar may be based on the contemplated aims. High significant differences (at p<0.001) were found among the clones.Research was supported by the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (I.W.O.N.L.), Brussels.     

Interactions of plant stress and herbivory: intraspecific variation in the susceptibility of a palatable versus an unpalatable seaweed to sea urchin grazing

Summary Interactions among environmental stresses, plant defensive characteristics, and plant nutrient status may significantly affect an alga's susceptibility to herbivores. Following desiccation, the palatable seaweed Gracilaria tikvahiae was less susceptible to grazing by the sea urchin Arbacia punctulata while the unpalatable alga Padina gymnospora became more susceptible. Increased grazing on desiccated Padina appeared to result from a loss of chemical defenses following desiccation. Palatable plants treated with organic extracts from desiccated Padina plants were consumed at more than twice the rate of plants treated with extracts from undesiccated plants. Increased susceptibility of Padina did not correlate with changes in protein content of the alga; reduced grazing on desiccated Gracilaria was associated with a decrease in protein content. When Padina was grazed by Arbacia or mechanically damaged to mimic urchin grazing, its susceptibility to Arbacia decreased within 1 to 5 days. These results demonstrate that history of physical or biological stress may affect a plant's susceptibility to herbivory. We hypothesize that urchins cue primarily on attractiveness features (e.g. nutrient content) of highpreference algae and deterrent features (e.g. chemical defenses) of low-preference algae. Stresses may therefore increase, decrease, or not affect a plant's susceptibility to herbivory depending upon the primary feeding cues used by the herbivore, the defensive mechanisms used by the plant, and the way these are altered by various environmental stresses.