Construction and characterization of a bacterial artificial chromosome library of maize inbred line 77Ht2

Maize inbred line 77Ht2 contains agriculturally important genes and has been widely used in corn breeding in China. A bacterial artificial chromosome (BAC) library of 77Ht2 has been constructed in order to identify useful genes and to facilitate the study of the maize genome. The library contains 175104 clones with an average insert size of 57 kb and represents about 4 maize haploid genome equivalents. Characterization of the library showed less than 0.5% of clones to not contain large inserts. Significant contamination of chloroplast and mitochondria DNA was not detected. BAC clones (152 arrays) were stored in 96 microtiter plates, with each well containing 12 clones. This is the first maize BAC library constructed in China. It is well suited for map-based cloning of maize genes and genome physical mapping.     

Surface plasmon resonance studies of wild-type and AV77 tryptophan repressor resolve ambiguities in super-repressor activity

The interactions of wild-type (WT) and AV77 tryptophan repressor (TR) with several operators have been studied using surface plasmon resonance. The use of this real-time method has been able to settle several outstanding issues in the field, in a way that has heretofore not been possible. We resolve the issue of the super-repressor status of the AV77 aporepressor and find that in contrast to early studies, which found no significant difference in the binding constants in vitro to those of the WT, that there is indeed a clear difference in the binding constant that can simply account for the phenotype. Accordingly, there is no need for alternative proposals invoking complex equilibria with in vivo components not found in the in vitro experiments. In addition, we find that the AV77 holorepressor-DNA complex is much more stable than the equivalent WT complex, which has not been apparent from either in vitro or equilibrium binding experiments.     

Analysis of a substrate specificity switch residue of cephalosporin acylase

Residue Phe375 of cephalosporin acylase has been identified as one of the residues that is involved in substrate specificity. A complete mutational analysis was performed by substituting Phe375 with the 19 other amino acids and characterising all purified mutant enzymes. Several mutations cause a substrate specificity shift from the preferred substrate of the enzyme, glutaryl-7-ACA, towards the desired substrate, adipyl-7-ADCA. The catalytic efficiency ( [Formula: see text] (cat)/ [Formula: see text] (m)) of mutant SY-77(F375C) towards adipyl-7-ADCA was increased 6-fold with respect to the wild-type enzyme, due to a strong decrease of [Formula: see text] (m). The [Formula: see text] (cat) of mutant SY-77(F375H) towards adipyl-7-ADCA was increased 2.4-fold. The mutational effects point at two possible mechanisms by which residue 375 accommodates the long side chain of adipyl-7-ADCA, either by a widening of a hydrophobic ring-like structure that positions the aliphatic part of the side chain of the substrate, or by hydrogen bonding to the carboxylate head of the side chain.     

General Utility of the Chicken βB1-Crystallin Promoter to Drive Protein Expression in Lens Fiber Cells of Transgenic Mice

Transgenic mouse technology has been very valuable for the study of lens fiber cells since they can not be propagated in cell culture. The targeting of transgenes to the lens has traditionally been done with the A-crystallin promoter. However, while lens-specific, transgenic lines made with the A-crystallin promoter express the transgene at levels 100–300-fold lower than endogenous A-crystallin. Here we propose an alternative, the chicken B1-crystallin promoter (–432/+30). Transgenic mice made with this promoter have successfully expressed CAT, d/n m-calpain, Wee1, and B2-crystallin mRNA at levels comparable to the endogenous B1-crystallin gene and no eye abnormalities such as cataracts, have resulted. All of the transgenic lines made with the chicken B1-crystallin promoter have expressed the transgene in the lens fiber cells, and the best lines express at levels close to endogenous B1-crystallin. While RNA expression is very high, only moderate protein expression has been achieved, implying that the high protein expression of the crystallins is partially controlled at the level of translation. Thus, the chicken B1-crystallin promoter directs high level RNA expression to lens fiber cells, which may be especially useful for the expression of ribozyme and anti-sense RNAs in addition to ectopic proteins.     

The development of antenna complexes of barley (Hordeum vulgare cv. Akcent) under different light conditions as judged from the analysis of 77 K chlorophyll a fluorescence spectra

Using 77 K chlorophyll a (Chl a) fluorescence spectra in vivo, the development was studied of Photosystems II (PS II) and I (PS I) during greening of barley under intermittent light followed by continuous light at low (LI, 50 μmol m⁻² s⁻¹) and high (HI, 1000 μmol m⁻² s⁻¹) irradiances. The greening at HI intermittent light was accompanied with significantly reduced fluorescence intensity from Chl b excitation for both PS II (F685) and PS I (F743), in comparison with LI plants, indicating that assembly of light-harvesting complexes (LHC) of both photosystems was affected to a similar degree. During greening at continuous HI, a slower increase of emission from Chl b excitation in PS II as compared with PS I was observed, indicating a preferred reduction in the accumulation of LHC II. The following characteristics of 77 K Chl a fluorescence spectra documented the photoprotective function of an elevated content of carotenoids in HI leaves: (1) a pronounced suppression of Soret region of excitation spectra (410–450 nm) in comparison with the red region (670–690 nm) during the early stage of greening indicated a strongly reduced excitation energy transfer from carotenoids to the Chl a fluorescing forms within PS I and PS II; (2) changes in the shape of the excitation band of Chl b and carotenoids (460–490 nm) during greening under continuous light confirmed that the energy transfer from carotenoids to Chl a within PS II remained lower as compared with the LI plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

15N natural abundance studies in Australian commercial sugarcane

The measurement of natural ¹⁵N abundance is a well-established technique for the identification and quantification of biological N₂ fixation in plants. Associative N₂ fixing bacteria have been isolated from sugarcane and reported to contribute potentially significant amounts of N to plant growth and development. It has not been established whether Australian commercial sugarcane receives significant input from biological N₂ fixation, even though high populations of N₂ fixing bacteria have been isolated from Australian commercial sugarcane fields and plants. In this study, ¹⁵N measurements were used as a primary measure to identify whether Australian commercial sugarcane was obtaining significant inputs of N via biological N₂ fixation. Quantification of N input, via biological N₂ fixation, was not possible since suitable non-N₂ fixing reference plants were not present in commercial cane fields. The survey of Australian commercially grown sugarcane crops showed the majority had positive leaf ¹⁵N values (73% >3.00, 63% of which were >5.00), which was not indicative of biological N₂ fixation being the major source of N for these crops. However, a small number of sites had low or negative leaf ¹⁵N values. These crops had received high N fertiliser applications in the weeks prior to sampling. Two possible pathways that could result in low ¹⁵N values for sugarcane leaves (other than N₂ fixation) are proposed; high external N concentrations and foliar uptake of volatilised NH₃. The leaf ¹⁵N value of sugarcane grown in aerated solution culture was shown to decrease by approximately 5 with increasing external N concentration (0.5–8.0 mM), with both NO₃ ^– and NH₄ ⁺ nitrogen forms. Foliar uptake of atmospheric NH₃ has been shown to result in depleted leaf ¹⁵N values in many plant species. Acid traps collected atmospheric N with negative ¹⁵N value (–24.45±0.90) from above a field recently surface fertilised with urea. The ¹⁵N of leaves of sugarcane plants either growing directly in the soil or isolated from soil in pots dropped by 3.00 in the same field after the fertiliser application. Both the high concentration of external N in the root zone (following the application of N-fertilisers) and/or subsequent foliar uptake of volatilised NH₃ could have caused the depleted leaf ¹⁵N values measured in the sugarcane crops at these sites.     

THE ANTARCTIC PSYCHROPHILE,CHLAMYDOMONAS RAUDENSIS ETTL (UWO241) (CHLOROPHYCEAE,CHLOROPHYTA), EXHIBITS A LIMITED CAPACITY TO PHOTOACCLIMATE TO RED LIGHT1

The psychrophilic Antarctic alga, Chlamydomonas raudensis Ettl (UWO241), grows under an extreme environment of low temperature and low irradiance of a limited spectral quality (blue‐green). We investigated the ability of C. raudensis to acclimate to long‐term imbalances in excitation caused by light quality through adjustments in photosystem stoichiometry. Log‐phase cultures of C. raudensis and C. reinhardtii grown under white light were shifted to either blue or red light for 12 h. Previously, we reported that C. raudensis lacks the ability to redistribute light energy via the short‐term mechanism of state transitions. However, similar to the model of mesophilic alga, C. reinhardtii, the psychrophile retained the capacity for long‐term adjustment in energy distribution between PSI and PSII by modulating the levels of PSI reaction center polypeptides, PsaA/PsaB, with minimal changes in the content of the PSII polypeptide, D1, in response to changes in light quality. The functional consequences of the modulation in PSI/PSII stoichiometry in the psychrophile were distinct from those observed in C. reinhardtii. Exposure of C. raudensis to red light caused 1) an inhibition of growth and photosynthetic rates, 2) an increased reduction state of the intersystem plastoquinone pool with concomitant increases in nonphotochemical quenching, 3) an uncoupling of the major light‐harvesting complex from the PSII core, and 4) differential thylakoid protein phosphorylation profiles compared with C. reinhardtii. We conclude that the characteristic low levels of PSI relative to PSII set the limit in the capacity of C. raudensis to photoacclimate to an environment enriched in red light.     

Characterization of a predominantly pistil-expressed gene encoding a γ-thionin-like protein of Petunia inflata

We isolated a cDNA clone from a pistil cDNA library of Petunia inflata which encodes a protein, PPT, with sequence similarity to -thionins. Characterization of a genomic clone containing a PPT gene revealed the presence of a single intron. Northern analysis revealed that the PPT gene was predominantly expressed in the pistil during all stages of flower development. Since thionins have been implicated in plant defense against pathogens, PPT may play a role similar to that of other defense-related proteins found in the pistil, defending the pistil against pathogen infection.     

Red/far-red modulation in vitro of enzyme activity in a membrane fraction from Phaseolus aureus

In a membrane fraction isolated from hypocotyls of Phaseolus aureus Roxb. the activity of a number of enzymes was regulated by red and far-red irradiation in vitro, provided that the tissue received a brief red light treatment before extraction. Other enzymes showed no photoregulation. There were two types of photocontrol, neither of which could be detected in the solute fraction, nor in extracts from completely etiolated material. One (Type I) was a red/far-red reversible regulation of the rate of enzyme activity, depending on the light given (in vivo or in vitro) before the assay was begun. The second (Type II) was a promotion of enzyme activity by red or far-red light given during the assay. The action spectra for type II responses do not coincide with either the phytochrome absorption or difference spectra. However, the effectiveness of red and far-red was correlated with the P_fr/P ratio present at the beginning of the assay, such that far-red was more efficient at high P_fr/P and red at low P_fr/P ratios. All enzymes that were regulated involved ATP. In samples that showed enzyme regulation, small changes in fluorescence yield of tryptophan and the covalent probe Fluram (Roche) accompanied the photoconversion of phytochrome, but no fluorescence changes could be measured after briefly incubating the membrane fraction with ATP. The results indicate that light may affect the interaction of ATP with the membrane fraction.Abbreviations F far-red light - P_r and P_fr phytochrome in the red and far-red absorbing forms - P_tot total phytochrome - R red light - RNP ribonucleoprotein     

Geographical Variation in the Characteristics of Absorption Spectra and Fluorescence Spectra of Pinus armandi

By measuring the derivative absorption spectra of chloroplasts of different populations of Pinus armandi Franch., it was found that southern populations maintained higher absorption at 680 nm than at 670 nm, but some of the northern populations deviated the maximum absorption from 680 nm to 670 nm, which indicated that the activity of PS Ⅱ in some of the northern populations declined. Clear geographical differences also have been found in the positions of emission peaks of PS Ⅰ and PS Ⅱ in the fluorescence emission spectra at 77 K. Analysis of the fluorescence excitation spectra at 77 K revealed geographical changes in the absorption.status of Chl a. Besides, the experimental results indicated that the intact needles of Pinus armandi are not ideal materials to be used in detecting the geographical variation in photochemical reaction process because the presence of thicker coat, resin etc. can conceal the spectral differences in different populations.