Characterization of a highly structured domain in Tbp2 from Neisseria meningitidis involved in binding to human transferrin.

The binding of iron-loaded human transferrin at the surface of Neisseria meningitidis is mediated by two polypeptides, Tbp1 and Tbp2. Predicted Tbp amino acid sequences from N. meningitidis strains are highly divergent. This variability is particularly pronounced throughout the Tbp2 polypeptide. In this study, a highly structured and extremely stable Tbp2 domain of about 270 to 290 amino acids which is involved in the binding to transferrin and whose position is well conserved has been characterized. The conservation of such a remarkable structure in a very divergent protein domain (there is only 43% amino acid identity within this region) suggests that is plays an essential biological role and raises a number of questions regarding tbp2 evolution.     

Thiophene synthesis and distribution in young developing plants of Tagetes patula and Tagetes erecta

Thiophene synthesis and accumulation were investigated in organsof Tagetes patula and T. erecta. Thiophene accumulation startedrapidly in germinating seedlings of both species. Roots andhypocotyls were the major thiophene accumulating organs and5-(3-buten-1-ynyl)-2, 2-bithienyl (BBT) and 5-(4-acetoxy-1-butynyl)-2,2 -bithienyl (BBTOAc) were the major accumulated compounds.Higher thiophene concentrations were reached in Tagetes patulathan in T. erecta. Accumulation patterns for individual thiopheneswere different within organs, between organs and between bothspecies. Within hypocotyls of Tagetes patula, thiophene concentrationswere high in the epidermis and the vascular tissue and low inthe parenchymatic tissues of cortex and pith. Synthesis of thiopheneswas high in the roots and hypocotyls and very low in the leaves.Transport of thiophenes from the roots into the shoot occurred,but the rate of transport was too low to explain the high concentrationsin the hypocotyl. It is concluded that for the main part thiophenesare accumulated where they are synthesized. Key words: Tagetes, hiophenest, synthesis, accumulation, secondary metabolites     

Construction and characterisation of a yeast artificial chromosome library containing five haploid sugarbeet (Beta vulgaris L.) genome equivalents

A yeast artificial chromosome (YAC) genomic library of Beta vulgaris was constructed in the pYAC4 vector. High-molecular-weight DNA was prepared from agarose-embedded leaf protoplasts from a triploid cultivar. The library was found to contain 33,500 clones in an ordered array of microtiter plates. Mean size of the inserts was estimated to be 135 kb, and the library should therefore represent the equivalent of five haploid genomes. The library was characterised for the presence of highly repetitive, chloroplast and single-copy sequences. In order to isolate single-copy sequences, 18 pools of DNA, each from 1920 individual YAC clones, were prepared for rapid screening of the library by the polymerase chain reaction. The results of these screenings showed that the number of isolated clones was at or near the frequency expected.     

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)⁺-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses.The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5 non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5 sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3 sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site.This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.Abbreviations AK aspartate kinase - HSDH homoserine dehydrogenase - ID intermediate domain - Tp transit peptide     

Differential interactions of promoter elements in stress responses of the Arabidopsis Adh gene.

The Adh (alcohol dehydrogenase, EC 1.1.1.1.) gene from Arabidopsis thaliana (L.) Heynh. can be induced by dehydration and cold, as well as by hypoxia. A 1-kb promoter fragment (CADH: -964 to +53) is sufficient to confer the stress induction and tissue-specific developmental expression characteristics of the Adh gene to a beta-glucuronidase reporter gene. Deletion mapping of the 5' end and site-specific mutagenesis identified four regions of the promoter essential for expression under the three stress conditions. Some sequence elements are important for response to all three stress treatments, whereas others are stress specific. The most critical region essential for expression of the Arabidopsis Adh promoter under all three environmental stresses (region IV: -172 to -141) contains sequences homologous to the GT motif (-160 to -152) and the GC motif (-147 to -144) of the maize Adh1 anaerobic responsive element. Region III (-235 to -172) contains two regions shown by R.J. Ferl and B.H. Laughner ([1989] Plant Mol Biol 12: 357-366) to bind regulatory proteins; mutation of the G-box-1 region (5'-CCACGTGG-3', -216 to -209) does not affect expression under uninduced or hypoxic conditions, but significantly reduces induction by cold stress and, to a lesser extent, by dehydration stress. Mutation of the other G-box-like sequence (G-box-2: 5'-CCAAGTGG-3', -193 to -182) does not change hypoxic response and affects cold and dehydration stress only slightly. G-box-2 mutations also promote high levels of expression under uninduced conditions. Deletion of region I (-964 to -510) results in increased expression under uninduced and all stress conditions, suggesting that this region contains a repressor binding site. Region II (-510 to -384) contains a positive regulatory element and is necessary for high expression levels under all treatments.     

Visiting bags: a labile thermal environment.

OBJECTIVE--To define usual colour and site of storage of visiting bags in general practitioners'' cars and to investigate effect of these variables on temperature inside bag. DESIGN--Questionnaire to general practitioners; serial temperature measurements from paired black visiting bags at different storage sites and from bags of different colour. SETTING--South Devon coastal town during May and June. SUBJECTS--200 general practitioners, of whom 145 returned legible questionnaires. MAIN OUTCOME MEASURES--Bag colour, duration and site of storage, temperature inside black bags at defined storage sites, and effects of bag colour on internal temperature. RESULTS--111 (77%) of the general practitioners carried a black visiting bag, and 76 kept their bag in their car all day. The bag was coolest in the car boot, but irrespective of storage site, maximum internal temperature of the bag was always over 25 degrees C and reached up to 80 degrees C. Spraying a black bag silver significantly reduced the bag''s internal temperature (mean difference 8.37 degrees C (95% confidence interval 6.68 to 9.86 degrees C) df = 59, t = 10.29, P < 0.001). CONCLUSIONS--General practitioners should use a silver coloured visiting bag; when visiting, they should store it in their car boot; at other times they should remove it to a cooler site.     

A SEARCH FOR SOME ANG1OSPERM HORMONES AND THEIR METABOLITES IN CAULERPA PASPALOIDES (CHLOROPHYTA)1

The best available methods were used to search for an-giosperm hormones in the green alga Caulerpa paspa-loides (Bory) Greville. Solvent partitioning of methanol extracts led to acidic ethyl acetate fractions that it were run through high-performance liquid chromatography. Each of the resulting 25 fractions was divided in half so that one half was tested in the dwarf rice or lettuce hypocotyl assays for gibberellins and the other half was methylated and silylated for capillary gas chromatography combined with computerized mass spectrometry. By comparing algal mass spectra with spectra in the extensive library of mass spectra and associated Kovats Retention Indices that MacMillan's Bristol group has amassed, indole-3-acetic acid's presence in the alga was confirmed and dioxindole-3-acetic acid was found. However, despite the presence of several peaks of gibberellin-like activity in the bioassays, no gibberellin or gibberellin metabolite was found in the thousands of full mass spectra examined. The gibberellin-like activity in the acidic ethyl acetate fractions was therefore presumably not from any gibberellin known from vascular plants.     

Rhizome gravitropism precedes gravimorphogenesis after inversion of the green algal coenocyte Caulerpa prolifera (Caulerpales)

Previous investigations reported that the marine alga Caulerpa prolifera, a giant coenocyte, had a fast morphogenetic response to a change in position with regard to gravity: the next rhizoid formed approximately 1 day later on the new underside of the inverted rhizome (Jacobs and Olson, 1980, American Journal of Botany 67: 141–146). Preceding the change in site of rhizoid development was a striking accumulation of amyloplasts at the new prospective rhizoid-initiation site in the rhizome tip. Detailed monitoring with video equipment of inverted Caulerpa plants, growing under controlled conditions, now reveals that after the amyloplast accumulation, but before the gravimorphogenetic effect on rhizoid development, the inverted rhizome tip shows a negative gravitropism that restores it to its normally upturned position. Because the experiments were performed with two-sided illumination, this was clearly not a phototropic response.