Cloning of a pea cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I using a monoclonal antibody

A monoclonal antibody (MAb UB42) is described that binds to thylakoids in pea chloroplasts, as shown by EM-immunogold labelling. The antibody recognised proteins of ca. 23–29 kDa in western blots of a pea leaf homogenate. A cDNA library was prepared from pea epidermal cells in the vector ZAP II, and immunoscreening of the library with UB42 led to the isolation of a clone, pUB42. This was sequenced and had an open reading frame of 269 codons encoding a predicted polypeptide of 28.9 kDa. The sequence showed extensive homology with three closely related polypeptides belonging to a family of chlorophyll a/b-binding proteins from the light harvesting complex of photosytem I (LHCI). Collectively, the results suggest that MAb UB42 recognises an epitope on the type II chlorophyll a/b-binding protein from LHCI and that clone pUB42 encodes this protein.     

Lectin binding distinguishes between neuroendocrine and neuronal derivatives of the sympathoadrenal neural crest

Lectin cytochemistry was used to identify surface epitopes selectively expressed by chromaffin cell chemoreceptors (glomus cells) in the rat carotid body. Unexpectedly, these studies revealed that binding sites for peanut agglutinin (PNA; Arachis hypogea) were highly expressed by all neuroendocrine derivatives of the sympathoadrenal neural crest, including glomus cells, small, intensely fluorescent cells, and adrenal chromaffin cells in situ. In contrast, principal sympathetic neurons did not express PNA receptors. PNA binding was inhibited by 2% galactose. To determine whether expression of PNA receptors was selectively induced by neuroendocrine differentiation of sympathoadrenal precursors, we compared PNA labeling of embryonic sympathoblasts in the presence of either nerve growth factor (NGF) or the synthetic glucocorticoid dexamethasone (DEX). Dex-treated cells, which expressed several neuroendocrine traits, bound PNA, whereas NGF-treated neuronal derivatives did not. In addition, to examine whether expression of existing PNA receptors was down-regulated by neuronal differentiation of chromaffin cells, we compared labeling of PC12 cells, which normally bind PNA, in the presence and absence of NGF. Although PC12 cells acquired characteristic neuronal morphologies in the presence of NGF, they did not lose PNA labeling, even after 8 days of NGF treatment. These findings indicate that neuronal and neuroendocrine derivatives of the sympathoadrenal lineage can be distinguished by differential expression of carbohydrate epitopes and suggest that PNA receptors are induced by neuroendocrine differentiation. © 1995 John Wiley & Sons, Inc.     

Development of an identified serotonergic neuron in the antennal lobe of the moth and effects of reduction in serotonin during construction of olfactory glomeruli

Each olfactory (antennal) lobe of the moth Manduca sexta contains a single serotonin (5-HT) immunoreactive neuron whose processes form tufted arbors in the olfactory glomeruli. To extend our present understanding of the intercellular interactions involved in glomerulus development to the level of an individual, identified antennal lobe neuron, we first studied the morphological development of the 5-HT neuron in the presence and absence of receptor axons. Development of the neuron's glomerular tufts depends, as it does in the case of other multiglomerular neurons, on the presence of receptor axons. Processes of the 5-HT neuron are excluded from the region in which the initial steps of glomerulus construction occur and thus cannot provide a physical scaffolding on which the array of glomeruli is organized. Because the neuron's processes are present in the antennal lobe neuropil throughout postembryonic development, 5-HT could provide signals that influence the pattern of development in the lobe. By surgically producing 5-HT-depleted antennal lobes, we also tested the importance of 5-HT in the construction of olfactory glomeruli. Even in the apparent absence of 5-HT, the glomerular array initiated by the receptor axons was histologically normal, glial cells migrated to form glomerular borders, and receptor axons formed terminal branches in their normal region within each glomerulus. In some cases, 5-HT-immunoreactive processes from abnormal sources entered the lobe and formed the tufted intraglomerular branches typical of most antennal lobe neurons, suggesting that local cues strongly influence the branching patterns of developing antennal lobe neurons. © 1995 John Wiley & Sons, Inc.     

Cloning of a full-length symbiotic hemoglobin cDNA and in situ localization of the corresponding mRNA in Casuarina glauca root nodule

We have characterized a full-length cDNA ( hb -Cg1F) that represents symbiotic mRNA hemoglobin ( hb ) from Casuarina glauca root nodules. In situ hybridization was used to examine the correlation between hb -Cg1F mRNA and the state of the Frankia infection process. The efficiency of in situ hybridization using DIG-labeled vs [³⁵S]-labeled probes was compared. The expression of hb -Cg1F gene is induced in young infected host cells prior to the detection of Frankia nif H mRNA. Since Frankia does not form vesicles in C. glauca nodules, it is proposed that Hb is necessary to reduce the O₂ concentration in the cytoplasm of the host cells before the nif genes are expressed.     

Molecular Characterization of the Clusters of Genes Encoding the Botulinum Neurotoxin Complex in Clostridium botulinum (Clostridium argentinense) Type G and Nonproteolytic Clostridium botulinum Type B

The cluster of genes encoding components of the progenitor botulinum neurotoxin complex has been mapped and cloned in Clostridium botulinum type G strain ATCC 27322. Determination of the nucleotide sequence of the region has revealed open reading frames encoding nontoxic components of the complex, upstream of the gene encoding BoNT/G (botG). The arrangement of these genes differs from that in strains of other antigenic toxin types. Immediately upstream of botG lies a gene encoding a protein of 1198 amino acids, which shows homology with the nontoxic-nonhemagglutinin (NTNH) component of the progenitor complex. Further upstream there are genes encoding proteins with homology to hemagglutinin components (HA-17, HA-70) and a putative positive regulator of gene expression (P-21). Sequence comparison has shown that BoNT/G has highest homology with BoNT/B. The sequence of the BoNT-cluster of genes in non-proteolytic C. botulinum type B strain Eklund 17B has been extended to include the complete NTNH and HA-17, and partial HA-70 gene sequences. Comparison of NTNH/G with other NTNHs reveals that it shows highest homology with NTNH/B consistent with the genealogical affinity shown between BoNT/G and BoNT/B genes. Received: 28 January 1997 / Accepted: 24 March 1997     

Expansion of cultured Zinnia mesophyll cells in response to hormones and light

Mesophyll suspension cultures of Zinnia elegans L. have been used extensively to investigate the development of tracheary elements. Here we have modified the culture conditions to promote cell expansion and inhibit tracheary element differentiation and cell division. Cell expansion, measured by computer image analysis, was stimulated by auxin ( α -naphthyleneacetic acid), cytokinin (N⁶-benzylaminopurine), gibberellic acid, brassinosteroid (24-epibrassinolide), and light, all of which are known to promote cell expansion in whole plants or excised organs. Whereas light stimulated cell expansion primarily during the first 48 h of culture, auxin, cytokinin, gibberellic acid and brassinosteroid had little effect until after 48 h. Treatments also differed in their relative effects on cell elongation and radial cell expansion. Light and cytokinin had a greater effect on radial cell expansion, auxin and epibrassinolide promoted only cell elongation, and gibberellic acid had nearly equal effects on expansion in both directions. We have also shown by combining treatments that the effects of cytokinin and auxin are additive. Neither hormone treatment, however, was additive with the effect of light treatment. Finally, in contrast to xylogenic cultures where expansion occurs by tip growth, cell expansion in non-differentiating cells was due to diffuse growth. These data show that cell expansion can be induced by hormones in primary mesophyll cultures from Zinnia in contrast to serially transferred plant suspension cultures. Furthermore, they indicate that auxin, cytokinin, and light induce cell expansion by different mechanisms in these cultures.     

Conformational studies of oligomeric oxetane-based dipeptide isosteres derived from L-rhamnose or D-xylose.

Conformational investigations have been undertaken on oligomers (dimers, tetramers, hexamers) of five closely related oxetane-based dipeptide isosteres. All the oligomers were subjected to a range of studies by NMR, FT-IR and CD spectroscopy. The oligomers derived from methyl 2,4-anhydro-5-azido-3-O-tert-butyldimethylsilyl-5-deoxy-L-rhamnonate 'monomer' all exhibited evidence of ordered conformations in chloroform and 2,2,2-trifluoroethanol (TFE) solution. 5-Acetamido and N-methylamide derivatives of the L-rhamnonate 'monomer', along with a 'dimer' lacking silyl protection at C-3, were synthesized to ascertain the role of intramolecular interactions. This led to the conclusion that, for the L-rhamnonate oligomers, steric interactions govern the conformational preference observed. The equivalent silyl-protected D-lyxonate oligomers gave ordered CD spectra in TFE solution, but NMR and FT-IR spectroscopy in chloroform solution suggested an irregular, non-hydrogen bonded system. The remaining silyl-protected 6-deoxy-L-altronate, 6-deoxy-D-gulonate and D-fuconate oligomers appear to be characterized by their lack of ordered conformation in TFE and chloroform solution.     

Quantification of consumption of corn pollen by the predator Coleomegilla maculata (Coleoptera: Coccinellidae) during anthesis in an Illinois cornfield

Abstract 1 Coleomegilla maculata DeGeer feeds on corn pollen in the field, but the degree to which this predator relies on corn pollen as part of its diet is not well understood. We quantified the amount of pollen consumed by C. maculata second, third and fourth instars and adults in the field. 2 Laboratory experiments were conducted to determine the digestion rate and duration of different stadia or stages using temperature regimens that reflected field conditions during anthesis. Coleomegilla maculata larvae and adults were collected from the field and the amount of pollen in their digestive tracts was determined gravimetrically. The rate of digestion, duration of each life stage and the field observations were used to estimate the amount of pollen consumed by second, third and fourth instars and adults. 3 Our models estimate that larvae consume 0.66, 1.67 and 3.30 mg of pollen during the second, third and fourth stadia, respectively. Adults consumed an estimated 13.15 mg during anthesis. 4 The relevance of our results to ecological risk assessment of transgenic insecticidal corn and predator life history strategies is discussed. The results presented here are a first attempt to quantify pollen consumption by a predator, and future areas of research are suggested.     

Cell-free protein synthesis for proteomics.

The use of cell-free expression systems as an alternative to cell-based methods for protein production is greatly facilitating studies of protein functions. Recent improvements to cell-free systems, and the development of cell-free protein display and microarray technologies, have led to cell-free protein synthesis becoming a powerful tool for large-scale analysis of proteins. This paper reviews the most commonly used cell-free systems and their applications in proteomics.