Selected cell wall proteins from Zea mays: Assessment of their role in wall hydrolysis

Coleoptile cell wall proteins from Zea mays L. hybrid B 37 × Mo 17 were extracted and fractionated. Three enzymes identified in that extract were examined to determine their role in cell wall hydrolysis with a goal of evaluating the extent to which they participated in autohydrolytic reactions. Two separate proteins were identified as endo- and exo-glucanases. Incubation of these enzymes with heat inactivated cell walls, liberates products derived from the constitutive (1→3), (1→4)-β- d -glucan. The release of sugars from walls resembles that of cell wall autolysis. A third cell wall protein degraded polysaccharides in a more general manner, releasing carbohydrates containing xylose, arabinose, galactose and glucose. Polyclonal antibodies raised against the exoglucanase protein suppressed autolytic reactions of isolated cell wall.     

Structure and expression of the lignin O-methyltransferase gene from Zea mays L.

The isolation and characterization of cDNA and homologous genomic clones encoding the lignin O-methyltransferase (OMT) from maize is reported. The cDNA clone has been isolated by differential screening of maize root cDNA library. Southern analysis indicates that a single gene codes for this protein. The genomic sequence contains a single 916 bp intron. The deduced protein sequence from DNA shares significant homology with the recently reported lignin-bispecific caffeic acid/5-hydroxyferulic OMTs from alfalfa and aspen. It also shares homology with OMTs from bovine pineal glands and a purple non-sulfur photosynthetic bacterium. The mRNA of this gene is present at different levels in distinct organs of the plant with the highest accumulation detected in the elongation zone of roots. Bacterial extracts from clones containing the maize OMT cDNA show an activity in methylation of caffeic acid to ferulic acid comparable to that existing in the plant extracts. These results indicate that the described gene encodes the caffeic acid 3-O-methyltransferase (COMT) involved in the lignin biosynthesis of maize.     

Root-specific expression of a Zea mays gene encoding a novel glycine-rich protein,zmGRP3

The isolation and characterization of a cDNA clone from Zea mays coding for a novel glycine-rich protein (GRP) is described. The corresponding 1.4 kb mRNA accumulates exclusively in roots (primary, lateral seminal and crown roots) of young maize seedlings, following developmentally specific patterns. In agreement with previously described GRPs from other plant species the derived protein sequence exhibits a hydrophobic domain at the N-terminal region followed by repeated glycine-rich motifs. Genomic Southern analysis indicates that the zmGRP3 gene is present in the maize genome as one or two copies or at a low copy number.     

Molecular cloning of three cDNAs encoding putative larval cuticle protein expressed differentially after larval ecdysis from the mulberry longicorn beetle, Apriona germari

Three cDNAs encoding putative larval cuticle protein (LCP) were cloned from the mulberry longicorn beetle, Apriona germari. The three cDNA sequences were 309 bp, 396 bp and 408 bp in length, encoding 103, 132 and 136 amino acid residues, respectively. The predicted molecular masses for these LCPs were approximately 9.2 kDa (AgLCP9.2), 12.3 kDa (AgLCP12.3) and 12.6 kDa (AgLCP12.6). Pairwise identity among AgLCP9.2, AgLCP12.3 and AgLCP12.6 were relatively low. Each AgLCP contained a type-specific consensus sequence identifiable in other insect cuticle proteins. The deduced amino acid sequence of AgLCP9.2 is most similar to Bombyx mori LCP18 and those of AgLCP12.3 and AgLCP12.6 are both most similar to B. mori LCP17. Northern blot analysis revealed that the three AgLCPs showed epidermis-specific expression. The expression profile of AgLCPs after larval ecdysis revealed by Northern blot analysis that the high-level mRNA expression of AgLCPs was detected on the first day of larval ecdysis for AgLCP9.2, on the fifth day for AgLCP12.3 and from the first day of larval ecdysis to the fifth day after larval ecdysis for AgLCP12.6, demonstrating that AgLCP mRNAs are differentially expressed in epidermis after larval ecdysis.     

Molecular cloning, localization and characterization of a 40-kDa catecholamine-regulated protein

We have previously described catecholamine-regulated proteins of molecular masses 47, 40 and 26 kDa (CRP47/40/26). In mammals, these proteins are detected only in brain and have been implicated as playing a role in dopaminergic neurotransmission. In this report, we have cloned the cDNA encoding CRP40 from bovine brain. Analysis of the predicted amino acid sequence revealed that the CRP40 product contains an hsp70 motif and shares homology with heat-shock protein hsp70. Immunolocalization studies using mAbs to dopamine show that it colocalizes with CRP40 in the vesicles of dopaminergic neuroblastoma SH-SY5Y cells. The constitutive expression of CRP40 was increased by exposure to heat shock similar to inducible heat-shock protein hsp70 in SH-SY5Y cells. Dopamine significantly modulated the levels of CRP40, whereas, the expression of hsp70 remained unchanged upon dopamine treatment of these cells. Moreover, CRP40 is able to prevent the thermal aggregation of luciferase in vitro, similar to hsp70, suggesting that CRP40 encodes a dopamine-inducible protein with properties similar to heat-shock proteins. The immunofluorescence analyses show that in SH-SY5Y cells, CRP40 translocates to the nucleus during dopamine-induced apoptosis. These results suggest that CRP40 could play a protective role against the harmful effects of catecholamine metabolites.     

Barley aleurone cell development: molecular cloning of aleurone-specific cDNAs from immature grains

The cloning of 11 different homology groups of cDNAs representing genes expressed in aleurone, but not in starchy endosperm of 20-day-old barley grains is described. Among the cDNAs, four are aleurone-specific, while the remaining are also expressed in the embryo, but not in any other part of the plant.Sequence analysis of one of the aleurone-specific clones, B11E, reveals an open reading frame coding for an unidentified 10.4 kDa protein with a putative signal sequence and a possible metal-binding finger. The B11E gene has a high GC content in the 5 leader sequence (63%), as well as in the coding region (70%) compared to known cDNAs from the barley starchy endosperm. Northern analysis of B11E indicates maximum mRNA abundance around mid-phase of grain development.When isolated immature aleurone/pericarp is incubated in tissue culture medium (MS) the B11E message disappears, indicating a requirement for a diffusible factor from the intact grain for its continued presence.     

Molecular cloning of pea mRNAs encoding a shoot-specific polypeptide and light-induced polypeptides

Summary The molecular cloning of cDNA corresponds to pea seedling mRNA sequences encoding a shoot-specific polypeptide, the small subunit of the ribulose 1,5 biphosphate carboxylase and a component of the light-harvesting chlorophyll a/b complex is described. cDNA prepared from polysomal poly(A)RNA of light-grown shoots was enriched for shoot-specific and light-induced sequences by heterologous liquid hybridization with mercurated polysomal poly(A)RNA of dark-grown roots, followed by sulfhydryl chromatography. Cloned shoot-specific sequences were identified by 2D electrophoretic analysis of hybrid release translation products. The cloned shoot-specific sequence corresponded to a mRNA of 850 nt present both in light-and dark-grown shoots, and produced anin vitro translation product of M_r27 500 and isoelectric point of 4.7.     

Molecular cloning and characterization of anther-preferential cDNA encoding a putative actin-depolymerizing factor

A cDNA clone, LMP131A, which is preferentially expressed in mature anther was isolated from a lily cDNA library. Northern blot analysis and plaque hybridization expriments showed that the LMP131A mRNA is present at ca. 0.3% of the mRNA in mature pollen and is not detectable in carpel, petal, floral bud, leaf, or root. The clone contains an open reading frame of 139 amino acid residues which shows greater than 40% sequence identity in a 91 amino acid overlap to animal actin-depolymerizing factors (ADF), cofilin and destrin. The sequences at and near the actin-binding site are most conserved. Using the lily clone as a probe, a cDNA clone, BMP1, was isolated from a mature anther library of Brassica napus. The expression pattern of the BMP1 clone was the same as that of the lily clone. The Brassica anther-preferential clone contains an open reading frame which is 79% identical to the lily LMP131A protein. Southern blot analysis showed that there are one or a few copies of the putative ADF genes in B. napus and Arabidopsis thaliana.     

Molecular cloning of cDNAs encoding a range of digestive enzymes from a phytophagous beetle, Phaedon cochleariae

To gain better knowledge of the variety of digestive enzymes in phytophagous coleopteran pests, a sequencing screen of 76 random cDNAs from a gut library from Phaedon cochleariae larvae was performed. The screen yielded 21 cDNAs encoding amino-acid sequences homologous to known digestive enzymes, most of them were cell wall-hydrolysing enzymes. The deduced protein sequences of 7 cDNAs encoding putative -amylase, cysteine proteinase, trypsin, chymotrypsin, cellulase, pectinase and xylanase display all the structural features that characterize these enzymes in other eukaryotic organisms. Except the -amylase and chymotrypsin cDNAs, the other cDNAs probably derive from multigene families. The distribution of the corresponding enzymatic activities at various developmental stages of P. cochleariae was examined. -amylase activity is present in guts of larvae and adults, proteinases are abundant in guts of larvae and adults, but scarce in eggs and larval carcasses, xylanases are present in the guts of larvae and adults, as well as in carcasses of larvae, whereas cellulase and pectinase activities are distributed in larval and adult guts, larval carcasses, and eggs. Only a minor fraction of the cellulases is secreted by microorganisms, suggesting that P. cochleariae synthesizes most of its own cell-wall hydrolysing enzymes. The physiological role of the enzymes is discussed, as well as the significance of these results for pest management strategies involving transgenic plants expressing enzyme inhibitors.     

Molecular cloning and characterization of a novel V-ATPase associated protein, DVA9.2, from human dendritic cells

The vacuolar proton-ATPase (V-ATPase) is a ubiquitous ATP-driven H(+) transporter that functions in numerous cell processes. Accumulating evidence shows important roles of V-ATPase in tumor metastasis and antigen presentation of dendritic cells (DC). A novel V-ATPase associated protein, designated as DVA9.2 (dendritic cell-derived V-ATPase associated protein of 9.2 kDa), has been identified from a human DC cDNA library by large-scale random sequencing. Full length cDNA of DVA9.2 encodes an 81-residue protein that shares 70-80% homology with human V-ATPase subunit M9.2. Distant relationship is also found with Vma21p, a yeast protein required for V-ATPase assembly. DVA9.2 contains a conserved domain, ATP synthase subunit H (pafm05493), and two membrane-spanning helices. DVA9.2 mRNA is detectable in several human tumor cell lines as well as some human normal cells and tissues. Moreover, the inducible expression of DVA9.2 mRNA in DC during maturation is observed. DVA9.2 displays integration with membrane and main localization in lysosome, endoplasmic reticulum and Golgi-associated organelles, only less at the plasma membrane. In addition, DVA9.2 is co-localized with V(0)-sector subunit a. Silencing of DVA9.2 by small interfering RNA (siRNA) does not affect the V-ATPase activity in cell membrane fractions or attenuate the migration and invasion in breast cancer MDA-MB-231 cells. These results indicate that DVA9.2, as a novel V-ATPase-associated protein, is not essential for the activity of V-ATPase complex and may be involved in functions of DC.     

三疣梭子蟹蜕皮抑制激素cDNA的克隆与序列分析

甲壳动物的蜕皮是由位于头胸部前鳃腔的一对Y-器通过分泌蜕皮激素（Molting hormone）来控制的（Lachaise et al．,1993）,而蜕皮激素的分泌又受到蜕皮抑制激素（Molt-inhibiting hormone,MIH）的调控（Watson et al．,2001）。MIH和性腺抑制激素（Gonad-inhibiting hormone,GIH）、甲壳动物高血糖激素（Crustacean hyperglycemic hormone,CHH）、