Processing of Thionin Precursors in Barley Leaves by a Vacuolar Proteinase

Thionins are synthesized as precursors with a signal peptide and a long C-terminal acidic peptide that is post-translationally processed. A fusion protein including the maltose-binding protein from Eschrrichia coli (MalE), thionin DG3 froin barley leaves, and its acidic C-terminal peptide has been used to obtain antibodies that recognize both domains of the precursor. In barley leaf sections. mature thionins accuinulated in the vacuolar content, while the acidic peptide was not detected in any cell fraction. Brefeldin A and inonensin inhibited processing of the precursor but its export from the microsomal fraction was not inhibited. Both purified vacuoles aiid an acid (pH 5.5) extract from leaves processed the fusion protein into a MalE-thionin and an acidic peptide fragment. A 70-kDa proteinase that effected this cleavage was purified froin the acid extract. Processing of the fusion protein by both lysed vacuoles and the purified proteinase was inhibited by Zn2+ and by Cu2+, but not by inhibitors of the previously described vacuolar processing thiol or aspartic proteinases. In vivo processing of the thionin precursor in leaf sections was also inhibited by Zn+, and Cu2+, Variants of the fusion protein with altered processing sites that represented thme of thionin precursors from different taxa were readily processed by the proteinase, whereas changing the polarity of either the C-terminal or N-terminal residues of the processing site prevented cleavage by the proteinase

Subcellular localization of type-I thionins in the endosperms of wheat and barley.

Thionins are cysteine-rich polypeptides of about 5,000 Da. Localization at the subcellular level of type I endosperm thionins has been carried out by immunogold labeling, using an antibody that recognizes type I thionin variants. In developing wheat and barley caryopses, sectioned at different times between 13 and 24 days after flowering, this type of thionins was only detected around protein bodies from cells of the starchy endosperm, using light microscopy. Electron microscopy revealed that these proteins were located in electron-dense spheroids in the periphery of protein bodies, at the earlier stages, whereas later the label appeared also as a thin layer around these organelles

Two cold inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens

The barley genesHvLtp4.2 andHvLtp4.3 both encode the lipid transfer protein LTP4 and are less than 1 kb apart in tail-to-tail orientation. They differ in their non-coding regions from each other and from the gene corresponding to a previously reportedLtp4 cDNA (nowLtp4.1). Southern blot analysis indicated the existence of three or moreLtp4 genes per haploid genome and showed considerable polymorphism among barley cultivars. We have investigated the transient expression of genesHvLtp4.2 andHvLtp4.3 following transformation by particle bombardment, using promoter fusions to the-glucuronidase reporter sequence. In leaves, activities of the two promoters were of the same order as those of the sucrose synthase (Ss1) and cauliflower mosaic virus 35S promoters used as controls. Their expression patterns were similar, except thatLtp4.2 was more active thanLtp4.3 in endosperm, andLtp4.3 was active in roots, whileLtp4.2 was not. The promoters of both genes were induced by low temperature, both in winter and spring barley cultivars. Northern blot analysis, using theLtp4-specific probe, indicated thatXanthomonas campestris pv.translucens induced an increase over basal levels ofLtp4 mRNA, whilePseudomonas syringae pv.japonica caused a decrease. TheLtp4.3-Gus promoter fusion also responded in opposite ways to these two compatible bacterial pathogens, whereas theLtp4.2-Gus construction did not respond to infectio

Effect of T4 modification of host valyl-tRNA synthetase on enzyme action in vivo

It has previously been found that bacteriophage T4 modifies the valyl-tRNA synthetase of its host; in this work we have examined the effects of this modification on the action of the enzyme in vivo. For this purpose we compared the properties of T4-infected cells with those of uninfected cells or cells infected with the amber mutant vs2, which by all available criteria seems to leave the host enzyme completely unmodified.We find that, although the overall rate of transfer of valine into protein is reduced after infection, modification is apparently not the cause of this reduction. Also, modification does not alter the level of aminoacylation of the total valine tRNA pool nor of the invidividual species of valine tRNA. Nor does modification appear to be necessary to maintain these levels after infection. We found no evidence that modification affects any unknown valyl-tRNA synthetase reaction that yields novel valine-containing products. Although the modified enzyme is more stable in vitro than the unmodified enzyme, modification does not seem to facilitate phage growth at temperatures above the optimum.To all appearances, therefore, T4-directed modification of the host valyl-tRNA synthetase does not detectably affect the in vivo catalytic action of the enzyme in any straightforward way, at least under prevailing laboratory conditions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/22178/1/0000609.pd

Initiation of rrn transcription in chloroplasts of Euglena gracilis bacillaris

The site of initiation of chloroplast rRNA synthesis was determined by Sl-mapping and by sequencing primary rRNA transcripts specifically labeled at their 5′-end. Transcription initiates at a single site 53 nucleotides upstream of the 5'-end of the mature 16S rRNA under all growth conditions examined. The initiation site is within a DNA sequence that is highly homologous to and probably derived from a tRNA gene-region located elsewhere in the chloroplast genome. A nearly identical sequence (102 of 103 nucleotides) is present near the replication origin. The near identity of the two sequences suggests a common mode for control of transcription of the rRNA genes and initiation of chloroplast DNA replication. The related sequence in the tRNA gene-region does not appear to serve as a transcript initiation site.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46967/1/294_2004_Article_BF00521275.pd