Identification of an enhancer/silencer sequence directing the aleurone-specific expression of a barley chitinase gene.

Chitinases are expressed in various plant tissues where they are thought to play a role in defense against chitin-containing pathogens. Transient gene expression assays have been used in tissues of barley to delineate promoter sequences involved in the regulation of an aleurone-specific chitinase gene (Chi26), and of a vegetatively expressed chitinase gene (Chi33). The assays measured the activities of transcriptional fusions between chitinase 5' upstream sequences and GUS reporter genes after DNA delivery by particle bombardment. Analysis of Chi26 5' and 3' promoter deletions indicated that sequences between -200 and -140 confer developmental and aleurone-specific expression. Deletions/replacements covering this part of the promoter indicated that sequences between -179 and -147 (E-region) direct expression in aleurone cells. The ability of the 33bp E-region of the Chi26 promoter to activate transcription specifically in aleurone was confirmed by constructing and testing two types of chimeric promoters. The first type, which contained two copies of the E-region fused to the CaMV 35S TATA box, conferred aleurone-specific expression of a GUS reporter gene. The second type, which contained a single copy of the E-region inserted into a deleted, inactive Chi33 promoter derivative, was also capable of directing transcription in aleurone but not in leaves. The pattern of expression of this and other Chi26/Chi33 chimeric promoters suggest that the E-region contains cis-acting sequences which activate transcription in aleurone and silence transcription in leaves. DNA sequence motifs implicated in the regulation of Chi26 and Chi33 are described.

T-helper-cell determinants in protein antigens are preferentially located in cysteine-rich antigen segments resistant to proteolytic cleavage by cathepsin B, L, and D.

We report on a computer algorithm capable of predicting the location of T-helper-cell epitopes in protein antigen (Ag) by analysing the Ag amino acid sequence. The algorithm was constructed with the aim of identifying segments in Ag which are resistant to proteolytic degradation by the enzymes cathepsin B, L, and D. These are prominent enzymes in the endocytic pathway through which soluble protein Ag enter APC, and resistant segments in Ag may, therefore, be expected to contain more T-cell determinants than susceptible segments. From information available in the literature on the substrate specificity of the three enzymes, it is clear that a cysteine is not accepted in any of the S2, S1, S1', and S2' subsites of cathepsin B and L, and not in the S1 and S1' subsites of cathepsin D. Moreover, we have noticed that cysteine-containing T-cell determinants in a number of protein Ag are particularly rich in the amino acids alanine, glycine, lysine, leucine, serine, threonine, and valine. By searching protein Ag for clusters of amino acids containing cysteine and two of the other amino acids we were able to predict 17 out of 23 empirically known T-cell determinants in the Ag with a relatively low number of false (positive) predictions. Furthermore, we present a new principle for searching Ag for potential amphipatic alpha-helical protein segments. Such segments accord well with empirically known T-cell determinants and our algorithm produces a lower number of false positive predictions than the principle based on discrete Fourier transformations previously described.