Temporal and spatial control of expression of anthocyanin biosynthetic genes in developing flowers of Antirrhinum majus

The co-ordination of expression of anthocyanin biosynthetic genes was studied in developing flowers. Four genes encoding enzymes operating late in the anthocyanin biosynthetic pathway are induced together during flower development but the early steps appear to be induced more rapidly. Co-ordination of expression could imply a common regulatory mechanism controlling the expression of metabolically related genes. The data presented here show that while four genes may share such a mechanism for the control of their expression during flower development, different control processes regulate the early steps of the pathway. Spatially, gene expression is patterned across the flower and appears to be very similar for all the biosynthetic genes. However, the observed influence of the regulatory gene Delila shows that the spatial co-ordination of gene expression must involve more than one regulatory system. Delila itself appears to have a dual function, being required for activation of expression of the later genes in the flower tube but repressing chalcone synthase gene expression in the mesophyll of the corolla lobes. It is postulated that common signals induce the expression of genes in the pathway during flower development. The data presented here suggest that the same regulatory mechanism interprets these signals for four of the genes encoding the later biosynthetic enzymes, but that different or modified mechanisms interpret the signals to control expression of chalcone synthase and chalcone isomerase genes in Antirrhinum flowers.     

Peptides That Bind Specifically to an Antibody from a Chronic Lymphocytic Leukemia Clone Expressing Unmutated Immunoglobulin Variable Region Genes

Chronic lymphocytic leukemia (CLL) is a clonal disease of a subset of human B lymphocytes. Although the cause of the disease is unknown, its development and evolution appear to be promoted by signals delivered when B-cell receptors (BCRs) engage (auto)antigens. Here, using a peptide phage display library of enhanced size and diverse composition, we examined the binding specificity of a recombinant monoclonal antibody (mAb) constructed with the heavy chain and light chain variable domains of a CLL BCR that does not exhibit somatic mutations. As determined by testing the peptides identified in the selected peptide phage pool, this CLL-associated unmutated mAb bound a diverse set of sequences, some of which clustered in families based on amino acid sequence. Synthesis of these peptides and characterization of binding with the CLL-associated mAb revealed that mAb-peptide interactions were generally specific. Moreover, the mAb-peptide interactions were of lower affinities (micromolar K_D), as measured by surface plasmon resonance, than those observed with a CLL mAb containing somatic mutations (nanomolar K_D) and with immunoglobulin heavy chain variable (IGHV)-mutated antibodies selected by environmental antigens. This information may be of value in identifying and targeting B lymphocytes expressing specific BCRs in CLL patients and healthy subjects with monoclonal B lymphocytosis.     

Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid–derived cyanogenic glucosides (α-hydroxynitrile glucosides) by specific β-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the β-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related β-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants.