Site-directed mutagenesis and expression in Escherichia coli of WMAI-1, a wheat monomeric inhibitor of insect α-amylase

The wheat monomeric inhibitor WMAI-1 (syn. 0.28) produced inEscherichia coli using the pT7-7 expression ventor has the correct N-terminal sequence and the same electrophoretic mobility and specific activity towards the -amylase from the insectTenebrio molitor as the native WMAI-1 isolated from wheat. This confirms that the native inhibitor is not glycosylated and contradicts claims that a putative glycosyl moiety was essential for inhibition. Thirteen mutants have been obtained at six different sites. Substitution of the highly conserved N-terminal S by the sequence ARIRAR increased the pre-incubation time required for maximum activity. A similar result was obtained by insertion of GPRLPW after position 4, while insertion of EPRAPW at the same position rendered the inhibitor inactive. The substitution D/EGPRL and insertions DGP or D, at position 58, produced complete inactivation. All other mutations had only minor effects on activity

cDNAs of a wheat tetrameric inhibitor of a-amylases

In the laboratory of M. Metzlaff, genome specific DNA probes from Hordeum vulgare were cloned and characterized by H. Junghans. Regarding repeated DNA sequences our further investigations will concéntrate on the proofof alien chromatin in the wheat -Ae. markgrafii crossing material and on the enlargement of the investigation to their distribution in Poaceae species

Nucleotide sequence of a cDNA encoding an α/β-type gliadin from hexaploid wheat (Triticum aestivum).

A cDNA clone was isolated from a library obtained from developing endosperm of cv. Chinese Spring which encoded an α/β-type gliadin that differs from previously described ones. The nucleotide sequence and deduced amino acid sequence of the new clone, designated MM1, are presented

Extreme variations in the ratios of non-synonymous to synonymous nucleotide substitution rates in signal peptide evolution

Nucleotide sequences encoding signal peptidcs from the precursors of α-amylase/trypsin inhibitors from cereals are homologous to those corresponding to the precursors of thaumatin II and of plastocyanins. Non-synonymous (KA) and synonymous (KS) rates of nucleotide substitutions have been calculated for all possible binary combinations. Extreme variation in KA/KS ratios has been observed, from the 0.167 average found within the plastocyanin family to an average of 1.90 calculated for the inhibitors/thaumatin II transition. A similar calculation has been carried out for the signal peptide sequences of thionins. which are unrelated to those of the α-amylase trypsin inhibitor family, and an average KA/KS of 0.12 has been obtained. This variation can be largely explained in terms of an empirical index of stability related to amino acid composition and seems to be independent of functional constraints

Extreme variations in the ratios of non-synonymous to synonymous nucleotide substitution rates in signal peptide evolution

Nucleotide sequences encoding signal peptides from the precursors of 2-amylase/trypsin inhibitors from cereals are homologous to those corresponding to the precursors of thaumatin II and of plastocyanins. Non-synonymous (K A ) and synonymous {K % ) rates of nucleotide substitutions have been calculated for all possible binary combinations. Extreme variation in A' A^S ratios has been observed; from the 0.167 average found within the plastocyanin family to an average of 1.90 calculated for the inhibitors/thaumatin II transition. A similar calculation has been carried out for the signal peptide sequences of thionins. which are unrelated to those of the a-amylase trypsin inhibitor family. and an average /í A //w s of 0.12 has been obtained. This variation can be largely explained in terms of an empirical index of stability related to amino acid composition and seems to be independent of functional constraints. ot-Amylase;trypsin inhibitor; Thaumatin II; Thionin; Plastocyanin; Signal peptide evolutio

Trypsin/α-amylase inhibitors and thionins: possible defence proteins from barley

This chapter reviews recent work on the trypsin/α-amylase inhibitor and thionin protein families. The genomic distribution of protein genes in barley and related species, gene expression and in vitro activities are considered. Some of the evidence of a possible defence role against stored products pests for inhibitors and thionins is briefly discusse

DNA Sequencing Sensors: An Overview

The first sequencing of a complete genome was published forty years ago by the double Nobel Prize in Chemistry winner Frederick Sanger. That corresponded to the small sized genome of a bacteriophage, but since then there have been many complex organisms whose DNA have been sequenced. This was possible thanks to continuous advances in the fields of biochemistry and molecular genetics, but also in other areas such as nanotechnology and computing. Nowadays, sequencing sensors based on genetic material have little to do with those used by Sanger. The emergence of mass sequencing sensors, or new generation sequencing (NGS) meant a quantitative leap both in the volume of genetic material that was able to be sequenced in each trial, as well as in the time per run and its cost. One can envisage that incoming technologies, already known as fourth generation sequencing, will continue to cheapen the trials by increasing DNA reading lengths in each run. All of this would be impossible without sensors and detection systems becoming smaller and more precise. This article provides a comprehensive overview on sensors for DNA sequencing developed within the last 40 years