No abstract

We have purified a novel class of protein that can inhibit the activity of endo-beta-1,4-xylanases. The inhibitor from wheat (Triticum aestivum, var. Soisson) is a glycosylated, monomeric, basic protein with a pI of 8.7-8.9, a molecular mass of 29 kDa and a unique N-terminal sequence of AGGKTGQVTVFWGRN. We have shown that the protein can inhibit the activity of two family-11 endo-beta-1, 4-xylanases, a recombinant enzyme from Aspergillus niger and an enzyme from Trichoderma viride. The inhibitory activity is heat and protease sensitive. The kinetics of the inhibition have been characterized with the A. niger enzyme using soluble wheat arabinoxylan as a substrate. The Km for soluble arabinoxylan in the absence of inhibitor is 20+/-2 mg/ml with a kcat of 103+/-6 s-1. The kinetics of the inhibition of this reaction are competitive, with a Ki value of 0.35 microM, showing that the inhibitor binds at or close to the active site of free xylanase. This report describes the first isolation of a xylanase inhibitor from any organism.

The structural gene and the regulatory DNA sequence of the yeast Hansenula polymorpha methanol oxidase have been isolated. According to the nucleotide sequence data obtained, the structural gene encodes a 664 amino acids long protein, contains no intervening sequences, and the 5'- and 3'-non-coding region contains several sequences implicated in transcription initiation and termination in the yeast Saccharomyces cerevisiae. Although the methanol oxidase is translocated to the peroxisomes, no cleavable signal sequence was found at the N-terminus of the protein.

We have purified a novel class of protein that can inhibit the activity of endo-beta-1,4-xylanases. The inhibitor from wheat (Triticum aestivum, var. Soisson) is a glycosylated, monomeric, basic protein with a pI of 8.7-8.9, a molecular mass of 29 kDa and a unique N-terminal sequence of AGGKTGQVTVFWGRN. We have shown that the protein can inhibit the activity of two family-11 endo-beta-1, 4-xylanases, a recombinant enzyme from Aspergillus niger and an enzyme from Trichoderma viride. The inhibitory activity is heat and protease sensitive. The kinetics of the inhibition have been characterized with the A. niger enzyme using soluble wheat arabinoxylan as a substrate. The Km for soluble arabinoxylan in the absence of inhibitor is 20+/-2 mg/ml with a kcat of 103+/-6 s-1. The kinetics of the inhibition of this reaction are competitive, with a Ki value of 0.35 microM, showing that the inhibitor binds at or close to the active site of free xylanase. This report describes the first isolation of a xylanase inhibitor from any organism.

The sequences of the first 194 base pairs at both termini of adenovirus type 5 (Ad5) DNA have been determined, using the chemical degradation technique developed by Maxam and Gilbert (Proc. Nat. Acad. Sci. USA 74 (1977), pp. 560-564). The nucleotide sequences 1-75 were confirmed by analysis of labeled RNA transcribed from the terminal HhaI fragments in vitro. The sequence data show that Ad5 DNA has a perfect inverted terminal repetition of 103 base pairs long.

No abstract

SummaryThe BAP3 gene of Saccharomyces cerevisiae encodes a protein with a high similarity to the BAP2 gene product, a high-affinity permease for branched-chain amino acids. In this paper, we show that, like BAP2, the expression of the BAP3 gene in S. cerevisiae is induced by the addition of branched-chain amino acids to the medium. Unexpectedly, most other naturally occurring L-amino acids found in proteins (with the exception of proline, lysine, arginine and histidine) have the same effect on the expression of BAP3. The induction of BAP3 expression appears to be dependent on Stp1p, a nuclear protein, previously shown to be involved in pre-tRNA maturation and also required for the expression of BAP2, as induction is no longer observed in an stp1¹ mutant. The transcriptional regulator Leu3p is not involved in the induction of BAP3 expression, but may act as a repressor of BAP3 expression in the absence of leucine, as can be inferred from a transcriptional analysis in a ⌬leu3 mutant. By extensive deletion analysis of the BAP3 promoter fused to a GUS reporter, as well as by fusions of different parts of the BAP3 promoter to a LacZ reporter, we have found that a portion of the BAP3 promoter from ¹ 418 to ¹ 392 relative to the ATG start codon is both necessary and sufficient for the Stp1p-dependent induction of BAP3 expression by (most) amino acids. We have therefore named this sequence UAS aa (amino acid-dependent upstream activator sequence). Neither Stp1p nor Leu3p appear to bind to the UAS aa , at least in vitro, as judged from gel retardation assays. Sequences similar to the UAS aa can be found in the promoters of BAP2, PTR2 and TAT1 ; genes that, like BAP3, encode permeases inducible by amino acids, suggesting that amino acid induction of all these genes is exerted via a common mechanism.

A gene library from the methanol utilizing yeast Hansenula polymorpha, constructed in a lambda Charon4A vector, was used to clone the gene encoding a key methanol assimilating enzyme, dihydroxyacetone synthase (DHAS) by differential plaque hybridization. The nucleotide sequence of the 2106 bp structural gene and the 5' and 3' non-coding regions was determined. The deduced amino acid sequence of the protein is in agreement with the apparent molecular weight and amino acid composition of the purified protein. The codon bias is not so pronounced as in some Saccharomyces cerevisiae genes.