The exceptionally high rate of spontaneous mutations in the polymerase delta proofreading exonuclease-deficient Saccharomyces cerevisiae strain starved for adenine

Background

Mutagenesis induced in the yeast Saccharomyces cerevisiae by starvation for nutrilites is a well-documented phenomenon of an unknown mechanism. We have previously shown that the polymerase delta proofreading activity controls spontaneous mutagenesis in cells starved for histidine. To obtain further information, we compared the effect of adenine starvation on mutagenesis in wild-type cells and, in cells lacking the proofreading activity of polymerase delta (phenotype Exo^-, mutation pol3-01).

Results

Ade⁺ revertants accumulated at a very high rate on adenine-free plates so that their frequency on day 16 after plating was 1.5 × 10^-4 for wild-type and 1.0 × 10^-2 for the Exo^- strain. In the Exo^- strain, all revertants arising under adenine starvation are suppressors of the original mutation, most possessed additional nutritional requirements, and 50% of them were temperature sensitive.

Conclusions

Adenine starvation is highly mutagenic in yeast. The deficiency in the polymerase delta proofreading activity in strains with the pol3-01 mutation leads to a further 66-fold increase of the rate of mutations. Our data suggest that adenine starvation induces genome-wide hyper-mutagenesis in the Exo^- strain.     

A labeling, detection, and purification system based on 4-hydroxyazobenzene-2-carboxylic acid: an extension of the avidin-biotin system

We introduce a new nonradioactive, chromogenic label based on 4-hydroxyazobenzene-2-carboxylic acid (HABA), which is suitable for bioanalytical application, e.g., detection, localization, isolation, and purification. The HABA label is superior to other systems where it is difficult to separate labeled from unlabeled molecules or to determine the amount of label. HABA is readily detected spectroscopically by its absorption at 350 nm or by its interaction with avidin that results in a red shift to 500 nm. The HABA reagents described can be conjugated to a variety of functional groups on biomolecules and purified thereafter by affinity chromatography on an avidin column. The interaction of the HABAylated biomolecules with their corresponding targets is detected with high-affinity anti-HABA antibodies or with avidin. The nonradioactive, chromogenic HABA-based reagents form a homogeneous system that can complement or replace systems where facile quantification of the label is desired.     

Evolutionary Adaptations of Haemoglobins

Abstract

The properties of haemoglobin oxygen dissociation curves and the Bohr effect have, been studied in various poikiloterms animals living in different ecological conditions. The position of the curve and the direction and the intensity of the Bohr effect are perfectly correlated with the ecological habit of the organism under examination. Both these parameters are regulated by cofactors modifiyng the properties of the haemoglobin. An increase in the Bohr effect, probably with evolutionary adaptive value has been found in the indian peruvian population living from generation at high altitude. Also in this case the increase in Bohr effect is due to some factor presente in the red cell and different by the organic phosphates.     

Novel families of toxin-like peptides in insects and mammals: a computational approach

Most animal toxins are short proteins that appear in venom and vary in sequence, structure and function. A common characteristic of many such toxins is their apparent structural stability. Sporadic instances of endogenous toxin-like proteins that function in non-venom context have been reported. We have utilized machine learning methodology, based on sequence-derived features and guided by the notion of structural stability, in order to conduct a large-scale search for toxin and toxin-like proteins. Application of the method to insect and mammalian sequences revealed novel families of toxin-like proteins. One of these proteins shows significant similarity to ion channel inhibitors that are expressed in cone snail and assassin bug venom, and is surprisingly expressed in the bee brain. A toxicity assay in which the protein was injected to fish induced a strong yet reversible paralytic effect. We suggest that the protein may function as an endogenous modulator of voltage-gated Ca(2+) channels. Additionally, we have identified a novel mammalian cluster of toxin-like proteins that are expressed in the testis. We suggest that these proteins might be involved in regulation of nicotinic acetylcholine receptors that affect the acrosome reaction and sperm motility. Finally, we highlight a possible evolutionary link between venom toxins and antibacterial proteins. We expect our methodology to enhance the discovery of additional novel protein families.     

Lignin triggers irreversible cellulase loss during pretreated lignocellulosic biomass saccharification

Background

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.

Results

In this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion^a (AFEX?-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.

Conclusions

Our findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

     

Microbiological titration of proteins and of single amino acid content in biological materials without purification and hydrolysis

A method is described for the microbiological determination of the protein content of biological materials. This method can also be adopted to titrate the concentration of a single amino acid in the protein and has the following advantages: (1) titration can be done without purification and hydrolysis of proteins; (2) the titration graph is a straight line between 25 and 800 microgram/ml; (3) protein values agree with those obtained using the Kjeldhal method; and (4) each mutant requiring one amino acid may be used to titrate the concentration of a single amino acid of the protein. The leucine content of various kinds of flour was measured with this system.