Richard L. Weiss

A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors

The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.

Enabling a Community to Dissect an Organism: Overview of the Neurospora Functional Genomics Project

A consortium of investigators is engaged in a functional genomics project centered on the filamentous fungus Neurospora, with an eye to opening up the functional genomic analysis of all the filamentous fungi. The overall goal of the four interdependent projects in this effort is to accomplish functional genomics, annotation, and expression analyses of Neurospora crassa, a filamentous fungus that is an established model for the assemblage of over 250,000 species of non yeast fungi. Building from the completely sequenced 43-Mb Neurospora genome, Project 1 is pursuing the systematic disruption of genes through targeted gene replacements, phenotypic analysis of mutant strains, and their distribution to the scientific community at large. Project 2, through a primary focus in Annotation and Bioinformatics, has developed a platform for electronically capturing community feedback and data about the existing annotation, while building and maintaining a database to capture and display information about phenotypes. Oligonucleotide-based microarrays created in Project 3 are being used to collect baseline expression data for the nearly 11,000 distinguishable transcripts in Neurospora under various conditions of growth and development, and eventually to begin to analyze the global effects of loss of novel genes in strains created by Project 1. cDNA libraries generated in Project 4 document the overall complexity of expressed sequences in Neurospora, including alternative splicing alternative promoters and antisense transcripts. In addition, these studies have driven the assembly of an SNP map presently populated by nearly 300 markers that will greatly accelerate the positional cloning of genes.

Measurement of Pulmonary Blood Flow with Transesophageal Two-dimensional and Doppler Echocardiography

Transesophageal echocardiography permits measurement of the pulmonary artery diameter (two-dimensional echocardiography) and pulmonary artery blood flow velocity (pulsed-wave Doppler). These measurements considered with the heart rate allow for the determination of pulmonary artery blood flow, which is equivalent to cardiac output. This study compared the precision of transesophageal Doppler-derived cardiac output (DdCO) with the precision of thermodilution cardiac output (TdCO) and examined the agreement between DdCO and TdCO in 33 cardiac surgical patients. The proximal pulmonary artery diameter was measured in triplicate during systole and end expiration, and the local blood flow velocity was recorded on video tape. The instantaneous pulmonary artery blood flow velocity (centimeters per second) for three random cardiac beats was integrated with respect to time. DdCO was calculated as the product of the flow velocity integral (centimeters per beat), heart rate (beats per min), and the mean cross-sectional area (centimeters squared) of the main pulmonary artery. At the same time that the velocity recordings were made, three serial determinations of TdCO were made by an independent observer. Pulmonary blood flow could be measured in 25 of the 33 patients. The anatomical relationship among the esophagus, the left main stem bronchus, and the pulmonary artery did not allow adequate imaging of the pulmonary artery in 8 (24%) of the patients. A total of 45 sets of triplicate measurements were made. The range of cardiac outputs encountered was 1.7-6.6 l.min-1 by TdCO and 1.5-6.9 l.min-1 by DdCO. The 95% confidence limits for the difference between the two methods (agreement) was 0.030 +/- 0.987 l.min-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and characterization of the ornithine carbamoyltransferase gene from Aspergillus nidulans

An Aspergillus nidulans DNA fragment composed of two adjacent SalI subfragments (1.8 and 0.85 kb) that carries an argB gene complementing the yeast arg3 mutation has been isolated from two different gene libraries. Hybridization results and immunological tests indicate that the cloned fragment contains the A. nidulans structural gene coding for ornithine carbamoyltransferase (OTCase). Using the cloned gene as a probe, the specific mRNA was identified. The level of this RNA observed in A. nidulans strains grown under various conditions correlated with the level of the OTCase activity, suggesting transcriptional control of OTCase synthesis. Expression of the cloned gene in Saccharomyces cerevisiae does not depend on its orientation in the vector. In Escherichia coli, the cloned gene does not function; however arg- transformants revert to prototrophy with high frequency possibly due to DNA rearrangements within the recombinant plasmid.

Enzyme assays using permeabilized cells of Neurospora

Abstract A procedure is described for the preparation of homogeneous, steady-state cultures of germinated conidia of Neurospora crassa . Permeabilization of such cells has been accomplished by a combination of toluene-ethanol and freeze-thaw treatments. These permeabilized cells have been used for the determination of enzyme activities. The method has been shown to be rapid and rellable and is applicable to nuclear, mitochondrial, and cytosolic enzymes.

Validation of Plasma Biomarkers in Degenerative Calcific Aortic Stenosis

BACKGROUND Calcific aortic stenosis (CAS) is the most common acquired valvular disorder in industrialized countries. This study investigates the correlation of different known biomarkers for CAS as a first step towards the development of a panel of biomarkers that can be used in prognostic staging. METHODS Venous blood samples were obtained from both patients with CAS scheduled for surgery and healthy individuals. Plasma levels of fetuin-A, NT-proBNP, BNP, homocysteine and osteopontin were measured by enzyme-linked immunosorbent assay (ELISA). CAS was measured by echocardiography and was defined as an aortic valve area of less than 2.0 cm(2). Non-paired t-tests were used for comparison. RESULTS CAS was present in 33 subjects (mean age 75.9 y) and absent in 11 subjects (mean age 55.36 y). Individuals with CAS exhibited higher plasma levels of NT-proBNP (1.33 versus 0.73 pmol/mL, P < 0.05), BNP fragment (1.47 versus 0.34 ng/mL P < 0.05), and osteopontin (60.79 versus 25.42 ng/mL P < 0.05) compared with controls. Fetuin-A levels were lower in individuals with CAS than in healthy controls (0.25 versus 0.34g/L, P < 0.05). Asymmetric dimethylarginine (ADMA) were lower (1.08 versus 1.1 micromol/L, P > 0.05) while homocysteine levels (20.34 +/- 2.14 versus 19.23 +/- 4.19 P > 0.05) were higher in the CAS patients. CONCLUSION This study demonstrates a direct correlation of NT-pro-BNP, BNP, and osteopontin and the presence of CAS, while fetuin A showed an inverse correlation. Plasma ADMA and homocysteine levels were comparable in the CAS patients and healthy individuals. This is the first study in which several biomarkers previously studied independently in patients with CAS have been investigated simultaneously in the same study population.

Multiple forms of arginase are differentially expressed from a single locus in Neurospora crassa.

The Neurospora crassa catabolic enzyme, arginase (l-arginine amidinohydrolase, EC 3.5.3.1), exists in multiple forms. Multiple forms of arginase are found in many vertebrates, but this is the only reported example in a microbial organism. The two major forms are structurally similar with subunit sizes of 36 and 41 kDa, respectively. The larger form is produced by mycelia growing in arginine-supplemented medium. Both forms are localized in the cytosol. The structural gene for arginase,aga, has been cloned and sequenced; it contains a 358-codon open reading frame with three in-frame ATGs at the amino terminus. Mutagenesis of these ATGs revealed that the first ATG initiates the 41-kDa protein and the third ATG initiates the 36-kDa protein. Mutation of the second ATG has no effect on translation. Northern analysis demonstrated that a 1.4-kilobase (kb) transcript is synthesized in minimal medium and both a 1.4- and 1.7-kb transcript are produced in arginine-supplemented medium. Primer extension identified the 5′ ends of each transcript and demonstrated that the first and third ATG of the open reading frame are the initial AUGs of the 1.7- and 1.4-kb mRNA, respectively. The results suggest that a basal promoter produces the 1.4-kb transcript and an arginine “activated” promoter is responsible for the 1.7-kb transcript. Tandem promoters are rare in eukaryotic organisms, and they often regulate developmental or tissue-specific gene expression. The possibility that arginase has a role in differentiation in N. crassa is being investigated.

Novel mechanisms controlling arginine metabolism in Neurospora

Abstract Overproduction and compartmentalization of metabolites are recognized as elements of pathway control and pathway switching in the metabolism of arginine in Neurospora .

Acetylglutamate synthase from Neurospora crassa: structure and regulation of expression.

A DNA clone which complemented an arg‐14 mutation of Neurospora crassa was isolated by sib selection from a cosmid library (pMOcosX). Southern and restriction‐fragment‐length polymorphism (RFLP) analysis confirmed that the cloned DNA contained the arg‐14 gene. The arg‐14 gene was identified as the structural gene for acetylglutamate synthase by immunodepletion of enzyme activity with antibodies prepared against an arg‐14 fusion protein and by the thermal instability of acetylglutamate synthase in a temperature‐sensitive arg‐14 mutant. The fungal acetylglutamate synthase has little sequence homology to its bacterial counterpart, unlike other arginine biosynthetic enzymes. Expression of the arg‐14 gene is regulated by cross‐pathway control similar to many amino acid biosynthetic genes. However, expression of acetylglutamate synthase occurs throughout the developmental growth cycle, unlike other arginine biosynthetic enzymes.

Ornithine transcarbamylase from Neurospora crassa: purification and properties.

Ornithine transcarbamylase catalyzes the synthesis of citrulline from carbamyl phosphate and ornithine. This enzyme is involved in the biosynthesis of arginine in many organisms and participates in the urea cycle of mammals. The biosynthetic ornithine transcarbamylase has been purified from the filamentous fungus, Neurospora crassa. It was found to be a homotrimer with an apparent subunit molecular weight of 37,000 and a native molecular weight of about 110,000. Its catalytic activity has a pH optimum of 9.5 and Kms of about 5 and 2.5 mM for the substrates, ornithine and carbamyl phosphate, respectively, at pH 9.5. The Kms and pH optimum are much higher than those of previously characterized enzymes from bacteria, other fungi, and mammals. These unusual kinetic properties may be of significance with regard to the regulation of ornithine transcarbamylase in this organism, especially in the avoidance of a futile ornithine cycle. Polyclonal antibodies were raised against the purified enzyme. These antibodies and antibody raised against purified rat liver ornithine transcarbamylase were used to examine the structural similarities of the enzyme from a number of organisms. Cross-reactivity was observed only for mitochondrial ornithine transcarbamylases of related organisms.