Cloning and characterization of centromeric DNA from Neurospora crassa.

The centromere locus from linkage group VII of Neurospora crassa has been cloned, characterized, and physically mapped. The centromeric DNA is contained within a 450-kb region that is recombination deficient, A+T-rich, and contains repetitive sequences. Repetitive sequences from within this region hybridize to a family of repeats located at or near centromeres in all seven linkage groups of N. crassa. Genomic Southern blots and sequence analysis of these repeats revealed a unique centromere structure containing a divergent family of centromere-specific repeats. The predominantly transitional differences between copies of the centromere-specific sequence repeats and their high A+T content suggest that their divergence was mediated by repeat-induced point (RIP) mutations.     

Cloning of the ARO cluster gene of Neurospora crassa and its expression in Escherichia coli

Summary We have constructed a phage, Ncl, which comprises a 4.0 kb HindIII insert of Neurospora DNA into the immunity region of the vector 598. Ncl complements the aroD6 mutation of E. coli, permitting the formation of galaxy plaques on medium lacking aromatic supplements, and transforms an aro-9 qa-2 Neurospora mutant to prototrophy at a low frequency. Low levels of 5-dehydroquinate hydrolyase (E.C.4.2.1.10.), with properties unlike those of the catabolic isoenzyme that is coded by qa-2, are present in E. coli aroD6 cell lysates following infection with Ncl. Ncl does not hybridize with qa-2 DNA and it is concluded that it contains at least the aro-9 region of the pentafunctional aro cluster gene.     

Isolation of telomere DNA from Neurospora crassa.

The most distal known gene on Neurospora crassa linkage group VR, his-6, was cloned. A genomic walk resulted in isolation of the telomere at VR. It was obtained from a library in which the endmost nucleotides of the chromosome had not been removed by nuclease treatment before being cloned, and mapping indicates that the entire chromosome end has probably been cloned. Sequences homologous to the terminal 2.5 kilobases of DNA from VR from these Oak Ridge N. crassa strains are found at other sites in the genome. To characterize these sites, I crossed an Oak Ridge-derived his-6 strain with a wild-type strain of different genetic background (Mauriceville) and characterized the hybridization patterns seen in the progeny. It appears that the sequences homologous to the VR terminus are found at genetically different sites in the two parental strains, and no hybridization to the VR telomere from Mauriceville was detected. The other genomic copies identified in the Oak Ridge parent were not telomeres. I suggest that any repeating sequence blocks found immediately adjacent to the VR terminus in Oak Ridge strains must be small and that the repeating element identified in that background may be an N. crassa transposable element integrated near the the chromosome end at VR.     

Cloning and functional characterization of a eucaryotic DNA photolyase gene from Neurospora crassa.

We cloned a genomic fragment of a photolyase gene from Neurospora crassa by polymerase chain reaction using synthesized oligonucleotide primers designed from the most conserved amino acid sequences among photolyases of various organisms. Using the cloned fragment as a hybridization probe we isolated a genomic fragment and cDNA clones encoding the complete photolyase gene of this organism. The amino acid sequence of the photolyase deduced from the determined nucleotide sequence indicates a protein consisting of 615 amino acid residues (Mr 69,971), which is most similar to that of Saccharomyces cerevisiae. Like yeast photolyase it contains a protruding amino terminus which is missing in photolyases of bacterial origin. Comparison of amino acids sequences among six photolyases suggests that the Neurospora crassa photolyase is more similar to photolyases of pterin type than those of deazaflavin type.     

Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli.

The Neurospora crassa metallothionein (NC) synthesis gene was cloned and expressed in Escherichia coli in two different expression vectors (pING2 and pUA7), both under the regulation of the Salmonella typhimurium arabinose operon. Upon induction with arabinose, the pING2-NC vector expressed as inclusion body-localized AraB'::NC fusion protein of 21 kilodaltons. The pUA7-NC vector expressed a 5.3-kilodalton Lpp::NC fusion protein anchored to the outer membrane of the cell. Cells expressing the NC fusion proteins accumulated Cd2+ and Cu+ (between 2.3- and 11-fold) compared with nonexpressing cells. To generate novel forms of metal-binding peptides, a set of specific mutant genes for N. crassa NC was designed in which each cysteine residue was replaced with a subset of amino acids implicated in peptide-metal coordination (Asn, Asp, His, Lys, or Tyr residues). These mutant NC sequences were cloned into the two vectors and expressed in E. coli. One of the mutant proteins (containing His residues) showed accumulation of Cd2+ and Cu+ (threefold) from a mixture of 16 heavy metals species. None of the other heavy metals present in the culture was accumulated.     

H+/ATP stoichiometry of proton pumps from Neurospora crassa and Escherichia coli

A kinetic method has been used to measure the apparent stoichiometry of H+ ions translocated per ATP split by membrane-bound [H+]-ATPases. In this method, membrane vesicles are suspended in well-buffered medium, ATP is added, and a fluorescent probe of delta pH (acridine orange) is used to detect the formation of a steady-state pH gradient. At the steady state, it is assumed that proton pumping in one direction is exactly balanced by the leak of protons in the opposite direction. The pump is then rapidly turned off by the addition of an appropriate inhibitor, and the initial rate of relaxation of delta pH is used to infer the pump rate. This rate is divided by the rate of ATP hydrolysis, measured under the same condition, to give the apparent H+/ATP stoichiometry. The method has been applied to two different [H+]-ATPases, the plasma-membrane ATPase of Neurospora (a Mr = 100,000 integral membrane protein) and the ATPase of Escherichia coli (which belongs to the F0F1 group). The Neurospora ATPase displayed an apparent stoichiometry close to 1 H+/ATP (0.82-1.23), in agreement with previous estimates from electrophysiological measurements on whole cells. In contrast, the E. coli ATPase yielded an apparent stoichiometry close to 2 H+/ATP (1.90), consistent with several published values obtained by both kinetic and thermodynamic methods for bacterial, mitochondrial, and chloroplast ATPases.     

Cloning of the structural gene for orotidine 5'-phosphate carboxylase of Neurospora crassa by expression in Escherichia coli

A Neurospora gene bank in plasmid pRK9 was used to complement pyrimidine auxotrophs in E. coli. Two plasmids were obtained that complement a pyrF mutant of E. coli. These plasmids hybridise to Neurospora DNA and transform a pyr-4 strain of Neurospora. The promoter used in expressing the orotidine 5'-monophosphate carboxylase in E. coli is within the Neurospora sequence.     

Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli

The Neurospora crassa metallothionein (NC) synthesis gene was cloned and expressed in Escherichia coli in two different expression vectors (pING2 and pUA7), both under the regulation of the Salmonella typhimurium arabinose operon. Upon induction with arabinose, the pING2-NC vector expressed as inclusion body-localized AraB'::NC fusion protein of 21 kilodaltons. The pUA7-NC vector expressed a 5.3-kilodalton Lpp::NC fusion protein anchored to the outer membrane of the cell. Cells expressing the NC fusion proteins accumulated Cd2+ and Cu+ (between 2.3- and 11-fold) compared with nonexpressing cells. To generate novel forms of metal-binding peptides, a set of specific mutant genes for N. crassa NC was designed in which each cysteine residue was replaced with a subset of amino acids implicated in peptide-metal coordination (Asn, Asp, His, Lys, or Tyr residues). These mutant NC sequences were cloned into the two vectors and expressed in E. coli. One of the mutant proteins (containing His residues) showed accumulation of Cd2+ and Cu+ (threefold) from a mixture of 16 heavy metals species. None of the other heavy metals present in the culture was accumulated.     

Endocytic uptake of Escherichia coli spheroplasts by Neurospora crassa slime cells

Summary Interaction of Escherichia coli spheroplasts with Neurospora crassa slime cells was examined by transmission electron microscopy after treatment with polyvinyl alcohol followed by dilution with the high pH-high Ca buffer. Bacterial spheroplasts were found either adhering to the flat surface, associating with the invaginating surface, or residing within the intracellular vesicle of fungal protoplasts. In addition, bacterial spheroplasts free of the surrounding vesicles and those in the course of breakdown were observed in the fungal cytoplasm. It was concluded that Escherichia coli spheroplasts are taken up by Neurospora crassa protoplasts almost exclusively via endocytosis. This is the first cytological evidence for the endocytic activity of fungal cells.     

Redundant DNA of Neurospora crassa

Approximately 20% of the DNA of Neurospora crassa consists of redundant sequences. This is calculated from the reassociation rate of fragmented, denatured DNA as measured by hydroxyapatite column chromatography. The redundant DNA has a complexity of 10⁵ base pairs and a repetition frequency of up to 60 copies per genome. Its buoyant density in CsCl is 1.720 g/ml and its hypochromicity 20–24%. Base composition determination shows 54% GC content like Neurospora nuclear DNA. DNA-RNA hybridization studies indicate that rRNA and tRNA cistrons make up 2.3 and 1.2%, respectively, of the redundant fraction. Pulse-labeled RNA is shown to hybridize with both redundant and unique DNA fractions, suggesting that both fractions are transcribed.This work is supported by a grant from National Science Foundation (GB 8058) and National Institute of Health Research Career Development Award (K3GM31-238).     

一种粗糙脉孢霉基因组DNA的快速制备方法

粗糙脉孢霉基因组ＤＮＡ的制备方法一般很费工费时。ＷｅｎｄｌａｎｄＪＡ等人发展了一种丝状真菌的ＤＮＡ提取方法 ,应用在裂褶菌取得了良好的效果[1] 。本文基于该方法制备粗糙脉孢霉基因组ＤＮＡ也取得了成功 ,应用ＰＣＲ从基因组扩增出了一个与无机焦磷酸酶有同源性的基因。1　材料与方法1 1　菌种 :粗糙脉孢霉 (Ｎｅｕｒｏｓｐｏｒａｃｒａｓｓａ)菌种 490 7ｐｒｄ - 4 ,ｂｄＡ ,来自ＦｕｎｇａｌＧｅｎｅｔｉｃｓｓｔｏｃｋｃｅｎｔｅｒ,ＵｎｉｖｅｒｓｉｔｙｏｆＫａｎｓａｓＭｅｄｉｃａｌＣｅｎｔｅｒ,Ｋａｎｓａｓ ,ＵＳＡ。1 2…