The Neurospora crassa genome: cosmid libraries sorted by chromosome

A Neurospora crassa cosmid library of 12,000 clones (at least nine genome equivalents) has been created using an improved cosmid vector pLorist6Xh, which contains a bacteriophage lambda origin of replication for low-copy-number replication in bacteria and the hygromycin phosphotransferase marker for direct selection in fungi. The electrophoretic karyotype of the seven chromosomes comprising the 42.9-Mb N. crassa genome was resolved using two translocation strains. Using gel-purified chromosomal DNAs as probes against the new cosmid library and the commonly used medium-copy-number pMOcosX N. crassa cosmid library in two independent screenings, the cosmids were assigned to chromosomes. Assignments of cosmids to linkage groups on the basis of the genetic map vs. the electrophoretic karyotype are 93 +/- 3% concordant. The size of each chromosome-specific subcollection of cosmids was found to be linearly proportional to the size of the particular chromosome. Sequencing of an entire cosmid containing the qa gene cluster indicated a gene density of 1 gene per 4 kbp; by extrapolation, 11,000 genes would be expected to be present in the N. crassa genome. By hybridizing 79 nonoverlapping cosmids with an average insert size of 34 kbp against cDNA arrays, the density of previously characterized expressed sequence tags (ESTs) was found to be slightly <1 per cosmid (i.e., 1 per 40 kbp), and most cosmids, on average, contained an identified N. crassa gene sequence as a starting point for gene identification.     

Molecular cloning, characterization, and nucleotide sequence of nit-6, the structural gene for nitrite reductase in Neurospora crassa.

The Neurospora crassa assimilatory nitrite reductase structural gene, nit-6, has been isolated. A cDNA library was constructed from poly(A)+ RNA isolated from Neurospora mycelia in which nitrate assimilation had been induced. This cDNA was ligated into lambda ZAP II (Stratagene) and amplified. This library was then screened with a polyclonal antibody specific for nitrite reductase. A total of six positive clones were identified. Three of the six clones were found to be identical via restriction digests, restriction fragment length polymorphism mapping, Southern hybridization, and some preliminary sequencing. One of these cDNA clones (pNiR-3) was used as a probe in Northern assays and was found to hybridize to a 3.5-kb poly(A)+ RNA whose expression is nitrate inducible and glutamine repressible in wild-type mycelia. pNiR-3 was used to probe an N. crassa genomic DNA library in phage lambda J1, and many positive clones were isolated. When five of these clones were tested for their ability to transform nit-6 mutants, one clone consistently generated many wild-type transformants. The nit-6 gene has been subcloned to generate pnit-6. The nit-6 gene has been sequenced and mapped; its deduced amino acid sequence exhibits considerable levels of homology to the sequences of Aspergillus sp. and Escherichia coli nitrite reductases. Several pnit-6 transformants have been propagated as homokaryons. These strains have been assayed for the presence of multiple copies of the nit-6 gene, as well as nitrite reductase activity.     

A mitochondrial protein from Neurospora crassa detected both on ribosomes and in membrane fractions. Analysis of the gene, the message, and the protein

We have isolated clones representing at least three nuclear genes for mitochondrial ribosomal proteins from Neurospora crassa by screening a lambda gt11 cDNA library with an antiserum against a mixture of these proteins. The cDNA and genomic DNA sequence for one of these genes, mrp-3, was determined. The MRP3 protein was purified by immune-affinity chromatography, using a monoclonal antibody probe, and subjected to amino acid sequence analysis to identify the mature amino terminus and a prospective mitochondrial-targeting presequence. MRP3 was identified as the largest, least basic protein detected from the small subunit of ribosomes which had been salt-washed and fractionated on sucrose gradients. However, the mRNA and protein products of mrp-3 were found to be present in excess over those of other N. crassa mitoribosomal protein genes. Using a solution hybridization/S1 nuclease assay, we found three-fold- more mRNA for mrp-3 than for another mito-ribosomal protein gene. In addition, a 30- to 50-fold excess of non-ribosomal MRP3 protein was discovered. The additional protein was localized in mitochondrial membrane fractions; none was detected in matrix fractions after removal of the ribosomes. An immunologically related protein was detected in ribosome and membrane fractions of mitochondria from Saccharomyces cerevisiae. The functional significance of this dual localization remains an enigma.     

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by S1 nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the leu-5 mutation had been mapped before. We show that the leu-5 strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the leu-5 strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in leu-5 extracts, the leu-5 strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.     

Isolation and characterization of the complete chicken beta-globin gene region: frequent deletion of the adult beta-globin genes in lambda.

A library of bacteriophage lambda clones containing chicken chromosomal DNA was screened, using the adult beta-globin cDNA plasmid pHb 1001 as a probe. Sixteen overlapping clones were isolated containing 35 kilobase pairs (kbp) of chicken DNA. Characterization of these clones revealed four beta-like globin genes, some genomically repeated sequences, but no pseudo-genes. The four beta-like genes have an average intergenic distance of less than half of that found for the mammalian beta-like globin gene clusters so far characterized. The overall features of the map were confirmed by genomic Southern analysis. Frequent deletions were shown to occur between the various beta-like globin genes during phage propagation. The presumptive hatching gene in particular was always associated with abnormal lambda clones although we were able to find one such clone that did contain a normal copy of the hatching gene itself. Probably such deletions explain the failure to recover this gene in previous attempts.     

Isolation of the cDNA for human prostaglandin H synthase

Prostaglandin H Synthase (PGHS, cyclooxygenase) is a 67 kd protein which catalyzes the first step in prostaglandin synthesis. The primary amino acid sequence and the molecular mechanisms regulating expression are unknown. We report here isolation of a cDNA clone for the enzyme from human vascular endothelial cells for use in such studies. High titre, polyclonal antiserum against PGHS was developed in rabbits. The antiserum was monospecific, reacted with cyclooxygenase on Western blots at a limiting dilution of 1:500,000 and immunoprecipitated cyclooxygenase synthesized by in vitro translation of PGHS messenger RNA. It was used to screen a lambda gt11 cDNA expression library from human endothelial cells. Three positive clones were isolated. Following plaque purification, one clone reacted strongly with two other polyclonal antisera independently raised against highly purified cyclooxygenase and the aspirin-acetylated enzyme. Western blot analysis confirmed production of a large approximately 180 kd fusion protein of cyclooxygenase and beta-galactosidase. The cDNA insert of approximately 2.2 kilo base pairs was excised and subcloned into plasmid pUC8. A 24 nucleotide DNA probe, synthesized according to the amino acid sequence of the aspirin-acetylation site of cyclooxygenase, hybridized strongly with the 2.2 kbp cDNA insert. It is concluded that the 2.2 kbp cDNA insert represents a cDNA clone for human cyclooxygenase, which also expresses the aspirin-acetylation site. This is the first reported isolation of the cDNA for this enzyme, and will facilitate further studies on the primary sequence and on the regulation of the enzyme at the molecular level.     

Assignment of a novel zinc finger gene ZNF191 to human chromosome 18Q12.1 by human/rodent somatic cell hybrid panel and fluorescent in situ hybridization]

A novel human zinc finger gene, ZNF191, was assigned to chromosome 18 by hybridization of human/rodent hybrid cell panel to a full-length cDNA as a probe. Meanwhile, a human genomic DNA lambda/DASH library was screened using this cDNA probe and several positive clones were obtained. Fluorescence in situ hybridization (FISH) was performed by using one of these positive clones, 16-1, as a probe. Thus, the ZNF191 gene was precisely mapped in 18q12. 1. To date, some hereditary diseases and tumors have been found to be associated with this region by analysis of genetic linkage and loss of heterozygosity. Hence, it suggested that the gene ZNF191 can be taken as a candidate gene responsible for those diseases and tumors.     

The DNF15S2 locus at 3p21 is transcribed in normal lung and small cell lung cancer

Small cell lung cancer (SCLC) has been associated with a deletion of the short arm of chromosome 3. One SCLC cell line, H748, has an interstitial deletion of chromosome 3p and shows allele loss for the DNF15S2 locus detected by the probe lambda H3. Conservation of DNF15S2 sequences in mouse indicated that this human genomic fragment may contain coding sequences. Screening of a normal lung cDNA library with chromosome 3-specific fragments of the lambda H3 probe resulted in the isolation of 18 positive clones. The cDNA clones detect an additional DNA polymorphism that is in linkage disequilibrium with the HindIII polymorphism of the DNF15S2 locus. Sequence analysis indicated that the DNF15S2 locus could potentially code for a previously unreported protein of 67 kDa which has 26 cysteine residues. DNF15S2 is part of the coding region of a 3.3-kb mRNA expressed in lung. Northern analysis indicated that this mRNA was not detectable in one of five SCLC lines. This SCLC line, H128, also lacks the enzyme aminoacylase 1.     

hsp80 of Neurospora crassa: cDNA cloning, gene mapping, and studies of mRNA accumulation under stress.

Using mRNA isolated from Neurospora crassa mycelium, grown for 14 h at normal growth temperature of 28 degrees C, and heat shocked for 1 h at 48 degrees C, a cDNA library was prepared in the expression vector lambda gt11. Following immunoscreening of this library with a polyclonal antiserum raised against a 80-kilodalton heat-shock protein (HSP80), cDNA clones containing 1.1- and 1.4-kilobase inserts were selected. Analysis of the partial nucleotide sequence and the deduced amino acid sequence of the cDNA clones revealed a remarkable extent of homology with other eukaryotic stress-90 family proteins; 85% identity of the amino acid sequence with that of yeast HSP90(82) was seen. The C-terminal end of the sequence contained the MEEVD motif, characteristic of eukaryotic stress proteins with a predominantly cytosolic localization. The gene for N. crassa HSP80 was mapped to the right arm of linkage group V, using restriction fragment length polymorphism mapping. Its expression during heat shock and recovery was monitored by probing Northern blots of RNA isolated from mycelium grown under various stress conditions.     

A versatile phage lambda expression vector system for cloning in Escherichia coli

By integrating fragments from the expression plasmids pJK2 and pJK4 into a derivative of the bacteriophage lambda, we constructed the phage expression vectors lambda JK2 and lambda JK4, which allow efficient cloning of genomic or cDNA either into the 5' end or the 3' end of the lacZ gene of Escherichia coli. Expression of barrier-free DNA in phase may lead to fusion proteins consisting of active beta-galactosidase (beta Gal) plus an additional polypeptide encoded by the inserted DNA. Analysis of distinct recombinant clones is quick and easy, due to the reversible integration of the plasmid into the genome. As an example, we constructed an expression library of genomic Plasmodium falciparum DNA in lambda JK2. We polymerised (amplified) and expressed a synthetic DNA fragment, which codes for a potential antigenic determinant of the 11-1 gene of Plasmodium falciparum as a fusion to the N terminus of active beta Gal. We demonstrate that such chimeric molecules can be affinity-purified and that polypeptides can be separated from the beta Gal part by cleavage with the protease factor Xa.     

Nuclear genes for cytochrome c oxidase subunits of Neurospora crassa. Isolation and characterization of cDNA clones for subunits IV, V, VI, and possibly VII

We obtained cDNA clones for cytochrome oxidase subunits IV, V, VI, and possibly VII by constructing a lambda gt11 library of Neurospora crassa cDNA and probing it with antiserum directed against Neurospora cytochrome oxidase holoenzyme. Positive clones were further characterized with antisera directed against individual cytochrome oxidase subunits and subsequently by DNA sequencing. The clones for subunits IV and V encode proteins with regions matching the known N-terminal amino acid sequences of purified Neurospora cytochrome oxidase subunits IV and V, respectively. The sequences of these clones provide the first evidence that cytochrome oxidase subunits IV and V are made as precursors with N-terminal extensions in Neurospora. The N-terminal extensions encoded by these clones share homology, and are rich in arginine, as are signal sequences of other mitochondrially destined proteins. The subunit VI clone codes for the carboxyl terminus of a protein homologous to the carboxy termini of yeast cytochrome oxidase subunit VI and bovine cytochrome oxidase subunit Va. The subunit VII clone contains an open reading frame for a 47-residue protein, the expected size for subunit VII. However, the protein coded by this clone has an unusual amino acid composition. Whether this clone represents an authentic cytochrome oxidase subunit is not established.     

Detection of large cDNA inserts within crude lambda gt11 lysates: a rapid and sensitive method

A method is presented for the isolation of bacteriophage lambda DNA and the rapid identification of large cDNA inserts within crude phage lysates. The primary screening of a lambda gt11 cDNA library with a 32P-radiolabeled cDNA probe yielded 21 putative positive clones. A phage "spot-blot" analysis was employed to quickly screen these potential recombinants. This eliminated 9 of the 21 clones as the result of false positive signals. The remaining 12 recombinant phage were amplified on agarose-based media, and phage DNA was isolated using a modified plate lysate procedure. The DNA thus obtained from these crude lysates could be easily digested with EcoRI and examined by Southern blot analysis. The resulting blot was hybridized with the same cDNA probe used in the initial screening of the library. Thus, two clones harboring the longest cDNA insert were identified from a mixed phage population and were subsequently plaque purified. The procedure is rapid, sensitive, reproducible, inexpensive and allows the processing of several clones at once without sacrificing the quality or yields of the DNA preparation. Furthermore, the method obviates the need for plaque purifying all the positives obtained from the initial screening of a cDNA library.     

Primary hyperoxaluria type I due to a point mutation of T to C in the coding region of the serine:pyruvate aminotransferase gene

cDNA clones for serine:pyruvate aminotransferase (SPT, alternative name: alanine:glyoxylate aminotransferase) were obtained from a cDNA library constructed from the liver of a primary hyperoxaluria type I (PH1) case in which the SPT activity was approximately one-hundredth that in control liver. Six clones were isolated from 100,000 transformants and all of them contained an approximately 1.5 kbp insert which included the whole coding region for human SPT. Nucleotide sequence analysis revealed a point mutation of T to C at position 634 (relative to the 5'-end of the cDNA) encoding a Ser to Pro substitution at residue 205. The T to C conversion created a new SmaI site, which enabled us to demonstrate that the point mutation had occurred in the patient's SPT gene. SmaI digestion of genomic DNA may be useful for the diagnostic gene analysis of this type of PH1.     

NAD(P)H:menadione oxidoreductase. Novel purification of enzyme cDNA and complete amino acid sequence, and gene regulation

NAD(P)H:menadione oxidoreductase induction by polycyclic hydrocarbons is known to be governed by the aromatic hydrocarbon-responsive (Ah) locus. This cytosolic enzyme was isolated from 3-methylcholanthrene-treated rat liver by a rapid two-step procedure with the use of affinity gel purification and fast-protein liquid chromatography. Polyclonal antiserum to menadione reductase was raised in rabbits. On Western (immuno) blot analysis, large increases in this hepatic menadione reductase protein (NMOR1) of 3-methylcholanthrene-treated C57BL/6N but not DBA/2N mice confirmed that induction of this enzyme by 3-methyl-cholanthrene is regulated by the Ah receptor. A cDNA expression library was constructed in lambda gt11 and screened with antiserum. Positive cDNA clones were plaque purified and further characterized by showing enhanced hybridization to 3-methylcholanthrene-induced poly(A+) RNA from rats; the longest cDNA clone (1,501 base pairs) has an open reading frame (bases 75-899) and a nucleotide sequence consistent with a new gene family. On Northern blot analysis, a single 3-methylcholanthrene-inducible rat liver mRNA (approximately 1.6 kilobases) hybridizes to the cDNA probe. On Southern blot analysis a total of 14-16 kilobases of rat genomic DNA fragments hybridize to the cDNA probe, indicating one or a small number of menadione reductase genes in this family. The amino acid sequence (274 residues) and Mr of 30,946 compare well with the size of the rat enzyme (32 kDa) estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of two internal fragments of the trypsin-digested purified NMOR1 protein is in complete agreement with that deduced from the cDNA nucleotide sequence. This study represents the first cloning and sequencing of a cDNA encoding a Phase II drug-metabolizing enzyme under control of the Ah locus.     

The isolation and characterisation of cDNA and genomic clones for human lecithin: cholesterol acyltransferase

The protein sequencing of tryptic peptides from purified human lecithin: cholesterol acyltransferase (LCAT) identified sufficient amino-acid sequence to construct a corresponding mixed oligonucleotide probe. This was used to screen an adult human cDNA liver library, from which incomplete cDNA clones were isolated. The DNA sequence of these clones allows the prediction of the entire amino-acid sequence of the mature LCAT enzyme. The mature protein consists of 416 amino acids and contains several marked stretches of hydrophobic residues and four potential glycosylation sites. The cDNA probe detects LCAT mRNA sequences approx. 1500 bases long in human liver, but not intestine, RNA. The cDNA probe was used to isolate LCAT genomic recombinants from a human genomic library. Southern blotting data, and restriction site mapping, suggest that there is a single human LCAT structural gene between 4.3 and 5.5 kb in size.