Analysis of the pdx-1 (snz-1/sno-1) Region of the Neurospora crassa Genome: Correlation of Pyridoxine-Requiring Phenotypes With Mutations in Two Structural Genes

Corresponding author: Donald O. Natvig, Department of Biology, University of New Mexico, Albuquerque, NM 87131., dnatvig{at}unm.edu (E-mail)

Communicating editor: J. ARNOLD

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We report the analysis of a 36-kbp region of the Neurospora crassa genome, which contains homologs of two closely linked stationary phase genes, SNZ1 and SNO1, from Saccharomyces cerevisiae. Homologs of SNZ1 encode extremely highly conserved proteins that have been implicated in pyridoxine (vitamin B6) metabolism in the filamentous fungi Cercospora nicotianae and in Aspergillus nidulans. In N. crassa, SNZ and SNO homologs map to the region occupied by pdx-1 (pyridoxine requiring), a gene that has been known for several decades, but which was not sequenced previously. In this study, pyridoxine-requiring mutants of N. crassa were found to possess mutations that disrupt conserved regions in either the SNZ or SNO homolog. Previously, nearly all of these mutants were classified as pdx-1. However, one mutant with a disrupted SNO homolog was at one time designated pdx-2. It now appears appropriate to reserve the pdx-1 designation for the N. crassa SNZ homolog and pdx-2 for the SNO homolog. We further report annotation of the entire 36,030-bp region, which contains at least 12 protein coding genes, supporting a previous conclusion of high gene densities (12,000–13,000 total genes) for N. crassa. Among genes in this region other than SNZ and SNO homologs, there was no evidence of shared function. Four of the genes in this region appear to have been lost from the S. cerevisiae lineage.

ALTHOUGH efforts are underway to sequence and annotate the genomes of Neurospora crassa and other filamentous fungi, there remain few carefully annotated large regions of genomic DNA. Such analyses are required for accurate estimates of gene numbers, and they are extremely valuable for investigations in comparative genomics as well as in gene structure and function. We have sequenced and annotated a cosmid insert carrying N. crassa genes homologous to the SNZ and SNO genes (BRAUN et al. 1996 ; PADILLA et al. 1998 ) from Saccharomyces cerevisiae, which encode conserved proteins distantly related to proteins involved in amino acid and nucleotide biosynthesis (GALPERIN and KOONIN 1997 ). Recent evidence suggests that homologs of SNZ participate in pyridoxine (vitamin B6) metabolism in Cercospora nicotianae (EHRENSHAFT et al. 1999A , EHRENSHAFT et al. 1999B ) and Aspergillus nidulans (OSMANI et al. 1999 ). Results presented here indicate a role in pyridoxine metabolism for both SNZ and SNO homologs in N. crassa.

Initial interest in eukaryotic SNZ and SNO homologs on the part of several researchers stemmed from patterns of expression as well as from a possible role for SNZ homologs in avoidance of oxidative damage. The synthesis of the S. cerevisiae Snz1 protein increases dramatically when cells enter stationary phase (FUGE et al. 1994 ; BRAUN et al. 1996 ), and homologs of SNZ have been identified as ethylene-inducible mRNAs from the rubber tree plant, Hevea brasiliensis (SIVASUBRAMANIAM et al. 1995 ) and the marine sponge, Suberites domuncula (KRASKO et al. 1999 ). The SNZ homolog in the filamentous fungus C. nicotianae was discovered because mutations in this gene, designated SOR1, result in hypersenstitivity to singlet oxygen-generating agents (EHRENSHAFT et al. 1999B ).

In many organisms, genes encoding Snz homologs are closely linked to genes encoding Sno homologs, which are related to amidotransferases involved in amino acid and nucleotide biosynthesis (GALPERIN and KOONIN 1997 ). The SNZ and SNO homologs form an apparent operon in some prokaryotes (GALPERIN and KOONIN 1997 ) and are coregulated in S. cerevisiae (PADILLA et al. 1998 ). In addition to close genomic linkage of their respective genes, Snz and Sno proteins exhibit physical and genetic interactions that suggest they function as components of an oligomeric complex (PADILLA et al. 1998 ). It can be inferred, therefore, that SNZ and SNO cooperate in function, a conclusion strongly supported by results presented here.

This study afforded the opportunity to explore the relationship between the N. crassa SNZ and SNO homologs and mutations that result in a requirement for pyridoxine, and it allowed a detailed examination of a portion of the genome in which these genes reside. The N. crassa SNZ and SNO homologs were found to be closely linked, as is observed in other microorganisms, and they map to the pdx-1 (pyridoxine requiring) region of linkage group IVR (see NELSON et al. 1998 ). Results further indicate that pyridoxine auxotrophy in N. crassa can be caused by mutations in either structural gene.

The 36-kbp region examined contains at least 12 genes, including the homologs of SNZ and SNO. This reflects a gene density consistent with recent estimates (NELSON et al. 1997 ; KELKAR et al. 2001 ) suggesting high gene numbers (12,000–13,000) for N. crassa. With the exception of the SNZ and SNO homologs, there is no evidence for clustering of genes of shared function in this region. In fungi and prokaryotes, the clustering of genes of shared function can reflect dispensable function and a potential for horizontal transfer ("selfish" operons; LAWRENCE and ROTH 1996 ; KELLER and HOHN 1997 ). However, there is no evidence that the clustering of SNZ and SNO homologs of N. crassa and other organisms reflects either dispensable function or horizontal transfer.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Library:
Cosmid clone G6G8 from the Orbach/Sachs cosmid library (ORBACH 1994 ; KELKAR et al. 2001 ) was obtained from the Fungal Genetics Stock Center (FGSC), University of Kansas Medical Center, Kansas City. This clone has the alternative designation X137G08 (KELKAR et al. 2001 ). In preliminary experiments (not presented) it was found to contain N. crassa homologs of the SNZ and SNO genes of S. cerevisiae (BRAUN et al. 1996 ; PADILLA et al. 1998 ). Initial identification was made by colony blot hybridization employing ³²P-labeled DNA from a cDNA clone carrying the N. crassa SNZ homolog.

Subcloning of cosmid G6G8:
Escherichia coli cells containing the G6G8 cosmid were grown at 37° for 15 hr in 50 ml Terrific broth (SAMBROOK et al. 1989 ) with 50 µg/ml ampicillin, and cosmid DNA was isolated using the QIAGEN (Valencia, CA) Plasmid Midi kit.

Cosmid DNA was subcloned for shotgun sequencing using two different methods. First, Sau3AI partial digestion was performed (SAMBROOK et al. 1989 ). To reduce the number of chimeric clones, the partially digested DNA was dephosphorylated using shrimp alkaline phosphatase [United States Biochemical (Cleveland) and Amersham (Buckinghamshire, UK)] according to the manufacturer's recommendations. After purification using QIAquick PCR product clean-up (QIAGEN), the partially digested, dephosphorylated DNA was cloned into BamHI-digested pUC-18 using a standard ligation protocol (SAMBROOK et al. 1989 ). Ligated DNA was transformed into INVF' cells (Invitrogen, San Diego). In addition to using Sau3AI, fragments were produced for subcloning by complete digestion of the G6G8 cosmid DNA using four different restriction enzymes with 6-bp recognition sequences followed by dephosphorylation. One procedure used cosmid DNA digested with HindIII and EcoRI, while another used KpnI and PstI. Digestion products were ligated into pUC-18 cut with the corresponding enzymes.

Individual white colonies were transferred to 96-well block plates containing 1.5 ml Terrific broth with ampicillin (50 µg/ml), and cells were grown at 250 rpm for 20 hr at 37°. Template DNA for sequencing was purified using the alkaline lysis protocol of ROE et al. 1996 or the QIAprep spin Miniprep kit (QIAGEN) according to the manufacturer's instructions.

DNA sequencing:
DNA sequences were obtained with an ABI 377 automated sequencer using cycle-sequencing, dye-terminator procedures with ThermoSequenase (Amersham) and ABI PRISM BigDye chemistries. Sequence gaps left after assembly of random-clone sequences were closed by direct sequencing of cosmid template DNA (prepared as described above) using custom-synthesized oligonucleotide primers.

Sequence assembly:
Phred (GREEN and EWING 1997 ) was used to call bases in the raw data files on a SUN workstation. Cloning-vector DNA sequences were deleted from each raw sequencing file using Crossmatch. The insert sequence was assembled into contiguous fragments from 700 individual sequence reads using Phrap (GREEN 1996 ) running on an SGI workstation. We used Sequencher 3.0 (Gene Codes Corporation, 1995) as a quality check of the PHRAP assembly to design primers to fill gaps and improve sequence quality and to confirm sequence across the entire insert using representative chromatograms. The 36,030-nucleotide G6G8 insert sequence has been deposited at GenBank, with annotations, under accession no. AF309689.

Sequence analysis:
The nucleotide sequence was searched for homologs of previously identified genes by performing gapped BLAST searches (ALTSCHUL et al. 1997 ) using protein and nucleotide databases available from the National Center for Biotechnology Information (NCBI, Bethesda, MD). The databases examined included the nonredundant database (NR) and dbEST [espressed sequence tagged (EST) database] from NCBI, as well as the Saccharomyces genome database (Stanford University, Stanford, CA). The algorithms employed included BLASTX, BLASTP, TBLASTX, and BLASTN, as appropriate for specific databases and queries. MacDNASIS v. 3.2 (Hitachi) was used to find open reading frames (ORFs) using codon bias for N. crassa. Identification of open reading frames and determination of codon usage were also aided by services available from the Virtual Genome Center (http://alces.med.umn.edu/webtrans.html).

Analysis of pdx-1 mutants:
Strains carrying various pdx-1 alleles were obtained from the FGSC in the Department of Microbiology, University of Kansas Medical Center. Mycelium was grown in N medium, supplemented with 1.5 µg/ml pyridoxine (DAVIS and DESERRES 1970 ). Genomic DNA was prepared using the Puregene D-5000A plant DNA isolation kit (Gentra Systems, Research Triangle Park, NC). The N. crassa SNZ1 homolog was amplified from genomic DNA preparations by polymerase chain reaction (PCR) using forward primer 5'-ACAAACCTAAGCTCTCAATCGTGGT-3' and reverse primer 5'-TCCAAGCCCCTTTTTAGTTCGT-3'. Sequences were obtained using forward and reverse PCR primers along with internal primers 5'-GCGTCGACTACATCGACGAGA-3' and 5'-TTCTTGAGGAGCTCAACATCGG-3'. The N. crassa SNO1 homolog was amplified by PCR using forward primer 5'-CCTGGTGTAACCAAAAGACCTATCG-3' and reverse primer 5'-AACCGTGACCCTCATAGTCGC-3'. Sequences were obtained using forward and reverse PCR primers along with internal primers 5'-AGTCTTTTTTTCTCTTTTCCTAACCCG-3' and 5'-ACTCTGGAGCTGTGTGCCGTA-3'. Primers were tested and wild-type SNZ1 and SNO1 homolog sequences were confirmed with N. crassa strain 74-OR23-1A.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Genes represented in the cosmid insert:
Our annotation of the 36,030-bp insert from cosmid G6G8 includes 13 putative protein-coding genes (Table 1), 12 of which were deduced with a high level of certainty. The identification of coding regions employed a combination of analyses including BLAST searches, examination of ORFs for N. crassa codon preference (e.g., CHARY et al. 1990 ), and searches for consensus sequences associated with translational start sites (BRUCHEZ et al. 1993A ) and intron splicing (BRUCHEZ et al. 1993B ). With two exceptions, the validity of each gene was established by identification of a homologous genomic or cDNA sequence using BLAST searches. One exception, ORF G6G8.11, encodes 426 amino acids without interruption and exhibits strong N. crassa codon bias. It appears to represent a true protein-coding sequence, despite the fact that no homolog or cDNA was identified. The other exception, ORF G6G8.2, exhibits rather poor N. crassa codon preference and lacks similarity to known genes from other organisms. This ORF is contained within certain N. crassa cDNA sequences, but nevertheless there is some question whether this region encodes a protein (discussed below).

Six of the 13 annotated genes in Table 1 are represented by partial cDNA sequences at GenBank that are derived from N. crassa EST projects at the University of New Mexico and the University of Oklahoma (see footnote to Table 1). In addition, a partial cDNA sequence from E. nidulans encoding the probable ortholog of one gene (G6G8.9) has been identified (Table 1).

Two of the genes in G6G8 have paralogs previously identified in N. crassa. A different 3-hydroxyisobutyrate dehydrogenase (3HD) homolog was found earlier by the Neurospora Genome Project in a cDNA clone (NELSON et al. 1997 ). There is only moderate sequence similarity between the two predicted N. crassa 3HD proteins, and BLAST searches indicated that the sequence reported here is more closely related to 3HDs in other organisms (best BLAST match to Drosophila melanogaster). It is therefore possible that the gene identified previously encodes a dehydrogenase with a function different from that of characterized 3HDs. There also was a previously identified N. crassa thioredoxin. Again, the results of BLAST searches suggest a closer relationship between the protein reported here and thioredoxins from other organisms [best BLAST match to Emericella (=Aspergillus) nidulans].

There are four genes in G6G8 that appear to lack S. cerevisiae orthologs, despite evidence suggesting they were present in the common ancestor of N. crassa and S. cerevisiae. Three of these proteins (encoded by G6G8.5, G6G8.6, and G6G8.9, see Table 1) have homologs in other eukaryotic kingdoms but lack an S. cerevisiae homolog, the criterion used to establish gene loss by BRAUN et al. 2000 . Two of the proteins are probable structural enzymes [3HD and D-amino acid oxidase (DAO)]. The third appears distantly related to translation initiation factor 1A, a protein essential for transfer of the initiator tRNA to 40 S ribosomal subunits to form the 40 S preinitiation complex (CHAUDHURI et al. 1997 ). Although the loss of a translation factor within the fungi may seem surprising, previous analyses have established the loss of translation factor components in the S. cerevisiae lineage (BRAUN et al. 2000 ), and an ortholog of eIF1A is present in S. cerevisiae (Tif11p, see WEI et al. 1995 ), suggesting a distinct function for G6G8.9. The possible transcription factor encoded by G6G8.4 (Table 1) has a region of identity to a Schizosaccharomyces pombe protein that shows weak identity to the helix-turn-helix structure of S. cerevisiae Mbp1p (see TAYLOR et al. 1997 ) in profile searches. However, a protein much more closely related to G6G8.4 is present in S. pombe, an apparent outgroup to a clade containing N. crassa and S. cerevisiae (BRUNS et al. 1992 ), suggesting the absence of an S. cerevisiae G6G8.4 ortholog through gene loss.

The observation of 4 of 13 genes showing possible loss in S. cerevisiae is surprising, since a previous survey based on EST data suggested that 12% of N. crassa genes with detectable homologs were lost in the S. cerevisiae lineage (BRAUN et al. 2000 ). Although the higher proportion of genes in this category observed here may simply reflect sampling error, it is also possible that such predictions based upon EST data are biased in some manner.

The intergenic regions in the G6G8 portion of the N. crassa genome are substantially larger than comparable regions in the S. cerevisiae genome, as expected (KUPFER et al. 1997 ). However, the intergenic regions separating convergently transcibed genes are only slightly larger than comparable regions in the S. cerevisiae genome (Table 2), while those separating either divergently transcribed genes or genes transcribed in the same direction are substantially larger than comparable regions in the S. cerevisiae genome. Although there is substantial evidence linking the SNZ and SNO genes functionally (GALPERIN and KOONIN 1997 ; PADILLA et al. 1998 ; this work), their start codons do not appear to be unusually close for divergently transcribed genes (separated by 1992 bp).

The shortest intergenic region separates the NOT-56 homolog (G6G8.10) from a convergently transcribed gene related to an E. nidulans EST and a hypothetical S. pombe eIF1A-like ORF (G6G8.9). Surprisingly, a NOT-56 cDNA (SM1G12) shows substantial overlap (at least 180 nucleotides) with the adjacent G6G8.9 open reading frame. This overlap raises the possibility that these genes exhibit transcriptional interference similar to the convergently transcribed S. cerevisiae POT1 and YIL161w genes (PUIG et al. 1999 ).

The most closely spaced putative parallel transcription units (G6G8.2 and G6G8.3) may present an even more substantial transcriptional overlap. An mRNA (d4b03ne) that has a 3' end downstream of G6G8.2 extends into the second exon of G6G8.3 and lacks a putative intron present in G6G8.3 (Table 1). Thus, it is possible that G6G8.2 mRNAs actually correspond to the 3' untranslated region of G6G8.3, making our annotation of G6G8.2 as a protein coding region more tentative than the other genes in this region. On the basis of an in-frame stop codon in G6G8.3, verified in genomic and cDNA sequences, and similarity between G6G8.3 and known rho GDI homologs from other organisms, G6G8.2 apparently does not represent an extension of the G6G8.3 coding region.

Analysis of SNZ and SNO homologs:
The N. crassa SNZ and SNO homologs were first identified as cDNAs by the Neurospora Genome Project at the University of New Mexico (NELSON et al. 1997 ). The genomic sequence reported here reveals that the SNZ homolog contains no introns, while the SNO homolog contains a single intron. The two genes are divergently transcribed and separated by 2 kbp (Table 1). There are two overlapping ORFs between the two genes that could each encode a polypeptide >100 amino acids (not shown). However, these ORFs lack strong consensus sequences for translational start, they do not exhibit codon preference typical for N. crassa, and they lack homologs in other organisms or corresponding EST sequences from N. crassa. Therefore, neither ORF was included among the predicted genes for the region.

Given mapping results that placed this region close to the pdx-1 locus (linkage group IVR; NELSON et al. 1998 ), together with recent reports that SNZ homologs are involved in pyridoxine metabolism, we hypothesized that mutations in the N. crassa SNZ homolog were responsible for the pdx-1 phenotype. Sequences obtained from several known mutants, designated pdx-1, strongly suggest that this is the case. Five of nine pdx-1 mutants examined possessed mutations in the coding region of the SNZ homolog that either altered the amino acid sequence in highly conserved regions or caused a frameshift (Table 3). However, analysis of four pdx-1 mutants revealed no mutations in the SNZ homolog but, instead, demonstrated mutations in conserved regions of the SNO homolog (Table 3). This represents the first direct evidence that mutations in SNZ and SNO homologs disrupt a shared metabolic pathway.

The conclusion that the observed mutations in SNZ and SNO homologs cause the pyridoxine-requiring phenotypes of the mutants examined is supported by complementation studies reported by RADFORD 1966 for six of the strains—FGSC numbers 1407, 1409, 1411, 1413, 1415, and 4055 (alleles 35405, 39106, 44602, 44204, 39706, and 37803, respectively). Working with alleles that were presumed to represent a single locus, Radford failed to obtain complementation between strains carrying alleles for which we identified corresponding mutations in the SNZ homolog (35405, 37803), as well as among strains with alleles with mutations in the SNO homolog (39106, 39706, 44602, 44204). In contrast, Radford reported successful complementation in tests where one strain possessed a mutation in the SNZ homolog while the other possessed a mutation in the SNO homolog. The one exception was a reported failure to obtain complementation between strains with alleles 44204 (SNO) and 37803 (SNZ), an anomaly for which there is no obvious explanation.

Strains 1409 and 1415, carrying alleles designated 39106 and 39706, possess identical mutations in the SNO homolog. It is likely that this reflects confusion in allele labeling in the laboratory history of these strains.

A shared function for SNZ and SNO homologs is further supported by high-resolution "intragenic" mapping data obtained by RADFORD 1968 . Radford reported evidence for three separate clusters of mutations at the pdx-1 locus, and he designated these clusters , ß, and (Fig 1). Our sequence analysis agrees with the chromosomal order suggested by Radford for alleles in the group (35405, 37803), which possess mutations in the SNZ homolog, relative to alleles in the ß (39106) and (44602, 44204) groups combined, which possess mutations in the SNO homolog (Table 3, Fig 1). Also in agreement with sequence analysis, Radford's results indicated that all ß and mutations were closer to one another than to any mutations in the group. However, the Radford study tentatively placed the ß allele group proximal to the group. Sequence results indicate instead that the group is proximal to the group. The positions of , ß, and groups approximated by Radford were based in part on recombination frequencies between pdx alleles and genetic markers flanking the pdx region. However, considering only the frequencies of prototrophs recovered in crosses with alternative pdx alleles, one RADFORD study (1968) was inconclusive with respect to the positions of ß and relative to , while another study (RADFORD 1967 ) in fact supported the order indicated by our sequence analysis of mutants.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Comparison of high-resolution allele mapping results and sequence analysis of pyridoxine-requiring mutants. (A) Map adapted from RADFORD 1968 showing relative positions of , ß, and allele groups. Only alleles examined in both studies are shown. Note: this map was not represented by the author as being to scale. The direction of the centromere is indicated by an open circle. (B) Mutant sequence analysis (Table 3). Positions of mutations observed in pdx-1 (SNZ homolog) and pdx-2 (SNO homolog) coding regions, shown approximately to scale. Arrows indicate direction of transcription.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Comments on annotation:
Our attempt to identify genes in the G6G8 insert highlights the difficulties of annotation with filamentous fungi when only genomic sequence data are available, and it underscores the value of supplemental information. The 36-kbp region in question contains 106 ORFs that could encode peptides of at least 100 amino acids each. Thirty-eight of these ORFs begin with a start ATG. In contrast, the actual estimate for this region is 13 genes (Table 1). None of the ORFs excluded from the gene list in Table 1 exhibited strong N. crassa codon preference, nor did any produce a BLAST E-value < 10^-3. Several of the excluded ORFs overlapped verified genes, raising additional doubt with respect to possible protein-coding function. Eleven protein-coding genes could be verified by BLAST analyses revealing homology with known genes from other organisms or fungal ESTs (Table 1). An additional gene, not identified by BLAST analysis, was inferred from a long ORF (426 codons) with strong N. crassa codon preference. If additional protein-coding genes exist in this region, they were not identified, either because they do not exhibit strong codon preference or because they encode relatively short polypeptides. Further, the presence of an identifiable 5' start ATG is not a reliable criterion for ORF identification due to the frequent occurrence of introns in the 5' regions of N. crassa genes. This point is well illustrated by the genes identified in Table 1. Nine of the 13 annotated genes possess introns, 5 of which could be deduced by comparison with cDNA (EST) sequences. Among these 9 genes, 6 possess an initial intron within the first 100 codons, exemplifying the poor predictive value of a start ATG for gene finding in this organism.

Significance of observed gene density:
N. crassa is a multicellular fungus with a complex life cycle that involves both asexual and sexual reproduction. It possesses a genome size of 42.9 Mbp (ORBACH et al. 1988 ; ORBACH 1992), nearly three times that of its ascomycete relative S. cerevisiae. In N. crassa, asexual reproduction involves the generation of two different types of conidia, while sexual reproduction involves the development of ascospores within a morphologically complex perithecium (SPRINGER 1993 ). The developmental complexity and relatively large genome size of N. crassa suggest that it might possess a substantially larger number of genes than do unicellular fungi such as S. cerevisiae and S. pombe. Previous analyses suggested that at least some of these differences in genome complexity reflect gene loss in S. cerevisiae (BRAUN et al. 1998 , BRAUN et al. 2000 ).

Although all recent estimates suggest substantially larger gene numbers for N. crassa and other filamentous ascomycetes than for S. cerevisiae, specific estimates for N. crassa differ. KUPFER et al. 1997 estimated that filamentous fungi typically harbor 8000–9000 genes and suggested that N. crassa has 9200 genes based on a nonlinear extrapolation of gene number from genome size. In contrast, NELSON et al. 1997 estimated a larger number of genes for N. crassa, up to 13,000, on the basis of gene densities in the mating-type and qa-cluster regions.

There exists a minimum of 12 protein-coding genes in the region represented by the 36,030-bp insert in cosmid G6G8, corresponding to a genetic unit of 3000 bp. Assuming 39 x 10⁶ bp of genomic DNA, after subtracting rDNA repeats and other low complexity sequences (KRUMLAUF and MARZLUF 1980 ; NELSON et al. 1997 ), the predicted total gene number is 13,000. This estimate is consistent with our previous estimate (NELSON et al. 1997 ) and with another recent estimate based on analysis of a distinct cosmid sequence (KELKAR et al. 2001 ).

Function and evolution of the pdx-1 region:
Our results demonstrate that the pdx-1 mutant phenotype can derive from mutations in either the SNZ or SNO homolog of N. crassa. The coordinate function for these two genes was inferred for other organisms from previous studies of regulation and gene linkage. Our analysis of N. crassa pdx-1 mutants provides confirming experimental evidence in support of this inference.

A nomenclature problem exists with respect to mutant alleles currently designated pdx-1. The three separate allele clusters identified by RADFORD 1968 in high-resolution mapping studies—designated , ß, and —were interpreted as intragenic on the basis of close physical proximity and shared phenotype. Sequence analyses demonstrate that alleles possess mutations in the SNZ homolog, whereas ß and alleles possess mutations in the SNO homolog. Alleles from and ß groups alike were among those originally described (HOULAHAN et al. 1949 ; RADFORD 1968 ). Given that pyridoxine metabolism was first linked experimentally to SNZ homologs (EHRENSHAFT et al. 1999A ; OSMANI et al. 1999 ), we suggest that in N.crassa the pdx-1 designation is most appropriate for the SNZ homolog.

An allele from the Radford group, 44204, was at one time designated pdx-2 but was considered by RADFORD 1965 to belong to pdx-1. Allele 44204 and another allele from the Radford group, 44602, possess mutations in conserved regions of the SNO homolog (Table 3). We therefore suggest that the pdx-2 designation is appropriate for the SNO homolog (Fig 1).

SNZ and SNO homologs are closely linked in diverse prokaryotes and eukaryotes. It has been proposed that in general such clustering in prokaryotes occurs with "selfish operons," operons whose products provide functions that are under weak or sporadic positive selection (LAWRENCE and ROTH 1996 ). Because the functions encoded by such clusters are dispensable under certain environmental conditions, the genes encoding such functions may be subject to accumulation of deleterious mutations and loss. Gene clustering is thought to facilitate the horizontal transfer of an intact functional operon, allowing the acquisition or reacquisition of function. Support for a process analogous to the selfish operon theory exists for clustered genes in fungi. Often, clustered genes encode dispensable catabolic functions or components of secondary-metabolite pathways (KELLER and HOHN 1997 ; PRADE et al. 1997 ). Clustering may provide a mechanism for horizontal transfer or other forms of transfer not involving sexual reproduction among individuals of the same species.

The selfish operon model does not appear to provide an adequate explanation for the close linkage of SNZ and SNO homologs. Quite clearly, within the genera Neurospora and Emericella, SNZ homologs do not fit the profile of dispensable genes well. Mutations in the SNZ homologs in members of these genera create pyridoxine auxotropy, which to our knowledge has not been observed among thousands of wild-type strains. Furthermore, mutations in SNZ homologs increase susceptibility to oxidative stress (EHRENSHAFT et al. 1999A , EHRENSHAFT et al. 1999B ), and SNZ homologs have been observed in all ascomycetes for which substantial genome sequencing has been performed. Phylogenetic tree-building analyses using predicted amino-acid sequences for SNZ and SNO homologs suggest that the evolution of these genes is compatible with organismal phylogeny (results not presented). Together, these observations make it unlikely that the physical linkage of SNZ and SNO genes in fungi reflects a recent horizontal transfer from prokaryotes. Instead, this linkage likely reflects selection for coordinate regulation.

Conclusion:
Our analysis of this 36-kbp region of the N. crassa genome demonstrates that efforts in fungal genomics to identify coding regions and determine gene function will be most successful with combined approaches. Results illustrate the difficulties of annotation, given only genomic sequence data, and they reveal the added value of information from cDNA sequences, biochemistry, bioinformatics, and classical genetics.

This study also underscores the diversity of processes underlying genome evolution. Two genes were identified with N. crassa paralogs, despite the relative paucity of duplicated genes in N. crassa (NELSON et al. 1997 ; BRAUN et al. 2000 ). Four of 13 genes appear to have been lost in S. cerevisiae, emphasizing the contribution of gene loss to the S. cerevisiae lineage (see BRAUN et al. 1998 , BRAUN et al. 2000 ). Although the contribution of gene loss to the evolution of other small fungal genomes is not known at present, it is likely that gene loss has had a similar impact upon such genomes.

The close linkage of SNZ and SNO genes in many organisms, including N. crassa, signals the functional information present in the genomic context of genes. Although the correlation between location and function has long been appreciated in prokaryotes (reviewed by ARAVIND 2000 ), results such as those presented here suggest that this correlation will also be valuable in eukaryotes. The elucidation of evolutionary mechanisms driving correlation between gene location and function should aid in efforts to predict gene function.

	FOOTNOTES

¹ Present address: Cell and Molecular Biology Program, Michigan State University, East Lansing, MI.
² Present address: Department of Plant Biology, The Ohio State University, Columbus, OH.
³ Present address: Department of Plant Pathology, Cornell University, Ithaca, NY.

	ACKNOWLEDGMENTS

We thank Dr. Alan Radford for very helpful comments during the course of pdx-1 analyses. This work was supported by National Science Foundation grants HRD-9550649 (D.O.N., M.A.N., M.W.-W., and Robert K. Miller), MCB-9603902 (D.O.N.), IBN-9870878 (M.W.-W.) and MCB-9874488 (M.A.N.). A.E. and M.G. were supported in part by the Minority Biomedical Research Support program of the University of New Mexico (National Institutes of Health grant GM-52576). E.L.B. was supported in part by United States Department of Agriculture fellowship 1999-01582. G.S.S. was supported in part by a postdoctoral fellowship from the Ford Foundation. We gratefully acknowledge computer and computational support from the Albuquerque High Performance Computing Center at the University of New Mexico.

Manuscript received October 3, 2000; Accepted for publication December 15, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ALTSCHUL, S. F., T. L. MADDEN, A. A. SCHÄFFER, J. ZHANG, and Z. ZHANG et al., 1997  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3492[Abstract/Free Full Text].

ARAVIND, L., 2000  Guilt by association: contextual information in genome analysis. Genome Res. 10:1074-1077[Free Full Text].

BARRATT, R. W., D. NEWMEYER, D. D. PERKINS, and L. GARNJOBST, 1954  Map construction in Neurospora crassa. Adv. Genet. 6:1-93[Medline].

BRAUN, E. L., E. K. FUGE, P. A. PADILLA, and M. WERNER-WASHBURNE, 1996  A stationary phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family. J. Bacteriol. 178:6865-6872[Abstract/Free Full Text].

BRAUN, E. L., S. KANG, M. A. NELSON, and D. O. NATVIG, 1998  Identification of the first fungal annexin: analysis of annexin gene duplications and implications for eukaryotic evolution. J. Mol. Evol. 47:531-543[Medline].

BRAUN, E. L., A. L. HALPERN, M. A. NELSON, and D. O. NATVIG, 2000  Large scale comparison of fungal sequence information: mechanisms of innovation in Neurospora crassa and gene loss in Saccharomyces cerevisiae. Genome Res. 10:416-430[Abstract/Free Full Text].

BRUCHEZ, J. J. P., J. EBERLE, and V. E. A. RUSSO, 1993a  Regulatory sequences in the transcription of Neurospora crassa genes: CAAT box, TATA box, introns, poly(A) tail formation sequences. Fungal Genet. Newsl. 40:89-96.

BRUCHEZ, J. J. P., J. EBERLE, and V. E. A. RUSSO, 1993b  Regulatory sequences involved in the translation of Neurospora crassa mRNA: Kozak sequences and stop codons. Fungal Genet. Newsl. 40:85-88.

BRUNS, T. D., R. VILGALYS, S. M. BARNS, D. GONZALEZ, and D. S. HIBBETT et al., 1992  Evolutionary relationships within the fungi: analyses of nuclear small subunit rRNA sequences. Mol. Phylogenet. Evol. 1:531-543.

CHARY, P., R. A. HALLEWELL, and D. O. NATVIG, 1990  Structure, exon pattern and chromosome mapping of the gene for cytosolic copper-zinc superoxide dismutase (sod-1) from Neurospora crassa. J. Biol. Chem. 265:18961-18967[Abstract/Free Full Text].

CHAUDHURI, J., K. SI, and U. MAITRA, 1997  Function of eukaryotic translation initiation factor 1A (eIF1A) (formerly called eIF-4C) in initiation of protein synthesis. J. Biol. Chem. 272:7883-7891[Abstract/Free Full Text].

DAVIS, R. H. and F. J. DESERRES, 1970  Genetic and microbiological research techniques for Neurospora crassa. Methods Enzymol. 17:79-143.

DUJON, B., 1996  The yeast genome project: what did we learn? Trends Genet. 12:263-270[Medline].

EHRENSHAFT, M., P. BILSKI, M. Y. LI, C. F. CHIGNELL, and M. E. DAUB, 1999a  A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis. Proc. Natl. Acad. Sci. USA 96:9374-9378[Abstract/Free Full Text].

EHRENSHAFT, M., K.-R. CHUNG, A. E. JENNS, and M. E. DAUB, 1999b  Functional characterization of SOR1, a gene required for resistance to photosensitizing toxins in the fungus Cercospora nicotianae. Curr. Genet. 34:478-485[Medline].

FUGE, E. K., E. L. BRAUN, and M. WERNER-WASHBURNE, 1994  Protein synthesis in long-term stationary-phase cultures of Saccharomyces cerevisiae. J. Bacteriol. 176:5802-5813[Abstract/Free Full Text].

GALPERIN, M. Y. and E. V. KOONIN, 1997  Sequence analysis of an exceptionally conserved operon suggests enzymes for a new link between histidine and purine biosynthesis. Mol. Microbiol. 24:443-445[Medline].

GREEN, P., 1996 PHRAP ("phragment assembly program," or "Phil's revised assembly program"). Department of Molecular Biotechnology, University of Washington, Seattle.

GREEN, P., and B. EWING, 1997 PHRED. Department of Molecular Biotechnology, University of Washington, Seattle.

HOULAHAN, M. B., G. W. BEADLE, and H. G. CALHOUN, 1949  Linkage studies with biochemical mutants of Neurospora crassa. Genetics 34:493-507[Free Full Text].

KELKAR, H. S., J. GRIFFITH, M. E. CASE, S. F. COVERT, and R. D. HALL et al., 2001  The Neurospora crassa genome: cosmid libraries sorted by chromosome. Genetics 157:979-990[Abstract/Free Full Text].

KELLER, N. P. and T. M. HOHN, 1997  Metabolic pathway gene clusters in filamentous fungi. Fungal Genet. Biol. 21:17-29[Medline].

KRASKO, A., H. C. SCHRÖDER, S. PEROVIC, R. STEFFEN, and M. KRUSE et al., 1999  Ethylene modulates gene expression in cells of the marine sponge Suberites domuncula and reduces the degree of apoptosis. J. Biol. Chem. 274:31524-31530[Abstract/Free Full Text].

KRUMLAUF, R. and G. A. MARZLUF, 1980  Genome organization and characterization of the repetitive and inverted repeat DNA sequences in Neurospora crassa. J. Biol. Chem. 255:1138-1145[Free Full Text].

KUPFER, D. M., C. A. REECE, S. W. CLIFTON, B. A. ROE, and R. A. PRADE, 1997  Multicellular ascomycetous fungal genomes contain more than 8000 genes. Fungal Genet. Biol. 21:364-372[Medline].

LAWRENCE, J. G. and J. R. ROTH, 1996  Selfish operons: horizontal transfer may drive the evolution of gene clusters. Genetics 143:1843-1860[Abstract].

NELSON, M. A., S. KANG, E. L. BRAUN, M. E. CRAWFORD, and P. DOLAN et al., 1997  Expressed sequences from conidial, mycelial, and sexual stages of Neurospora crassa. Fungal Genet. Biol. 21:348-363[Medline].

NELSON, M. A., M. E. CRAWFORD, and D. O. NATVIG, 1998  Restriction polymorphism maps of Neurospora crassa: 1998 update. Fungal Genet. Newsl. 45:44-54.

ORBACH, M. J., 1994  A cosmid with a Hy^R marker for fungal library construction and screening. Gene 150:159-162[Medline].

ORBACH, M. J., D. VOLLRATH, R. W. DAVIS, and C. YANOFSKY, 1988  An electrophoretic karyotype of Neurospora crassa. Mol. Cell. Biol. 8:1469-1473[Abstract/Free Full Text].

OSMANI, A. H., G. S. MAY, and S. A. OSMANI, 1999  The extremely conserved pyroA gene of Aspergillus nidulans is required for pyridoxine synthesis and is required indirectly for resistance to photosensitizers. J. Biol. Chem. 274:23565-23569[Abstract/Free Full Text].

PADILLA, P. A., E. K. FUGE, M. E. CRAWFORD, A. ERRETT, and M. WERNER-WASHBURNE, 1998  The highly conserved, coregulated SNO and SNZ gene families in Saccharomyces cerevisiae respond to nutrient limitation. J. Bacteriol. 180:5718-5726[Abstract/Free Full Text].

PRADE, R. A., J. GRIFFITH, K. KOCHUT, J. ARNOLD, and W. E. TIMBERLAKE, 1997  In vitro reconstruction of the Aspergillus (= Emericella) nidulans genome. Proc. Natl. Acad. Sci. USA 94:14564-14569[Abstract/Free Full Text].

PUIG, S., J. E. PÉREZ-ORTÍN, and E. MATALLANA, 1999  Transcriptional and structural study of a region of two convergent overlapping yeast genes. Curr. Microbiol. 39:369-373[Medline].

RADFORD, A., 1965  Heterokaryon complementation among the pyridoxine auxotrophs of Neurospora crassa. Can. J. Genet. Cytol. 7:472-477[Medline].

RADFORD, A., 1966  Further studies on complementation at the pdx-1 locus of Neurospora crassa. Can. J. Genet. Cytol. 8:672-676.

RADFORD, A., 1967  Prototroph frequencies from crosses between pyridoxine auxotrophs. Neurospora Newsl. 11:4.

RADFORD, A., 1968  High resolution recombination analysis of the pyridoxine-1 locus of Neurospora. Can. J. Genet. Cytol. 10:893-897[Medline].

ROE, B. A., J. S. CRABTREE and A. S. KHAN, 1996 DNA Isolation and Sequencing. John Wiley & Sons, New York.

SAMBROOK, J., E. F. FRITSCH and T. MANIATIS, 1989 Molecular Cloning: A Laboratory Manual, Ed. 2. Cold Spring Harbor Laboratory Press, Plainview, NY.

SIVASUBRAMANIAM, S., V. M. VANNIASINGHAM, C.-T. TAN, and N.-H. CHUA, 1995  Characterization of HEVER, a novel stress-induced gene from Hevea brasiliensis. Plant Mol. Biol. 29:173-178[Medline].

SPRINGER, M. L., 1993  Genetic control of fungal differentiation: the three sporulation pathways of Neurospora crassa. Bioessays 15:365-374[Medline].

TATUM, E. L., R. W. BARRATT, N. FRIES, and D. BONNER, 1950  Biochemical mutant strains of Neurospora produced by physical and chemical treatment. Am. J. Bot. 37:38-46.

TAYLOR, I. A., M. K. TREIBER, L. OLIVI, and S. J. SMERDON, 1997  The X-ray structure of the DNA-binding domain from the Saccharomyces cerevisiae cell-cycle transcription factor Mbp1 at 2.1 Å resolution. J. Mol. Biol. 272:1-8[Medline].

WEI, C. L., M. KAINUMA, and J. W. HERSHEY, 1995  Characterization of yeast translation initiation factor 1A and cloning of its essential gene. J. Biol. Chem. 270:22788-22794[Abstract/Free Full Text].

This article has been cited by other articles:

				M. Gengenbacher, T. B. Fitzpatrick, T. Raschle, K. Flicker, I. Sinning, S. Muller, P. Macheroux, I. Tews, and B. Kappes Vitamin B6 Biosynthesis by the Malaria Parasite Plasmodium falciparum: BIOCHEMICAL AND STRUCTURAL INSIGHTS J. Biol. Chem., February 10, 2006; 281(6): 3633 - 3641. [Abstract] [Full Text] [PDF]

				J. Zhu, J. W. Burgner, E. Harms, B. R. Belitsky, and J. L. Smith A New Arrangement of ({beta}/{alpha})8 Barrels in the Synthase Subunit of PLP Synthase J. Biol. Chem., July 29, 2005; 280(30): 27914 - 27923. [Abstract] [Full Text] [PDF]

				J. Stolz, H. J. P. Wohrmann, and C. Vogl Amiloride Uptake and Toxicity in Fission Yeast Are Caused by the Pyridoxine Transporter Encoded by bsu1+ (car1+) Eukaryot. Cell, February 1, 2005; 4(2): 319 - 326. [Abstract] [Full Text] [PDF]

				T. Tanaka, Y. Tateno, and T. Gojobori Evolution of Vitamin B6 (Pyridoxine) Metabolism by Gain and Loss of Genes Mol. Biol. Evol., February 1, 2005; 22(2): 243 - 250. [Abstract] [Full Text] [PDF]

				Z. W. Luo, R. M. Zhang, and M. J. Kearsey Theoretical basis for genetic linkage analysis in autotetraploid species PNAS, May 4, 2004; 101(18): 7040 - 7045. [Abstract] [Full Text] [PDF]

				K. A. Borkovich, L. A. Alex, O. Yarden, M. Freitag, G. E. Turner, N. D. Read, S. Seiler, D. Bell-Pedersen, J. Paietta, N. Plesofsky, et al. Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism Microbiol. Mol. Biol. Rev., March 1, 2004; 68(1): 1 - 108. [Abstract] [Full Text] [PDF]

				B. R. Belitsky Physical and Enzymological Interaction of Bacillus subtilis Proteins Required for De Novo Pyridoxal 5'-Phosphate Biosynthesis J. Bacteriol., February 15, 2004; 186(4): 1191 - 1196. [Abstract] [Full Text] [PDF]

				J. A. Bauer, E. M. Bennett, T. P. Begley, and S. E. Ealick Three-dimensional Structure of YaaE from Bacillus subtilis, a Glutaminase Implicated in Pyridoxal-5'-phosphate Biosynthesis J. Biol. Chem., January 23, 2004; 279(4): 2704 - 2711. [Abstract] [Full Text] [PDF]

				J. Stolz and M. Vielreicher Tpn1p, the Plasma Membrane Vitamin B6 Transporter of Saccharomyces cerevisiae J. Biol. Chem., May 23, 2003; 278(21): 18990 - 18996. [Abstract] [Full Text] [PDF]

				M. Ehrenshaft and M. E. Daub Isolation of PDX2, a Second Novel Gene in the Pyridoxine Biosynthesis Pathway of Eukaryotes, Archaebacteria, and a Subset of Eubacteria J. Bacteriol., June 1, 2001; 183(11): 3383 - 3390. [Abstract] [Full Text]

				H. S. Kelkar, J. Griffith, M. E. Case, S. F. Covert, R. D. Hall, C. H. Keith, J. S. Oliver, M. J. Orbach, M. S. Sachs, J. R. Wagner, et al. The Neurospora crassa Genome: Cosmid Libraries Sorted by Chromosome Genetics, March 1, 2001; 157(3): 979 - 990. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bean, L. E. Articles by Natvig, D. O. Search for Related Content PubMed PubMed Citation Articles by Bean, L. E. Articles by Natvig, D. O.