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The Ustilago maydis nar 1 gene encoding nitrate reductase activity: sequence and transcriptional regulation
Gene, I3 1 (1993) 69-78 1.T)1993 Elsevier Science Publishers
GENE
B.V. All rights
reserved.
69
0378-I 119/93/SO6.00
07244
The Ustilago maydis narl gene encoding nitrate reductase activity: sequence and transcriptional regulation (Recombinant DNA; basidiomycete fungus; gene disruption; inducible transcript)
G.R. Banks*, P.A. Shelton*, N. Kanuga*, D.W. Holden* and A. Spanos* Genetics Dioision, National Institutefor Medical Research, The Ridgeway, Mill Hill, London NW7 IAA. UK Received by J.R. Kinghorn:
10 December
1992; Revised/Accepted:
19 March/28
March
1993; Received at publishers:
6 May 1993
SUMMARY
The narl gene was cloned from Ustilago maydis and the 90%amino-acid (aa) sequence of the encoded protein found to have strong identities with other nitrate reductases from fungi and plants. This was especially so in three domains which define enzyme cofactor-binding sites. The gene was isolated alone and in association with the nirl gene, suggesting that the two genes are closely linked on the chromosome. The phenotype of a strain in which narl had been disrupted was consistent with the only role of narl being in nitrate reduction. Nitrate ions induced a 90-fold increase in narl transcript levels, while ammonium ions repressed transcript levels.
INTRODUCTION
The ability to assimilate nitrate ions as the sole nitrogen source for growth is possessed by many bacteria, fungi, algae and plants (reviewed in Dunn-Coleman et al., 1984; Wray and Kinghorn, 1989). Assimilation is effected Correspondence to: Dr. G.R. Banks at his present
address:
Laboratory
of Yeast Genetics, National Institute for Medical Research, Ridgeway, Mill Hill, London NW7 IAA. UK. Tel. (44-81)959-3666; (44-8 1)906-4477.
The Fax
*Present addresses: (G.R.B., N.K. and A.S.) Laboratory of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Tel. (44-81)959-3666; (P.A.S) ICI/University Joint Laboratory, School of Biological Sciences, University of Leicester, Leicester LEI 7RH, UK. Tel. (44-81)533-523364; (D.W.H) Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Hammersmith Hospital, London W 12 ONN, UK. Tel. (44-8 1)740-3222.
Du Cane Road,
Abbreviations: A., Aspergillus; aa, amino acid(s); bp, base pair(s); CM, Ustilago complete medium; FAD, flavin adenine dinucleotide; Hy. hygromycin B; kb, kilobase or 1000 bp; N., Neurospora; NADH. reduced form of nicotinamide-adenine dinucleotide; narl, gene encoding nitrate reductase of U. maydis; nirl, gene encoding nitrite reductase of U. maydis; NM, U&ago nitrate minimal medium; nt, nucleotide(s); ORF, open reading frame; R, resistance/resistant: tsp. transcription start point(s); U.. Ustilago: wt, wild type.
by the reduction of nitrate to ammonium ions and involves a substantial investment in metabolic energy. It is not surprising that this pathway is tightly regulated in several ways. Studies with the filamentous fungi Aspergillus nidulans (Scazzocchio and Arst, 1989) and Neurospora crassa (Marzluf and Fu, 1989) in particular have made major contributions to our understanding of the regulation of nitrate metabolism. In outline, nitrate ions are transported into the cell by a permease, encoded by the crnA gene of A. nidulans (Brownlee and Arst, 1983) to be reduced to nitrite ions by nitrate reductase, the apoprotein of which is encoded by the niaD and nit-3 genes of A. nidulans and N. crassa, respectively. Nitrite ions are reduced to ammonium ions by nitrite reductase, the niiA and nit-6 genes of A. nidulans and N. crassa respectively encoding the apoprotein of this enzyme (reviewed in Cove, 1979; Scazzocchio and Arst, 1989; Marzluf and Fu, 1989). The pathway is regulated through at least two genetic systems. In the first, which is pathway specific, nitrate and nitrite reductase activities are transcriptionally induced by exogenous nitrate ions. The A. nidulans nirA and the N. crassa nit-415 genes are responsible for this regulation (Cove, 1979; Marzluf, 1981). In addition, the nitrate reductase gene product appears to
70 autoregulate
its own gene expression
of the nitrite
reductase
and regulate
(Cove, 1979). Nitrate
that
assimilation
to nirl and a one step disruption
linkage
is also regulated as part of a number of genes under the control of nitrogen metabolite repression. Expression of
apoenzymes
these
transcriptional
genes
is reduced
by ammonium
a process
by the areA and nit-2 genes of A. nidu-
which is mediated
lans and N. crassa, respectively and Arst,
ions,
(reviewed
1989; Arst and Cove,
1973; Marzluf
nitrate cloned
and
nitrite
revealing
and conservation peptides, studies
reductase
genetic
of the sequences
and confirming (Crawford
fungal
apoproteins
organisation,
organisms
inducibility
are presented
and the
of the narl gene by nitrate
ions.
and Fu, RESULTS
years, the genes of several
of other
in Scazzocchio
1989). In recent
of a chromo-
somal copy of the narl gene. Amino acid sequence similarities of the encoded protein to nitrate reductase
domain
have
been
structure
of the encoded
poly-
results from earlier biochemical
et al, 1986; Daniel-Vedele
AND DISCUSSION
and plant
et a1.,1989;
Johnstone et al., 1990; Gruber et al., 1992). Thus the A. nidulans niaD gene encodes an apoprotein of 97 kDa which exists as a homodimer, each monomer carrying the three cofactors FAD, haem, and molybdenum in the form of a complex with pterin. The electron flow in these redox prosthetic groups is NADPH+FAD+haem-+ molybdenum+nitrate. The domains of the nitrate reductase apoprotein which interact with the cofactors have been identified from the activities found in proteolytic products, and by comparison with the amino acid sequences of nitrate reductases from different organisms. In addition, aa identities between nitrate reductases and enzymes involved in similar electron transfers, but which have unrelated physiological functions, have been described (Crawford et al., 1988; Kinghorn and Campbell, 1989; Campbell and Kinghorn, 1990; Caboche and Rouze, 1990; Unkles at al., 1992). There is conservation of these domains between nitrate reductase apoproteins from fungi and plants. Although not characterised in detail, the genetics of nitrate assimilation by the basidiomycete fungus U. maydis are similar in outline to assimilation in A. niduIans (Lewis and Fincham, 1970a,b). Mutations in the narl and nirl genes define the structural genes for the nitrate and nitrite reductase apoproteins respectively, and other mutations define genes probably involved in molybdenum cofactor biosynthesis. Nitrate ions as sole nitrogen source induce nitrate reductase enzyme activity whilst ammonium ions repress it (Lewis and Fincham, 1970b; Smith et al., 1988). A diploid homozygous for a nirl mutation is able to reduce nitrate to nitrite ions, which accumulate because further reduction is blocked. This observation has led to a biochemical assay for recombination in a homozygous nirl mutant diploid using a sensitive assay for nitrite ions which accumulated after recombination between heteroalleles of narl mutants to give a wt allele (Holliday, 1971). We report here the cloning of the narl gene by direct complementation, suggestions of its close
(a) Isolation of the narl and nirl genes The nitrate and the nitrite reductase were isolated The cosmid DNA
from cosmid library
of about
apoprotein
and plasmid-based
comprises
fragments
40 kb inserted
into
genes
libraries.
of wt U. maydis
the BamHI
site of
cosmid pCUl3 (Wang et al., 1989). Transformants are generated by ectopic integration of cosmid DNA into chromosomes. The plasmid library has U. maydis wt DNA of 5- 15 kb inserted into the BamHI site of plasmid pCM54 (Banks and Taylor, 1988; D.W.H., unpublished). Transformants arise by autonomous replication of plasmids in U. maydis by virtue of the UARSI sequence in pCM54 (Tsukuda et al., 1988). Each library was transformed into U. maydis strain 95 (narl-I panl-1 Ab) and into 363 (nirl-1 panl-1 inosl-3 reel-I), HyR transformants being selected. These transformants were replicated to select those which grew on media with nitrate or nitrite ions as sole nitrogen source. All transformants, as well as the non-transformed mutant parents, displayed limited growth on these media because of the presence of trace quantities of ammonium ions (Lemontt, 1976), but transformants in which complemetation of the narl- 1 and nirl1 mutations occurred grew much more vigorously and were easily identified. Cosmid H7 complemented the nirl1 mutation and was isolated from the cosmid library by the sib selection procedure (Akins and Lambowitz, 1985). H7 was subsequently found to complement the narl-1 mutation in addition to nirl-1. Plasmid pMH3051, containing an 8.5-kb insert of U. maydis DNA, was a member of the plasmid library and complemented the narl- 1, but not the nirl-1 mutation. It was rescued from the primary transformant by virtue of its autonomous maintenance within the cell. A 6.7-kb EcoRI DNA fragment (Fig. 1A; the left-hand EcoRI site shown in the figure originates from the polylinker of the vector pCM54) from the insert in pMH305 1 was cloned into the vector pBluescript KS + to give pMH3055. A 4.7-kb EcoRI-XbaI fragment from the insert of pMH305 1 (Fig. 1A) was cloned into the vectors pBluescript KS+ and pCM54 to yield plasmids pMH3057 and pMH3053, respectively. Plasmid pMH3061 contained 9.5 kb of U. maydis DNA, was a member of the plasmid library and complemented the nirl-1, but not narl-I mutation. In summary, cosmid H7
71
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.
.
.
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.
.
.
.
. -503
.
.
GAATTCGAGCTCGCCCGGGGATC
ATCACTCTGTTTGCGCTCGATTACTTTGCMGCCCTTACACACTTGTTGCTCTCCTTCTGCTAGCTTGTGCGTCGCMGTGGCGAGCTCTTCTTGAGACCGTTTACMCAGTGCGATTCA MCGCAAAGGCATCAGCATCTTAGCTTGCTTCCC3CMTTTGTCMGCGTTCGGCGTCATTTGATTCTCTCACTCTCAGCTAAACGG3TTGAGTATTCTTTCTTTTCCCCGATCCCCATC GCTCTCTCACTTCCACCACACGAGCAGTTAGGAAlrGATGACGATCTCCTCMCCTCGAGCTCTACATCATC~GACATCCTCTGMMCCCCGACTTGAAAGGTTTCTATCGTCGTCAT CACCGGCATCCTCGCGATCCTCGTCGGCTACCAC~CCAGAGCCTTCGAGTCCGACCATCTTGGCGGC~GACCATCGAGTCGATCGATCCCTTTCAGCCTCGCMGGACTACMCGAGA ATGTCCTACCCGCCATCCCAGCCACGGMGGCGGTGCMCCATTGACTAGTGACGCCMCACTCCGGATCACTGGATCGCTCGAGATGAGCGCATGATCCGTCTCACTGGCAAACATCCC S Q P R K AV Q P L T? DAN T P D HW I AR DE R M I R L T Y P P M S TTCAACAGCGMGCACCTCTCTCAGAGCTCTTTTCCAAACGGTTTCTCACTCCTCAGMCCTCTTTTACGTACGTTCGCAGGGTGACACGCCGCGGGTTACTCGTGAGCMGCTG~C G D T PRVTREQAEN FYVRSQ K G F L T P Q N L E L F S FNSEAPLS TGGMGCTCAAGGTTCATGGTCTCGTAGAGCMGAGGTTGA~TCAGTATCMGGATCTCMGGAAAAGTTTTCCTACAGTCACCCCCAAACCATCACCCTCGTATGTGCCGGTMCAGA TLVCAGNR V E L S IKDLKEKFSYSHPQTI WKLKVHGLVEQZ
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CGAAAAGAGCAAAACATGGTCGCTAAAGGCCTCGOTTTCMCTGGGGCGCCGCOOGTGTCTCGACGGGTCTATTTACCGGAGTCTACCTCGCCGACATTCTCGATTACTGCMGCCCMG LDYCKPK TGLFTGVYLADI KGLGFNWGAAGVS RKEQNMVA MTCCACTTCTCTCTTCTTTCCCTTCATACGATGTGGCTGTGCTGGATCOGGCGAGACATGTCGTCTTTGAGGGCGCCGACGAGCTGCCTMGOGAAAGTACGGCACTTCGCAGCGACTC KYGTSQRL EGADELPKG SYDVAVLDRARHVVF NPLLSSFP MCTGGGCGCTCGACAGGTGCMGGGCATGCTGATCGCTTGGGGTCTCMTffiTGMGACCTCTCTCCGGATCATGOCTACCCTTTGCGTCTCGTAGTGCCAGGTCAGATCAGTG~AGA LRLVVPGQ DLSPDHGYP KGMLIAWGLNGE NWALDRC ATGGTCMGTGGCTCGAGCGCATCGMGTTTCGGATCOCGAGAGCCAGCATCATCTGCACTTTCACGACMCCAGGTGCTTCCCACCGMGTGACCGCCGATCAGGCCAGMGCG~TG TEVTADQARSEM DNQVLP EVS3RE SQHHLHFH MVKWLERI CACTGGTGGTACGATCCAAAGTACATTATCMCG~TCTCMCGTCMTTCTGCCATCTGCTCGCCTGATCATGATCAGGTCGTCATGTTGCCGMCCATCTACGTCCAGTCGAGATGCTC VVMLPNHLRP INDLNVNSAICS P D B D Q HWWYDPKYI CCTCTTGMGGCTATGCGTACACGGGCGOTGGCCGTGTATCCACCAGAGTCGAAATCTCACTCGACGATGGTCGTAGCTGGMGTGTGCCTCGATTCACTACCCTGMGATCTGTACCGC PEDLYR SWKCASIHY T R V E ISLDDGR EGYAYTGGGRVS P L ATGTATCCGATCCAGCGGCACGAGTACTTTGGCACGCTGGATCTCAGCGCCACCGAAATGAGCTTCTCCTGGTGCTTCTGGCGTCTCGATGTCGATGTCGMGCGGACATCATCGCCMG FWRLDVDVEADI F S W C LDLSATEMS GHEYFGT M Y P I Q GATGTCMGGTGATCTCGGTTCGAGCACTGGACGMGTTCTGGCGACTCAGCCCCGCGACATGTACTffiMCGCCACTTCGATGATG4ACTCATGGTGOTTCCGTGTGTGCATCGCGMG YWNATSMMNSWWFRVC DEVLATQPRDM S V R A L D V K V I GCGAGAACGGCMCCMGATCCGCTTTGAGCATCCGACCTCGCCGCMTGCCCTGGCGGCTGGATGCAGAGGATGMCGAGGCTGGTCTCMCCCTCGCTATCCTCAGTTTGTCGMGCC PGGWMQRMNEAGLNPRYPQFVEA IRFEHP T S P Q C A R T A T K AAGGCCGTCGAGTCTTGCAAAACGGATGCGMCACACTACCGCGGCCMGGMGCCMGTGGGTCCCCGAAAGCGATCATGATCGACCCATCC~GCTGATACCATMTCACGGCGCCG SKADTI R P R K P SGSPKAIMIDP KTDANTLP KAVESC ATCTTGGCTGCTCACGGTGATGGCGAGGGTCCTGAGCCGTGGTTTGTCGTCCATGGTCATCTGTATGACGGGACTGGCTTCTTGAAAGACCACCCCGGAGGAGATCMTCGATCGTCTTG G G D Q DGTGFLKDHP PWFVVHGHLY E G P E ILAAHGDG TCGCCGGGAGMGACGACGCTACCGMGATTTCATGGCMTCCACTCGATGGACGCCAAAAAGATGCTTCGAGATTTCCACCTCGGTAGACTCGAGAAACMGATGCAGCGCCTCCTGCA DAAPPA HLGRLEKQ IHSMDAKKMLRDF DDATEDFMA S P G E GCCACGGAGCGGGMGTCTTGGATCTCAGCMGCCGTTCCTCGATCCCAAGAAATGGCGAGCTACACGACTCGGTGAGCMGCAAAT~ACTCACCAGATGCGAGGATCTTCCGCTTTGCT PDARIFRFA 0 E Q A N H S FLDPKKWRATRL D L S K P ATEREVL CTAGGMGCGMGATCAAGAGCTTGGGCTTCCGT3GCCAGCAGCTCTGCGTCTGTCGCTTGAAAAACGCAGMCAGGCGMGCAGAGATGGTACAAAGAGCTTACACACCATACAGTffiT RAYTPYSG KRRTGEAEMVQ B P A A L R L S L E E L G L P LGSEDQ MCACACAGCGTGGCTTCTTGGACATCTTGATCAAGGTCTACTTCCCGAGCGATGCAGCTGCTACGTCGGCACCCGCATTCGMGGA~30AAAGATGACGATGCTTCTCGAAAAGATCGAT PAFEGGKMTMLL KVYFPS DAAATSA NTQRGFLDILI GTGTCTTCGCCCTCAGACGATCTGACGATCGMCTCMG~ACCTCTTGGATCCTTCACCTATCTTGGTCA~AGCAGATCCGCT~~CCCGCCA~~GT~TCGCTTCGCMGCTC S IRWKPASAC IELKGPLGSFTYLGQQQ PSDDLT v s s
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GCCATGATCGCCGGTGGATCGGGCATCACGCCCATCTGOTCGACCCTCAAAGCCATCGCCGACGMGTGCTCGATGCCTCGMTCCTTCGAGCCCTGCTTTAGACCCCATCCAGATCTGG PALDPIQIW S N P S S IADEVLDA I W ‘S T L K A G I T P AMIAGGS ATCGTGTACGGTMCCGCACGGMCAGGATATCCTGATCCGCGMGAGCTAGACGCATTGAGTAGGTTGAAAGGCMTCTTAAAGTCTGGCATGTGCTCAGCMTTGCACTCCTGAAAAC TEQDILIREELDALSRLKGNLKVWHVLSNCTPEN IVYGNR GMGCCAACTGGAGTATGGGCCGCGGCATATCTCTGCCMTGTCCTTAGGAGCACCTGCCACCMCCACCGGCAAAGCTGCTTCGGAMAGACGAGCTCGAGGATACTTTGGCGCTGGTC DTLALV FGGDELE PMSLGAPATNHRQSC EANWSMGRGISL TGCGGTCCCCCTCCGATGGAAAAAGCTGTCTCGGACGGTCTCMCGAGTTGGGCTGGOACCTGCMCGTTGCGTCGTGTTTTTCTGATTTCATACGTTTGTTCTCGTCATTATTTGCMT F 908 D G L N E LGWDLQRCVVF PMEKAVS C G P P CTTACCTMCCTAACCTACTTACTCGTGACTGTAAGTTATGGCGCTGACTCGATTGTCGAGATAGGATCC 2830
-481 -361 -241 -121 -1 120 40 240 80 360 120 480 160 600 200 720 240 840 280 960 320 1080 360 1200 400 1320 440 1440 480 1560 520 1680 560 1800 600 1920 640 2040 680 2160 720 2200 760 2400 800 2520 840 2640 860 2760
Fig. 1. Restriction map, nt and derived aa sequence of the narl gene. (A) Restriction map of the narl locus in plasmid pMH3055-solid line, sequenced DNA; broken line, DNA cloned and mapped but not sequenced; thick line, narl ORF. Plasmid pMH3055 comprises the 6.7-kb EcoRI-EcoRI DNA fragment from pMH305 1 cloned into pBluescript KS +. Plasmid pMH3051 was isolated from the primary transformant. Restriction enzyme abbreviations are B, E,g[II; H, BarnHI; P, PstI; R, EcoRI; S, SacI; X, XbaI. Distances shown below the map are in kb. (B) The nt and aa sequences of the ORF (EMBL accession no. X67687). The aa are aligned with the second nt of each codon. Methods: The 4.5-kb EcoRI-XbaI fragment from pMH3051 was subcloned into pBluescript KS + to give plasmid pMH3057. The EcoRI site is derived from the pCM54 polylinker present in pMH305 1 (Tsukuda et al., 1988). U. maydis DNA in pMH3055 and pMH3057 Biochemicals. Cleveland, OH, USA). Oligodeoxynucleotide
and subclones derived primers were employed
complemented both the narl-1 and nirl-1 mutations, pMH3051, pMH3053, pMH3055 plasmids and pMH3057 complemented the narl-1 but not the Cl-1 mutation, whereas plasmid pMH3061 complemented the nirl-1 but not the nurl-1 mutation. This pattern of complementation was also found for the nurl-6 allele and the nirl-1 allele in different genetic backgrounds. The results suggest that primary plasmid pMH3051 from the library carries the nurl gene, pMH3061 the nirl gene and cosmid H7 both genes. This suggestion was confirmed by hybridisation analysis of plasmid, cosmid and chromosomal DNAs. Plasmid pMH3051, pMH3061, H7 cosmid and wt chromosomal DNAs were digested with PstI and Sac1
from them was sequenced on both to sequence gaps in the subclones.
strands
(Sequenase
kit, US
(shown in Fig. 2) and with XbaI, PuuI and Sal1 (G.R.B., unpublished) and the resulting blot probed with a 1.14kb BamHI-BglII fragment from within the nurl ORF. As predicted from the restriction map of the nurl locus (Fig. lA), the probe hybridised to a 2.4-kb PstI and to a 2.3-kb Sac1 fragment respectively, common to pMH305 1, cosmid H7 and chromosomal DNAs. In addition, there was a 7.2-kb PstI fragment in pMH3051, replaced by fragments of equal mobility but > 12 kb in H7 and chromosomal DNAs (Fig. 2A and 2B). These results are consistent with the structure of the nur-I locus cloned into pMH3051. No hybridisation was found to pMH3061 restriction fragments (Fig. 2A, lane 2 and Fig 2B, lane 2).
72
kb
1.0,
0.5,
1 Fig. 3. Analysis of cloned
2
3
4
narf and nirl gene-containing
constructs
by Southern
hybridisation.
(A) DNAs digested
by Pstl and probed
uith
a I.14
kb &III-BamHI fragment within the narf ORF (Fig. 1A). (B) DNAs digested by Sac1 and probed by the same DNA fragment. (C) DNAs digested by Sac1 and probed with a 5.2-kb EcoRI fragment from the nirl locus. Lanes: (1) Plasmid pMH3051 DNA containing the narl gene. (2) Plasmid pMH3061 containing the nirl gene. (3) Cosmid H7 containg both narl and nirl genes. (4) Chromosomal DNA from the U. maydis wt strain 521 (Holliday. 1974). Chromosomal DNA was extracted as described by Holden et al. (1989). All DNA was digested with the indicated restriction endonucleases and electrophoresed in 0.8% agarose. The gels were stained with ethidium bromide to ensure that equal amounts of plasmid DNAs were transferred Kit, Amersham
to nitrocellulose (Hybond C-extra, Amersham International). International) with [a-s2P]dTTP (NEN Research Products).
I-kb DNA ladder (GIBCO-BRL) being shown on the left margin.
was used as the DNA size markers.
The probes were labelled by the random primer method (Multiprime Hybridisation was essentially as described (Sambrook at al., 1989). A
The positions
Although the nirl complementing DNA in pMH3061 has not been characterised in detail, we used a 5.2-kb EcoRI fragment necessary for genetic complementation of the nirl-1 mutation (P.A.S., unpublished) to probe the digests of the DNAs just described. This probe hybridised to a Sac1 fragment of 3.9 kb common to pMH3061, cosmid H7 and chromosomal DNAs, and to two fragments of 1.6 and 1.4 kb common to H7 and chromosomal DNAs (Fig. 2C). No hybridisation to pMH3051 sequences was detected (Fig. 2C, lane 1). Similar results were obtained for hybridisation of this probe to PuuI and Sal1 fragments of these DNAs (G.R.B., unpublished). These results confirm the complementation data and show that pMH3051 and 3061, each contain gene sequences common to cosmid H7 and to chromosomal DNAs, but not to each other. The results do not prove that the narl and nirl genes are closely linked on the chromosome because it is possible that two fragments of different chromosomal origin were cloned together in the same cosmid molecule, rather than one continuous sequence containing both genes which are adjacent on the chromosome. However it is more likely that there is close linkage because it has been demonstrated genetically (Holliday. 1971). Close
of some of these are indicated
by the arrowheads,
their sizes (kb)
linkage was found for the A. nidulans niiA and niaD genes which are separated by 1272 bp, and from which transcription of the two genes occurs divergently (Tomsett and Cove, 1979; Johnstone et al., 1990). In N. crassa, however, the two analogous genes are on different chromosomes (Tomsett and Garrett, 1980). In a previous publication, we reported the isolation by differential hybridisation of U. maydis DNA sequences in plasmid pMH3007, the transcript of which was induced by nitrate ions (Smith et al., 1988). This transcript hybridised at low stringency to the A. nidulans niaD nitrate reductase gene. We have now attempted to hybridise sequences from the narl gene isolated in the present work to restriction digests of pMH3001 and vice versa, without success (G.R.B., unpublished). Thus although the transcript encoded by the U. maydis DNA in pMH3007 is induced by growth of cells in the presence of nitrate ions, this transcript clearly does not originate from the narf gene. (b) Disruption of the narl gene To confirm that the complementing DNA was the narl gene itself, a one step disruption of a chromosomal narl
73
gene was carried out. The 1.33-kb BglII fragment within the ORF of the cloned DNA (Fig. 1A) was replaced in pMH3057 by a 2.8kb BumHI-BglI fragment able to express a bacterial HyR gene from the U. maydis urns2 promoter sequence (Wang et al., 1988). Plasmid pMH3040 containing this cassette resulted and was cleaved with SpeI+BamHI to give a linear DNA molecule with the urns2 gene cassette flanked by the narl sequences on either side of the disruption (Fig. 3A). This DNA was transformed into competent wt U. maydis cells and HyR transformants then selected. Transformants carrying disruptions of the chromosomal narl gene resulting from homologous recombination should be unable to grow on medium containing nitrate as the sole nitrogen source, but able to do so on ammonium salts. Ectopic integration of the transforming DNA at sites other than the narl gene in the genome should generate transformants able to grow on both salts by virtue of possessing an intact as well as disrupted narl gene. Ten HyR transformants were transferred to CM + Hy, NM +Hy and NM plates and five failed to grow on the NM media. Furthermore, although these five transformants were unable to grow on NM medium containing nitrate, they were able to do so on NM media containing nitrite or ammonium ions as expected for potential disruptants of a chromosomal narl gene. DNA was extracted from both classes of transformants, digested by PstI or by BglII, electrophoresed and transferred for hybridisation analysis with labelled pMH3057 as the probe. This probe should hybridise to a wt PstI fragment of 2.12 kb and to wt BgZII fragments of 1.32 and 2.82 kb, in addition to junction fragments (Fig. 3A). Disruption of the narl locus should result in the loss of the PstI fragment and gain of one of 1.53 kb; the BglII fragments should be replaced by one of 5.6 kb (Fig. 3A). Ectopic transformants should retain the wt fragments but gain others because of integration of transforming DNA elsewhere in the genome resulting in the generation of novel restriction fragments. Representative hybridisation analyses are shown in Fig. 3B, and in addition, that for a transformant in which tandom duplications of transforming DNA had disrupted the narl gene (Fig. 3B, panels a and b, lane 3), a situation which is not uncommon with other genes in U. maydis (Banks et al., 1992). These results are in agreement with the expectations described above and show that the narl gene was disrupted in all transformants which grew on nitrite and ammonium, but not on nitrate ions; all transformants which grew on all three nitrogen sources had retained wt restriction fragments and had suffered ectopic integration. The results confirm the no-2 identity of the cloned ORF, and also that the narl function is dispensable for growth of cells in the presence of nitrite or ammonium ions.
(c) Coding sequence of the narl gene
The nt sequence contains an ORF of 2724 nt, able to encode a protein of 908 aa (Fig. 1B). Similar to aa sequences of other U. maydis proteins, there is no marked codon useage bias. All codons are used, the codons of 309/721 aa terminate in C where possible and side by side GC pairs are not avoided, 128/376 codons possessing them. However, third position A’s are avoided, only 1441674 codons ending in A. These properties are characteristic of filamentous fungi (Ballance, 1986; Spanos et al., 1992). Although the evidence is not conclusive, the gene does not appear to contain introns-the proportion of U. maydis genes containing them has yet to be established. However, the genomic nt sequence contains none of the consensus intron signals found in filamentous fungi (Ballance, 1986). Nitrate reductases are functionally complex enzymes with multiple redox centers and cofactors, a situation reflected in the strong domain structure of their polypeptides which is conserved from fungi to plants (Kinghorn and Campbell, 1989; Campbell and Kinghorn, 1990; Caboche and Rouze, 1990). The aa sequences of these domains also bear homologies to those in proteins with different functions, but with similar cofactor requirements. Domains in nitrate reductases have been identified which define the cofactor binding sites for molybdenumpterin, iron-heme and FAD (Crawford et al., 1988; Campbell and Kinghorn, 1990). This conservation of domain structure is maintained in the U. maydis nitrate reductase apoprotein (Fig. 4). In particular, aa sequences 33-47 and 203-231 are very similar to those of domains which define the molybdenum-pterin binding site in other nitrate reductases and in mammalian sulfite oxidase (Kinghorn and Campbell, 1989; Campbell and Kinghorn, 1990). In the 43 aa shown (Fig. 4), 27 are identical to those in N. tabacum and in A. nidulans nitrate reductases. The sequence of narl from aa 531-591 is very similar to the iron-heme binding domains identified in other nitrate reductases and in mammalian cytochrome b5 and yeast flavo-cytochrome b2 (Kinghorn and Campbell,1 989). In this 61 nt sequence of the narl gene encoded protein, there are 36 aa identical with those in A. nidulans and 35 in N. tabacum nitrate reductases, and 26 in mammalian cytochrome b, (Fig. 4). The FAD-binding domain, homologous with mammalian NADH:cytochrome b5 reductase, has been localised to the C-terminal region of nitrate reductase (Kinghorn and Campbell, 1989; Campbell and Kinghorn, 1990). The sequence aa 6 13-908 of the narl encoded protein has marked similarities with these domains (Fig. 4). In particular, the sequence FRFALP, which is conserved in plant nitrate reductases and mammalian cytochrome b,, appears as FTLSLE in
74
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P
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4
34
Fig. 3. Disruption of the narf gene. (A) Structures of the gene and its disruptant. Single line, rlurl locus; tilled bar. rlur/ ORk; open bar, IUU.\? sequences; hatched bar, HyR gene. Restriction endonuclease abbreviations as for Fig. I; fragment sizes are in bp. A 1325.bp BglII fragment (a) in pMH3057 was replaced by a 2800-kb &/II-BamHI fragment (c) containing the HyR cassette. This cassette fragment originated as the 2.8-kb XhoIHind111 fragment from pHLl (Wang et al., 1988) and was then recloned into the vector pIC19RHL (J. Kronstad, unpublished; Marsh et al., 1984). The structure of the plasmid resulting from the replacement, pMH3040 and (b+c), was verified by restriction analysis. Plasmid pMH3057 comprises a 4.5-kb EcoRI-XhaI fragment, (a+ b), derived from pMH305I and ligated into pBluescript KS+. Note that the EcoRI site is derived from the pCM54 polylinker (MCS; Tsukuda et al., 1988). Plasmid pMH3040 was digested with SpeI and BamHI to give linear DNA of 4.52-kb incorporating the HyR cassette surrounded by narl sequences and then transformed into competent cells of wt 521 strain of U. maydis, essentially as described by Wang et al. (1988). Transformants were selected in the presence of 200 pg Hy/ml (Boehringer, Mannheim). Ten colonies were transferred to CM and NM agar media containing Hy, and also to NM agar. All ten grew on the CM +Hy medium, and the same five failed to grow on both of the NM media. (B) Hybridisation analysis of DNA from transformants carrying disruptions of narl. Chromosomal DNA was digested by PstI (a) or by &III (b),electrophoresed in 0.8% agarose, transferred to Hybond C-extra nitrocellulose membrane (Amersham International) before Southern hybridisation analysis with 32P-labelled pMH3057, essentially as described by Sambrook et al. (1989). Lane (1) wt 521 DNA: (2) example of a disruption of the narl gene; (3) transformant generated by ectopic integration of transforming DNA randomly in the genome; (4) disruption of narl gene by tandom copies of transforming at the left margin.
DNA. A I-kb DNA ladder
(Gibco-BRL)
was the size marker.
A. nidulans (Kinghorn and Campbell, 1989) and FRFALG in the U. may&s nitrate reductase (Fig. 4). Therefore, not only does the U. maydis narl encoded protein maintain the sequence conservation of other nitrate reductases, but it also maintains the same relative locations of the functional cofactor domains along the polypeptide.
Arrowheads
identify
some markers,
with their sizes (kb) shown
(d) Transcription of the narl gene 503 nt of S-untranslated DNA was sequenced (Fig 1B). Although the tsp has not yet been determined, this upstream region does contain four S-CTTT and eleven 5’-CATC motifs, both of which may be associated with the tsp of some U. maydis genes (Spanos et al., 1992). In addition, the sequences 5’-TCTCTCACT (ST at - 209
L’.tnuydlJ
33
“;.1
A.nidulans
67
249
N. tabacum
103
288
sulphite oxidase
113
299
Umaydis
531
A.nidulans
528
N.tabacum
547
606
domain
26
86
Umnydis
613
677
A.nidulans
612
676
N.lbaCUm
643
703
Ham
Hmpms
cyt b,
Umaydis
678
A.nidulans
677
N.tabacum
704
H.sapiens cyt b,
95
35
96
Umaydis
743
A.nidulans
730
N.:abacum
752
H.sapiem cyt b, 152
YsGNTQRGFLDItIXWFPSDAAATSAPAFEf~~~KIDVS~PSDDLTXELf((lP~~S~~L III I III I I I I I PSIPaQ@&TA%DTLPLG@J ____~$%CK!?@XJRFEYL ISPS~L6MVD1LXXIYAET~ Ill I III I I I I I LDVK'X'LBHIEYQ TSTIDEVGYFELW~IYFKGIHPKFPN~~GQMSQY~~DSMPLG~_ III I III I I I I I X$FRQPSGLLWQ IgSDDDK@!VDLV~~DTHPKFPA_!%~SQYtESMQIGDT_____
742
_cQQQrR~PASAC_SLR~~~~~~~IWST~~AD~L~SNPSSPALDPIQIWI~ I II II IIII I I I VKSFVMfCQCTCtISPVFQVtRr4VMQDEQDETKCVMLD DRGRVLISGKERFI II II Ill1 I I I GK@NFLVHGEtQKF_AK%%i~~T@X~PVYQVMQ~~LKDPEODTYWYA I II II III1 I I I GK#FAIRPDXXSNPIIRT"KSVGUS;RqET&'~~$‘,?MLQVIR&~MKDP~HTVCHLLFA
803
751 151
773 810 208
Umaydis
804
867
A.nidulam
180
835
N.tabacum
811
863
H.sapiens cyt bS 2 0 9 lhaydis
868
A.nidulans
836
N.tabacum
864
H.sapiens cyt b, 2 6 1
I-lg. 4. The aa sequcncc in all sequences shown;
729
260
~DEL~D~~VC~PPP~_~VS~~~~L_Q~~F I I I III1 I I _~TPDRE_~LVCGPEA?@KJSKKI~LS@KE_ENLHY-% I I I IIII I I _IPEPSHTX'@rjACGPPP~IQFhVNPN~EKNGYDIKDSLL@?_ I III I _LPPPEEEp~vLMCGPBP~IQY~cLPN~DHV~HPT_~d~_
908 873 904 300
comparrsons of the rrurl gene encoded protcm. The aa positrons of the regrona shown arc md~cated; vertical IIIU, aa nknt~cal shading, aa identical in two or three sequences. The sequences of the proteins compared with the U. maydis narl sequences
are the A. nidulans niaD (Johnstone et al., 1990) gene; the N. tahacum nitrate reductase (Calza et al.. 1987); mammalian sulfite oxidase (Crawford et al., 1988); rat liver cytochrome b, reductase heme domain (Calza et al., 1987); human NADH-cytochrome b, reductase (Yabisui et al.. 1984). Other data are from Kinghorn and Campbell (1989). The sequences were compared using the Molecular and Genetics Sequencing (MGS) suite of programs developed by W. Greer and P. Gillett, National Institute for Medical Research. London, and refined by eye.
and - 266) and S-ATCCTCG (5-A at - 39 1 and - 400) are each repeated directly. The sequence 5’CAAAGACATC (5’-C at -334) has a repeat, but modified by the insertion of C between nt 7 and 8 (5’-C at -450). The sequence 5’-ATCGAGTCGAT (5’-A at nt -46) has a potential to form a hair-pin structure, albeit with only a 5-bp stem and one nt loop. Adjacent to this potential structure, the sequence 5’-AGACCATCGAGTCGATCGATCCCTTTCA (5’-A at - 5 1) has several motifs in common the the U. maydis reel sequence 5’-
AGACCATCACATGCTTTCT (5-A at - 151; Holden et al., 1989) and glyceraldehyde-3-phosphate dehydrogenase gene sequence 5’-CCACCATCGAATCTTTCT (5’-C at -52; Smith and Leong, 1990). 106 nt of 3’untranslated DNA have also been sequenced (Fig. 1B). As reported for the U. maydis pyr3 gene, no defined sequence elements can be discerned (Spanos et al., 1992). Induction by nitrate and repression by ammonium ions appear to be a common feature of assimilatory nitrate reductase transcripts (Dunn-Coleman et al., 1984).
76 Nitrate reductase enzyme activity in U. maydis is markedly increased when cells are grown in media containing nitrate ions as sole nitrogen source (Fincham and Lewis, 1970b). We therefore used a fragment of the cloned narl gene to determine by Northern hybridisation analysis if this increase is transcriptional in nature (Fig. 5). Aliquots of a culture growing in CM were switched to NM media containing nitrate or ammonium plus nitrate ions, or maintained in CM. The cells were grown for 2 h, a time
A
kb 5-o4,03*02.0-
l.O-
o-5-
B
Fig. 5. The nature
of the narl
transcript
and its induction
by nitrate
ions. (A) Northern blot probed with the 32P-labelled 1.14-kb BglIIBamHI narl DNA fragment (Fig. IA). (B) The same blot probed with the urns2 gene in plasmid pHLI. Only the 2.2-kb urns2 transcript is shown (Holden et al., 1989). Lane (1) 20 ug total RNA from cells grown in CM. (2) 20 ug total RNA from cells grown in NM. (3) 60 ug total RNA from cells grown in NM containing ammonium sulfate. Methods: 30 ml aliquots of an overnight culture of U. maydis 521 at 10’ cells/ml in CM medium were harvested by sterile filtration. Cells from one aliquot were briefly washed with pre-warmed CM and then resuspended in 30 ml CM at 30°C. Cells from a second aliquot were washed in prewarmed NM and suspended in 30 ml NM, which contains 0.3% potassium nitrate. A third aliquot was washed in pre-warmed NM containing 0.3% w/v ammonium sulfate and suspended in 30 ml of the same medium. The cultures were vigorously shaken at 30°C for 2 h and then rapidly cooled before centrifugation. Total RNA was extracted from the cells, quantitated by spectrophotometry and its integrity verified by gel electrophoresis (Fonzi and Sypherd, 1985). RNA samples were glyoxylated and electrophoresed in 1% agarose (McMaster and Carmichael, 1977) and transferred to GeneScreen (NEN Research Products) for Northern hybridisation analysis. The filter was probed with 32P-labelled 1.14-kb BgIII-BamHI narl fragment under conditions recommended by the filter manufacturer. After autoradiography, the filter was then stripped by holding in 96% formamide, 10 mM EDTA and TrisHCl pH 8 at 60’C for 30 min. It was then hybridised to 32Plabelled pHLl DNA to detect the urns2 transcript (Holden et al.. 1989).
shown to produce maximum induction of nitrate reductase enzyme activity (Fincham and Lewis, 1970a). Total RNA was then extracted from the cells and nitrate reductase transcripts analysed by Northern hybridisation, using a BglII-BumHI 1.14-kb fragment contained within the narl ORF (Fig. 1A). A major transcript of 3.0 kb was detected (Fig. 5A), consistent with an ORF of 2724 nt. In cells grown for 2 h in the presence of nitrate ions, densitometry of the autoradiograms revealed that the abundance of this transcript was 90-fold greater than in cells grown in the presence of ammonium plus nitrate ions or in CM. The hybridisation filter was stripped of the narl probe and then probed with the urns2 gene (Fig. 5B), which encodes a U. maydis stress/heat shock protein, in order to provide an RNA loading control (Holden at al., 1989). It was apparent that transcription of this gene, or the stability of its transcript, was altered by the media changes although not apparently by the nature of the source of the nitrogen available to cells. Thus cells growing in CM contained about a fivefold increase in steady state levels of urns2 transcript over cells transferred to NM media containing either nitrate or ammonium plus nitrate ions. We have repeated these experiments four times, each time with identical results showing that when equal amounts of total RNA are analysed, cells grown in NM medium containing nitrate ions contained the very high levels of the narl transcript compared to cells growing in the presence of ammonium plus nitrate ions or in CM medium. Conversely, steady state levels of the urns2 transcript are higher in cells grown in CM than in the two NM-based media. It is clear, however, that the U.maydis narl gene transcript is induced very significantly by nitrate, and repressed by ammonium ions. (e) Conclusions (I) The U. maydis narl and nirl genes, encoding nitrate and nitrite reductases respectively, have been isolated separately on plasmid vectors (plasmids pMH3051 and 3061 respectively) and together on a cosmid (cosmid H7). Because these genes have been shown to be linked by genetical methods, we propose that they are molecularly linked such that a single segment of DNA containing them was cloned into the cosmid H7. (2) Disruption of a chromosomal copy of the narl gene by homologous recombination with a disrupted copy of the cloned sequence which complements the narl-I mutation, yielded transformants with the expected phenotypes and chromosomal DNA restriction patterns. The narl gene function is dispensable when cells are grown in the presence of nitrite or ammonium ions. (3) The ORF encodes a 90%aa polypeptide. Its sequence maintains the sequence conservation found in fungal and plant nitrate reductases. This is particularly
77 true for those sequences which define the molybdenumpterin, iron-heme and FAD-binding domains, cofactors for the electron transfer reactions. The relative localisation of these domains along the polypeptide is also conserved. (4) The 2724-nt ORF encodes a transcript of 3.0 kb. It is induced some 90-fold when nitrate ions become the only source of nitrogen for cells. When ammonium ions are also present, transcription is repressed.
domain
structure
and amino acid homologies
in higher plants. Gene
85 (1989) 371-380. Dunn-Coleman, ilation Fonzi,
N.S., Smarrelli
in eukaryotic
Jr.. J. and Garrett,
W.A. and Sypherd,
P.S.: Expression
decarboxylase of Saccharomyces Cell. Biol. 5 (1985) 161-166. Gruber.
R.H.: Nitrate
H.. Goetinck,
of the gene for ornithine
cereoisiae
S.D., Kirk,
in Escherichin
D.L. and Schmitt.
D.W., Kronstad,
regulated hsp70 192771934.
J.W. and Leong.
gene
of Uslilago
S.A.: Mutation
ntuJdis.
coli. Mol.
R.: The nitrate
reductase-encoding gene of Voloou curter? map location, and induction kinetics. Gene I20 (1992) 75-83. Holden,
assim-
cells. Int. Rev. Cytol. 92 (1984) l-50.
EMBO
sequence in a heat-
J. 8 (1989)
Holden, D.W.. Spanos, A. and Banks. G.R.: Nucleotide sequence of the RECI gene of Usrilago maydis. Nucleic Acids Res. I7 (1989) 10489.
ACKNOWLEDGEMENTS
We are indebted to Steve Sedgwick for critical reading of the manuscript and for his computer graphics wizardry; to Jim Kronstad for the urns2 gene on pIC19RL; to Tony Johnson for Molecular Dynamics ImageQuantTM densitometry and to Jim Kinghorn for information before publication.
Holliday, R.: Biochemical measure radiation-induced recombination
of the time and the frequency of in U.stilclgSo.Nature New Biol. 232
(1971) 233-236. Holliday, Johnstone,
maydis.
R.: Ustilugo
Genetics.
Vol. I., Plenum, I.L.. McCabe,
In: King.
R.C. (Ed.),
Handbook
of
New York, 1974. pp. 5755595.
P.C., Greaves.
P.. Gurr. S.J.. Cole, G.E.. Brow,
M.A.D.. Unkles. SE.. Clutterbuck, A.J.. Kinghorn, J.R. and Innis. M.A.: Isolation and characterisation of the cmA-niiA-nicrD gene cluster for nitrate (1990) 181-192.
in Aspergillus
assimilation
niduluns. Gene
90
Kinghorn, J.R. and Campbell, E.I.: Amino acid sequence relationships between bacterial. fungal and plant nitrate reductase and nitrite REFERENCES Akins. R.A. and Lambowitz, A.M.: General method Neurospora crassa nuclear genes by complementation Mol. Cell. Biol. 5 (1985) 227222278. Arst
for cloning of mutants.
Jr., H.N. and Cove, D.J.: Nitrogen metabolite repression Aspergillus niduluns. Mol. Gen. Genet. 126 (1973) I I ll141.
Ballance, D.J.:Sequences important fungi. Yeast 2 ( 1986) 2299236.
for gene expression
in
in filamentous
Banks, G.R.. and Taylor, S.Y.: Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation. Mol. Cell. Biol. 8 (1988) 5417-5424. Banks, G.R.. Kanuga, influence of the chromosome
N., Ealing. J., Spanos, A. and Holden, D.W.:The Ustilago maydis RECI gene on plasmidrecombination. Curr. Genet. 22 (1992) 4833489.
Brownlee. A.G. and Arst Jr., H.N.: Nitrate uptake in Aspergillus and involvement of the third gene of the nitrate assimilation J. Bact. II5 (1983) 1138-1146.
nidulans cluster.
Caboche, M. and Rouze, P.: Nitrate reductase; a target for molecular and cellular studies in higher plants. Trends Genet. 6 (1990) 1877192. Calza. R.. Hutter, E.. Vincentre, M., RouzC, P. Galangue, F.. Vaucheret, H., Chevel. I., Mezer. C.. Kronenberger. J. and Caboche, M.: Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductase from higher plants. Mol. Gen. Genet. 209 (1987) 5522562. Campbell, W.H. and Kinghorn, J.R.: Functional tory nitrate reductases and nitrite reductases. I5 (1990) 315-319.
domains of assimilaTrends Biochem. Sci.
Crawford, N., Campbell, W. and Davis, R.W.: Nitrate reductase from squash: cDNA cloning and nitrate regulation. Proc. Natl. Acad. Sci. USA 83 (1986) 8073-8076. Crawford, N.M.. Smith, M., Bellissimo, D. and Davis. R.W.: Sequence and nitrate regulation of the Arabidopsis thuliana mRNA encoding nitrate reductase. a metalloflavoprotein with three functional domains. Proc. Natl. Acad. Sci. USA 85 (1988) 500665012. Cove, D.J.: Genetic studies of nitrate Biol. Rev. 54 (1979) 291-327.
assimilation
in Aspeygillus nidulans
Daniel-Vedele, F.. Dorbe, M.-F., Caboche, M. and Rouze. P.: Cloning and analysis of the tomato nitrate reductase-encoding gene: protein
reductase Molecular University Lemontt.
proteins. In: Wray. and Genetic Aspects Press. Oxford, 1989.
J.: Induced
mutagenesis
J.L. and Kinghorn. J.R. (Eds) of Nitrate Assimilation. Oxford pp. 3855402.
in Ustilago muydis. I. Isolation
characterisation of a radiation-revertible for nitrate reductase. Mol. Gen. Genet.
allele of the structural 145 (1976) 1355138.
Lewis, C.M. and Fincham. J.R.S.: Genetics of nitrate Ustilugo muydis. Genet. Res. I6 ( 1970a) I5 I 163.
reductase
and gene in
Lewis. C.M. and Fincham, J.R.S.: Regulation of nitrate reductase in the basidiomycete U&ago maydis. J. Bact. 103 (1970b) 55-61. Marsh. J.L.. Erfle. M and Wykes, E.J.: The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32 (1984) 4X1-485. Marzluf, G.A.: Regulation of nitrogen metabolism and gene expression in fungi. Microbial. Rev. 45 (1981) 4377467. Marzluf, G.A. and Fu. Ying-Hui.: Genetics. regulation. and molecular studies of nitrate assimilation in Neurospora c’rasscc. In: Wray, J.L. and Kinghorn, J.R. (Eds) Molecular and Genetic Aspects of Nitrate Assimilation. Oxford University Press, Oxford. 1989, pp. 314-327. McMaster. G.K. and Carmichael, G.G.: Analysis of single and double stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74 (1977) 4835-4838. Sambrook, J.. Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. 1989. Scazzocchio, C. and Arst Jr.. H.N.: Regulation of nitrate assimilation in Aspergillus nidukzns. In: Wray. J.L. and Kinghorn, J.R. (Eds.) Molecular and Genetic Aspects of Nitrate Assimilation. Oxford University Press, Oxford, 1989. pp. 29993 13. Smith, T.M., Spanos, A. and Banks, G.R.: Differential expression of Usrilago maydis DNA sequences during induction of nitrate reductase enzyme activity. Curr. Genet. I4 (1988) 4577460. Smith, T.L. and Leong. S.A.: Isolation and characterization of a Usrilago mclydis glyceraldehyde-3-phosphate Gene93 (1990) 111-117.
dehydrogenase-encoding
gene.
Spanos, A., Kanuga. N.. Holden. D.W. and Banks, G.R.: The Usrilugo maydis pyr3 gene: sequence and transcriptional analysis. Gene I I7 (1992) 73-79.
78 Tomsett, A.B. and Garrett, R.H.: The isolation mutants defective in nitrate assimilation Genetics 95 (1980) 649-660. Tsukuda,
T.. Carleton,
Isolation sequence
and
S., Fotheringham.
characterization
from Us&go
of an
and characterization of in Neurospera crassa.
S. and
Holloman.
autonomously
W.K.:
replicating
maydis. Mol. Cell. Biol. 8 (1988) 3703-3709.
Unkles, S.E., Campbell, E.J., Punt, P.J., Hawker, Hawkins, A.R.. Van den Handel. C.A.M.J.J. The Aspergihs niger niaD gene encoding upstream nucleotide and amino acid sequence Ill (1992) 1499155.
K.L., Contreras, R.. and Kinghorn. J.R.: nitrate reductase: comparisons. Gene
Yabisui, T., Miyata, T., Iwanaga, S., Tamura. M., Yosheda. S., Takeshita, M. and Nakajima, H.: Amino acid sequence of NADH-cytochrome b, reductase of human erythrocytes. J. Biochem. 96 (1984) 579-582. Wang, J., Holden,
D.W. and Leong,
phytopathogenic fungus 85 (1988) 8655869.
U&ago
S.A.: Gene transfer
system for the
maydis. Proc. Nat]. Acad. Sci. USA
Wang, J., Budde, A.D. and Leong, S.A.: Analysis
of ferrichrome
biosyn-
thesis in the phytopathogenic fungus Usfilago maydis: cloning of an ornithine-N5-oxygenase gene. J. Bact. 17 I (1989) 28 I l-28 18. Wray, J.L. and Kinghorn, J.R.: Molecular and Genetic Aspects of Nitrate
Assimilation.
Oxford
University
Press, Oxford,
1989.
GENE
B.V. All rights
reserved.
69
0378-I 119/93/SO6.00
07244
The Ustilago maydis narl gene encoding nitrate reductase activity: sequence and transcriptional regulation (Recombinant DNA; basidiomycete fungus; gene disruption; inducible transcript)
G.R. Banks*, P.A. Shelton*, N. Kanuga*, D.W. Holden* and A. Spanos* Genetics Dioision, National Institutefor Medical Research, The Ridgeway, Mill Hill, London NW7 IAA. UK Received by J.R. Kinghorn:
10 December
1992; Revised/Accepted:
19 March/28
March
1993; Received at publishers:
6 May 1993
SUMMARY
The narl gene was cloned from Ustilago maydis and the 90%amino-acid (aa) sequence of the encoded protein found to have strong identities with other nitrate reductases from fungi and plants. This was especially so in three domains which define enzyme cofactor-binding sites. The gene was isolated alone and in association with the nirl gene, suggesting that the two genes are closely linked on the chromosome. The phenotype of a strain in which narl had been disrupted was consistent with the only role of narl being in nitrate reduction. Nitrate ions induced a 90-fold increase in narl transcript levels, while ammonium ions repressed transcript levels.
INTRODUCTION
The ability to assimilate nitrate ions as the sole nitrogen source for growth is possessed by many bacteria, fungi, algae and plants (reviewed in Dunn-Coleman et al., 1984; Wray and Kinghorn, 1989). Assimilation is effected Correspondence to: Dr. G.R. Banks at his present
address:
Laboratory
of Yeast Genetics, National Institute for Medical Research, Ridgeway, Mill Hill, London NW7 IAA. UK. Tel. (44-81)959-3666; (44-8 1)906-4477.
The Fax
*Present addresses: (G.R.B., N.K. and A.S.) Laboratory of Yeast Genetics, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Tel. (44-81)959-3666; (P.A.S) ICI/University Joint Laboratory, School of Biological Sciences, University of Leicester, Leicester LEI 7RH, UK. Tel. (44-81)533-523364; (D.W.H) Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Hammersmith Hospital, London W 12 ONN, UK. Tel. (44-8 1)740-3222.
Du Cane Road,
Abbreviations: A., Aspergillus; aa, amino acid(s); bp, base pair(s); CM, Ustilago complete medium; FAD, flavin adenine dinucleotide; Hy. hygromycin B; kb, kilobase or 1000 bp; N., Neurospora; NADH. reduced form of nicotinamide-adenine dinucleotide; narl, gene encoding nitrate reductase of U. maydis; nirl, gene encoding nitrite reductase of U. maydis; NM, U&ago nitrate minimal medium; nt, nucleotide(s); ORF, open reading frame; R, resistance/resistant: tsp. transcription start point(s); U.. Ustilago: wt, wild type.
by the reduction of nitrate to ammonium ions and involves a substantial investment in metabolic energy. It is not surprising that this pathway is tightly regulated in several ways. Studies with the filamentous fungi Aspergillus nidulans (Scazzocchio and Arst, 1989) and Neurospora crassa (Marzluf and Fu, 1989) in particular have made major contributions to our understanding of the regulation of nitrate metabolism. In outline, nitrate ions are transported into the cell by a permease, encoded by the crnA gene of A. nidulans (Brownlee and Arst, 1983) to be reduced to nitrite ions by nitrate reductase, the apoprotein of which is encoded by the niaD and nit-3 genes of A. nidulans and N. crassa, respectively. Nitrite ions are reduced to ammonium ions by nitrite reductase, the niiA and nit-6 genes of A. nidulans and N. crassa respectively encoding the apoprotein of this enzyme (reviewed in Cove, 1979; Scazzocchio and Arst, 1989; Marzluf and Fu, 1989). The pathway is regulated through at least two genetic systems. In the first, which is pathway specific, nitrate and nitrite reductase activities are transcriptionally induced by exogenous nitrate ions. The A. nidulans nirA and the N. crassa nit-415 genes are responsible for this regulation (Cove, 1979; Marzluf, 1981). In addition, the nitrate reductase gene product appears to
70 autoregulate
its own gene expression
of the nitrite
reductase
and regulate
(Cove, 1979). Nitrate
that
assimilation
to nirl and a one step disruption
linkage
is also regulated as part of a number of genes under the control of nitrogen metabolite repression. Expression of
apoenzymes
these
transcriptional
genes
is reduced
by ammonium
a process
by the areA and nit-2 genes of A. nidu-
which is mediated
lans and N. crassa, respectively and Arst,
ions,
(reviewed
1989; Arst and Cove,
1973; Marzluf
nitrate cloned
and
nitrite
revealing
and conservation peptides, studies
reductase
genetic
of the sequences
and confirming (Crawford
fungal
apoproteins
organisation,
organisms
inducibility
are presented
and the
of the narl gene by nitrate
ions.
and Fu, RESULTS
years, the genes of several
of other
in Scazzocchio
1989). In recent
of a chromo-
somal copy of the narl gene. Amino acid sequence similarities of the encoded protein to nitrate reductase
domain
have
been
structure
of the encoded
poly-
results from earlier biochemical
et al, 1986; Daniel-Vedele
AND DISCUSSION
and plant
et a1.,1989;
Johnstone et al., 1990; Gruber et al., 1992). Thus the A. nidulans niaD gene encodes an apoprotein of 97 kDa which exists as a homodimer, each monomer carrying the three cofactors FAD, haem, and molybdenum in the form of a complex with pterin. The electron flow in these redox prosthetic groups is NADPH+FAD+haem-+ molybdenum+nitrate. The domains of the nitrate reductase apoprotein which interact with the cofactors have been identified from the activities found in proteolytic products, and by comparison with the amino acid sequences of nitrate reductases from different organisms. In addition, aa identities between nitrate reductases and enzymes involved in similar electron transfers, but which have unrelated physiological functions, have been described (Crawford et al., 1988; Kinghorn and Campbell, 1989; Campbell and Kinghorn, 1990; Caboche and Rouze, 1990; Unkles at al., 1992). There is conservation of these domains between nitrate reductase apoproteins from fungi and plants. Although not characterised in detail, the genetics of nitrate assimilation by the basidiomycete fungus U. maydis are similar in outline to assimilation in A. niduIans (Lewis and Fincham, 1970a,b). Mutations in the narl and nirl genes define the structural genes for the nitrate and nitrite reductase apoproteins respectively, and other mutations define genes probably involved in molybdenum cofactor biosynthesis. Nitrate ions as sole nitrogen source induce nitrate reductase enzyme activity whilst ammonium ions repress it (Lewis and Fincham, 1970b; Smith et al., 1988). A diploid homozygous for a nirl mutation is able to reduce nitrate to nitrite ions, which accumulate because further reduction is blocked. This observation has led to a biochemical assay for recombination in a homozygous nirl mutant diploid using a sensitive assay for nitrite ions which accumulated after recombination between heteroalleles of narl mutants to give a wt allele (Holliday, 1971). We report here the cloning of the narl gene by direct complementation, suggestions of its close
(a) Isolation of the narl and nirl genes The nitrate and the nitrite reductase were isolated The cosmid DNA
from cosmid library
of about
apoprotein
and plasmid-based
comprises
fragments
40 kb inserted
into
genes
libraries.
of wt U. maydis
the BamHI
site of
cosmid pCUl3 (Wang et al., 1989). Transformants are generated by ectopic integration of cosmid DNA into chromosomes. The plasmid library has U. maydis wt DNA of 5- 15 kb inserted into the BamHI site of plasmid pCM54 (Banks and Taylor, 1988; D.W.H., unpublished). Transformants arise by autonomous replication of plasmids in U. maydis by virtue of the UARSI sequence in pCM54 (Tsukuda et al., 1988). Each library was transformed into U. maydis strain 95 (narl-I panl-1 Ab) and into 363 (nirl-1 panl-1 inosl-3 reel-I), HyR transformants being selected. These transformants were replicated to select those which grew on media with nitrate or nitrite ions as sole nitrogen source. All transformants, as well as the non-transformed mutant parents, displayed limited growth on these media because of the presence of trace quantities of ammonium ions (Lemontt, 1976), but transformants in which complemetation of the narl- 1 and nirl1 mutations occurred grew much more vigorously and were easily identified. Cosmid H7 complemented the nirl1 mutation and was isolated from the cosmid library by the sib selection procedure (Akins and Lambowitz, 1985). H7 was subsequently found to complement the narl-1 mutation in addition to nirl-1. Plasmid pMH3051, containing an 8.5-kb insert of U. maydis DNA, was a member of the plasmid library and complemented the narl- 1, but not the nirl-1 mutation. It was rescued from the primary transformant by virtue of its autonomous maintenance within the cell. A 6.7-kb EcoRI DNA fragment (Fig. 1A; the left-hand EcoRI site shown in the figure originates from the polylinker of the vector pCM54) from the insert in pMH305 1 was cloned into the vector pBluescript KS + to give pMH3055. A 4.7-kb EcoRI-XbaI fragment from the insert of pMH305 1 (Fig. 1A) was cloned into the vectors pBluescript KS+ and pCM54 to yield plasmids pMH3057 and pMH3053, respectively. Plasmid pMH3061 contained 9.5 kb of U. maydis DNA, was a member of the plasmid library and complemented the nirl-1, but not narl-I mutation. In summary, cosmid H7
71
.
.
.
.
.
.
.
.
.
. -503
.
.
GAATTCGAGCTCGCCCGGGGATC
ATCACTCTGTTTGCGCTCGATTACTTTGCMGCCCTTACACACTTGTTGCTCTCCTTCTGCTAGCTTGTGCGTCGCMGTGGCGAGCTCTTCTTGAGACCGTTTACMCAGTGCGATTCA MCGCAAAGGCATCAGCATCTTAGCTTGCTTCCC3CMTTTGTCMGCGTTCGGCGTCATTTGATTCTCTCACTCTCAGCTAAACGG3TTGAGTATTCTTTCTTTTCCCCGATCCCCATC GCTCTCTCACTTCCACCACACGAGCAGTTAGGAAlrGATGACGATCTCCTCMCCTCGAGCTCTACATCATC~GACATCCTCTGMMCCCCGACTTGAAAGGTTTCTATCGTCGTCAT CACCGGCATCCTCGCGATCCTCGTCGGCTACCAC~CCAGAGCCTTCGAGTCCGACCATCTTGGCGGC~GACCATCGAGTCGATCGATCCCTTTCAGCCTCGCMGGACTACMCGAGA ATGTCCTACCCGCCATCCCAGCCACGGMGGCGGTGCMCCATTGACTAGTGACGCCMCACTCCGGATCACTGGATCGCTCGAGATGAGCGCATGATCCGTCTCACTGGCAAACATCCC S Q P R K AV Q P L T? DAN T P D HW I AR DE R M I R L T Y P P M S TTCAACAGCGMGCACCTCTCTCAGAGCTCTTTTCCAAACGGTTTCTCACTCCTCAGMCCTCTTTTACGTACGTTCGCAGGGTGACACGCCGCGGGTTACTCGTGAGCMGCTG~C G D T PRVTREQAEN FYVRSQ K G F L T P Q N L E L F S FNSEAPLS TGGMGCTCAAGGTTCATGGTCTCGTAGAGCMGAGGTTGA~TCAGTATCMGGATCTCMGGAAAAGTTTTCCTACAGTCACCCCCAAACCATCACCCTCGTATGTGCCGGTMCAGA TLVCAGNR V E L S IKDLKEKFSYSHPQTI WKLKVHGLVEQZ
G
K
H
P
I
S
G
R
V
E
M
L
I
A
K
I
A
K
I
T
A
P
s
I
v
L
CGAAAAGAGCAAAACATGGTCGCTAAAGGCCTCGOTTTCMCTGGGGCGCCGCOOGTGTCTCGACGGGTCTATTTACCGGAGTCTACCTCGCCGACATTCTCGATTACTGCMGCCCMG LDYCKPK TGLFTGVYLADI KGLGFNWGAAGVS RKEQNMVA MTCCACTTCTCTCTTCTTTCCCTTCATACGATGTGGCTGTGCTGGATCOGGCGAGACATGTCGTCTTTGAGGGCGCCGACGAGCTGCCTMGOGAAAGTACGGCACTTCGCAGCGACTC KYGTSQRL EGADELPKG SYDVAVLDRARHVVF NPLLSSFP MCTGGGCGCTCGACAGGTGCMGGGCATGCTGATCGCTTGGGGTCTCMTffiTGMGACCTCTCTCCGGATCATGOCTACCCTTTGCGTCTCGTAGTGCCAGGTCAGATCAGTG~AGA LRLVVPGQ DLSPDHGYP KGMLIAWGLNGE NWALDRC ATGGTCMGTGGCTCGAGCGCATCGMGTTTCGGATCOCGAGAGCCAGCATCATCTGCACTTTCACGACMCCAGGTGCTTCCCACCGMGTGACCGCCGATCAGGCCAGMGCG~TG TEVTADQARSEM DNQVLP EVS3RE SQHHLHFH MVKWLERI CACTGGTGGTACGATCCAAAGTACATTATCMCG~TCTCMCGTCMTTCTGCCATCTGCTCGCCTGATCATGATCAGGTCGTCATGTTGCCGMCCATCTACGTCCAGTCGAGATGCTC VVMLPNHLRP INDLNVNSAICS P D B D Q HWWYDPKYI CCTCTTGMGGCTATGCGTACACGGGCGOTGGCCGTGTATCCACCAGAGTCGAAATCTCACTCGACGATGGTCGTAGCTGGMGTGTGCCTCGATTCACTACCCTGMGATCTGTACCGC PEDLYR SWKCASIHY T R V E ISLDDGR EGYAYTGGGRVS P L ATGTATCCGATCCAGCGGCACGAGTACTTTGGCACGCTGGATCTCAGCGCCACCGAAATGAGCTTCTCCTGGTGCTTCTGGCGTCTCGATGTCGATGTCGMGCGGACATCATCGCCMG FWRLDVDVEADI F S W C LDLSATEMS GHEYFGT M Y P I Q GATGTCMGGTGATCTCGGTTCGAGCACTGGACGMGTTCTGGCGACTCAGCCCCGCGACATGTACTffiMCGCCACTTCGATGATG4ACTCATGGTGOTTCCGTGTGTGCATCGCGMG YWNATSMMNSWWFRVC DEVLATQPRDM S V R A L D V K V I GCGAGAACGGCMCCMGATCCGCTTTGAGCATCCGACCTCGCCGCMTGCCCTGGCGGCTGGATGCAGAGGATGMCGAGGCTGGTCTCMCCCTCGCTATCCTCAGTTTGTCGMGCC PGGWMQRMNEAGLNPRYPQFVEA IRFEHP T S P Q C A R T A T K AAGGCCGTCGAGTCTTGCAAAACGGATGCGMCACACTACCGCGGCCMGGMGCCMGTGGGTCCCCGAAAGCGATCATGATCGACCCATCC~GCTGATACCATMTCACGGCGCCG SKADTI R P R K P SGSPKAIMIDP KTDANTLP KAVESC ATCTTGGCTGCTCACGGTGATGGCGAGGGTCCTGAGCCGTGGTTTGTCGTCCATGGTCATCTGTATGACGGGACTGGCTTCTTGAAAGACCACCCCGGAGGAGATCMTCGATCGTCTTG G G D Q DGTGFLKDHP PWFVVHGHLY E G P E ILAAHGDG TCGCCGGGAGMGACGACGCTACCGMGATTTCATGGCMTCCACTCGATGGACGCCAAAAAGATGCTTCGAGATTTCCACCTCGGTAGACTCGAGAAACMGATGCAGCGCCTCCTGCA DAAPPA HLGRLEKQ IHSMDAKKMLRDF DDATEDFMA S P G E GCCACGGAGCGGGMGTCTTGGATCTCAGCMGCCGTTCCTCGATCCCAAGAAATGGCGAGCTACACGACTCGGTGAGCMGCAAAT~ACTCACCAGATGCGAGGATCTTCCGCTTTGCT PDARIFRFA 0 E Q A N H S FLDPKKWRATRL D L S K P ATEREVL CTAGGMGCGMGATCAAGAGCTTGGGCTTCCGT3GCCAGCAGCTCTGCGTCTGTCGCTTGAAAAACGCAGMCAGGCGMGCAGAGATGGTACAAAGAGCTTACACACCATACAGTffiT RAYTPYSG KRRTGEAEMVQ B P A A L R L S L E E L G L P LGSEDQ MCACACAGCGTGGCTTCTTGGACATCTTGATCAAGGTCTACTTCCCGAGCGATGCAGCTGCTACGTCGGCACCCGCATTCGMGGA~30AAAGATGACGATGCTTCTCGAAAAGATCGAT PAFEGGKMTMLL KVYFPS DAAATSA NTQRGFLDILI GTGTCTTCGCCCTCAGACGATCTGACGATCGMCTCMG~ACCTCTTGGATCCTTCACCTATCTTGGTCA~AGCAGATCCGCT~~CCCGCCA~~GT~TCGCTTCGCMGCTC S IRWKPASAC IELKGPLGSFTYLGQQQ PSDDLT v s s
E
K
I
D
L
R
K
L
GCCATGATCGCCGGTGGATCGGGCATCACGCCCATCTGOTCGACCCTCAAAGCCATCGCCGACGMGTGCTCGATGCCTCGMTCCTTCGAGCCCTGCTTTAGACCCCATCCAGATCTGG PALDPIQIW S N P S S IADEVLDA I W ‘S T L K A G I T P AMIAGGS ATCGTGTACGGTMCCGCACGGMCAGGATATCCTGATCCGCGMGAGCTAGACGCATTGAGTAGGTTGAAAGGCMTCTTAAAGTCTGGCATGTGCTCAGCMTTGCACTCCTGAAAAC TEQDILIREELDALSRLKGNLKVWHVLSNCTPEN IVYGNR GMGCCAACTGGAGTATGGGCCGCGGCATATCTCTGCCMTGTCCTTAGGAGCACCTGCCACCMCCACCGGCAAAGCTGCTTCGGAMAGACGAGCTCGAGGATACTTTGGCGCTGGTC DTLALV FGGDELE PMSLGAPATNHRQSC EANWSMGRGISL TGCGGTCCCCCTCCGATGGAAAAAGCTGTCTCGGACGGTCTCMCGAGTTGGGCTGGOACCTGCMCGTTGCGTCGTGTTTTTCTGATTTCATACGTTTGTTCTCGTCATTATTTGCMT F 908 D G L N E LGWDLQRCVVF PMEKAVS C G P P CTTACCTMCCTAACCTACTTACTCGTGACTGTAAGTTATGGCGCTGACTCGATTGTCGAGATAGGATCC 2830
-481 -361 -241 -121 -1 120 40 240 80 360 120 480 160 600 200 720 240 840 280 960 320 1080 360 1200 400 1320 440 1440 480 1560 520 1680 560 1800 600 1920 640 2040 680 2160 720 2200 760 2400 800 2520 840 2640 860 2760
Fig. 1. Restriction map, nt and derived aa sequence of the narl gene. (A) Restriction map of the narl locus in plasmid pMH3055-solid line, sequenced DNA; broken line, DNA cloned and mapped but not sequenced; thick line, narl ORF. Plasmid pMH3055 comprises the 6.7-kb EcoRI-EcoRI DNA fragment from pMH305 1 cloned into pBluescript KS +. Plasmid pMH3051 was isolated from the primary transformant. Restriction enzyme abbreviations are B, E,g[II; H, BarnHI; P, PstI; R, EcoRI; S, SacI; X, XbaI. Distances shown below the map are in kb. (B) The nt and aa sequences of the ORF (EMBL accession no. X67687). The aa are aligned with the second nt of each codon. Methods: The 4.5-kb EcoRI-XbaI fragment from pMH3051 was subcloned into pBluescript KS + to give plasmid pMH3057. The EcoRI site is derived from the pCM54 polylinker present in pMH305 1 (Tsukuda et al., 1988). U. maydis DNA in pMH3055 and pMH3057 Biochemicals. Cleveland, OH, USA). Oligodeoxynucleotide
and subclones derived primers were employed
complemented both the narl-1 and nirl-1 mutations, pMH3051, pMH3053, pMH3055 plasmids and pMH3057 complemented the narl-1 but not the Cl-1 mutation, whereas plasmid pMH3061 complemented the nirl-1 but not the nurl-1 mutation. This pattern of complementation was also found for the nurl-6 allele and the nirl-1 allele in different genetic backgrounds. The results suggest that primary plasmid pMH3051 from the library carries the nurl gene, pMH3061 the nirl gene and cosmid H7 both genes. This suggestion was confirmed by hybridisation analysis of plasmid, cosmid and chromosomal DNAs. Plasmid pMH3051, pMH3061, H7 cosmid and wt chromosomal DNAs were digested with PstI and Sac1
from them was sequenced on both to sequence gaps in the subclones.
strands
(Sequenase
kit, US
(shown in Fig. 2) and with XbaI, PuuI and Sal1 (G.R.B., unpublished) and the resulting blot probed with a 1.14kb BamHI-BglII fragment from within the nurl ORF. As predicted from the restriction map of the nurl locus (Fig. lA), the probe hybridised to a 2.4-kb PstI and to a 2.3-kb Sac1 fragment respectively, common to pMH305 1, cosmid H7 and chromosomal DNAs. In addition, there was a 7.2-kb PstI fragment in pMH3051, replaced by fragments of equal mobility but > 12 kb in H7 and chromosomal DNAs (Fig. 2A and 2B). These results are consistent with the structure of the nur-I locus cloned into pMH3051. No hybridisation was found to pMH3061 restriction fragments (Fig. 2A, lane 2 and Fig 2B, lane 2).
72
kb
1.0,
0.5,
1 Fig. 3. Analysis of cloned
2
3
4
narf and nirl gene-containing
constructs
by Southern
hybridisation.
(A) DNAs digested
by Pstl and probed
uith
a I.14
kb &III-BamHI fragment within the narf ORF (Fig. 1A). (B) DNAs digested by Sac1 and probed by the same DNA fragment. (C) DNAs digested by Sac1 and probed with a 5.2-kb EcoRI fragment from the nirl locus. Lanes: (1) Plasmid pMH3051 DNA containing the narl gene. (2) Plasmid pMH3061 containing the nirl gene. (3) Cosmid H7 containg both narl and nirl genes. (4) Chromosomal DNA from the U. maydis wt strain 521 (Holliday. 1974). Chromosomal DNA was extracted as described by Holden et al. (1989). All DNA was digested with the indicated restriction endonucleases and electrophoresed in 0.8% agarose. The gels were stained with ethidium bromide to ensure that equal amounts of plasmid DNAs were transferred Kit, Amersham
to nitrocellulose (Hybond C-extra, Amersham International). International) with [a-s2P]dTTP (NEN Research Products).
I-kb DNA ladder (GIBCO-BRL) being shown on the left margin.
was used as the DNA size markers.
The probes were labelled by the random primer method (Multiprime Hybridisation was essentially as described (Sambrook at al., 1989). A
The positions
Although the nirl complementing DNA in pMH3061 has not been characterised in detail, we used a 5.2-kb EcoRI fragment necessary for genetic complementation of the nirl-1 mutation (P.A.S., unpublished) to probe the digests of the DNAs just described. This probe hybridised to a Sac1 fragment of 3.9 kb common to pMH3061, cosmid H7 and chromosomal DNAs, and to two fragments of 1.6 and 1.4 kb common to H7 and chromosomal DNAs (Fig. 2C). No hybridisation to pMH3051 sequences was detected (Fig. 2C, lane 1). Similar results were obtained for hybridisation of this probe to PuuI and Sal1 fragments of these DNAs (G.R.B., unpublished). These results confirm the complementation data and show that pMH3051 and 3061, each contain gene sequences common to cosmid H7 and to chromosomal DNAs, but not to each other. The results do not prove that the narl and nirl genes are closely linked on the chromosome because it is possible that two fragments of different chromosomal origin were cloned together in the same cosmid molecule, rather than one continuous sequence containing both genes which are adjacent on the chromosome. However it is more likely that there is close linkage because it has been demonstrated genetically (Holliday. 1971). Close
of some of these are indicated
by the arrowheads,
their sizes (kb)
linkage was found for the A. nidulans niiA and niaD genes which are separated by 1272 bp, and from which transcription of the two genes occurs divergently (Tomsett and Cove, 1979; Johnstone et al., 1990). In N. crassa, however, the two analogous genes are on different chromosomes (Tomsett and Garrett, 1980). In a previous publication, we reported the isolation by differential hybridisation of U. maydis DNA sequences in plasmid pMH3007, the transcript of which was induced by nitrate ions (Smith et al., 1988). This transcript hybridised at low stringency to the A. nidulans niaD nitrate reductase gene. We have now attempted to hybridise sequences from the narl gene isolated in the present work to restriction digests of pMH3001 and vice versa, without success (G.R.B., unpublished). Thus although the transcript encoded by the U. maydis DNA in pMH3007 is induced by growth of cells in the presence of nitrate ions, this transcript clearly does not originate from the narf gene. (b) Disruption of the narl gene To confirm that the complementing DNA was the narl gene itself, a one step disruption of a chromosomal narl
73
gene was carried out. The 1.33-kb BglII fragment within the ORF of the cloned DNA (Fig. 1A) was replaced in pMH3057 by a 2.8kb BumHI-BglI fragment able to express a bacterial HyR gene from the U. maydis urns2 promoter sequence (Wang et al., 1988). Plasmid pMH3040 containing this cassette resulted and was cleaved with SpeI+BamHI to give a linear DNA molecule with the urns2 gene cassette flanked by the narl sequences on either side of the disruption (Fig. 3A). This DNA was transformed into competent wt U. maydis cells and HyR transformants then selected. Transformants carrying disruptions of the chromosomal narl gene resulting from homologous recombination should be unable to grow on medium containing nitrate as the sole nitrogen source, but able to do so on ammonium salts. Ectopic integration of the transforming DNA at sites other than the narl gene in the genome should generate transformants able to grow on both salts by virtue of possessing an intact as well as disrupted narl gene. Ten HyR transformants were transferred to CM + Hy, NM +Hy and NM plates and five failed to grow on the NM media. Furthermore, although these five transformants were unable to grow on NM medium containing nitrate, they were able to do so on NM media containing nitrite or ammonium ions as expected for potential disruptants of a chromosomal narl gene. DNA was extracted from both classes of transformants, digested by PstI or by BglII, electrophoresed and transferred for hybridisation analysis with labelled pMH3057 as the probe. This probe should hybridise to a wt PstI fragment of 2.12 kb and to wt BgZII fragments of 1.32 and 2.82 kb, in addition to junction fragments (Fig. 3A). Disruption of the narl locus should result in the loss of the PstI fragment and gain of one of 1.53 kb; the BglII fragments should be replaced by one of 5.6 kb (Fig. 3A). Ectopic transformants should retain the wt fragments but gain others because of integration of transforming DNA elsewhere in the genome resulting in the generation of novel restriction fragments. Representative hybridisation analyses are shown in Fig. 3B, and in addition, that for a transformant in which tandom duplications of transforming DNA had disrupted the narl gene (Fig. 3B, panels a and b, lane 3), a situation which is not uncommon with other genes in U. maydis (Banks et al., 1992). These results are in agreement with the expectations described above and show that the narl gene was disrupted in all transformants which grew on nitrite and ammonium, but not on nitrate ions; all transformants which grew on all three nitrogen sources had retained wt restriction fragments and had suffered ectopic integration. The results confirm the no-2 identity of the cloned ORF, and also that the narl function is dispensable for growth of cells in the presence of nitrite or ammonium ions.
(c) Coding sequence of the narl gene
The nt sequence contains an ORF of 2724 nt, able to encode a protein of 908 aa (Fig. 1B). Similar to aa sequences of other U. maydis proteins, there is no marked codon useage bias. All codons are used, the codons of 309/721 aa terminate in C where possible and side by side GC pairs are not avoided, 128/376 codons possessing them. However, third position A’s are avoided, only 1441674 codons ending in A. These properties are characteristic of filamentous fungi (Ballance, 1986; Spanos et al., 1992). Although the evidence is not conclusive, the gene does not appear to contain introns-the proportion of U. maydis genes containing them has yet to be established. However, the genomic nt sequence contains none of the consensus intron signals found in filamentous fungi (Ballance, 1986). Nitrate reductases are functionally complex enzymes with multiple redox centers and cofactors, a situation reflected in the strong domain structure of their polypeptides which is conserved from fungi to plants (Kinghorn and Campbell, 1989; Campbell and Kinghorn, 1990; Caboche and Rouze, 1990). The aa sequences of these domains also bear homologies to those in proteins with different functions, but with similar cofactor requirements. Domains in nitrate reductases have been identified which define the cofactor binding sites for molybdenumpterin, iron-heme and FAD (Crawford et al., 1988; Campbell and Kinghorn, 1990). This conservation of domain structure is maintained in the U. maydis nitrate reductase apoprotein (Fig. 4). In particular, aa sequences 33-47 and 203-231 are very similar to those of domains which define the molybdenum-pterin binding site in other nitrate reductases and in mammalian sulfite oxidase (Kinghorn and Campbell, 1989; Campbell and Kinghorn, 1990). In the 43 aa shown (Fig. 4), 27 are identical to those in N. tabacum and in A. nidulans nitrate reductases. The sequence of narl from aa 531-591 is very similar to the iron-heme binding domains identified in other nitrate reductases and in mammalian cytochrome b5 and yeast flavo-cytochrome b2 (Kinghorn and Campbell,1 989). In this 61 nt sequence of the narl gene encoded protein, there are 36 aa identical with those in A. nidulans and 35 in N. tabacum nitrate reductases, and 26 in mammalian cytochrome b, (Fig. 4). The FAD-binding domain, homologous with mammalian NADH:cytochrome b5 reductase, has been localised to the C-terminal region of nitrate reductase (Kinghorn and Campbell, 1989; Campbell and Kinghorn, 1990). The sequence aa 6 13-908 of the narl encoded protein has marked similarities with these domains (Fig. 4). In particular, the sequence FRFALP, which is conserved in plant nitrate reductases and mammalian cytochrome b,, appears as FTLSLE in
74
A
B
0.73
,P 0.59
y
4
b)
c) 1.1
I3
B
P
P
b
a
I
1,
i 12
HI
b
I
1
123
4
34
Fig. 3. Disruption of the narf gene. (A) Structures of the gene and its disruptant. Single line, rlurl locus; tilled bar. rlur/ ORk; open bar, IUU.\? sequences; hatched bar, HyR gene. Restriction endonuclease abbreviations as for Fig. I; fragment sizes are in bp. A 1325.bp BglII fragment (a) in pMH3057 was replaced by a 2800-kb &/II-BamHI fragment (c) containing the HyR cassette. This cassette fragment originated as the 2.8-kb XhoIHind111 fragment from pHLl (Wang et al., 1988) and was then recloned into the vector pIC19RHL (J. Kronstad, unpublished; Marsh et al., 1984). The structure of the plasmid resulting from the replacement, pMH3040 and (b+c), was verified by restriction analysis. Plasmid pMH3057 comprises a 4.5-kb EcoRI-XhaI fragment, (a+ b), derived from pMH305I and ligated into pBluescript KS+. Note that the EcoRI site is derived from the pCM54 polylinker (MCS; Tsukuda et al., 1988). Plasmid pMH3040 was digested with SpeI and BamHI to give linear DNA of 4.52-kb incorporating the HyR cassette surrounded by narl sequences and then transformed into competent cells of wt 521 strain of U. maydis, essentially as described by Wang et al. (1988). Transformants were selected in the presence of 200 pg Hy/ml (Boehringer, Mannheim). Ten colonies were transferred to CM and NM agar media containing Hy, and also to NM agar. All ten grew on the CM +Hy medium, and the same five failed to grow on both of the NM media. (B) Hybridisation analysis of DNA from transformants carrying disruptions of narl. Chromosomal DNA was digested by PstI (a) or by &III (b),electrophoresed in 0.8% agarose, transferred to Hybond C-extra nitrocellulose membrane (Amersham International) before Southern hybridisation analysis with 32P-labelled pMH3057, essentially as described by Sambrook et al. (1989). Lane (1) wt 521 DNA: (2) example of a disruption of the narl gene; (3) transformant generated by ectopic integration of transforming DNA randomly in the genome; (4) disruption of narl gene by tandom copies of transforming at the left margin.
DNA. A I-kb DNA ladder
(Gibco-BRL)
was the size marker.
A. nidulans (Kinghorn and Campbell, 1989) and FRFALG in the U. may&s nitrate reductase (Fig. 4). Therefore, not only does the U. maydis narl encoded protein maintain the sequence conservation of other nitrate reductases, but it also maintains the same relative locations of the functional cofactor domains along the polypeptide.
Arrowheads
identify
some markers,
with their sizes (kb) shown
(d) Transcription of the narl gene 503 nt of S-untranslated DNA was sequenced (Fig 1B). Although the tsp has not yet been determined, this upstream region does contain four S-CTTT and eleven 5’-CATC motifs, both of which may be associated with the tsp of some U. maydis genes (Spanos et al., 1992). In addition, the sequences 5’-TCTCTCACT (ST at - 209
L’.tnuydlJ
33
“;.1
A.nidulans
67
249
N. tabacum
103
288
sulphite oxidase
113
299
Umaydis
531
A.nidulans
528
N.tabacum
547
606
domain
26
86
Umnydis
613
677
A.nidulans
612
676
N.lbaCUm
643
703
Ham
Hmpms
cyt b,
Umaydis
678
A.nidulans
677
N.tabacum
704
H.sapiens cyt b,
95
35
96
Umaydis
743
A.nidulans
730
N.:abacum
752
H.sapiem cyt b, 152
YsGNTQRGFLDItIXWFPSDAAATSAPAFEf~~~KIDVS~PSDDLTXELf((lP~~S~~L III I III I I I I I PSIPaQ@&TA%DTLPLG@J ____~$%CK!?@XJRFEYL ISPS~L6MVD1LXXIYAET~ Ill I III I I I I I LDVK'X'LBHIEYQ TSTIDEVGYFELW~IYFKGIHPKFPN~~GQMSQY~~DSMPLG~_ III I III I I I I I X$FRQPSGLLWQ IgSDDDK@!VDLV~~DTHPKFPA_!%~SQYtESMQIGDT_____
742
_cQQQrR~PASAC_SLR~~~~~~~IWST~~AD~L~SNPSSPALDPIQIWI~ I II II IIII I I I VKSFVMfCQCTCtISPVFQVtRr4VMQDEQDETKCVMLD DRGRVLISGKERFI II II Ill1 I I I GK@NFLVHGEtQKF_AK%%i~~T@X~PVYQVMQ~~LKDPEODTYWYA I II II III1 I I I GK#FAIRPDXXSNPIIRT"KSVGUS;RqET&'~~$‘,?MLQVIR&~MKDP~HTVCHLLFA
803
751 151
773 810 208
Umaydis
804
867
A.nidulam
180
835
N.tabacum
811
863
H.sapiens cyt bS 2 0 9 lhaydis
868
A.nidulans
836
N.tabacum
864
H.sapiens cyt b, 2 6 1
I-lg. 4. The aa sequcncc in all sequences shown;
729
260
~DEL~D~~VC~PPP~_~VS~~~~L_Q~~F I I I III1 I I _~TPDRE_~LVCGPEA?@KJSKKI~LS@KE_ENLHY-% I I I IIII I I _IPEPSHTX'@rjACGPPP~IQFhVNPN~EKNGYDIKDSLL@?_ I III I _LPPPEEEp~vLMCGPBP~IQY~cLPN~DHV~HPT_~d~_
908 873 904 300
comparrsons of the rrurl gene encoded protcm. The aa positrons of the regrona shown arc md~cated; vertical IIIU, aa nknt~cal shading, aa identical in two or three sequences. The sequences of the proteins compared with the U. maydis narl sequences
are the A. nidulans niaD (Johnstone et al., 1990) gene; the N. tahacum nitrate reductase (Calza et al.. 1987); mammalian sulfite oxidase (Crawford et al., 1988); rat liver cytochrome b, reductase heme domain (Calza et al., 1987); human NADH-cytochrome b, reductase (Yabisui et al.. 1984). Other data are from Kinghorn and Campbell (1989). The sequences were compared using the Molecular and Genetics Sequencing (MGS) suite of programs developed by W. Greer and P. Gillett, National Institute for Medical Research. London, and refined by eye.
and - 266) and S-ATCCTCG (5-A at - 39 1 and - 400) are each repeated directly. The sequence 5’CAAAGACATC (5’-C at -334) has a repeat, but modified by the insertion of C between nt 7 and 8 (5’-C at -450). The sequence 5’-ATCGAGTCGAT (5’-A at nt -46) has a potential to form a hair-pin structure, albeit with only a 5-bp stem and one nt loop. Adjacent to this potential structure, the sequence 5’-AGACCATCGAGTCGATCGATCCCTTTCA (5’-A at - 5 1) has several motifs in common the the U. maydis reel sequence 5’-
AGACCATCACATGCTTTCT (5-A at - 151; Holden et al., 1989) and glyceraldehyde-3-phosphate dehydrogenase gene sequence 5’-CCACCATCGAATCTTTCT (5’-C at -52; Smith and Leong, 1990). 106 nt of 3’untranslated DNA have also been sequenced (Fig. 1B). As reported for the U. maydis pyr3 gene, no defined sequence elements can be discerned (Spanos et al., 1992). Induction by nitrate and repression by ammonium ions appear to be a common feature of assimilatory nitrate reductase transcripts (Dunn-Coleman et al., 1984).
76 Nitrate reductase enzyme activity in U. maydis is markedly increased when cells are grown in media containing nitrate ions as sole nitrogen source (Fincham and Lewis, 1970b). We therefore used a fragment of the cloned narl gene to determine by Northern hybridisation analysis if this increase is transcriptional in nature (Fig. 5). Aliquots of a culture growing in CM were switched to NM media containing nitrate or ammonium plus nitrate ions, or maintained in CM. The cells were grown for 2 h, a time
A
kb 5-o4,03*02.0-
l.O-
o-5-
B
Fig. 5. The nature
of the narl
transcript
and its induction
by nitrate
ions. (A) Northern blot probed with the 32P-labelled 1.14-kb BglIIBamHI narl DNA fragment (Fig. IA). (B) The same blot probed with the urns2 gene in plasmid pHLI. Only the 2.2-kb urns2 transcript is shown (Holden et al., 1989). Lane (1) 20 ug total RNA from cells grown in CM. (2) 20 ug total RNA from cells grown in NM. (3) 60 ug total RNA from cells grown in NM containing ammonium sulfate. Methods: 30 ml aliquots of an overnight culture of U. maydis 521 at 10’ cells/ml in CM medium were harvested by sterile filtration. Cells from one aliquot were briefly washed with pre-warmed CM and then resuspended in 30 ml CM at 30°C. Cells from a second aliquot were washed in prewarmed NM and suspended in 30 ml NM, which contains 0.3% potassium nitrate. A third aliquot was washed in pre-warmed NM containing 0.3% w/v ammonium sulfate and suspended in 30 ml of the same medium. The cultures were vigorously shaken at 30°C for 2 h and then rapidly cooled before centrifugation. Total RNA was extracted from the cells, quantitated by spectrophotometry and its integrity verified by gel electrophoresis (Fonzi and Sypherd, 1985). RNA samples were glyoxylated and electrophoresed in 1% agarose (McMaster and Carmichael, 1977) and transferred to GeneScreen (NEN Research Products) for Northern hybridisation analysis. The filter was probed with 32P-labelled 1.14-kb BgIII-BamHI narl fragment under conditions recommended by the filter manufacturer. After autoradiography, the filter was then stripped by holding in 96% formamide, 10 mM EDTA and TrisHCl pH 8 at 60’C for 30 min. It was then hybridised to 32Plabelled pHLl DNA to detect the urns2 transcript (Holden et al.. 1989).
shown to produce maximum induction of nitrate reductase enzyme activity (Fincham and Lewis, 1970a). Total RNA was then extracted from the cells and nitrate reductase transcripts analysed by Northern hybridisation, using a BglII-BumHI 1.14-kb fragment contained within the narl ORF (Fig. 1A). A major transcript of 3.0 kb was detected (Fig. 5A), consistent with an ORF of 2724 nt. In cells grown for 2 h in the presence of nitrate ions, densitometry of the autoradiograms revealed that the abundance of this transcript was 90-fold greater than in cells grown in the presence of ammonium plus nitrate ions or in CM. The hybridisation filter was stripped of the narl probe and then probed with the urns2 gene (Fig. 5B), which encodes a U. maydis stress/heat shock protein, in order to provide an RNA loading control (Holden at al., 1989). It was apparent that transcription of this gene, or the stability of its transcript, was altered by the media changes although not apparently by the nature of the source of the nitrogen available to cells. Thus cells growing in CM contained about a fivefold increase in steady state levels of urns2 transcript over cells transferred to NM media containing either nitrate or ammonium plus nitrate ions. We have repeated these experiments four times, each time with identical results showing that when equal amounts of total RNA are analysed, cells grown in NM medium containing nitrate ions contained the very high levels of the narl transcript compared to cells growing in the presence of ammonium plus nitrate ions or in CM medium. Conversely, steady state levels of the urns2 transcript are higher in cells grown in CM than in the two NM-based media. It is clear, however, that the U.maydis narl gene transcript is induced very significantly by nitrate, and repressed by ammonium ions. (e) Conclusions (I) The U. maydis narl and nirl genes, encoding nitrate and nitrite reductases respectively, have been isolated separately on plasmid vectors (plasmids pMH3051 and 3061 respectively) and together on a cosmid (cosmid H7). Because these genes have been shown to be linked by genetical methods, we propose that they are molecularly linked such that a single segment of DNA containing them was cloned into the cosmid H7. (2) Disruption of a chromosomal copy of the narl gene by homologous recombination with a disrupted copy of the cloned sequence which complements the narl-I mutation, yielded transformants with the expected phenotypes and chromosomal DNA restriction patterns. The narl gene function is dispensable when cells are grown in the presence of nitrite or ammonium ions. (3) The ORF encodes a 90%aa polypeptide. Its sequence maintains the sequence conservation found in fungal and plant nitrate reductases. This is particularly
77 true for those sequences which define the molybdenumpterin, iron-heme and FAD-binding domains, cofactors for the electron transfer reactions. The relative localisation of these domains along the polypeptide is also conserved. (4) The 2724-nt ORF encodes a transcript of 3.0 kb. It is induced some 90-fold when nitrate ions become the only source of nitrogen for cells. When ammonium ions are also present, transcription is repressed.
domain
structure
and amino acid homologies
in higher plants. Gene
85 (1989) 371-380. Dunn-Coleman, ilation Fonzi,
N.S., Smarrelli
in eukaryotic
Jr.. J. and Garrett,
W.A. and Sypherd,
P.S.: Expression
decarboxylase of Saccharomyces Cell. Biol. 5 (1985) 161-166. Gruber.
R.H.: Nitrate
H.. Goetinck,
of the gene for ornithine
cereoisiae
S.D., Kirk,
in Escherichin
D.L. and Schmitt.
D.W., Kronstad,
regulated hsp70 192771934.
J.W. and Leong.
gene
of Uslilago
S.A.: Mutation
ntuJdis.
coli. Mol.
R.: The nitrate
reductase-encoding gene of Voloou curter? map location, and induction kinetics. Gene I20 (1992) 75-83. Holden,
assim-
cells. Int. Rev. Cytol. 92 (1984) l-50.
EMBO
sequence in a heat-
J. 8 (1989)
Holden, D.W.. Spanos, A. and Banks. G.R.: Nucleotide sequence of the RECI gene of Usrilago maydis. Nucleic Acids Res. I7 (1989) 10489.
ACKNOWLEDGEMENTS
We are indebted to Steve Sedgwick for critical reading of the manuscript and for his computer graphics wizardry; to Jim Kronstad for the urns2 gene on pIC19RL; to Tony Johnson for Molecular Dynamics ImageQuantTM densitometry and to Jim Kinghorn for information before publication.
Holliday, R.: Biochemical measure radiation-induced recombination
of the time and the frequency of in U.stilclgSo.Nature New Biol. 232
(1971) 233-236. Holliday, Johnstone,
maydis.
R.: Ustilugo
Genetics.
Vol. I., Plenum, I.L.. McCabe,
In: King.
R.C. (Ed.),
Handbook
of
New York, 1974. pp. 5755595.
P.C., Greaves.
P.. Gurr. S.J.. Cole, G.E.. Brow,
M.A.D.. Unkles. SE.. Clutterbuck, A.J.. Kinghorn, J.R. and Innis. M.A.: Isolation and characterisation of the cmA-niiA-nicrD gene cluster for nitrate (1990) 181-192.
in Aspergillus
assimilation
niduluns. Gene
90
Kinghorn, J.R. and Campbell, E.I.: Amino acid sequence relationships between bacterial. fungal and plant nitrate reductase and nitrite REFERENCES Akins. R.A. and Lambowitz, A.M.: General method Neurospora crassa nuclear genes by complementation Mol. Cell. Biol. 5 (1985) 227222278. Arst
for cloning of mutants.
Jr., H.N. and Cove, D.J.: Nitrogen metabolite repression Aspergillus niduluns. Mol. Gen. Genet. 126 (1973) I I ll141.
Ballance, D.J.:Sequences important fungi. Yeast 2 ( 1986) 2299236.
for gene expression
in
in filamentous
Banks, G.R.. and Taylor, S.Y.: Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation. Mol. Cell. Biol. 8 (1988) 5417-5424. Banks, G.R.. Kanuga, influence of the chromosome
N., Ealing. J., Spanos, A. and Holden, D.W.:The Ustilago maydis RECI gene on plasmidrecombination. Curr. Genet. 22 (1992) 4833489.
Brownlee. A.G. and Arst Jr., H.N.: Nitrate uptake in Aspergillus and involvement of the third gene of the nitrate assimilation J. Bact. II5 (1983) 1138-1146.
nidulans cluster.
Caboche, M. and Rouze, P.: Nitrate reductase; a target for molecular and cellular studies in higher plants. Trends Genet. 6 (1990) 1877192. Calza. R.. Hutter, E.. Vincentre, M., RouzC, P. Galangue, F.. Vaucheret, H., Chevel. I., Mezer. C.. Kronenberger. J. and Caboche, M.: Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductase from higher plants. Mol. Gen. Genet. 209 (1987) 5522562. Campbell, W.H. and Kinghorn, J.R.: Functional tory nitrate reductases and nitrite reductases. I5 (1990) 315-319.
domains of assimilaTrends Biochem. Sci.
Crawford, N., Campbell, W. and Davis, R.W.: Nitrate reductase from squash: cDNA cloning and nitrate regulation. Proc. Natl. Acad. Sci. USA 83 (1986) 8073-8076. Crawford, N.M.. Smith, M., Bellissimo, D. and Davis. R.W.: Sequence and nitrate regulation of the Arabidopsis thuliana mRNA encoding nitrate reductase. a metalloflavoprotein with three functional domains. Proc. Natl. Acad. Sci. USA 85 (1988) 500665012. Cove, D.J.: Genetic studies of nitrate Biol. Rev. 54 (1979) 291-327.
assimilation
in Aspeygillus nidulans
Daniel-Vedele, F.. Dorbe, M.-F., Caboche, M. and Rouze. P.: Cloning and analysis of the tomato nitrate reductase-encoding gene: protein
reductase Molecular University Lemontt.
proteins. In: Wray. and Genetic Aspects Press. Oxford, 1989.
J.: Induced
mutagenesis
J.L. and Kinghorn. J.R. (Eds) of Nitrate Assimilation. Oxford pp. 3855402.
in Ustilago muydis. I. Isolation
characterisation of a radiation-revertible for nitrate reductase. Mol. Gen. Genet.
allele of the structural 145 (1976) 1355138.
Lewis, C.M. and Fincham. J.R.S.: Genetics of nitrate Ustilugo muydis. Genet. Res. I6 ( 1970a) I5 I 163.
reductase
and gene in
Lewis. C.M. and Fincham, J.R.S.: Regulation of nitrate reductase in the basidiomycete U&ago maydis. J. Bact. 103 (1970b) 55-61. Marsh. J.L.. Erfle. M and Wykes, E.J.: The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32 (1984) 4X1-485. Marzluf, G.A.: Regulation of nitrogen metabolism and gene expression in fungi. Microbial. Rev. 45 (1981) 4377467. Marzluf, G.A. and Fu. Ying-Hui.: Genetics. regulation. and molecular studies of nitrate assimilation in Neurospora c’rasscc. In: Wray, J.L. and Kinghorn, J.R. (Eds) Molecular and Genetic Aspects of Nitrate Assimilation. Oxford University Press, Oxford. 1989, pp. 314-327. McMaster. G.K. and Carmichael, G.G.: Analysis of single and double stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74 (1977) 4835-4838. Sambrook, J.. Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. 1989. Scazzocchio, C. and Arst Jr.. H.N.: Regulation of nitrate assimilation in Aspergillus nidukzns. In: Wray. J.L. and Kinghorn, J.R. (Eds.) Molecular and Genetic Aspects of Nitrate Assimilation. Oxford University Press, Oxford, 1989. pp. 29993 13. Smith, T.M., Spanos, A. and Banks, G.R.: Differential expression of Usrilago maydis DNA sequences during induction of nitrate reductase enzyme activity. Curr. Genet. I4 (1988) 4577460. Smith, T.L. and Leong. S.A.: Isolation and characterization of a Usrilago mclydis glyceraldehyde-3-phosphate Gene93 (1990) 111-117.
dehydrogenase-encoding
gene.
Spanos, A., Kanuga. N.. Holden. D.W. and Banks, G.R.: The Usrilugo maydis pyr3 gene: sequence and transcriptional analysis. Gene I I7 (1992) 73-79.
78 Tomsett, A.B. and Garrett, R.H.: The isolation mutants defective in nitrate assimilation Genetics 95 (1980) 649-660. Tsukuda,
T.. Carleton,
Isolation sequence
and
S., Fotheringham.
characterization
from Us&go
of an
and characterization of in Neurospera crassa.
S. and
Holloman.
autonomously
W.K.:
replicating
maydis. Mol. Cell. Biol. 8 (1988) 3703-3709.
Unkles, S.E., Campbell, E.J., Punt, P.J., Hawker, Hawkins, A.R.. Van den Handel. C.A.M.J.J. The Aspergihs niger niaD gene encoding upstream nucleotide and amino acid sequence Ill (1992) 1499155.
K.L., Contreras, R.. and Kinghorn. J.R.: nitrate reductase: comparisons. Gene
Yabisui, T., Miyata, T., Iwanaga, S., Tamura. M., Yosheda. S., Takeshita, M. and Nakajima, H.: Amino acid sequence of NADH-cytochrome b, reductase of human erythrocytes. J. Biochem. 96 (1984) 579-582. Wang, J., Holden,
D.W. and Leong,
phytopathogenic fungus 85 (1988) 8655869.
U&ago
S.A.: Gene transfer
system for the
maydis. Proc. Nat]. Acad. Sci. USA
Wang, J., Budde, A.D. and Leong, S.A.: Analysis
of ferrichrome
biosyn-
thesis in the phytopathogenic fungus Usfilago maydis: cloning of an ornithine-N5-oxygenase gene. J. Bact. 17 I (1989) 28 I l-28 18. Wray, J.L. and Kinghorn, J.R.: Molecular and Genetic Aspects of Nitrate
Assimilation.
Oxford
University
Press, Oxford,
1989.