Identification of a cosmid clone containing the Neurospora crassa lys-5 and un-4 genes, isolation of a partial lys-5 cDNA and associated chromosome walking.
Thomas J. Schmidhauser, Dan Chen and Yihong Wan* - Department of Biology, The
University of Southwestern Louisiana, Lafayette, LA and *University of Colorado Health
Sciences Center, Denver, Colorado
Fungal Genetics Newsletter 46:23-24
The un-4 gene of Neurospora crassa was cloned to determine the limits of a chromosome walk on linkage group VI (LGVI) and to allow analysis of un loci on LGVI. Subsequent analysis identified the lys-5 locus on the same cosmid clone as un-4. We have isolated and sequenced a partial lys-5 cDNA clone and initiated a chromosome walk from the lys-5, un-4 cosmid clone.
DNA sequence analysis of pYW19-2 identified an open reading frame encoding a deduced polypeptide with strong similarity to homocitrate synthases and isopropylmalate synthases from other organisms (Figure 1). Neurospora lys-5 mutants lack homocitrate synthase activity. G13:8:G DNA complements lys-5 spheroplasts allowing growth on minimal medium. lys-5 maps 2% away from un-4 and un-4, by definition, is irreparable by supplementation at the restrictive temperature. Thus, un-4+ and lys-5+ are separate loci and both are present in G13:8:G. pYW19-2 likely represents a partial lys-5 cDNA clone. The partial deduced Lys-5 polypeptide has highest similarity to the homocitrate synthase of Penicillium chrysogenum with 80% identity in an optimized alignment (Figure 2).
1 CGTTATTGAG TATGTCAAGT CCAAGGGACT TGAGGTTCGC TTCTCCTCCG AGGATTCCTT
V I E Y V K S K G L E V R F S S E D S F
61 CCGCTCCGAT CTCGTCGATC TCCTTTCCCT TTACCGCGCT GTTGACAAGG TCGGCGTCCA
R S D L V D L L S L Y R A V D K V G V H
121 CCGTGTCGGT ATCGCCGATA CCGTCGGCTG CGCTTCTCCC CGCCAGGTCT ATGACCTCGT
R V G I A D T V G C A S P R Q V Y D L V
181 CCGTACCCTT CGCGGCGTCG TTTCGTGCGA TATCGAGACC CACTTCCACG ACGACACTGG
R T L R G V V S C D I E T H F H D D T G
241 CTGCGCCATT GCCAACGCCT ACTGTGCTCT CGAGGCTGGT GCCACCCACA TCGACACCTC
C A I A N A Y C A L E A G A T H I D T S
301 CGTTCTCGGT ATCGGCGAGC GTAACGGTAT CACCCCTCTC GGTGGCTTGA TGGCTCGCAT
V L G I G E R N G I T P L G G L M A R M
361 GATCGTTACC AGCCCCGACT ACGTCAAGAG CAAGTACAAG CTCCACAAGC TCAAGGAGCT
I V T S P D Y V K S K Y K L H K L K E L
421 CGAGGATTTG GTTGCCGAGG CTGTTGAGAT CAACACCCCC TTCAACAACC CCATCACTGG
E D L V A E A V E I N T P F N N P I T G
481 TTTCTGCGCC TTCACCCACA AGGCTGGCAT CCACGCCAAG GCCATCCTCA ACAACCCCAG
F C A F T H K A G I H A K A I L N N P S
541 CACCTATGAA ATTCTCAACC CTGCCGACTT CGGTCTCACC CGCTACGTCC ACTTCGCTTC
T Y E I L N P A D F G L T R Y V H F A S
601 GCGCTTGACT GGCTGGAACG CCGTCAAGAC CCGTGTCGGC CAGCTTGGTC TCGAGATGAC
R L T G W N A V K T R V G Q L G L E M T
661 CGACGACCAG GTCAAGGAAT GTACCGCCAA GATCAAGGCC CTTGCCGACG TGCGCCCAAT
D D Q V K E C T A K I K A L A D V R P I
721 CGCCATCGAC GACGCCGATT CGATCATCCG TACTTTCCAC CTCGGTCTTC ACGAGCAGAA
A I D D A D S I I R T F H L G L H E Q N
781 CAAGGTCCAG CCTCCCGCTG TTGTCGAGAA CTAAGCGGAA GCAGAGCGTT CGACCAACGG
K V Q P P A V V E N *
841 AGTTGTCCTT TAGCATGAAG GGGAATATAC CAGGATTTTT ACGAGGAGAG ATGCGGGCAT
901 CATGACGATT TTCTTTTTAC TTGTGTTTGG GGTCATTTTT CACACATCCA CCGGAGTTCT
961 TTGAGTACTA TAATCTCCCT GTTTGGGGAG CAAAAAGGGG GTTGATTGGG TTAACTGGGG
1021 ATGACTGAGC AGGCCAATAT TGCCGACTGT GTTCCTAATC AGGGGGAATG CTCGTCGAAA
1081 AATGAGCATG AGATAGACAA AATCAACGGG AGACGAAAGT AACAACGTCA CCTGATTGTC
1141 CTTCAAAAAA AAAAAAAAAA AA
Figure 1. Nucleotide sequence of the cDNA insert of pYW19-2 and the deduced polypeptide product (GenBank AF142777). The stop codon is indicated by a *. Several isolates, including NC4A2-T7, from the Neurospora Genome Project, University of New Mexico, overlap pYW19-2 from position 549 to the polyadenylation site.
The pYW19-2 insert was used to probe a Southern blot of G13:8:G restriction digests. Results suggest that an approximately 6.3-kb EcoR1 fragment contains the lys-5 gene. As cosmid G13:8:G was not identified in the cpc-1 walk we initiated a chromosome walk from the lys-5/un-4 region in an attempt to link up to our cpc-1 walk. A G13:8:G based probe identified cosmid X6:6:F. A X6:6:F based probe identified cosmid X22:2:B. No new cosmid clones were identified with a X22:2:B based probe.
Penicillium c. HC Synthase 181 IEVIEFVKSK GIEIRFSSED
|||+|||| |+|+||||||
Neurospora Lys-5 VIEYVKSK GLEVRFSSED
Pc HC SFRSDLVDLL SIYSAVDKVG VNRVGIADTV GCASPRQVYE LVRVLRGVVG
|||||||||| |+| |||||| | |||||||| |||||||||+ |||+|||||+
Lys-5 SFRSDLVDLL SLYRAVDKVG VHRVGIADTV GCASPRQVYD LVRTLRGVVS
Pc HC CDIETHFHND TGCAIANAFC ALEAGATHID TSVLGIGERN GITPLGGLMA
||||||||+| ||||||||+| |||||| ||| |||||||||| ||||||||||
Lys-5 CDIETHFHDD TGCAIANAYC ALEAGAYHID TSVLGIGERN GITPLGGLMA
Pc HC RMMVADREYV KSKYKLEKLK EIEDLVAEAV EVNIPFNNYI TGFCAFTHKA
||+| +|| |||||| ||| |+|||||||| |+| |||| | ||||||||||
Lys-5 RMIVTSPDYV KSKYKLHKLK ELEDLVAEAV EINTPFNNPI TGFCAFTHKA
Pc HC GIHAKAILNN PSTYEIINPA DFGMSRYVHF ASRLTGWNAI KSRAQQLKLE
|||||||||| ||||||+||| |||++||||| |||||||||+ |+|+ || ||
Lys-5 GIHAKAILNN PSTYEILNPA DFGLTRYVHF ASRLTGWNAV KTRVGQLGLE
Pc HC MTDTQYKECT AKIKAMADIR PIAVDDADSI IRAYHRNLKS GENKPLLDLT
||| | |||| |||||+||+| |||+|||||| || +| |+ + ++
Lys-5 MTDDQVKECT AKIKALADVR PIAIDDADSI IRTFHLGLHE QNKVQPPAVV
Pc HC AEEQAAFAAK EKELLEAQAA GLPV
+
Lys-5 EN
Figure 2. Comparison of the amino acid sequence of the homocitrate synthase of Penicillium chrysogenum (Gene Bank AJ223630) and Lys-5. Identical residues are indicated by a vertical line. Residues of similar chemical properties are indicated by a +. A motif conserved in all known homocitrate synthases is underlined.
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