In work initially intended to use the am gene coding sequences as a reporter gene, 5’ RACE PCR (Frohman et al., 1988 Proc. Natl. Acad. Sci. USA. 85:8998-9002) with three gene specific nested primers was performed. The product was cloned and sequenced, but found not to represent the am gene. Comparison to sequences in Genbank revealed that the product could encode a product homologous to glyceraldehyde-3-phosphate dehydrogenase (GPD) from a variety of other organisms. Consequently the PCR product was used to screen a lambda gt-11 expression library (Sachs et al. 1986 J. Biol. Chem 261:869-873). The 1.3 kb insert from one cDNA clone was sequenced (Figure 1) and used to screen a Neurospora genomic library made in an EMBL-3 vector by E. Cambareri. All of the positive clones had a 7 kb BamHI fragment. Relevant portions of one of the genomic clones was sequenced (Figure 1) revealing two introns. Although the complete genomic clone was not sequenced, comparison of restriction fragments from the cDNA and genomic clones indicated that no other introns are present in the Neurospora gpd-1 gene.
Southern blot analysis of restriction enzyme digested DNA from Oak Ridge and Mauriceville strains revealed a polymorphism of kpnI sites at or near the gpd-1 locus, allowing RFLP mapping using the small set of tester progeny as described by Metzenberg et al. (Metzenberg et al. 1984, Neurospora Newsl. 31:35-39). The results shown in Table 1 indicate that gpd-1 is located on linkage group IIR near the arg-12 locus. Northern blot analysis using gpd-1 cDNA as probe revealed a single strong band of 1.3 kb in length (data not shown).
One interesting question is how did we clone the gpd-1 fragment by 5’ RACE when we were using a nested set of three specific am primers for the amplification? When the sequence was analyzed it became apparent that each of the primers had 3’ ends with five-to-six base pairs of perfect complementarity to sequences near the 5’ end of the gpd-1 message and that these sequences appeared in the same order in the gpd-1 message as did the “specific” sequences in the am message. Given the abundance of gpd-1 message this made amplification of the 5’ end of the gpd-1 gene probable during the 5’ RACE experiment. Clones with either cDNA or genomic inserts are available from the Fungal Genetics Stock Center.
Table 1. RFLP mapping of gpd-1a.
GENE 11 12 13 14 15 16 17 18 19 20
arg-12 (O) 0 0 M M (M) M M O O
gpd-1 (O) O O M M (M) M M O O
21 22 23 24 25 26 27 28 29 30
arg-12 M O M O O M O O M M
gpd-1 M O M O O M O O M M
aA comparison of the segregation of the gpd-1 KpnI RFLP with segregation data for arg-12 which is located on LGIIR; strains numbered 11-30 represent FGSC strains 4411-4430. O or M in a particular strain indicates a fragment identical to that of the Oakridge or Mauriceville strain respectively. Strain 4411 is the Oak Ridge (O) parent and strain 4416 is the Mauriceville (M) parent.
CCCGGTGACG GAGTGCTCTG GCTGCTTGTT GGGAATTGCC GAGGCTCGCA ACTGGAGCAG 60
TCAGCAATGT CAGCATCGAC ATGTTCAAGT TGACTCATTT CAGTTGGTAT TACAAAGACT 120
GAACCCGTGA AGCACATAGC GTGACCGAAT CACGGATTCT CCGGCAAGGA GCTTGTTTCA 180
TTGTTGCCTC TTGTCGGCGG CTTTCAAAGC AAAAAAGGAT GGGAATCTCT TCATGCCAAG 240
GGCGCGGCCG AGTACTGCGC TAACACTAGA CGCCAAGCCA TTGGAGAGTG GCCCCACCTC 300
ATCCCACCAT GTCCCACCAC CACAGCCCAC CATGGAGCAA AGCGTATGAT GCAACCACGA 360
TGGGAGGCGG CTGGTGGGAT GGAAGGAACG AGCAAAACCA CCCACCCATT GACCACCCCA 420
CCCTCAAACC AAATTTATGT CGCTCATGCC ACCACGGTGA CATTTGGCAG GCATTGAGAG 480
CGTTCAGGGG GGTGATGAGG AGCTCCCCTC CTCTTTTGCC CCTCCTTGCC GACTGGGGAT 540
TACCACAGGC TGATAACCAG ACTGGACGCG AGCAGGGCAG CTGGAGTCGG CTGGGAAACT 600
AGATAATAGA TAGTACAAGA ATCTCCTCCT GCCTCCCAAC TTTTTTCTTT CTTTCTCTTG 660
CTTCATCATC ATCCTCGCGA TACCAAGTTC ACTTCCAACC AAAACCCTTC TTCCAAACCA
720
------------------intron I-------------------------
CATCAGGTAT GTTGTGACTG CCCTCGCATT TACAGAAACC GAGCTTCCTT CCTCAACACT 780
----------------------------------------------------------------
TCCAATCATC GTCACTTCCC TTGTCAGCGG CGGCGGCAGC AGCAGCAGTA GCAGAAGCAG 840
----------------------------------------------------------------
AAGCAGAAGC AGCAGCTACC CCGCACCTTC CTGACCCCGT CCCGACCCCG TCCCATCTCA 900
----------------------------------------------------------------
TCCTCAGTCA GTTCCTCCCG CCTCGCTGCC AAGCTGCGCA CAGCATCTGG TGTCTGCGTC 960
----------------------------------------------------------------
TGTTTCCCCC CAAGAGGAAG TGGACGAGAC TCAGATCGGA CTGGCATGGA TGCTGGTGGT 1020
----------------------------------------------------------------
GGTGGCGGCA TTGGAAGGGT TCCTCGGAAT CGCTCCTCCC CGATCCTACC TGCAGTCGGT 1080
----------------------------------------------------------------
CCCTCCGTGT TTTGGGCGCT CCTCGTGTCC AATTGTTCTG CCACGCAAAC ATGTGAACAG 1140
----------------------------------------------------------------
ACGAGACCGA ACAGGATAAG GAAGGGCAGG CAGACGAGTC CGGCTTTAAA ACCCAGACTT 1200
------------------------------------------------------
TCCTTCATCC TACCACTCAT CATCATCTTA CAACCTTCAA CAACTTGCTT CACAAGGTCT 1260
-------------------------
TGATACTTAC TCGTCTTCAC TCCAACAGTC AAC ATG GTC GTC AAG GTC GGC ATC
AAC 1318
M V V K V G I N
GGT TTC GGC CGT ATC GGT CGC ATT GTC TTC CGC AAT GCC ATT GAG CAC GAT GAC
1371
G F G R I G R I V F R N A
I E H D D
---
ATC CAC ATC GTC GCT GTC AAC GAC CCC TTC ATT GAG CCC AAG TAC GCT GTAAGTT
1425
I H I V A V N D P F I E P
K Y A
-------------------intron 2-----------------------------
GGCC TCGCTCACAT AGATCCCTTG TCTCATATGACAACTCAGAC TCTGACCATC ATCCCT 1486
------
CTTA CAG GCT TAC ATG CTC CGC TAC GAC ACC ACC CAC GGC AAC TTC AAG GGC ACC
1541
A Y M L R Y D T T H G
N F K G T
ATC GAG GTT GAC GGT GCT GAC CTC GTC GTC AAC GGC AAG AAG GTC AAG TTC TAC 1595
I E V D G A D L V V N G K
K V K F Y
ACT GAT GCC GAC CCC GCT GCC ATC CCC TGG TCC GAG ACC GGT GCC GAC TAC ATT 1649
T D A D P A A I P W S E T
G A D Y I
GTC GAG TCC ACT GGT GTC TTC ACC ACC ACC GAG AAG GCC TCC GCC CAC TTG AAG
1703
V E S T G V F T T T E K A
S A H L K
GGT GGT GCC AAG AAG GTC ATC ATC TCT GCC CCC TCT GCT GAT GCC CCC ATG TAC 1757
G G A K K V I I S A P S A
D A P M Y
GTT ATG GGT GTC AAC AAC GAG ACC TAC GAT GGC TCC GCC GAC GTC ATC TCC AAC 1811
V M G V N N E T Y D G S A D
V I S N
GCC TCT TGC ACC ACC AAC TGC TTG GCT CCC CTC GCC AAG GTC ATC CAC GAC AAC 1865
A S C T T N C L A P L A K
V I H D N
TTC ACC ATC GTC GAG GGT CTC ATG ACC ACC GTC CAC TCC TAC ACC GCC ACC CAG 1919
F T I V E G L M T T V H S
Y T A T Q
AAG ACC GTC GAT GGT CCT TCC GCC AAG GAC TGG CGC GGT GGC CGC ACT GCT GCT 1973
K T V D G P S A K D W R G G
R T A A
CAG AAC ATC ATT CCC AGC AGC ACT GGT GCC GCC AAG GCC GTC GGC AAG GTC ATC 2027
Q N I I P S S T G A A K A
V G K V I
CCC GAC CTC AAC GGC AAG CTC ACT GGT ATG GCC ATG CGT GTC CCC ACC GCC AAC 2081
P D L N G K L T G M A M R V
P T A N
GTC TCC GTT GTC GAT CTT ACT GCC CGC ATC GAG AAG GGT GCT ACC TAC GAT GAG 2135
V S V V D L T A R I E K G
A T Y D E
ATC AAG GAG GTC ATC AAG AAG GCC TCT GAG GGT CCC CTC GCT GGC ATC CTT GCC 2189
I K E V I K K A S E G P L
A G I L A
TAC ACC GAG GAT GAG GTT GTC TCT TCC GAC ATG AAC GGC AAC CCC GCC TCC TCC 2243
Y T E D E V V S S D M N G N
P A S S
ATC TTC GAT GCC AAG GCT GGT ATC TCC CTC AAC AAG AAC TTC GTC AAG CTT GTC 2297
I F D A K A G I S L N K N F
V K L V
TCC TGG TAC GAC AAC GAG TGG GGC TAC TCT CGC CGT GTC CTC GAC CTC ATC TCC 2351
S W Y D N E W G Y S R R V L
D L I S
TAC ATC TCC AAG GTC GAT GCC AAG AAG GCT TAA ATCGGT TGCGTACCCGCACGGTTA 2408
Y I S K V D A K K A
TG AAGTAATGGT CTTTTCCTAG ATATGAAGAA AAAAAAAGGG CAATGATTCC GTGGGATT 2468
GAACTCGAGCAT GTTGGATCTC GGGCAGTCCT GCTTAAAGTA AAATAATATC CGAACTCAA 2528
ATAG ATACCAAGTTCACTTCG 2552
Figure 1. Sequence of the gpd-1 gene. The sequence presented
represents a combination of sequences from cDNA and genomic DNA. The first
nucleotide of the cDNA sequenced is at 677. This is 5 nucleotides downstream
of a consensus fungal transcriptional start site at position 666-673 (Bruchez
et al. 1993 Fungal Genet. Newsl. 40:89-96). The pyrimidine box
characteristically found upstream of the transcriptional start sites of fungal
genes is underlined. The two introns are indicated by dashed overlining.
There was no polyadenylated tract in the cDNA sequenced