Final published version
Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans
AU - Trueman, Laurence J.
AU - Richardson, Anna
AU - Forde, Brian G.
PY - 1996/10/10
Y1 - 1996/10/10
N2 - The crnA nitrate transporter from Aspergillus nidulans was identified as belonging to the major facilitator superfamily (MFS) of membrane transporters. Degenerate oligonucleotides corresponding to the crnA sequences at the locations of two conserved sequence motifs were designed and used in the polymerase chain reaction (PCR) to amplify related sequences from barley root poly(A)+ RNA. A 130 bp cDNA fragment with sequence similarities to crnA was amplified and used as a probe to screen a barley root cDNA library. Two full-length clones (pBCH1 and pBCH2) were isolated. The nt sequences of pBCH1 and pBCH2 are closely related (80% identical) and potentially encode hydrophobic polypeptides of 54.7 and 55.0 kDa respectively, with twelve predicted transmembrane domains. The encoded polypeptides are 41-43% identical to the A. nidulans CRNA protein and 56-57% identical to NAR-3, a high-affinity nitrate transporter from the eukaryotic alga Chlamydomonas reinhardtii. Phylogenetic analysis indicated that crnA, nar-3 and the barley homologues belong to a new family within the MFS, a family that also includes narK, the gene for a nitrite efflux pump in Escherichia coli. In northern blots, BCH1 hybridised to a mRNA species of 1.9 kb which is rapidly induced in barley roots by NO3-, but not by NH4+, and genomic Southern blots indicated that there may be seven to ten BCH1-related genes in the barley genome.
AB - The crnA nitrate transporter from Aspergillus nidulans was identified as belonging to the major facilitator superfamily (MFS) of membrane transporters. Degenerate oligonucleotides corresponding to the crnA sequences at the locations of two conserved sequence motifs were designed and used in the polymerase chain reaction (PCR) to amplify related sequences from barley root poly(A)+ RNA. A 130 bp cDNA fragment with sequence similarities to crnA was amplified and used as a probe to screen a barley root cDNA library. Two full-length clones (pBCH1 and pBCH2) were isolated. The nt sequences of pBCH1 and pBCH2 are closely related (80% identical) and potentially encode hydrophobic polypeptides of 54.7 and 55.0 kDa respectively, with twelve predicted transmembrane domains. The encoded polypeptides are 41-43% identical to the A. nidulans CRNA protein and 56-57% identical to NAR-3, a high-affinity nitrate transporter from the eukaryotic alga Chlamydomonas reinhardtii. Phylogenetic analysis indicated that crnA, nar-3 and the barley homologues belong to a new family within the MFS, a family that also includes narK, the gene for a nitrite efflux pump in Escherichia coli. In northern blots, BCH1 hybridised to a mRNA species of 1.9 kb which is rapidly induced in barley roots by NO3-, but not by NH4+, and genomic Southern blots indicated that there may be seven to ten BCH1-related genes in the barley genome.
KW - Barley
KW - crnA
KW - Major facilitator superfamily
KW - Multigene family
KW - narK
KW - Nitrate induction
KW - Roots
U2 - 10.1016/0378-1119(96)00154-0
DO - 10.1016/0378-1119(96)00154-0
M3 - Journal article
C2 - 8917103
AN - SCOPUS:0030579205
VL - 175
SP - 223
EP - 231
JO - Gene
JF - Gene
SN - 0378-1119
IS - 1-2
ER -