The isolation and characterisation of a cDNA clone encoding L-asparaginase from developing seeds of lupin (Lupinus arboreus).
An L-asparaginase cDNA clone, BR4, was isolated from a Lupinus arboreus Sims developing seed expression library by screening with polyclonal antibodies to the seed asparaginase. The cDNA hybridised with an oligonucleotide probe designed from amino acid sequence data and was found on sequencing to be 947 bp in length. Six polypeptide sequences obtained previously could be placed along the longest open reading frame. Computer-aided codon use analysis revealed that the cDNA sequence was consistent with other plant genes in terms of codon use. The cDNA insert was used to analyse asparaginase transcription in various tissues by northern blot analysis. A transcript size of approximately 1.2 kb was detected in L. arboreus seed total and poly(A)+ RNA. The level of this transcript declined from 30 days after anthesis to an undetectable level by day 55. Furthermore, under the high stringency conditions used, the seed asparaginase cDNA did not hybridise with total or poly(A)+ RNA isolated from root tips, suggesting that the asparaginase known to be present in this tissue may be the product of a different gene. Southern analysis suggested the seed asparaginase is a single-copy gene. The plant asparaginase amino acid sequence did not have any significant homology with microbial asparaginases but was 23% identical and 66% similar (allowing for conservative substitutions) to a human glycosylasparaginase.
Lough
T J
TJ
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Reddington
B D
BD
Grant
M R
MR
Hill
D F
DF
Reynolds
P H
PH
Farnden
K J
KJ
D000595:Amino Acid Sequence; D001215:Asparaginase / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D010946:Plants, Medicinal; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D012639:Seeds / Q000201:enzymology; D012689:Sequence Homology, Nucleic Acid; D012984:Software
D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA; D001215:Asparaginase