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Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha
P G Bruinenberg1, M Evers, H R Waterham
1Laboratory of Biochemistry, Groningen University, The Netherlands.
Abstract:
We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.
Insights
Researchers cloned the amine oxidase (AMO) gene from Hansenula polymorpha yeast. This enzyme is crucial for microbody function, and its gene sequence provides insights into protein import mechanisms.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Enzymology
Background:
- Microbody matrix enzymes play vital roles in cellular metabolism.
- Amine oxidase (AMO) is a key microbody enzyme.
- Understanding the genetic basis of AMO is essential for elucidating microbody biogenesis.
Purpose of the Study:
- To clone and characterize the AMO gene from Hansenula polymorpha.
- To determine the nucleotide sequence and genetic structure of the AMO gene.
- To investigate potential signals for protein targeting to microbodies.
Main Methods:
- Differential screening of a cDNA library.
- Immunoselection techniques.
- Screening of a genomic library and DNA sequencing.
Main Results:
- The AMO gene from Hansenula polymorpha was successfully cloned.
- A 3.6 kilobase DNA sequence containing the AMO gene was determined.
- The gene encodes a 692-amino acid protein with a molecular mass of 77,435 Da, and a potential topogenic signal sequence was identified.
Conclusions:
- The AMO gene has been isolated and sequenced from H. polymorpha.
- The deduced amino acid sequence provides information on the enzyme's structure and potential function.
- A specific tripeptide sequence suggests a role in targeting the enzyme to microbodies.