Cloning of the homogentisate 1,2-dioxygenase gene, the key enzyme of alkaptonuria in mouse

Cloning of the homogentisate 1,2-dioxygenase gene, the key enzyme of alkaptonuria in mouse We determined 48 amino acid residues from five pep-tides from the homogeneous monomer of homogentisate 1,2-dioxygenase (HGO; E.C. 1.13.11.15) of mouse liver. After digestion with trypsin, peptides were separated by reversed phase chromatography and amino acid sequenced. The deduced codon sequence of three peptides was used to derive degenerated oligo-meres. By combining these oligos, we were able to amplify fragments from 100 to 300 bases (b) from mouse liver cDNA by polymerase chain reaction after reverse transcription (RT-PCR). A fragment of 200 b was cloned and used as a probe to screen a mouse liver cDNA library. One clone from this library contained the complete cDNA-insert for HGO as determined by sequencing. The cDNA encodes for a protein of 50 kDa, as predicted. The cDNA of mouse HGO has an overall identity of 41% to the corresponding gene hmgA from Aspergillus. Sequence similarities to human expressed sequence tags (EST) clones ranged from 70% to 20%. The positions of 122 conserved amino acids could be determined by multiple sequence alignment. We identified one first intron of 928 b in the mouse gene. The gene for HGO seems to be expressed in various tissues, as shown by RT-PCR on different cDNAs. FISH experiments with the whole murine cDNA as probe clearly revealed signals at the human chromosomal band 3ql3.3–q21. This corresponds well to the previous assignment of the locus for the human alkaptonuria gene (AKU) to the same chromosomal region by multipoint linkage analysis. We therefore conclude that the HGO cDNA encodes the gene responsible for alkaptonuria. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Mammalian Genome Springer Journals

Cloning of the homogentisate 1,2-dioxygenase gene, the key enzyme of alkaptonuria in mouse

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Publisher
Springer-Verlag
Copyright
Copyright © 1997 by Springer-Verlag
Subject
Life Sciences; Cell Biology; Anatomy; Zoology
ISSN
0938-8990
eISSN
1432-1777
D.O.I.
10.1007/s003359900383
Publisher site
See Article on Publisher Site

Abstract

We determined 48 amino acid residues from five pep-tides from the homogeneous monomer of homogentisate 1,2-dioxygenase (HGO; E.C. 1.13.11.15) of mouse liver. After digestion with trypsin, peptides were separated by reversed phase chromatography and amino acid sequenced. The deduced codon sequence of three peptides was used to derive degenerated oligo-meres. By combining these oligos, we were able to amplify fragments from 100 to 300 bases (b) from mouse liver cDNA by polymerase chain reaction after reverse transcription (RT-PCR). A fragment of 200 b was cloned and used as a probe to screen a mouse liver cDNA library. One clone from this library contained the complete cDNA-insert for HGO as determined by sequencing. The cDNA encodes for a protein of 50 kDa, as predicted. The cDNA of mouse HGO has an overall identity of 41% to the corresponding gene hmgA from Aspergillus. Sequence similarities to human expressed sequence tags (EST) clones ranged from 70% to 20%. The positions of 122 conserved amino acids could be determined by multiple sequence alignment. We identified one first intron of 928 b in the mouse gene. The gene for HGO seems to be expressed in various tissues, as shown by RT-PCR on different cDNAs. FISH experiments with the whole murine cDNA as probe clearly revealed signals at the human chromosomal band 3ql3.3–q21. This corresponds well to the previous assignment of the locus for the human alkaptonuria gene (AKU) to the same chromosomal region by multipoint linkage analysis. We therefore conclude that the HGO cDNA encodes the gene responsible for alkaptonuria.

Journal

Mammalian GenomeSpringer Journals

Published: Mar 23, 2009

References

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