Cloning of rabbit HPRT gene using the recombineering system.
Hypoxanthine phosphoribosyltransferase (HPRT) plays an important role in the metabolic salvage of purines, and been used as an alternative pathway for mutant selection in many studies. To facilitate its application in rabbits, we have cloned the cDNA and genomic DNA of the rabbit HPRT gene using an approach that combines bioinformatics and recombineering methods. The cDNA is comprised of 1449 bp containing a coding sequence for a protein of 218 amino acids. The deduced amino acid sequence of the rabbit HPRT gene shares 98%, 97%, 98% and 94% identity with human, mouse, pig and cattle HPRT genes, respectively. Reverse transcription-polymerase chain reaction analysis showed that this gene is ubiquitously expressed in tissues of adult rabbit. The rabbit HPRT gene spans approximately 48 kb in length and consists of nine exons. The cloning of the rabbit HPRT gene shows the usefulness of the recombineering system in cloning genes of large size. This system may facilitate the subcloning of DNA from bacterial artificial chromosomes for cloning genes of large size or filling big gaps in genomic sequencing.
Program for Graduation Studies, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Jianjun
J
Shi
Donghui
D
Cai
Xuejin
X
Chen
Huizheng
H
Sheng
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular; D003198:Computer Simulation; D018076:DNA, Complementary; D030541:Databases, Genetic; D053595:Embryonic Stem Cells / Q000166:cytology; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D020224:Expressed Sequence Tags; D015870:Gene Expression; D016678:Genome; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007438:Introns; D008957:Models, Genetic; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D018076:DNA, Complementary; D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase