Alternative splicing of canine glutamine synthetase gene

Abstract

Glutamine synthetase (GS; EC 6.3.1.2) catalyzes the ATP-dependent conversion of ammonia and glutamate to glutamine. It is one of the key enzymes in nitrogen metabolism, and primarily used for ammonia detoxification and glutamine biosynthesis. Besides its role in nitrogen metabolism, GS is also known to play an important role in neurotransmitter recycling, converting neurotransmitter glutamate to glutamine, thereby preventing possible neurotoxicity. A GS genomic clone was obtained in Beagle genomic library screening. Genomic structure of GS was determined, showing seven exons and six introns as reported in other mammalians. In our study of canine GS gene, two forms of GS 5’ UTRs were found by 5’-cRACE. RT-PCR analysis and sequencing of the first intron revealed that these UTRs arose by the alternative splicing of the first intron. These two 5’ UTRs are exactly the same except that the longer 5’ UTR has a 120 bp insert in the middle. Sequence comparison of canine GS intron 1 with human GS intron 1 showed that human GS intron 1 has a similar 120bp-like insert, but RT-PCR analysis of human and mouse tissues showed that, contrary to dogs, they have only one form of 5’ UTR having no alternative splicing in intron 1. Semi-quantitative RT-PCR showed that both of the two 5’ UTRs are expressed in 12 tissues(frontal lobe, parietal lobe, occipital lobe, thalamus, medulla oblongata, cerebellum, lung, liver, heart, skeletal muscle, kidney, spleen), but their ratios are different. To study the influence of two UTRs on translational efficiency, plasmids containing either of two UTRs with a firefly luciferase gene were constructed and transfected into 293T cells. The luciferase assay didn’t show a significant difference between them.