Differential expression of multiple glutaminase mRNAs in LLC-PK1-F+ cells.
LLC-PK1-F+ cells are porcine proximal tubule-like cells that have been used to model the renal ammoniagenic response to metabolic acidosis. A 3.2-kb porcine glutaminase (GA) cDNA (pGA201) containing 528 bp of coding sequence and 2.7 kb of 3'-untranslated region was cloned and sequenced. Probes derived from both porcine and rat GA cDNAs were used to characterize the expression of putative GA mRNAs in LLC-PK1-F+ cells. Two larger putative GA mRNAs (approximately 5.0 and 4.5 kb in length) were resolved and a smaller 2.5-kb species was also observed. The level of the 5.0-kb mRNA is detectable in freshly split LLC-PK1-F+ cells and increases as the cells reach confluence. In contrast, the amount of the 4.5-kb GA mRNA is greatest in freshly split cells and decreases gradually as the cells approach confluence. The levels of the 5.0- and 2.5-kb mRNAs are also affected by refeeding the cells, and the 2.5-kb mRNA accumulates to high levels if cells are retained in the same media for 4 days. Exposure to acidic media had little or no effect on the levels of GA mRNAs expressed in confluent or postconfluent cells, whereas, in growing and undifferentiated cells, this treatment did affect the level of the 4.5-kb mRNA. Thus the putative GA mRNA species are differentially expressed. Given this complexity, a careful assessment of GA mRNA species, of basal expression, and of growth conditions are essential for a meaningful analysis of GA mRNA levels in cultured cells.
Porter
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Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins 80523, USA.
Hansen
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Taylor
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Curthoys
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D000143:Acids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002454:Cell Differentiation; D002455:Cell Division; D003470:Culture Media / Q000494:pharmacology; D018076:DNA, Complementary / Q000032:analysis; D005972:Glutaminase / Q000235:genetics*; D018374:LLC-PK1 Cells; D008297:Male; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine; D014018:Tissue Distribution
D000143:Acids; D003470:Culture Media; D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D005972:Glutaminase