The human hyaluronan synthase genes: genomic structures, proximal promoters and polymorphic microsatellite markers

Identification and Analysis of the Promoter Region of the Human Hyaluronan Synthase 2 Gene

“…Previously, we carried out 5Ј-RACE analysis on HAS2 from total RNA (17). In the present study, mRNA was purified from HK-2, HEK293t, and TE-671 cells and used as the template for 5Ј-RACE reactions.…”

“…PCR amplification of promoter fragments was carried out from gDNA by standard means (23). PCR products were gel-purified as described above, digested (restriction endonucleases were from New England Biolabs, Hitchin, Hertfordshire, UK), cloned into a modified pGL-3 luciferase reporter vector (17), and sequenced to ensure fidelity of amplification using vector-specific primers RV3 (5Ј-CTAGCAAAATAGGCTGTCCC-3Ј) and GL-2 (5Ј-CTTTATGTTTTTGGCGTCTTCC-3Ј) according to the manufacturer's instructions. The ability of each HAS2 promoter fragment to drive transcription of the luciferase gene was tested transiently in human cell lines HEK293t and TE-671 as described in detail previously (17)(18)(19), with the following amendment to maximize luciferase output.…”

“…PCR products were gel-purified as described above, digested (restriction endonucleases were from New England Biolabs, Hitchin, Hertfordshire, UK), cloned into a modified pGL-3 luciferase reporter vector (17), and sequenced to ensure fidelity of amplification using vector-specific primers RV3 (5Ј-CTAGCAAAATAGGCTGTCCC-3Ј) and GL-2 (5Ј-CTTTATGTTTTTGGCGTCTTCC-3Ј) according to the manufacturer's instructions. The ability of each HAS2 promoter fragment to drive transcription of the luciferase gene was tested transiently in human cell lines HEK293t and TE-671 as described in detail previously (17)(18)(19), with the following amendment to maximize luciferase output. The growth medium used was a 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's F-12 nutrient mixture without L-glutamine (Invitrogen) supplemented with 20 mM HEPES and 2 mM Lglutamine (both from Invitrogen) together with 5 ng/ml sodium selenite, 5 g/ml insulin, 5 g/ml transferrin, and 0.4 g/ml hydrocortisone (all from Sigma).…”

“…1, specific to sequence AJ604570 and comprising the 5Ј-UTR and the N-terminal amino acid codons of HAS2. The smaller band of 282 bp amplified codons from the glycosyltransferase domain of HAS2 using primers H2-F and -R. To obviate the possibility of amplification from contaminating gDNA, both sets of RT-PCR primers amplified across one or more intron/exon boundaries spanning at least 11.5 kbp of intronic sequence (17). Orthologous products from the total RNA of mouse and rat kidneys were also amplified.…”

“…We have recently described the genomic structures of the human HAS genes (17) and demonstrated that ϳ500 bp upstream of each HAS reference mRNA sequence showed constitutive promoter activity in a high-throughput luciferase analysis system (18,19). The sequence upstream of HAS2 was the least active in this assay but still conformed to our criteria for promoter activity (18).…”