Structure and chromosomal localization of the human renal kallikrein gene

BA Evans, ZX Yun, JA Close, GW Tregear… - Biochemistry, 1988 - ACS Publications
BA Evans, ZX Yun, JA Close, GW Tregear, N Kitamura, S Nakanishi, DF Callen, E Baker…
Biochemistry, 1988ACS Publications
Materials and Methods Screening of a Human Parotid Gland cDNA Library. Poly (A) RNA
was isolated from human parotid gland ac-cording to the procedures of Chirgwin et al.(1979)
and Aviv and Leder (1972). A cDNA library of 50 000 clones in the XgtlO vector was
constructed as described by van Leeuwen et al.(1986). The library was screened with an
oligodeoxyribonucleotide probe (HK-1) designed to detect all human kallikrein genes. The
probe corresponds to amino acids 85-107 predicted from the renal kallikrein cDNA …
Materials and Methods
Screening of a Human Parotid Gland cDNA Library. Poly (A) RNA was isolated from human parotid gland ac-cording to the procedures of Chirgwin et al.(1979) and Aviv and Leder (1972). A cDNA library of 50 000 clones in the XgtlO vector was constructed as described by van Leeuwen et al.(1986). The library was screened with an oligodeoxyribonucleotide probe (HK-1) designed to detect all human kallikrein genes. The probe corresponds to amino acids 85-107 predicted from the renal kallikrein cDNA (Fukushima et al., 1985). All oligodeoxyribonucleotide probes were synthesized in an Applied Biosystems Inc. Model 380A DNA synthesizer and purified by polyacrylamide gel electrophoresis. Identification of the Human Renal Kallikrein Gene. The probe used (HRK-1) was a 30-mer corresponding to amino acids 142-151 of human renal kallikrein (Fukushima et al., 1985). The oligodeoxyribonucleotide was labeled with T4 polynucleotide kinase (Pharmacia) and [y-32P] ATP (Am-ersham Corp.).
Phage suspensions (1 yL) corresponding to 54 kallikrein-positive genomic clones (Fukushima et al., 1985) were spotted onto a lawn of LE392 cells and incubated at 37 C overnight. Phage DNA was transferred to nitrocellulose, and the filters were prehybridized at 42 C for 4 h in the 20% formamide buffer described by Ullrich et al.(1984). Following addition of the probe, hybridization was continued overnight at 42 C; then the filters were washed in 2 X standard saline citrate at 60 C and exposed at-80® C with intensifying screens to Kodak XAR-5 film.
ACS Publications