Abstract
The metabolism of low density lipoprotein (LDL) in the hamster is substantially similar to that of the human. To extend the usefulness of the hamster as an experimental model, the hamster LDL receptor gene was isolated and characterized. The gene is composed of 18 exons and 17 introns which span 26 kilobases. The introns occur at precisely the same positions as those previously determined for the human LDL receptor gene. The 18 exons of the hamster gene predict an LDL receptor protein of 854 amino acids that is similar in organization and sequence to those predicted from the cDNAs of rat, rabbit, cow, and human. Within the 5'-flanking region of the hamster LDL receptor gene are three highly conserved imperfect direct repeat sequences of 16 nucleotides each that in the human gene have been demonstrated to regulate transcription. In addition, a similar arrangement of direct repeat sequences was also isolated from the 5'-flanking region of the rat LDL receptor gene using the polymerase chain reaction. These results indicate a strong sequence and structural conservation of the LDL receptor among several species and further support the hamster as an experimental model for the study of human LDL-cholesterol metabolism.
Highlights
The metabolism of low density lipoprotein (LDL) in the hamster is substantially similar to that of the human
Probes derived from the human LDL receptor complementary DNA (cDNA), pLDLR-2 [19], were used to obtain clone hU4 from approximately 1x lo6 clones of a Chinese hamster-derived UT-1 cell genomic library [23]
A total of four genomic clones spanned all 18 exons and 17 introns of the gene and included 9.6 kb of the 5'-flanking region; the 3' untranslated region was not represented in the isolated clones (Fig. 1)
Summary
All methods were performed according to standard procedures [22]. Probes derived from the human LDL receptor cDNA, pLDLR-2 [19], were used to obtain clone hU4 from approximately 1x lo clones of a Chinese hamster-derived UT-1 cell genomic library [23]. A primer-specific, size-fractionated cDNA library was constructed from CHO cell poly(A)+RNA by standard methodologies [22] using an antisense oligonucleotide primer, complementary to nucleotide positions 106 to 140 of Fig. 2, in place of oligo(dT). Positive clones were identified from approximately 2 x lo recombinants using a "P-labeled antisense oligonucleotide complementary to nucleotide positions 68 to 104 of Fig. 2 and subcloned into bacteriophage M13 vectors for DNA sequencing as described above. The annealing reaction was carried out at 65°C using a 32P-labeled oligonucleotide complementary to nucleotides 68 to 104 of Fig. 2 and either 5 pg of poly(A)+RNA or 10 pg of total RNA isolated from CHO cells grown in the presence or absence of sterols [29]
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