Demonstration of physical linkage between the promoter region and the polymorphic kringle IV domain in the Apo(a) gene by pulsed-field gel electrophoresis
| Autor: | Nicoletta Malgaretti, Silvano Köchl, Francesco Acquati, Hg Kraft, Roberto Taramelli, Gerd Utermann |
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| Rok vydání: | 1993 |
| Předmět: |
Gel electrophoresis
Apolipoprotein B biology Genetic Linkage Promoter Apoprotein(a) Molecular biology Electrophoresis Gel Pulsed-Field genomic DNA Apolipoproteins Restriction map Genes Kringles Biochemistry Complementary DNA Genetics biology.protein Humans lipids (amino acids peptides and proteins) Promoter Regions Genetic Gene Alleles Lipoprotein(a) Southern blot |
| Popis: | Apolipoprotein(a) is a large glycoprotein that together with a low-density lipoprotein (LDL) particle forms lipoprotein(a) or Lp(a) is a quantitative genetic trait in human plasma. High concentrations of Lp(a) are a genetic risk factor for premature CHD and stroke. Knowledge of apo(a) gene structure and understanding the regulation of Lp(a) plasma levels may therefore be of practical importance. To demonstrate physical linkage of the 5[prime] apo(a) promoter region with the kringle IV repeat region in genomic DNA, the authors have capitalized from the size variation in the apo(a) gene. Here they demonstrate that the 5[prime] promoter-linked sequence is on a polymorphic DNA fragment that also contains the variable number of kringle IV repeats. Apo(a) protein phenotypes were determined by immunoblotting and the corresponding apo(a) KpnI DNA phenotypes by pulsed-field gel electrophoresis (PFGE) in a total of 235, subjects as described. PFGE Southern blotting was performed using a panel of rare-cutting enzymes and mixtures thereof (BssHII, FspI, NruI, KspI, NotI, and SfiI). The blots were probed with a digoxigenin (DIG)-labeled apo(a) kringle IV cDNA probe and then with a radioactively labeled probe for the 5[prime] region. The results allowed the construction of a long-range restriction map of the apo(a) gene region.more » From this map it can be seen that the smallest fragments containing physically linked apo(a) promoter an the apo(a) kringle IV region resulted from double digests with NruI + KspI or NruI + FspI. The size of the KspI-NruI fragments ([approx]100 kb in the case of a KpnI allele of 66 kb) demonstrates close physical linkage and suggests that the investigated promoter indeed belongs to the apo(a) structural gene.« less |
| Databáze: | OpenAIRE |
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