The techniques of molecular biology will be used to determine normal apo B gene structure as well as variation in the apo B gene that might underlie certain dyslipoproteinemias as well as atherosclerosis susceptibility.
The specific aims are in two sections: 1) To determine the structure of the apo B gene. Utilizing a liver cDNA expression library and polyclonal antisera to LDL, cDNA clones corresponding to apo B 1,500-1,600bp in length have been isolated. Northern blot analysis showed hepatic apo B mRNA to be 22kb in length, which means that the cDNA clones represent only 7% of apo B mRNA. Preliminary analysis indicates that the cDNA clones correspond to the region coding for the carboxy terminal 300-350 amino acids of apo B as well as the 3' untranslated region. To identify total apo B mRNA structure, these initial cDNA clones will be used as primers for extension on a template of HepG2 cell mRNA to allow cloning of additional cDNA sequences. Due to the length of apo B mRNA, this will have to be done in multiple steps to obtain cDNA corresponding to the most 5' portion of the apo B mRNA. These studies will allow us to deduce the primary amino acid sequence of apo B. The isolated cDNA clones will also be used to isolate from bacteriophage lambda and cosmid libraries clones containing apo B genomic regions. In this manner, the length of the apo B gene and its exon/intron configuration will be determined. Two subprojects will be emphasized: a) determining the relationship of B-48 and B-100, b) identification and characterization of the receptor-binding region. 2) to define human genetic variation in apo B. We intend to map apo B in the human genome and to use various probes for different regions of the apo B gene to identify restriction fragment length polymorphisms (RFLPs) that might be useful in clinical studies. We intend to see if any of these polymorphisms are associated with dyslipoproteinemia and/or atherosclerosis susceptibility. These RFLPs will also be useful in family studies to determine linkage of the apo B gene to a parameter that might segregate in a given family family such as atherosclerosis susceptibility or lipoprotein abnormality. Apo B gene abnormalities will be specifically sought in several human lipoprotein disorders, such as hyperapobetalipoproteinemia, abetalipoproteinemia, and normotriglyceridemic abetalipoproteinemia. If the abnormality in these disorders resides in the apo B gene, their specific molecular basis will be determined.
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