A polymorphism in a region with enhancer activity in the second intron of the human apolipoprotein B gene

A 443-base pair fragment (+622 to +1064) from the second intron of the human apolipoprotein B gene was shown to contain a tissue-specific enhancer when placed in front of an apolipoprotein B promoter-chloramphenicol acetyltransferase construct in transfection experiments. To identify potential regulatory mutations in this region of the gene, DNA from various subjects was examined for the presence of point mutations by means of chemical cleavage of mismatched heteroduplexes. An A----G substitution within the second intron of the gene at position +722 was identified in three unrelated subjects and confirmed by DNA sequencing. Although the base substitution was contained within a nuclear protein-binding site, as determined by DNase I footprinting, it did not appear to affect the protein/DNA interaction in its vicinity, as shown by gel retardation experiments. The single base substitution at position +722 abolishes a StyI restriction site, thus creating a StyI polymorphism. Using allele-specific oligonucleotides, we screened the DNA of 172 subjects for the presence of this polymorphism: two other subjects carrying the polymorphism were found. In each of the five unrelated subjects, the polymorphism was associated with the same haplotype.     

Identification of three sequence-specific DNA-binding proteins which interact with the Rous sarcoma virus enhancer and upstream promoter elements.

Three avian nuclear proteins which bind to the Rous sarcoma virus long terminal repeat have been detected. Two of the proteins bind to sequences within the enhancer, and the third protein binds to a sequence spanning the enhancer and an upstream promoter region.     

The 5' boundary of the human apolipoprotein B chromatin domain in intestinal cells

The 5' boundary of the chromosomal domain of the human apolipoprotein B (apoB) gene in intestinal cells has been localized and characterized. It is composed of two kinds of boundary elements; the first, functional boundary is an insulator activity exhibited by a 1.8 kb DNA fragment located between -58 and -56 kb upstream of the human apoB promoter. In this region, an enhancer-blocking activity has been mapped to a CTCF binding site that is located upstream of two apoB intestinal enhancers (IEs), the 315 IE and the 485 IE. The CTCF site represents a boundary between two types of chromatin structure: an open, DNaseI-sensitive region 3' of the CTCF site containing the intestinal regulatory elements and a closed, DNaseI-resistant region 5' of the CTCF site. The 1.8 kb fragment harboring the CTCF site also insulated mini-white transgenes against position effects in Drosophila melanogaster. The second, structural boundary is represented by a nuclear matrix attachment region (MAR), situated about 3 kb 5' of the CTCF site. This MAR may represent the 5' anchorage site for a chromosomal loop that functions to bring the intestinal regulatory elements closer to the apoB promoter.     

Single base substitution between human intestinal and hepatic apolipoprotein B mRNA detected by ribonuclease cleavage analysis

The molecular mechanism of human intestinal apolipoprotein (apo) B-48 synthesis has been elucidated by a combination of sequencing of cloned complementary DNAs and RNase cleavage analysis of RNA heteroduplex. All intestinal cDNA clones contained a single C to T base substitution in the codon CAA encoding Gln2153 in apoB-100 cDNA, resulting in a translational stop. One of the our intestinal apoB cDNA clones was polyadenylated 106 bases downstream from the stop codon, possibly producing a 7-kb apoB message in the intestine. RNase cleavage analysis of the RNA heteroduplex between hepatic or intestinal RNA and apoB cDNA-directed anti-sense RNA showed that this single C to U substitution may occur in most of intestinal apoB mRNA. These results suggested that human apoB-48 is mostly produced by apoB mRNA with an in-frame stop codon in the intestine.     

The origin recognition complex marks a replication origin in the human TOP1 gene promoter

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the human TOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation.     

Analysis of two different tandem repetitive elements within the human apolipoprotein B gene.

A large number of copies of the sequence (dTG-dAC)n, where n is between 10 and 60, exist in the human genome, and many are useful as polymorphic markers. One of these sequences occurs about 3 kilobases 5' of the human apolipoprotein (apo) B gene as seven distinguishable alleles containing from (TG)12 to (TG)18. This repeat is also present in the DNA of other primates. A second alternating purine-pyrimidine sequence with nine dinucleotide repeats and located in intron 4 is not polymorphic. Together with the apoB hypervariable repeat immediately 3' of the gene, the (TG)n sequence will provide a useful haplotype marker capable of distinguishing a large number of human apoB alleles, some of which may be associated with disease states.