The atherogenic macromolecule lipoprotein(a) [Lp(a)] has resisted in vvo analyses partly because it is found in a limited number of experimental animals. Although transgenic mice expressing human apolipoprotein (a) [apo(a)] have previously been described, they faied to assemble Lp(a) particles because of the inability of human apo(a) to associate with mouse apolipoprotein B (apoB). We isolated a 90-kilobase P1 paemid containing the human apoB gene and with this DNA generated 13 lines of tnsgec mice of which 11 expressed human apoB. The human apoB transcript was expressed and edited in the liver of the transgenic mice. In humans, the association between apo(a) and LDL to form the Lp(a) particle is the result of a disulfide linkage between apo(a) and apolipoprotein B (apoB) of LDL (4, 5, 7). Transgenic mice that express human apo(a) have been engineered, but, unlike in humans, the apo(a) in these animals was found in the lipoprotein-free plasma fraction and was not associated with mouse LDL (8, 9). Infusion ofisolated human LDL, but not mouse LDL, into apo(a) transgenic animals results in the appearance of Lp(a) particles (8). This suggests that the failure to form Lp(a) in the apo(a) transgenic mice is caused by the inability of mouse apoB-containing LDL to properly interact with human apo(a). Thus, the development of mice to examine the biological properties of Lp(a) in vivo requires the creation of animals that express both human apoB and apo(a) transgenes to facilitate assembly of Lp(a).ApoB is one ofthe largest known proteins, with 4536 amino acids (550 kDa) (10). In addition to the full-length molecule, a truncated version of apoB (B48) is produced from edited apoB mRNA transcripts exclusively in the intestines of nonrodent mammals and in the intestines plus the livers of rodents (11). The large size of the apoB gene, >43 kb, has made it difficult to clone an intact apoB genomic fragment in A phage or cosmid vectors for introduction into the mouse genome.A recently developed cloning system using P1 phagemids allows the cloning of 80-to 90-kb DNA inserts (12, 13). We have isolated a clone containing the entire human apoB gene plus extensive 5' and 3' regions from a human P1 library. This DNA was introduced into fertilized mouse embryos, and nearly all of the resulting transgenic mice had significant plasma levels of human apoB and increased plasma levels of LDL-cholesterol. Mice containing both human apo(a) and human apoB transgenes efficiently assembled Lp(a) particles.

The araC gene encodes a positive regulatory protein required for L-arabinose utilization in Escherichia coli. Transcription from the araC promoter has been shown to be under positive control by cAMP receptor protein and under negative control by its protein product (autoregulation). This work describes the identification of the region of the araC promoter that interacts with the cAMP receptor protein to mediate catabolite repression. A 3-base-pair deletion centered 60 base pairs from the transcriptional initiation site results in a mutant araC promoter that, in the absence of araC protein, reduces transcriptional activity when compared with the wildtype promoter and is unresponsive to various concentrations of intracellular cAMP in vivo. The same deletion results in a lowered affinity of the araC promoter for cAMP receptor protein in vitro. However, this lowered affinity for the mutant araC promoter does not result in substantial reduction of intracellular araC protein because autoregulation of the araC gene dominates catabolite repression. The 3-base-pair deletion in the cAMP receptor protein binding site of the araC promoter does not affect catabolite repression of the adjacent araBAD operon. The implications of these results on current models for expression of the araBAD operon and the araC gene are discussed.L-Arabinose utilization in the bacterium Escherichia coli B/r requires the activation of three unlinked genetic loci by a single regulatory gene, araC (1, 2). The araBAD operon encodes three enzymes that are responsible for the initial catabolism of L-arabinose; the araE and araF genes encode proteins that are responsible for the transport of L-arabinose into the bacterium. In the presence of L-arabinose, araC protein is an activator of transcription for the araBAD operon and the araE and araF genes (1, 2). In the presence or absence of L-arabinose, araC protein is a repressor of its own synthesis (3, 4). In addition to regulation by its own protein product, araC gene expression is under positive control of the cAMP receptor protein (CRP) (3).The regulatory region for the araC gene is adjacent to the regulatory region for the araBAD operon. Their respective promoters are transcribed in opposite directions (5) and their transcriptional initiation sites are separated by 147 base pairs (bp) (6). The nucleotide sequence of this region of DNA has been determined (7,8) and it will be referred to as the ara regulatory region. Binding sites for regulatory proteins have been identified between the two transcriptional initiation sites (refs. 4 and 9; see Fig. 1). The localization of the regulatory protein binding sites in the promoter region has been the basis for several proposed models of the regulation of the araBAD operon and the araC gene (4, 9, 10). The portions of these models that are relevant to this work are the following. The DNase I protection studies form the basis for the most recent models for araBAD and araC expression (4, 9). Although consistent with physiological data, these models have...

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Human carriers of apolipoprotein (apo) A-IMilano are heterozygous for an Arg173-->Cys substitution in the apoA-I primary sequence; despite severe reductions in HDL cholesterol concentrations, affected individuals do not develop coronary heart disease, suggesting that apoA-IMilano may possess antiatherogenic properties. As the beneficial effects of wild-type apoA-I are linked to its role in HDL cholesterol transport, we examined the capacity of apoA-IMilano to recruit cell cholesterol and activate lecithin:cholesterol acyltransferase (LCAT) (two key events in the antiatherogenic reverse cholesterol transport pathway). ApoA-IMilano and wild-type apoA-I were expressed in Chinese hamster ovary cells, and their ability to recruit membrane phospholipid and cholesterol for the assembly of nascent HDL was compared. Both clonal cell lines exhibited similar levels of apolipoprotein accumulation in serum-free medium (approximately 2 micrograms/mg cell protein per 24 hours), and 15% of each apolipoprotein was associated with membrane lipids to form nascent HDL (d = 1.063 to 1.21 g/mL). SDS-PAGE showed that a majority (66 +/- 12%) of the lipidated apoA-IMilano was in the homodimer form. Compositional analyses revealed that apoA-IMilano nascent HDL had a significantly lower (P < .001) unesterified cholesterol/phospholipid mole ratio (0.47 +/- 0.10) than wild-type apoA-I complexes (1.29 +/- 0.14), indicating that apoA-IMilano had a reduced capacity to recruit cell cholesterol. In addition to the reduced unesterified cholesterol/phospholipid ratio, apoA-IMilano nascent HDL consisted mostly of small 7.4-nm particles compared with wild-type apoA-I, in which 11- and 9-nm particles predominated. Despite these changes in nascent HDL particle size and composition, apoA-IMilano activated LCAT normally. We conclude that, even though apoA-IMilano is a normal activator of LCAT, it is less efficient that wild-type apoA-I in recruiting cell cholesterol, suggesting that the putative antiatherogenic properties attributed to apoA-IMilano may be unrelated to the initial stages of reverse cholesterol transport.