We report the cloning of cDNAs for rat liver apolipoprotein B (apo B) and the use of the cloned sequences to examine apo B expression at the level of mRNA in rat tissues. Fifteen putative apo B clones were identified by antibody screening of a rat liver cDNA library in the Xgtll expression vector. The identity of the clones was confirmed by immunological studies of the fusion protein products. All clones appear to contain sequences found only in apo B PI, the high molecular weight form of rat liver apo B. Blotting studies show that the clones hybridize to a single 20-kilobase liver mRNA species, sufficiently large to encode the entire apo B PI peptide, which is estimated to be 400 kDa in size. Apo B PI mRNA is abundant in liver and present in lower amounts in intestine but is absent in a variety of other tissues examined. This tissue distribution is consistent with that expected from studies on the in vivo synthesis of apo B. One clone, corresponding to a 240-base segment of the apo B PI mRNA, was sequenced and found to exhibit homology with a short region of rat apo E mRNA. Analysis of the secondary structure of the corresponding peptide did not show the preponderance of amphipathic a-helical structures characteristic of other apolipoproteins examined thus far.Apolipoprotein B (apo B) is a major protein component of mammalian very low density lipoproteins (VLDL) and chylomicrons and essentially the sole protein in low density lipoproteins (LDL). It is essential for the assembly/secretion of chylomicrons and VLDL, since these lipoproteins are absent in individuals with abetalipoproteinemia, a genetic disorder in which apo B is not produced (1). Apo B also functions as the ligand for the removal of LDL from the circulation by receptor-mediated uptake into a variety ofcells (2, 3).Despite intensive study, the structure of apo B has remained an enigma. It is among the largest peptides known, with estimates ranging from 250 to 550 kDa (1, 4-10), and is extremely hydrophobic as well as very sensitive to proteolytic degradation, and only recently has limited amino acid sequence information become available (11). Moreover, at least two major forms ofapo B are present in the circulation (8,9,(12)(13)(14)(15). In humans and most other mammals, the liver incorporates a 400-kDa species into VLDL, whereas the intestine incorporates a 210-kDa species into chylomicrons. However, the rat and mouse are exceptional in that their livers produce VLDL with comparable amounts ofboth these peptides (9,(13)(14)(15). We call these high and low molecular weight forms of apo B P(eptide)I and P(eptide)III, respectively; they are the B-100 and B-48 species in Kane's nomenclature (1,8,9). Although in the rat, PI is also accompanied by a slightly smaller component, PII, the two are very similar (9) and will be collectively referred to as PI. Peptide fingerprinting and immunological analysis suggest that PI encompasses a unique, PI-specific moiety joined to a second moiety which is identical or very similar to PIII (9,(16)(17)(18). Th...

Apolipoprotein B (apoB) is a major protein component of low density and very low density lipoproteins. Because of its large size and heterogeneity, molecular studies of apoB have been difficult, and its structure and regulation remain poorly understood. We now report the identification of human apoB cDNA clones by antibody screening of hepatoma libraries in the expression vector lambda gt11. Both oligo(dT) primed and random primed libraries were constructed and screened with polyclonal antibodies to intact apoB, as well as with antibodies raised against a synthetic peptide based on the limited amino acid sequence available for apoB. The identity of the clones was unambiguously established by comparisons of the cloned cDNA sequences with apoB amino acid sequences. The clones hybridize to an exceptionally large 20 kb mRNA that is present in liver and intestine but not other tissues examined, consistent with the distribution expected from protein biosynthetic studies. The properties of the mRNA have implications for the biogenesis of the multiple apoB molecular weight forms secreted by liver and intestine.

A library of the 8000 tripeptides derivable from coded amino acids was prepared in 20 sets of 400 using solid phase synthesis on a benzhydrylamine resin. The peptide mixtures, as C-terminal amides, were screened for inhibition of secreted alkaline phosphatase expression in a cellular (COS) system wherein a transfected SeAP gene construct was under control of the HIV-1 LTR promoter, activated by the product of a cotransfected HIV Tat gene construct. Thus, YPG-NH2 was discovered as an inhibitor of HIV-1 Tat function and then shown to block HIV replication in a CD4+ T-cell line (CEM) with IC50 = 35μM.