Secretion from cell culture of HDL and VLDL bearing apoB-33 with a large internal deletion.

Rat hepatoma McA-RH7777 cells synthesize and secrete two populations of apoB-containing lipoproteins: a larger, VLDL-sized population floating in the Sf 40-150 range and a smaller, LDL and HDL-sized population. Three permanently transfected cell lines of McA-RH7777 cells secreted (in addition to the endogenous lipoproteins) lipoproteins containing 1) a carboxyl-terminally truncated human apoB-53 (2377 amino acids in length); 2) a carboxyl-terminally truncated human apoB-31 (1420 amino acids in length); or 3) an internally deleted human apoB protein, apoB-18/95, containing a total of 1490 amino acid residues, equivalent in length to an apoB33. The apoB-18/95 protein contained amino acid residues 1-782 joined to 708 residues near the C-terminus of apoB (residues 36364343).

Chromosome Microdissection-Based Techniques for Genome Analysis.

It is now possible to start at a cytogenetically defined position in any eucaryotic genome and proceed toward isolation and identification of candidate genes known to map to that position, taking advantage of the new PCR amplification technology to produce position-specific DNA. The starting material for this very useful exercise is DNA microdissected from standard cytogenetic preparations. Here we describe techniques for acquiring useful samples of such DNA and strategies for the use of chromosome region-specific DNA for gene discovery projects.

Selection of hybrids by affinity capture (SHAC): a method for the generation of cDNAs enriched in sequences from a specific chromosome region.

We have established a method for preparing cDNA sublibraries enriched in sequences from specific chromosome regions, called selection of hybrids by affinity capture (SHAC). This procedure can be described in two stages. In the first stage, a particular chromosome region, in this study mouse chromosome 11, was microdissected, followed by PCR amplification with a universal degenerate primer.

Lipoprotein assembly. Apolipoprotein B size determines lipoprotein core circumference.

Apolipoprotein B (apoB) is an essential structural protein for the two triglyceride-rich lipoproteins synthesized by humans: chylomicrons and very low density lipoproteins. Although much is known about the role of apoB in clearance of lipoproteins from the circulation, relatively little is known about its role in the assembly of nascent lipoproteins. Therefore, we have investigated the relationship between the length of various N-terminal apoB fragments and the characteristics of the lipoproteins with which these fragments were associated.