Lipoprotein(a) is an LDL-like lipoprotein whose concentration in plasma is correlated with atherosclerosis. The characteristic protein component of lipoprotein(a) is apolipoprotein(a) which is disulphide-linked to apolipoprotein B-100. Sequencing of cloned human apolipoprotein(a) complementary DNA shows that it is very similar to human plasminogen. It contains a serine protease domain and two types of plasminogen-like kringle domains, one of which is present in 37 copies.

The deduced amino acid sequence of human factor VIII, obtained from the DNA sequence, predicts a mature polypeptide of 2,332 amino acids containing a triplicated domain structure. The polypeptide has 35% sequence homology with the copper-binding plasma protein, ceruloplasmin. Determination of the thrombin cleavage sites in plasma-derived factor VIII polypeptides allows prediction of the domains involved in the associated activation and inactivation of the protein.

No abstract

The complete 186,000 base-pair (bp) human factor VIII gene has been isolated and consists of 26 exons ranging in size from 69 to 3,106 bp and introns as large as 32.4 kilobases (kb). Nine kb of mRNA and protein-coding DNA has been sequenced and the mRNA termini have been mapped. The relationship between internal duplications in factor VIII and evolution of the gene is discussed.

Lipid-poor high density lipoprotein apolipoproteins remove cholesterol and phospholipids from cells by an active secretory pathway controlled by an ABC transporter called ABCA1. This pathway is induced by cholesterol and cAMP analogs in a cell-specific manner. Here we provide evidence that increased plasma membrane ABCA1 accounts for the enhanced apolipoprotein-mediated lipid secretion from macrophages induced by cAMP analogs. Treatment of RAW264 macrophages with 8-bromo-cAMP caused parallel increases in apoA-I-mediated cholesterol efflux, ABCA1 mRNA and protein levels, incorporation of ABCA1 into the plasma membrane, and binding of apoA-I to cellsurface ABCA1. All of these parameters declined to near base-line values within 6 h after removal of 8-bromocAMP, indicating that ABCA1 is highly unstable and is degraded rapidly in the absence of inducer. Thus, ABCA1 is likely to be the cAMP-inducible apolipoprotein receptor that promotes removal of cholesterol and phospholipids from macrophages.

No abstract

DNA clones encoding the complete 2,351 amino acid sequence for human factor VIII have been isolated and used to produce biologically active factor VIII in cultured mammalian cells. The recombinant protein corrects the clotting time of plasma from haemophiliacs and has many of the biochemical and immunological characteristics of serum-derived factor VIII.

An oligonucleotide hybridization procedure has been developed that eliminates the preferential melting of A'T versus G C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. This technique, which uses tetramethylammonium chloride, is especially helpful whenever a highly complex library is screened with a pool of oligonucleotide probes, which usually vary widely in base composition. The procedure can also be applied advantageously whenever an exact match to an oligonucleotide probe is desired, such as in screening for clones having as little as a single-base alteration generated by in vitro mutagenesis.Short oligonucleotide probes [range, 14-20 base pairs (bp)] are commonly used to screen libraries of cloned DNA for genes of interest (1-3). Typically, these probes are pools representing all possible codon choices for a short amino acid sequence. Although this method has been successful, there is considerable uncertainty in the hybridization conditions because the binding of the oligonucleotides depends on two factors: (i) the length of the hybrid formed and (ii) the G-C content of the probe. Empirically determined formulas allow for estimation of the oligonucleotide dissociation temperature (Td) (4); however, these methods can be unsatisfactory when screening with pools of oligonucleotides. Although the length of the probes in the pool is constant, the individual probes differ considerably in G-C content, making suitably stringent and selective hybridization conditions difficult to find for all members of the pool. Thus, a large number of false positives can occur when screening highly complex libraries for genes of low abundance.We describe here the use of tetramethylammonium chloride (Me4NCl) in the hybridization of oligonucleotide probes to eliminate the dependence of Td on the G-C content of the probe, reducing the problem to a simple dependence on length of the hybrid. Tetraalkylammonium salts were found some years ago to bind to A+T-rich polymers of DNA (5) and have been used to abolish the preferential melting of A-T versus G-C base pairs for fragments of DNA (6). Me4NCl binds selectively to A-T base pairs, thus displacing the dissociation equilibrium and raising the melting temperature. At 3.0 M Me4NCl, this displacement is sufficient to shift the melting temperature of A-T base pairs to that of G-C base pairs (6). For the melting of long DNA, this shift results in a remarkable sharpening of the melting profile. Natural DNAs that melt over a range of 5 to 10°C in the presence of Na+ melt within 1°C in Me4NCl (6)(7)(8). The data presented here show the utility of hybridizations with Me4NCl when oligonucleotide probes are used. The Td values of probes from 11 to >1000 bp have been determined so that hybridization conditions for probes of various lengths can be chosen easily. This method is applicable to a variety of circumstances in which an exact match to a probe is desired. METHODS DNA Synthesis, Binding, and Labeling. In this procedure, nit...