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Osteoporosis is a complex disease that affects >10 million people in the United States and results in 1.5 million fractures annually. In addition, the high prevalence of osteopenia (low bone mass) in the general population places a large number of people at risk for developing the disease. In an effort to identify genetic factors influencing bone density, we characterized a family that includes individuals who possess exceptionally dense bones but are otherwise phenotypically normal. This high–bone-mass trait (HBM) was originally localized by linkage analysis to chromosome 11q12-13. We refined the interval by extending the pedigree and genotyping additional markers. A systematic search for mutations that segregated with the HBM phenotype uncovered an amino acid change, in a predicted β-propeller module of the low-density lipoprotein receptor–related protein 5 (LRP5), that results in the HBM phenotype. During analysis of >1,000 individuals, this mutation was observed only in affected individuals from the HBM kindred. By use of in situ hybridization to rat tibia, expression of LRP5 was detected in areas of bone involved in remodeling. Our findings suggest that the HBM mutation confers a unique osteogenic activity in bone remodeling, and this understanding may facilitate the development of novel therapies for the treatment of osteoporosis.

Elucidation of the changes in gene expression associated with biological processes is a central problem in biology. Advances in molecular and computational biology have led to the development of powerful, high-thoughput methods for the analysis of differential gene expression. These tools have opened up new opportunities in disciplines ranging from cell and developmental biology to drug development and pharmacogenomics. In this review, the attributes of five commonly used differential gene expression methods are discussed: expressed sequence tag (EST) sequencing, cDNA microarray hybridization, subtractive cloning, differential display, and serial analysis of gene expression (SAGE). The application of EST sequencing and microarray hybridization is illustrated by the discovery of novel genes associated with osteoblast differentiation. The application of subtractive cloning is presented as a tool to identify genes regulated in vivo by the transcription factor pax-6. These and other examples illustrate the power of genomics for discovering novel genes that are important in biology and which also represent new targets for drug development. The central theme of the review is that each of the approaches to identifying differentially expressed genes is useful, and that the experimental context and subsequent evaluation of differentially expressed genes are the critical features that determine success. J. Cell. Biochem. Suppls. 30/31:286-296, 1998. © 1998 Wiley-Liss, Inc.

The avian pathogen Newcastle disease virus (NDV) is a member of the Paramyxoviridae, a family which includes Sendai virus, simian virus 5 (SV5) and the human virus parainfluenza 3 (para-3). These enveloped viruses contain a single-stranded RNA genome of negative sense, 15-18 kb in length, encased in a helical nucleocapsid. Six major messenger RNA species are transcribed sequentially by the virion-associated RNA polymerase. They are capped, methylated, and polydenylated by virion proteins (Huang et a/., 1971; Colonno and Stone, 1976). Organization of the NDV genome has been investigated indirectly by both uv-inactivation mapping and by the analysis of polycistronic transcripts (Collins et a/., 1980; Wilde and Morrison, 1984). Both studies indicate that the gene order is identical to that of Sendai virus, which has been determined to be 3’-NP-P-M-F-HN-L-5’ by nucleotide sequence analysis of genomic clones (Blumberg et a/., 1984, 1985a,b). NP and the phosphoprotein P are components of the viral nucleocapsid, L is an RNAdependent RNA polymerase, and M is a protein of undetermined function associated with the inner surface of the viral membrane (reviewed in Kingsbury, 1985). Paramyxovirus glycoproteins are functionally similar to the well-studied surface glycoproteins of influenza (Ward, 1981; Colman and Ward, 1985). They possess cell fusion, hemagglutination and neuraminidase activities (Hsu et a/., 1979; Scheid and Choppin, 1974; Merz et a/., 1981), and constitute the spikes which protrude from the viral envelope (Choppin and Compans, 1975). The F glycoprotein, which is responsible for the paramyxovirus’ ability to fuse with the host cell membrane, is activated by a proteolytic cleavage event (Scheid and

Total nucleic acid extraction and selection of poly A-containing molecules yield preparative quantities of undegraded mRNA from adult and fetal pancreas. Using a stringent immunoassay, this mRNA is found to direct the synthesis of an immunoreactive insulin polypeptide in the wheat germ translation system. On sodium dodecyl sulfate polyacrylamide gels, this polypeptide (12,000-13,000 daltons) is larger than proinsulin (9,000 daltons).

The functional assay of a eukaryotic mRNA, into which additional AUG codons have been introduced by in vitro mutagenesis, shows that a translational initiation site need not necessarily be the nearest AUG codon to the 5' end of a mRNA. Hence, the sequence surrounding the AUG, and not simply its position relative to the 5' end of mRNA, appears to be important in determining initiation efficiency.

Interleukin-1 (IL-1), a peptide hormone produced by activated macrophages, possesses the ability to modulate the proliferation, maturation and functional activation of a broad spectrum of cell types1–7 and may play a major role in the initiation and amplification of immune and inflammatory responses through its action on these diverse cell populations8. IL-1 exhibits microheterogeneity in terms of its relative molecular mass (Mr, 13,000–19,000) and charge properties8, and although murine IL-1 has been purified9,10 and some of its basic structure–function relationships have been elucidated8, it has proved difficult to prepare sufficient amounts of IL-1 for direct and detailed sequence and structural studies. Here we report the cloning, sequence analysis and expression of murine IL-1 cDNA in Escherichia coli. The IL-1 cDNA codes for a polypeptide precursor of 270 amino acids. Biologically active IL-1 was produced in E. coli by expressing the carboxy-terminal 156 amino acids of the IL-1 precursor.

The downstream (3') long terminal repeat (LTR) of an avian retroviral provirus is unable to act as an efficient promoter of transcription when a transcriptionally active upstream (5') LTR is present. This transcriptional interference may explain the observation that only deleted proviruses have been observed inserted adjacent to c-myc in avian leukosis virus induced lymphomas of chickens.

In vivo, glucose preferentially stimulates proinsulin biosynthesis; at least part of this process is independent of new RNA synthesis and is accompanied by increases in the overall rate of polypeptide chain initiation. The cell-free translation of proinsulin messenger RNA is very sensitive to changes in the protein-synthesizing system. Proinsulin synthesis is preferentially inhibited by the addition of increasing quantities of polyadenylate-containing RNA from the fetal bovine pancreas or by the addition of the drug, aurintricarboxylic acid, which blocks polypeptide chain initiation. These results suggest that proinsulin messenger RNA completes less efficiently for rate controlling initiation factors. We propose that glucose stimulates proinsulin biosynthesis by allowing the less competitive proinsulin messenger RNA to be translated more efficiently.

Publisher Summary This chapter discusses the biochemical characterization and the physiological role of interleukin 1 (IL-1). IL-1 is a molecule that possesses a wide variety of biological activities. Biochemical studies reveal that IL-1 produced by stimulated murine macrophages and macrophage tumor cell lines is a single polypeptide chain. Human IL-1 has been shown to be produced by stimulated normal human peripheral blood leukocytes or monocytes, leukemic cells, and a number of human tumor cell lines. Murine and human keratinocytes have been shown to produce an IL-1-like activity designated epidermal T-cell activating factor. This factor exhibits some of the activities of IL-1. IL-1 appears to play a key role in the activation of T-lymphocytes. The processes triggered by IL-1 may include both the secretion of IL-2 and induction of receptors for this growth factor. IL-1 can also modulate B-cell function. However, the nature of the effect can vary depending on the presence of other lymphocyte-derived signals and the type of B-cell stimulus. An effect of IL-1 on fibrinogen and haptoglobin synthesis has also been discussed in the chapter. IL-1 may also be a modulator of the fibroblast proliferation. IL 1 has also significant effects on the cartilage, bone, and synovial membrane. IL-1 is thought to be an important mediator of immune cell interactions and to contribute to a number of disease manifestations and physiological processes. Thus, agents that modulate either IL-1 production or IL-1 activity on the target tissues may be therapeutically beneficial and have been the focus of a number of investigations. This information and additional developments in IL-1 research should lead to an understanding of the mechanism, by which IL-1 induces each of its effector activities, of the role of IL-1 in various disease states and of the means, by which specific activities of IL-1 can be modulated.

We have constructed a set of expression vectors which contain synthetic DNA sequences comprising a computer-generated model ribosomal binding site located downstream from the tightly regulated phage λ pL promoter. These vectors have been used in several laboratories to produce significant amounts of eukaryotic and prokaryotic gene products in Escherichia coli, either as fusion proteins (with two to nine extra N-terminal amino acids) or as proteins containing the naturally occurring amino terminus. For inserting DNA sequences downstream of an initiation codon, we used synthetic oligonucleotides to introduce multiple-use restriction sites recognized by EcoRI, Bam HI and ClaI which generate termini complementary to those of a variety of enzymes (e.g., Eco RI∗, MboI, TaqI, and HpaII), in addition to their own. A set of three of these vectors was made to accommodate all three translational reading frames. In combination, the features of these vectors afford useful advantages over expression vectors previously described, especially for the application of shot-gun cloning of genomic DNA to generate expression libraries.