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Thyroid hormone (T3) plays important roles during vertebrate development (1). In humans, T3 is detected in the embryonic plasma by 6 mo and rises to high levels around birth (2). During this postembryonic period, extensive tissue remodeling and organogenesis take place. T3 deficiency during ...

Recent molecular genetic studies in cardiac and skeletal muscle have revealed mutations in a battery of sarcomeric muscle-restricted genes that appear to be associated with various myopathies (1,2). In sharp contrast, no mutations in smooth muscle cell (SMC)-restricted genes have be ...

Protein kinases play pivotal roles in almost all signal transduction pathways in eukaryotic cells (1–4) and are implicated in most major human diseases, including atherosclerosis and associated vasculoproliferative disorders of arteries such as restenosis and graft stenosis ...

The development of new and effective techniques to study differential gene expression has revolutionized biomedical research during the last decade. Such techniques include differential display reverse transcription polymerase chain reaction (ddRTPCR) (see Chapter 9), f ...

The vast majority of past and present efforts in the molecular cloning of expressed sequences involve isolation of clones from cDNA libraries constructed in bacteriophage lambda (1,2). As discussed in Chapter 6, screening these cDNA libraries using labeled probes remains the most strai ...

The purpose of screening cDNA libraries is to isolate a particular cDNA clone encoding a mRNA and by implication, a protein, of interest. The screening is based on identification of the desired clone among a large number of recombinant clones within the library selected (1,2). As an example of both the u ...

Changes in gene expression underlie many biological phenomena, including cellular differentiation and activation, embryonic development, and pathological processes. In recent years, much attention has been focused on the identification of genes that are differentially e ...

The isolation of full-length cDNAs remains a frequent task undertaken in many laboratories. A full-length cDNA is often desirable for one of the following purposes: 1) to complete the sequence of a partial cDNA cloned by library screenings or the yeast one- or two-hybrid system; 2) to derive the cDNA se ...

In 1992 a new approach for identifying differentially expressed genes was described by Liang and Pardee (1). Their method allowed the simultaneous differential display of mRNA from two or more cell types by means of the polymerase chain reaction (PCR). In a subsequent study demonstrating the t ...

The principle of the technique presented in this chapter is illustrated in Fig. 1. As with S1 mapping or riboprobe mapping, this technique can be used to determine precisely the start site of transcription of a mRNA sequence (1–3). Since this technique is relatively easier than other techniques, it is ...

The transfer and expression of DNA plasmids containing promoter fragments of heterologous genes linked to reporter cDNAs in mammalian cells has become an invaluable technique for studying the regulation of gene expression. Several reporter genes such as luciferase, β-galactosi ...

An accurate assessment of gene transcription is important for understanding the mechanism of gene expression. The nuclear run-on assay measures the relative in situ transcription rate of specific genes in intact nuclei (1,2). It provides information on the synthesis of a specific gene th ...

The nuclear factor-kappa B (NFκB) family of transcription factors has emerged as a signaling pathway that figures prominently in a cell’s initial response to a plethora of inflammatory stimuli. Modified lipids, oxidative stress, bacterial endotoxins, growth factors and cytokines, s ...

The developments of the polymerase chain reaction (PCR) analysis (1–3) and quantitative PCR by Gilliland (4) has provided researchers with a unique tool to analyze the expression of various genes in very small amounts of tissue samples. The sensitivity of PCR allows measurement of the expres ...

To understand the molecular mechanisms of vascular diseases, studies are designed to process gene function from the discovery of relevant genes to examination of their expression and association with disease. Once a gene has been cloned, Northern blot analysis can directly monitor the g ...

The technique of in situ hybridization was developed in the late 1960s (1) and is based on the ability of a single-stranded sequence of nucleotides to hybridize specifically to a complementary sequence to form stable double-stranded duplexes or hybrids. Incorporating a readily detectab ...

Kerr and Wyllie (1) have introduced the term apoptosis to separate this special form of cell death from necrosis. When a cell receives a signal to die an apoptotic death, it goes through a series of morphological changes that can be easily observed with the light microscope. Starting from shrinkage of t ...

Ribonuclease protection assay (RPA) is a sensitive solution hybridization method for quantitation of specific RNAs (1–3). The method is based on the ability of single-strand specific ribonuclease to degrade single-stranded RNA while leaving intact fragments of labeled antisense ...

Phenotypic modulation of arterial smooth muscle cells (SMC) is essential for the evolution of atheromatosis and restenosis after angioplasty (1). During these pathological phenomena, SMC express numerous genes such as those responsible for cell migration and proliferation (2). T ...

Apoptosis is a programmed cell death process in which surplus or damaged cells are eliminated through a highly regulated procedure. The first description of apoptosis relied upon morphological differences among apoptotic, necrotic, and healthy cells (1). Indeed, there are usually a nu ...