Chapter 062. Principles of Human Genetics (Part 8)

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Transcriptional activation can be divided into three main mechanisms: 1. Events that alter chromatin structure can enhance the access of transcription factors to DNA. For example, histone acetylation generally opens chromatin structure and is correlated with transcriptional activation. 2. Posttranslational modifications of transcription factors, such as phosphorylation, can induce the assembly of active transcription complexes. As an example, phosphorylation of CREB protein on serine 133 induces a conformational change that allows the recruitment of CREBbinding protein (CBP), a factor that integrates the actions of many transcription factors, including proteins, with histone acetyltransferase activity. 3. Transcriptional activators can displace a repressor protein. This mechanism is particularly common during development when...

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Chapter 062. Principles of Human Genetics (Part 8) Transcriptional activation can be divided into three main mechanisms: 1. Events that alter chromatin structure can enhance the access of transcription factors to DNA. For example, histone acetylation generally opens chromatin structure and is correlated with transcriptional activation. 2. Posttranslational modifications of transcription factors, such as phosphorylation, can induce the assembly of active transcription complexes. As an example, phosphorylation of CREB protein on serine 133 induces a conformational change that allows the recruitment of CREB- binding protein (CBP), a factor that integrates the actions of many
transcription factors, including proteins, with histone acetyltransferase activity. 3. Transcriptional activators can displace a repressor protein. This mechanism is particularly common during development when the pattern of transcription factor expression changes dynamically. Of course, these mechanisms are not mutually exclusive, and most genes are activated by some combination of these events. Suppression of gene expression is as important as gene activation in the control of cell differentiation and function. Some mechanisms of repression are the corollary of activation. For example, repression is often associated with histone deacetylation or protein dephosphorylation. For nuclear hormone receptors, transcriptional silencing involves the recruitment of repression complexes that contain histone deacetylase activity. Aberrant expression of repressor proteins is sometimes associated with neoplasia. The t(15;17) chromosomal translocation that occurs in promyelocytic leukemia fuses the PML gene to a portion of the retinoic acid receptor α (RAR α) gene (Table 62-2). This event causes unregulated transcriptional repression in a manner that precludes normal cellular differentiation. The addition of the RAR ligand, retinoic acid, activates the receptor, thereby relieving repression and allowing cells to differentiate and ultimately undergo apoptosis. This mechanism has therapeutic importance as the
addition of retinoic acid to treatment regimens induces a higher remission rate in patients with promyelocytic leukemia (Chap. 104). Methylation of promoter regions is frequently found in neoplasms and silences gene expression. Cloning and Sequencing DNA A description of recombinant DNA techniques, the methodology used for the manipulation, analysis, and characterization of DNA segments, is beyond the scope of this chapter. As these methods are widely used in genetics and molecular diagnostics, however, it is useful to review briefly some of the fundamental principles of cloning and DNA sequencing. Cloning of Genes Cloning refers to the creation of a recombinant DNA molecule that can be propagated indefinitely. The ability to clone genes and cDNAs therefore provides a permanent and renewable source of these reagents. Cloning is essential for DNA sequencing, nucleic acid hybridization studies, expression of recombinant proteins, and other recombinant DNA procedures. The cloning of DNA involves the insertion of a DNA fragment into a cloning vector, followed by the propagation of the recombinant DNA in a host cell. The most straightforward cloning strategy involves inserting a DNA fragment into bacterial plasmids. Plasmids are small, autonomously replicating, circular
DNA molecules that propagate separately from the chromosome in bacterial cells. The process of DNA insertion relies heavily on the use of restriction enzymes, which cleave DNA at highly specific sequences (usually 4–6 bp in length). Restriction enzymes generate complementary, cohesive sequences at the ends of the DNA fragment, which allow them to be efficiently ligated to the plasmid vector. Because plasmids contain genes that confer resistance to antibiotics, their presence in the host cell can be used for selection and DNA amplification. A variety of vectors (e.g., plasmids, phage, bacterial, or yeast artificial chromosomes) are used for cloning. Many of these are used for creating libraries, a term that refers to a collection of DNA clones. A genomic library represents an array of clones derived from genomic DNA. These overlapping DNA fragments represent the entire genome and can ultimately be arranged according to their linear order. cDNA libraries reflect clones derived from mRNA, typically from a particular tissue source. Thus, a cDNA library from the heart contains copies of mRNA expressed specifically in cardiac myocytes, in addition to those that are expressed ubiquitously. For this reason, a heart cDNA library will be enriched with cardiac-specific gene products and will differ from cDNA libraries generated from liver or pituitary mRNAs. As an example of the complexity of a genomic library, consider that the human genome contains 3 x 10 9 bp and the average genomic insert in a λ phage library is ~104 bp. Therefore, it requires at least 3 x
105 clones to represent all genomic DNA. Specific clones are isolated from the several hundred thousand clones by using DNA hybridization. With completion of the HGP, all human genes have been cloned and sequenced. As a result, many of these cloning procedures are now unnecessary or greatly facilitated by the extensive information concerning DNA markers and the sequence of DNA (see below).