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 accessof transcription factors to DNA. For example, histone acetylation generally opens chromatin structure and is correlated with transcriptional activation.2.Posttranslational modifications of transcription factors, suchas 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 manytranscription factors, including proteins, with histone acetyltransferase activity.3.Transcriptional activators can displace a repressor protein.This mechanism is particularly common during development when...
Nội dung trích xuất từ tài liệu:
Chapter 062. Principles of Human Genetics (Part 8) 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 accessof transcription factors to DNA. For example, histone acetylation generallyopens chromatin structure and is correlated with transcriptional activation. 2. Posttranslational modifications of transcription factors, suchas phosphorylation, can induce the assembly of active transcriptioncomplexes. As an example, phosphorylation of CREB protein on serine 133induces 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 genesare activated by some combination of these events. Suppression of gene expression is as important as gene activation in thecontrol of cell differentiation and function. Some mechanisms of repression are thecorollary of activation. For example, repression is often associated with histonedeacetylation or protein dephosphorylation. For nuclear hormone receptors,transcriptional silencing involves the recruitment of repression complexes thatcontain histone deacetylase activity. Aberrant expression of repressor proteins issometimes associated with neoplasia. The t(15;17) chromosomal translocation thatoccurs in promyelocytic leukemia fuses the PML gene to a portion of the retinoicacid receptor α (RAR α) gene (Table 62-2). This event causes unregulatedtranscriptional repression in a manner that precludes normal cellulardifferentiation. The addition of the RAR ligand, retinoic acid, activates thereceptor, thereby relieving repression and allowing cells to differentiate andultimately undergo apoptosis. This mechanism has therapeutic importance as theaddition of retinoic acid to treatment regimens induces a higher remission rate inpatients with promyelocytic leukemia (Chap. 104). Methylation of promoterregions is frequently found in neoplasms and silences gene expression. Cloning and Sequencing DNA A description of recombinant DNA techniques, the methodology used forthe manipulation, analysis, and characterization of DNA segments, is beyond thescope of this chapter. As these methods are widely used in genetics and moleculardiagnostics, however, it is useful to review briefly some of the fundamentalprinciples of cloning and DNA sequencing. Cloning of Genes Cloning refers to the creation of a recombinant DNA molecule that can bepropagated indefinitely. The ability to clone genes and cDNAs therefore providesa permanent and renewable source of these reagents. Cloning is essential for DNAsequencing, nucleic acid hybridization studies, expression of recombinantproteins, and other recombinant DNA procedures. The cloning of DNA involves the insertion of a DNA fragment into acloning vector, followed by the propagation of the recombinant DNA in a hostcell. The most straightforward cloning strategy involves inserting a DNA fragmentinto bacterial plasmids. Plasmids are small, autonomously replicating, circularDNA 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 ofthe DNA fragment, which allow them to be efficiently ligated to the plasmidvector. Because plasmids contain genes that confer resistance to antibiotics, theirpresence in the host cell can be used for selection and DNA amplification. A variety of vectors (e.g., plasmids, phage, bacterial, or yeast artificialchromosomes) 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 anarray of clones derived from genomic DNA. These overlapping DNA fragmentsrepresent the entire genome and can ultimately be arranged according to theirlinear order. cDNA libraries reflect clones derived from mRNA, typically from aparticular tissue source. Thus, a cDNA library from the heart contains copies ofmRNA expressed specifically in cardiac myocytes, in addition to those that areexpressed ubiquitously. For this reason, a heart cDNA library will be enrichedwith cardiac-spe ...