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How Do Genes Work?

In this chapter, we will discuss fundamental concepts central to molecular biology and to understanding epigenetics. We will briefly discuss DNA, genes and proteins, mutation, genotype and phenotype and the relationships between each, and finally provide an overview as to how genes are controlled, outlining gene regulation and repression. We will outline the concept of molecular homeostasis and the interaction between genome and environment, briefly differentiating between epigenetics and genetics.

1.1 DNA, Genes and Proteins

DNA is comprised of four nucleotide bases: adenine, guanine, cytosine and thymine. DNA is a stable double helix with guanine always pairing with cytosine and adenine always pairing with thymine, or uracil in the case of RNA. The human genome is diploid, so it is comprised of 2 sets of 23 chromosomes, 1 inherited from each parent. Every nucleated cell contains the same 46 chromosomes with the same genetic information - this is our genome. Approximately 10% of the human genome is estimated to be coding, that is, specifically encodes for proteins; the remainder is noncoding DNA including repetitive sequences such as microsatellites, minisatellites, transposable elements (SINES, LINES), satellite DNA and triplet repeats.

Type of region Description Protein-coding genes Segments of DNA that encode proteins. Noncoding RNA genes Segments of DNA that encode RNA molecules that do not translate into proteins. Examples include microRNAs and long noncoding RNAs. Regulatory regions DNA sequences that control gene expression, such as promoters, enhancers, silencers and insulators. These regions can be located upstream or downstream of the gene, or even within the gene itself. Repetitive DNA Segments of DNA that are repeated many times throughout the genome, such as transposable elements, satellite DNA and minisatellites. Intergenic regions DNA sequences located between genes that do not have any known function. These regions make up the majority of the human genome. Introns Segments of DNA located within genes that do not encode proteins. Introns are transcribed into RNA but are removed during the process of RNA splicing, which generates the final mRNA transcript that is used to produce proteins.

The human genome project suggests that there are around 20 000 genes, with each human chromosome on average containing 1300 genes. Genes are transcribed and translated to produce their encoded protein. This two-stage process takes the message embedded in double-stranded DNA and transcribes the coding sequence as single-stranded mRNA, which is then translated by the ribosome using tRNA and rRNA to produce the protein. Genes range in size from 1 kb, as in the case of insulin, to 2.5 Mb for larger genes, such as dystrophin. Almost all genes contain introns and exons. Exons are expressed coding regions and introns are non-expressed intervening sequences. Introns are transcribed into primary RNA and then spliced out of mature RNA in the cytoplasm. The average number of exons for a human gene is 9, and therefore introns are 8. However, there is considerable variation, e.g. 79 for dystrophin and 3 for beta-globin. The average size of an exon is 145 bp.

In addition to introns and exons, genes also have an adjacent upstream (5´) regulatory promoter region as well as other regulatory sequences such as enhancers, silencers and sometimes a locus control region. The promoter region contains specific conserved sequences such as TATA box, CG box and CAAT box, which provide binding sites for transcription factors. The first and last exons also contain untranslated regions (UTRs) known as the 5´UTR and 3´UTR. The 5´UTR signals the start of transcription and contains ATG, the initiator codon that initiates the site of the start of translation. The 3´UTR contains a termination codon, which marks the end of translation, plus nucleotides that encode a sequence of adenosine residues known as the poly (A) tail; the addition of a poly (A) tail is an essential step in the process of transcription that enables the pre-mRNA to exit the nucleus and move into the cytoplasm for translation.

Gene regulatory sequence Function Promoters DNA sequences located upstream of the transcription start site that recruit the transcriptional machinery to initiate gene expression Enhancers DNA sequences that can be located far away from the gene they regulate and can enhance gene expression by increasing transcription rates and/or making expression more specific to certain cells or conditions Silencers DNA sequences that can be located near or far from the gene they regulate and can reduce or turn off gene expression Insulators DNA sequences that act as boundaries between different gene regulatory regions, preventing their influence on each other Scaffold/matrix attachment regions DNA sequences anchor the chromatin to the nuclear matrix, thereby organising and stabilising the structure of chromatin and regulating gene expression CpG islands Regions of DNA that have a high density of CpG dinucleotides are often associated with gene promoters and are involved in the regulation of gene expression through DNA methylation miRNA target sites Specific RNA sequences within the 3´ UTR of messenger RNAs that are recognised by microRNAs and lead to translational repression or mRNA degradation Cis-regulatory modules Clusters of enhancers, silencers and promoter sequences that work together to regulate the expression of a specific gene or set of genes in response to different signalling pathways

Pre-mRNA contains the entire transcribed sequences of exons and introns; the introns must be removed to produce mRNA that comprises the precise code for translation into a functional protein product. The removal of introns occurs through splicing, which occurs in a spliceosome, itself composed of hundreds of proteins and 5 RNAs. Once a transcript has been spliced and its 5´ and 3´ ends modified, a piece of mature functional mRNA has been produced, suitable for translation into a protein. There are specific nucleotide recognition sequences that aid in splicing. These sequences are present: towards the end of an exon, at the beginning of an intron, at the end of an intron, at the beginning of the next exon and at a region within the intron but close to the 3´ end, which provides a binding site for intron removal. Such sequences are recognised by small nuclear ribonucleoproteins (snRNPs), and they cut the RNA at the intron-exon borders and connect the exons together. Alternative splicing is a mechanism that allows more information to be packed into a single gene. That is, from a single gene, through splicing exons together in different combinations, multiple RNAs and functional proteins can be produced; this allows cells to produce related but distinct proteins from a single gene. For example, one type of protein may be produced in one tissue, whereas another form may be produced in another tissue (Figure 1.1).

Figure 1.1 The gene is shown in a linear arrangement, with the promoter at the beginning, the transcribed region in the middle and the terminator at the end. The transcribed region is shown with a series of exons (which code for protein) separated by introns (which do not code for protein). The regulatory elements, which can be located upstream or downstream of the promoter, are also shown at the beginning of the gene. At the end of the transcribed region, there is a stop codon that signals the end of the protein-coding sequence.

The estimated 20 000 protein-coding genes comprising the human genome are spread between the lengths of the human chromosomes. Each gene has a promoter where transcription of mRNA by RNA polymerase II is initiated by transcription factors. There are also remote elements called enhancers that will modulate the activity of the promoter. Different cell types activate gene expression in a different manner by making use of the genome's extensive system of regulatory cis-acting elements. The mechanisms underlying this process involve physical changes to the chromatin that either promote or inhibit gene expression. This is fundamental to understanding epigenetics, as it is how gene expression is suppressed or enhanced.

1.2 Gene Control, Homeostasis and Epigenetics

In complex multicellular organisms, differential gene expression is fundamental during embryonic development and in the maintenance of the adult state. It is key to understand that different cells make different proteins, which means different genes are switched on in different tissues even though all cells carry the same comprehensive set of genetic instructions. Therefore, there must be a way in which the body controls which gene is switched on, to what extent and when. Unused genetic information is not discorded - just not switched; for this to...