Structure of the Human Genome The Human Genome Project was initiated in the mid-1980s as an ambitious effort to characterize the entire human genome and culminated in the completion of the DNA sequence for the last of the human chromosomes in 2006. The scope of a WGS analysis can be illustrated by the following analogy. Human DNA consists of ~3 billion base pairs (bp) of DNA per haploid genome, which is nearly 1000-fold greater than that of the Escherichia coli genome. If the human DNA sequence were printed out, it would correspond to about 120 volumes of Harrison’s Principles of Internal Medicine.

In addition to the human genome, the genomes of thousands of organisms have been sequenced completely or partially (Genomes Online Database [GOLD]; Table 1). They include, among others, eukaryotes such as the mouse (Mus musculus), Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster; bacteria (e.g., E. coli); and archaea, viruses, organelles (mitochondria, chloroplasts), and plants (e.g., Arabidopsis thaliana). Genomic information of infectious agents has significant impact for the characterization of infectious outbreaks and epidemics. Other ramifications arising from the availability of genomic data include, among others, (1) the comparison of entire genomes (comparative genomics); (2) the study of large-scale expression of RNAs (functional genomics), proteins (proteomics), or protein families (e.g., the kinome, the complete set of protein kinases) to detect differences between various tissues in health and disease; (3) the characterization of the variation among individuals by establishing catalogues of sequence variations and SNPs; and (4) the identification of genes that play critical roles in the development of polygenic and multifactorial disorders.

Table1. Selected Databases Relevant for Genomics and Genetic Disorders

CHROMOSOMES The human genome is divided into 23 different chromosomes, including 22 autosomes (numbered 1–22) and the X and Y sex chromosomes (Fig.1). Adult cells are diploid, meaning they contain two homologous sets of 22 autosomes and a pair of sex chromosomes. Females have two X chromosomes (XX), whereas males have one X and one Y chromosome (XY). As a consequence of meiosis, germ cells (sperm or oocytes) are haploid and contain one set of 22 autosomes and one of the sex chromosomes. At the time of fertilization, the diploid genome is reconstituted by pairing of the homologous chromosomes from the mother and father. With each cell division (mitosis), chromosomes are replicated, paired, segregated, and divided into two daughter cells.

Fig1. Structure of chromatin and chromosomes. Chromatin is composed of double-strand DNA that is wrapped around histone and nonhistone proteins forming nucleosomes. The nucleosomes are further organized into solenoid structures. Chromosomes assume their characteristic structure, with short (p) and long (q) arms at the metaphase stage of the cell cycle.

STRUCTURE OF DNA, DNA is a double-stranded helix composed of four different bases: adenine (A), thymidine (T), guanine (G), and cytosine (C). Adenine is paired to thymidine, and guanine is paired to cytosine, by hydrogen bond interactions that span the double helix (Fig. 1). DNA has several remarkable features that make it ideal for the transmission of genetic information. It is relatively stable, and the double-stranded nature of DNA and its feature of strict base-pair complementarity permit faithful replication during cell division. Complementarity also allows the transmission of genetic information from DNA → RNA → protein (Fig. 2). mRNA is encoded by the so-called sense or coding strand of the DNA double helix and is translated into proteins by ribosomes.

Fig2. Flow of genetic information. Multiple extracellular signals activate intracellular signal cascades that result in altered regulation of gene expression through the interaction of transcription factors with regulatory regions of genes. RNA polymerase transcribes DNA into RNA that is processed to mRNA by excision of intronic sequences. The mRNA is translated into a polypeptide chain to form the mature protein after undergoing posttranslational processing. CBP, CREB-binding protein; CoA, co-activator; COOH, carboxyterminus; CRE, cyclic AMP responsive element; CREB, cyclic AMP response element–binding protein; GTF, general transcription factors; HAT, histone acetyl transferase; NH2, aminoterminus; RE, response element; TAF, TBP-associated factors; TATA, TATA box; TBP, TATA-binding protein.

The presence of four different bases provides surprising genetic diversity. In the protein-coding regions of genes, the DNA bases are arranged into codons, a triplet of bases that specifies a particular amino acid. It is possible to arrange the four bases into 64 different triplet codons (4³). Each codon specifies 1 of the 20 different amino acids, or a regulatory signal such as initiation and stop of translation. Because there are more codons than amino acids, the genetic code is degenerate; that is, most amino acids can be specified by several different codons. By arranging the codons in different combinations and in various lengths, it is possible to generate the tremendous diversity of primary protein structure.

DNA length is normally measured in units of 1000 bp (kilobases, kb) or 1,000,000 bp (megabases, Mb). In the human genome, only ~1% of DNA accounts for protein-coding sequences. The noncoding DNA has multiple functional and structural roles including (1) sequences that form introns; (2) regulatory elements (promoters, enhancers, silencers, insulators); (3) sequences that generate RNAs that do not code for proteins; (4) centromeres and telomeres; (5) regions defining chromatin structure and histone modifications; (6) various forms of repetitive sequences of variable length; and (7) pseudogenes and regions without currently discernible functional or structural roles (Fig. 1).

GENES, A gene is a functional unit that is regulated by transcription (see below) and encodes an RNA product, which is most commonly, but not always, translated into a protein that exerts activity within or outside the cell (Fig. 3). Historically, genes were identified because they conferred specific traits that are transmitted from one generation to the next. Now, they are frequently characterized based on expression in various tissues (transcriptome). The size of genes is quite broad; some genes are only a few hundred base pairs, whereas others are extraordinarily large (2.3 Mb). The number of genes greatly underestimates the complexity of genetic expression, because single genes can generate multiple spliced messenger RNA (mRNA) products (isoforms), which are translated into proteins that are subject to com plex posttranslational modification such as phosphorylation. Exons refer to the portion of genes that are eventually spliced together to form mRNA. Introns refer to the spacing regions between the exons that are spliced out of precursor RNAs during RNA processing. The gene locus also includes regions that are necessary to control its expression (Fig. 2). Current estimates predict roughly 20,000 protein-coding genes in the human genome with an average of about four different coding transcripts per gene. Remarkably, the exome only constitutes 1.14% of the genome. Of note, the number of transcripts is close to 200,000 and includes thousands of noncoding transcripts (RNAs of various length such as microRNAs [miRNA] and long noncoding RNAs [lncRNA]). These noncoding RNAs are involved in numerous cellular processes such as transcriptional and posttranscriptional regulation of gene expression, chromatin remodeling, and protein trafficking, among others. Not surprisingly, aberrant expression and/or mutations in these RNAs play a pathogenic role in numerous diseases.

Fig3. Chromosome 7 is shown with the density of single nucleotide polymorphisms (SNPs) and genes above. A 200-kb region in 7q31.2 containing the CFTR gene is shown below. The CFTR gene contains 27 exons. Close to 2000 mutations in this gene have been found in patients with cystic fibrosis. A 20-kb region encompassing exons 4–9 is shown further amplified to illustrate the SNPs in this region.

SINGLE-NUCLEOTIDE POLYMORPHISMS On average, a typical genome differs from the reference human genome at 4 to 5 million sites. Some of these variants have no impact on health, whereas others may increase or lower the risk for developing a specific disease. Remarkably, however, the primary DNA sequence of humans has ~99.9% similarity compared to that of any other human. An SNP is a variation of a single base pair in the DNA. Across human populations from distinct ethnic backgrounds, there are more than 1 billion validated SNPs (Fig. 3). SNPs are the most common type of sequence variation and account for >90% of all sequence variation. They occur on average every 100–300 bases and are the major source of genetic heterogeneity. SNPs that are in proximity are inherited together (e.g., they are linked) and are referred to as haplotypes (Fig. 4). Haplotype maps describe the nature and location of these SNP haplotypes and how they are distributed among individuals within and among populations, information that has been facilitating GWAS designed to elucidate the complex interactions among multiple genes and lifestyle factors in multifactorial disorders. Moreover, haplotype analyses are useful to assess variations in responses to medications (pharmacogenomics) and environmental factors, as well as the prediction of disease predisposition.

Fig4. The origin of haplotypes is due to repeated recombination events occurring in multiple generations. Over time, this leads to distinct haplotypes. These haplotype blocks can often be characterized by genotyping selected Tag single nucleotide polymorphisms (SNPs), an approach that facilitates performing genome wide association studies (GWAS).

COPY NUMBER VARIATIONS Copy number variations (CNVs) are relatively large genomic regions (1 kb to several Mb) that have been duplicated or deleted on certain chromosomes and hence alter the dip loid status of the DNA (Fig. 5). It has been estimated that 5–10% of the genome can display CNVs. When comparing the genomes of two individuals, ~0.4–0.8% of their genomes differ in terms of CNVs scattered throughout the genome. Some CNVs can increase or decrease gene dosage, potentially leading to detrimental effects if essential genes are impacted. Of note, de novo CNVs have been observed between monozygotic twins, who otherwise have identical genomes.

Fig5. Copy number variations (CNV) encompass relatively large regions of the genome that have been duplicated or deleted. Chromosome 8 is shown with a CNV detected by genomic hybridization. An increase in the signal strength indicates a duplication, whereas a decrease reflects a deletion of the covered chromosomal regions.

Replication of DNA and Mitosis Genetic information in DNA is transmitted to daughter cells under two different circumstances: (1) somatic cells divide by mitosis, allowing the diploid (2n) genome to replicate itself completely in conjunction with cell division; and (2) germ cells (sperm and ova) undergo meiosis, a process that enables the reduction of the diploid (2n) set of chromosomes to the haploid state (1n).

Prior to mitosis, cells exit the resting, or G0 state, and enter the cell cycle. After traversing a critical checkpoint in G1, cells undergo DNA synthesis (S phase), during which the DNA in each chromosome is replicated, yielding two pairs of sister chromatids (2n → 4n). The process of DNA synthesis requires stringent fidelity in order to avoid transmit ting errors to subsequent generations of cells. Genetic abnormalities of DNA mismatch/repair include xeroderma pigmentosum, Bloom’s syndrome, ataxia telangiectasia, and hereditary nonpolyposis colon cancer (HNPCC), among others. Many of these disorders strongly predispose to neoplasia because of the rapid acquisition of additional mutations. After completion of DNA synthesis, cells enter G2 and progress through a second checkpoint before entering mitosis. At this stage, the chromosomes condense and are aligned along the equatorial plate at metaphase. The two identical sister chromatids, held together at the centromere, divide and migrate to opposite poles of the cell. After formation of a nuclear membrane around the two separated sets of chromatids, the cell divides and two daughter cells are formed, thus restoring the diploid (2n) state.

Assortment and Segregation of Genes During Meiosis, Meiosis occurs only in germ cells of the gonads. It shares certain features with mitosis but involves two distinct steps of cell division that reduce the chromosome number to the haploid state. In addition, there is active recombination that generates genetic diversity. During the first cell division, two sister chromatids (2n → 4n) are formed for each chromosome pair and there is an exchange of DNA between homologous paternal and maternal chromosomes. This process involves the formation of chiasmata, structures that correspond to the DNA segments that cross over between the maternal and paternal homologues (Fig. 6). Usually there is at least one crossover on each chromosomal arm; recombination occurs more frequently in female meiosis than in male meiosis. Subsequently, the chromosomes segregate randomly. Because there are 23 chromosomes, there exist 223 (>8 million) possible combinations of chromosomes. Together with the genetic exchanges that occur during recombination, chromosomal segregation generates tremendous diversity, and each gamete is genetically unique. The process of recombination and the independent segregation of chromosomes provide the foundation for performing linkage analyses, whereby one attempts to correlate the inheritance of certain chromosomal regions (or linked genes) with the presence of a disease or genetic trait.

Fig6. Crossing-over and genetic recombination. During chiasma formation, either of the two sister chromatids on one chromosome pairs with one of the chromatids of the homologous chromosome. Genetic recombination occurs through crossing-over and results in recombinant and nonrecombinant chromosome segments in the gametes. Together with the random segregation of the maternal and paternal chromosomes, recombination contributes to genetic diversity and forms the basis of the concept of linkage.

After the first meiotic division, which results in two daughter cells (2n), the two chromatids of each chromosome separate during a second meiotic division to yield four gametes with a haploid state (1n). When the egg is fertilized by sperm, the two haploid sets are combined, thereby restoring the diploid state (2n) in the zygote.

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مواضيع ذات صلة

Origins and Types of Mutations

Regulation of Gene Expression

The Polymerase Chain Reaction (PCR)

Additional Nonhistone Proteins Regulate Transcription and Replication

Nonhistone Proteins Organize Long Chromatin Loops

Modifications of Histone Tails Control Chromatin Condensation and Function

Chromatin Exists in Extended and Condensed Forms

Fetal DNA Sequencing and Fetal Genome Analysis

Globin Gene Clusters

Molecular Analysis of Nucleic Acid Sequences

Genes and Genome Complexity

Structure of Nucleic Acids