Ribosomal RNA

Ribosomal RNAs (rRNAs) are functional RNA transcripts that constitute one of the main components of cytoplasmic ribosomes. The genes coding for the major form of cytoplasmic rRNA are located in multiple copies on the short arms of the acrocentric chromosomes: 13, 14, 15, 21, and 22. They code for a single large 45S primary transcript that is cleaved into 28S, 18S, and 5.8S rRNA classes, designated by their separation in centrifugation gradients and by several associated proteins. Multiple copies of another gene on chromosome 1 produce 5S rRNA.

Transfer RNA

Transfer RNAs (tRNAs) are small RNA transcripts that bind specific amino acids and transport them to ribosomes for use during protein synthesis. More than 40 subfamilies of tRNA genes are known, dispersed across the genome. The mitochondrial genome uses a separate set of tRNAs.

Messenger RNA

Messenger RNAs (mRNAs) are the RNA transcripts of all genes that encode polypeptides and some other genes that encode unprocessed functional RNA molecules. Most are large. All mRNA transcripts undergo further processing, including excision of large segments of noncoding RNA known as introns, the addition of 7-methylguanosine to the first 5′ nucleotide, forming a CAP structure, cleavage of the 3′ end at a specific point downstream from the end of the coding sequence, and addition of the polyA tail at a site specified in part by the sequence AAUAAA, which is located in the 3′ untranslated region (3′ UTR) of the gene. The polyA tail appears to increase the stability of mRNA. The fully processed mRNA is transported to the cytoplasm, where translation occurs.

Small Nuclear RNA

Small nuclear RNA (snRNA) transcripts are small, uridine-rich RNA transcripts that associate with specific proteins to form ribonucleoprotein particles (RNPs). Some of them function in RNA splicing (removing introns from mRNA). They comprise a large family of genes dispersed across the genome.

Small Nucleolar RNA

Small nucleolar RNAs (snoRNAs) are small RNA transcripts that are present in the nucleolus and have important roles in specific cleavage reactions and base-specific modifications during maturation of ribosomal RNA. About 200 snoRNA genes have been identified.

MicroRNA

MicroRNAs (miRNAs) are another class of small noncoding genes that regulate the expression of protein-encoding genes at the post-transcriptional RNA level [Denli et al., 2004]. The process begins with transcription (synthesis) of primary RNA transcripts that range in size from several hundred to several thousand kb. These transcripts are recognized and cut into precursor miRNAs in the nucleus by a protein known as Dicer, moved to the cytoplasm, and processed into mature miRNAs. The mature miRNAs join the RNA-induced silencing complex (RISC), which recognizes and cleaves (or otherwise silences) a target gene. This process has been demonstrated in many organisms, including mammals, and appears likely to play a key role in regulation of many genes.

Genes

Genes are composed of a continuous length of DNA with definable start and end points, which include the sequence that codes for the RNA or polypeptide product and is thus known as the coding region. It has become clear, however, that the structure of a gene is complex and includes much more than the coding sequence of the protein. All genes include additional sequences on either end of the coding region – designated the 5′ and 3′ UTRs – that do not code for an RNA product or polypeptide. These regions function to regulate transcription and RNA stability. The gene is considered to include the entire sequence represented in the RNA product because some mutations within noncoding regions can impair gene function.

A model of a typical human gene is shown in Figure 30-3. Promoter sequences required for regulation and initiation of RNA transcription (red diamonds in Figure 30-3) are present at the 5′ end of the gene, such as the CAT and TATA boxes whose sequences are tightly conserved among many different genes and species. Downstream from the promoter sequences is a specific sequence that signals the start of transcription. A short way further downstream is an initiator codon, AUG, which codes for methionine. This triplet is the translation start site, which signals the start of the coding sequence for the polypeptide product. The region between the transcription and translation start sites is the 5′ UTR.

Fig. 30-3 The structure of a typical human gene.

The gene includes a primary regulatory region known as the promoter just upstream of the transcription start site that is required for binding of both DNA and RNA polymerases (red diamonds), as well as several types of distant regulatory elements that protect the gene from regulation of other nearby genes (insulator), increase or decrease gene expression (enhancers and silencers), or regulate several genes in the region (locus control region).

The next segment of the gene is the coding region. The coding regions of most genes in prokaryotes and lower eukaryotes are colinear, which means that the coding sequence corresponds exactly to the sequence of amino acids in the polypeptide. By contrast, most higher eukaryotic genes, including human genes, contain additional sequences that lie within the coding region, interrupting the sequence that represents the polypeptide. The regions that code for the final polypeptide (or functional RNA) product are known as exons, whereas the regions that are missing from the final mRNA product are introns. The removal of introns from the final mRNA product is known as splicing, a complex process that is regulated by a large number of proteins and functional RNA transcripts.

The coding sequence ends at one of three specific stop codons: UAA, UAG, or UGA. The last segment of the gene is the 3′ UTR, which contains a polyadenylation signal and presumably a signal to end transcription, although no transcription stop sequence has been identified. The length of a gene may vary, ranging from less than 1 kb to several hundred kb. The longest gene known, which codes for dystrophin, spans more than 2000 kb of genomic sequence, although this is not the largest protein produced in the cell.

Regulatory Regions

Many genes have highly conserved sequences, a longer distance upstream and downstream of the transcribed gene, that are involved in regulating expression, including enhancers, silencers, locus control regions, and insulators (see Figure 30-3). Enhancer elements function to increase gene expression, while silencers reduce gene expression. Locus control regions may regulate expression of several genes within a chromosome region, while insulators prevent co-regulation of more distant genes and gene regions. All of these are sequences that bind proteins called transcription factors, which can be ubiquitous, tissue-specific, and/or temporally expressed. Promoters are located immediately 5′ of the gene and bind to RNA polymerase II, a necessary step for transcription. Other transcription factors bind upstream of the promoter and activate transcription. Enhancers and silencers are often located at a distance from the promoter, and increase or decrease transcription in a tissue-specific or temporal manner. Overall, the transcription of each gene is tightly regulated, with multiple transcription factors involved.

Imprinting

The process by which certain genes in specific chromosomal regions are expressed from only one chromosome, depending on the parental origin of the chromosome, is known as “imprinting.” Although the mechanism is only partly understood, a key component involves allele-specific DNA methylation, found predominantly at the carbon 5 position of about 80 percent of all cytosines that are part of symmetrical cytosine-guanine (CpG) dinucleotides [Jiang et al., 2004; Strachan and Read, 2010; Weksberg et al., 2003].

This process is controlled by regulatory imprinting “centers,” located nearby on the same chromosome as that of the silenced or “imprinted” gene. In effect, then, two alleles of the same gene that are identical in nucleotide sequence but derived from opposite parents are regulated differently in the same nucleus. This process is reversible, so that the silent, imprinted allele can be reactivated and the active allele silenced when passed through the germline of the opposite-sex parent. Most imprinted genes are found in large clusters of greater than 1 Mb (megabase pairs) in length. Imprinted clusters have been identified in chromosomes 6q24, 7p11.2, 11p15.5, 14q32, 15q11–q13, and 20q13.2, and others may exist as well [Cavaille et al., 2002; Gardner et al., 2000; Hall, 1990; Jiang et al., 2004; Weksberg et al., 2003; Wylie et al., 2000]. Imprinted regions share several common characteristics, including differential DNA methylation, allele-specific RNA transcription, antisense transcripts, histone modifications, and differences in timing of replication.

X-Inactivation

In mammalian cells with two (or more) X chromosomes, all but one undergo widespread gene silencing by methylation. This phenomenon, known as X-chromosome inactivation (Xi), causes one of the two X chromosomes in cells of female mammals to become transcriptionally inactive early in embryonic development, a phenomenon known as the Lyon hypothesis [Lyon, 1961, 2002]. In mutant cells with more than two X chromosomes, all but one become inactivated. This has the effect of balancing gene dosage of X-linked genes between male and female cells. The process of Xi is random, so that on average the maternally and paternally derived X chromosomes are each inactivated in approximately 50 percent of cells. Changes in this pattern are seen in female carriers of some X-linked diseases, resulting in skewing of Xi. This alteration can be favorable, with decreased severity of the phenotype, or unfavorable, with increased severity of the phenotype [Dobyns et al., 2004].

Mitosis

Somatic cell division, or mitosis, is an elaborate mechanism that distributes one chromatid of each duplicated chromosome to each of the two daughter cells. The process is continuous but has been divided into the following five stages: prophase, prometaphase, metaphase, anaphase, and telophase (Figure 30-5).

Fig. 30-5 Diagram of mitosis demonstrating two chromosome pairs.

In prophase, the chromatin begins to condense, the nucleolus disappears, and the mitotic spindle begins to form. Prophase is followed by prometaphase, during which the nuclear membrane disappears, allowing the chromosomes to disperse in the cell and attach to the spindle by paired kinetochores located at the centromere. In metaphase, the chromosomes are maximally contracted and arranged at the equatorial plane of the cell. In anaphase, the replicated chromosomes separate at the centromere, allowing the two chromatids to become daughter chromosomes, which move to opposite ends of the cell. In telophase, the chromosomes decondense, the nuclear membrane reforms, and the nucleus returns to the interphase appearance. Shortly afterward, the cytoplasm divides to form two daughter cells. For routine studies, chromosomes are examined during metaphase. For high-resolution studies, they are examined before the point of maximal contraction, during prophase or prometaphase.

Meiosis

Reproductive cell division, or meiosis, is an even more complex mechanism in which two successive cell divisions, known as meiosis 1 and meiosis 2, give rise to the haploid germ cells (Figure 30-6). Meiosis is of critical importance in understanding many of the methods of modern molecular genetics and the pathogenesis of many genetic diseases.

Fig. 30-6 Diagram of meiosis depicting two chromosome pairs.

In meiosis 1, the chromosome number is reduced from the diploid to the haploid number. The key step consists of close pairing of homologous chromosomes during prophase 1, which is further divided into several stages. During leptotene, the chromosomes first become visible, with homologs located close together. During zygotene, the homologs begin to pair closely along their entire length, held together by a thin protein-containing structure known as a synaptonemal complex. During pachytene, synapsis or pairing is completed, and the homologs appear as a bivalent. Pachytene is the stage during which exchange of homologous segments between nonsister chromatids occurs, which is known as recombination or crossing over. The remaining steps are similar to mitosis, except that it is the paired homologs that are pulled apart rather than the centromeres. In meiosis 2, which closely resembles mitosis, the chromatids separate at the centromere to form daughter chromosomes. Ova and sperm have remarkably different timing, but the sequence of meiosis is the same.

Chromosome Structure

In humans, the nuclear DNA is dispersed among 46 separate linear structures or chromosomes, each of which consists of a single, uninterrupted double helix that contains 50–250Mb of DNA, and a group of associated proteins that form the support structure or scaffolding. The scaffolding consists of five basic proteins called histones and several more acidic nonhistone proteins. Two copies of each of four histones – H2A, H2B, H3, and H4 – join to form an octamer. The DNA double helix wraps almost twice around the octamer, which involves about 140 bp. Adjacent octamers are separated by a short spacer segment of 20–60bp that is associated with histone H1. The complex of DNA and core histones is known as a nucleosome (see Figure 30-2).

Strings of nucleosomes are further compacted into a secondary helical structure known as a solenoid. These structures have a diameter of about 30 nm (see Figure 30-2) and contain six nucleosomes per turn. The solenoids are packed into large loops of 10–100 kb of DNA, which are attached to a nonhistone protein scaffolding. These loops pack together loosely to form interphase chromosomes. During early prophase, they pack together more closely to form knoblike thickenings known as chromomeres, which then coalesce further to form the bands observed in prometaphase and metaphase chromosomes when stained with appropriate dyes.

The alternating light and dark bands that characterize all nuclear chromosomes with a variety of staining methods likely reflect the compartmentalization of the genome into isochores, defined as large regions with variation in base composition or variable spacing of scaffold attachment regions. The dark bands observed with Giemsa staining are AT-rich, replicate late in the DNA synthesis phase of the cell cycle, and contain relatively few genes. The light bands observed with Giemsa are GC-rich, replicate early, and contain many genes. Some are greatly enriched for GC and contain high concentrations of genes. Most, although not all, such bands are located near the ends or telomeres of chromosomes and therefore are known as T bands.

Specialized Regions

All nuclear chromosomes have specialized regions that are required for chromosome integrity and function, including centromeres, telomeres, and origins of replication. Centromeres are DNA sequences that act in cis. That is, they act on the chromosome on which they are located and are responsible for the segregation of chromosomes during cell division. Centromeres contain extensive repeats of an approximately 171-bp unit known as alpha-satellite DNA, the sequence of which differs slightly between each chromosome. Fragments of chromosomes that lack a centromere, known as acentric fragments, are lost during cell division.

The two ends of a chromosome are called telomeres and also are required for chromosome stability. In humans, they consist of long arrays of tandem repeats of the sequence TTAGGG, which extend about 5–20 kb. DNA polymerases are unable to replicate the telomeres because of the lack of a template. This problem is resolved by the enzyme telomerase, which contains an RNA component to serve as a template to prime further synthesis on the leading strand. Further extension of the leading strand provides the needed template for the lagging strand.

Origins of replication are specialized sequences where DNA replication begins, and thus are important in maintaining chromosome number and integrity. They consist of autonomously replicating sequence elements that contain a core consensus sequence and some imperfect copies with a length of about 50 nucleotides. A consensus human autonomously replicating sequence has been identified [Strachan and Read, 2010].

Regions of variable staining known as heterochromatin consist of long arrays of repeat sequences as short as 5 bp. These regions are located primarily in the pericentromeric regions of chromosomes 1, 9, and 16, and in distal Yq. The five human acrocentric chromosomes have small satellites attached to the short arm by short stalks or secondary constrictions that contain the rRNA genes.

Chromosome Number

Each human somatic cell contains 46 chromosomes that consist of 22 matched pairs known as autosomes and two sex chromosomes: XX in females and XY in males (Figure 30-7). In contrast, human germ cells contain only 23 chromosomes, consisting of 22 unpaired autosomes and a single sex chromosome. The former is known as the diploid or 2n number, and the latter is known as the haploid or 1n number. The autosomes were numbered according to length, with chromosome 1 the longest and chromosome 22 thought to be the shortest. Although chromosome 21 later proved to be shorter than chromosome 22, the numbers were retained for historical reasons. The two members of each pair of autosomes and the two X chromosomes in females carry the same genes and are known as homologous chromosomes, or homologs. Although they appear similar under the microscope, homologs are not strictly identical. They contain the same genes, but the nucleotide sequence differs at thousands of positions.

Fig. 30-7 Standardized diagram or idiogram of human chromosomes at the 400-band stage.

Chromosome Identification

Individual chromosomes may be seen only when tightly contracted during cell division. Since DNA replication is complete, each chromosome consists of two chromatids that are joined at the primary constriction or centromere. In standard cytogenetic nomenclature, the centromere divides the chromosome into two arms, with the shorter designated the “p” arm and the longer the “q” arm. The tip of each arm is the telomere. Human chromosomes are classified into three types according to the position of the centromere:

Genes and Conserved Noncoding DNA

Somewhat surprisingly, recent estimates predict that the human genome contains less than 30,000 protein-coding genes (possibly closer to 20,000) and an uncertain number of other genes producing functional RNA products. This is far fewer than earlier estimates, and accounts for only about 1.2 percent of nuclear DNA [Lander et al., 2001; Venter et al., 2001]. Another 5 percent of the human genome is more conserved than would be expected from estimates of neutral evolution, which suggests that many of these regions have specific, regulatory functions [Chiaromonte et al., 2003; Waterston et al., 2002]. Studies of these highly conserved regions of DNA have used different thresholds, such as stretches of more than 100 bp with 70–80 percent conservation between mouse and human. Some of these regions have been found to contain important noncoding elements [Dermitzakis et al., 2002, 2003; Frazer et al., 2004; Hardison, 2000]. More stringent analysis demonstrates that the human genome contains 481 sequences of 200 or more bp that are 100 percent conserved among human, mouse, and rat [Bejerano et al., 2004]. These segments were designated “ultra-conserved elements,” and are preferentially located near genes involved in RNA processing or regulation of transcription and development. Similarly, about 5000 sequences of 100 bp or more are conserved among these three species, which emphasizes that noncoding sequences are common and important.

Repetitive DNA

Repetitive DNA in the human genome consists of several classes of DNA whose nucleotide sequence is repeated, either exactly or with minor variations, hundreds to millions of times. Some classes are clustered, whereas others are dispersed throughout the genome. Clustered, repeated sequences constitute 10–15 percent of the genome and are collectively called satellite DNA because of their separation from other DNA on density centrifugation. Satellite DNA consists of head-to-tail or tandem arrayed repeat sequences that can extend for several thousand kb. Dispersed, repeat sequences constitute 6–10 percent of the genome and belong to several different classes. Minisatellite or variable number of tandem repeat (VNTR) sequences are dispersed, intermediate-length (15–65 bp) repeats that usually span only several kb. The Alu family of DNA repeats includes about 500,000 related sequences that are each about 300 bp in length and together make up about 3 percent of the genome. The L1 family of repeats includes about 10,000 related sequences that extend up to 6 kb in length and make up another 3 percent of the genome. Although the origin of these sequences is not known, no functions have been identified, and it appears likely that they simply exploit cellular processes to propagate themselves. Several classes have been useful as polymorphic DNA markers.

Low Copy Repeats

Segmental duplications, also known as low copy repeats (LCRs), are DNA sequences of 10–250 kb, present in multiple copies with greater than 95 percent sequence identity, that make up approximately 5 percent of the human genome [Babcock et al., 2003; Bailey et al., 2002; Cheung et al., 2001; Stankiewicz and Lupski, 2002]. LCRs are dynamic regions of the genome because specific repeats tend to cluster within the same genomic regions, where they mediate unequal nonhomologous recombination events, producing segmental deletions and duplications that are collectively designated “copy number variants” (CNVs). Several of these have been associated with well-known developmental disorders in humans, such as Williams’ syndrome in 7q11.23, Angelman’s syndrome and Prader–Willi syndrome in 15q12, hereditary neuropathy with predisposition to pressure palsies and Charcot–Marie–Tooth neuropathy type 1A in 17p12, Smith–Magenis syndrome in 17p11.2, and DiGeorge’s syndrome in 22q11.2 [Babcock et al., 2003]. Many new CNV-associated devlopmental brain disorders have been described over the past few years.

Minisatellites

One of the most useful classes of polymorphisms in the genome is that of the minisatellite or VNTR DNA sequences. These are intermediate-length (15–65 bp) DNA sequences that are repeated one to several dozen times in tandem and usually span several kb in total length. They are highly polymorphic, and their extreme polymorphic nature, coupled with the complexity of multilocus minisatellites, makes them valuable for DNA fingerprinting applications, such as forensic, paternity, and zygosity testing and linkage mapping. They also are inherently unstable and susceptible to mutation at a higher rate than observed for other sequences of DNA.

Microsatellites

Microsatellites, also known as satellite DNA or short tandem repeats, are segments of DNA 2–5 nucleotides in length (dinucleotide, trinucleotide, tetranucleotide, or pentanucleotide repeats) that are scattered throughout the genome in noncoding regions between genes or within genes (in introns). They often are used as markers for linkage analysis because of the naturally occurring high variability in repeat number between individuals. These regions are inherently unstable and susceptible to mutations.

The most common microsatellite family consists of 50,000–100,000 cytosine-adenine (CA) repeats, which consist of short tandem repeats of the dinucleotide CA on one strand and guanine-thymine on the complementary strand. They thus take the form (CA)n/(GT)n, with n in the range of 6–30 [Weber and May, 1989]. The number of repeats within a (CA)n block varies greatly among different members of a species, producing a set of alleles that always differ in size by multiples of two bases. About 70 percent of the human population is heterozygous at any given (CA)n repeat locus, making these highly polymorphic. The human genome contains about 50,000–100,000 interspersed (CA)n blocks, which is enough to place 1 block every 30–60 kb, if evenly spaced.

For both VNTR and CA repeat sequences, the combination of high frequency in the genome and a high rate of polymorphism has made them very useful for genetic mapping and association studies. Some microsatellite repeats, most often trinucleotide repeats, present within coding regions of genes or, less often, the 5′ or 3′ UTR, can expand to an abnormal length and are the basis of triplet repeat diseases such as Huntington’s disease, some forms of spinocerebellar ataxia, and fragile X syndrome.

Single-Nucleotide Polymorphisms

Single-nucleotide polymorphisms (SNPs, pronounced “snips”) are DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is changed. For example, a SNP might change the DNA sequence TCACG to TTACG. The most common sequence change involves replacement of cytosine (C) with thymidine (T), which accounts for about two-thirds of all SNPs. As with other types of sequence variation, a SNP must occur in at least 1 percent of the population to be classified as a polymorphism. SNPs occur in both unique-sequence (coding and noncoding) and repetitive DNA, and are responsible for about 90 percent of human genetic variation. On average, SNPs are found approximately every 100–300 bases along the entire human genome. Although most SNPs likely have no function, some are known to influence disease predisposition or responses to drugs, and thus are proving to be very valuable in studying the causes of common human diseases. The current inventory of known SNPs can be found in the Human SNP database (dbSNP) on the NCBI Entrez website (see Table 30-1).

Restriction enzymes are DNA-cutting enzymes or endonucleases derived from bacteria that cut DNA at specific short sequences found at locations across the entire genome. SNPs can alter the sequences recognized by restriction enzymes, thus adding or removing a cutting site. This is the biological basis for restriction enzyme fragment length polymorphisms (RFLPs). Depending on the location of restriction enzyme sites, specific DNA fragment lengths are obtained on digestion with restriction endonucleases. The presence of a SNP at one of these restriction enzyme sites will affect cleavage and produce two DNA fragments of different sizes that is the RFLP. RFLPs also can be produced by any change that alters the size of the DNA fragment on which the restriction site is located, such as deletions or duplications. RFLPs are a measure of naturally occurring variations or polymorphisms of normal DNA, and are inherited according to mendelian principles. RFLPs have been useful for gene mapping.