Having reviewed the relationship between transcription units and genes, we now consider the organization of genes on chromosomes and the relationship of noncoding DNA sequences to coding sequences.

Genomes of Many Organisms Contain Nonfunctional DNA

Comparisons of the total chromosomal DNA per cell in various species first suggested that much of the DNA in certain organisms does not encode functional RNA or have any apparent regulatory function. For example, yeasts, fruit flies, chickens, and humans have successively more DNA in their haploid chromosome sets (12.5, 180, 1300, and 3300 Mb, respectively), in keeping with what we perceive to be the increasing complexity of these organisms. Yet the vertebrates with the greatest amount of DNA per cell are amphibians, which are surely less complex than humans in their structure and behavior. Even more surprising, the unicellular protozoan Amoeba dubia has 200 times more DNA per cell than humans. Many plant species also have considerably more DNA per cell than humans have; tulips, for example, have 10 times as much DNA per cell as humans. The DNA content per cell also varies considerably between closely related species. All insects or all amphibians would appear to be similarly complex, but the amount of haploid DNA in species within each of these phylogenetic classes varies by a factor of 100.

Sequencing and identification of exons in chromosomal DNA have provided direct evidence that the genomes of higher eukaryotes contain large amounts of noncoding DNA. For instance, only a small portion of the β-globin gene cluster of humans, which is about 80 kb long, encodes protein (see Figure 1a). In contrast, a typical 80-kb stretch of DNA from the yeast S. cerevisiae, a single-celled eukaryote, contains many closely spaced protein-coding sequences without introns and relatively much less noncoding DNA (see Figure 1b). Moreover, the introns in globin genes are considerably shorter than those in most human genes. Glo bin proteins comprise about 50 percent of the total protein in developing red blood cells (erythroid progenitors), and the globin genes are expressed at maximum rates (i.e., a new RNA polymerase initiates transcription as soon as the previous polymerase transcribes far enough from the promoter to allow it to do so). Consequently, there has been selective pressure on globin genes for small introns that are compatible with the required high rate of globin mRNA transcription and processing. However, the vast majority of human genes are expressed at much lower levels, which require production of one encoded mRNA on a time scale of only tens of minutes or hours. Consequently, there has been little selective pressure to reduce the sizes of introns in most human genes.

Fig1. Comparison of gene density in higher and lower eukaryotes. (a) In this diagram of the β-globin gene cluster on human chromosome 11, the green boxes represent exons of β-globin– related genes. Exons spliced together to form one mRNA are connected by caret-like spikes. The human β-globin gene cluster contains two pseudogenes (white); these regions are related to the functional β-globin genes but are not transcribed. Each red arrow indicates the location of an Alu sequence, a roughly 300-bp noncoding repeated sequence that is abundant in the human genome. See F. S. Collins and S. M. Weissman, 1984, Prog. Nucl. Acid Res. Mol. Biol. 31:315. (b) In this diagram of yeast DNA from chromosome III, the green boxes indicate open reading frames. Most of these potential protein-coding sequences are functional genes without introns. Note the much higher proportion of noncoding to coding sequences in the human DNA than in the yeast DNA. See S. G. Oliver et al., 1992, Nature 357:28.

The density of genes varies among regions of human chromosomal DNA, from “gene-rich” regions, where a few hundred base pairs separate transcription units, to large gene-poor “gene deserts,” where intergenic regions are a few million base pairs long. Of the 96 percent of human genomic DNA that has been sequenced, only about 2.9 percent corresponds to exons, and only about 1.2 percent encodes proteins. (The fraction of the genome that corresponds to exons is much larger than the fraction that encodes proteins because many protein-coding genes include exons for long 3′ untranslated regions and because there are many exons in nonprotein-coding lncRNAs.) We learned in the previous section that the intron sequences of most human genes are significantly longer than the exon sequences. Approximately 55 percent of human genomic DNA is thought to be transcribed into pre-mRNAs, pre lncRNAs, or other nonprotein-coding RNAs in one cell or another, but some 95 percent of this sequence is intronic and is thus removed by RNA splicing. The remaining 45 per cent of human DNA constitutes noncoding DNA between genes as well as the regions of repeated DNA sequences that make up the centromeres and telomeres of the human chromosomes. Consequently, about 97 percent of human DNA does not encode proteins, functional noncoding RNAs, or potentially functional lncRNAs.

Different selective pressures may account, at least in part, for the remarkable difference in the amount of nonfunctional DNA in different organisms. For example, many microorganisms must compete with other species of microorganisms in the same environment for limited amounts of available nutrients, and metabolic economy is thus a critical characteristic for these organisms. Because synthesis of nonfunctional (i.e., noncoding) DNA requires time, nutrients, and energy, presumably there was selective pressure to lose nonfunctional DNA during the evolution of rapidly growing microorganisms such as the yeast S. cerevisiae. On the other hand, natural selection in vertebrates depends largely on their behavior. The energy invested in DNA synthesis is trivial compared with the metabolic energy required for the movement of muscles and the function of the nervous system; thus there may have been little selective pressure on vertebrates to eliminate nonfunctional DNA. Furthermore, the replication time of cells in most vertebrates and plants is much longer than in rapidly growing microorganisms, so there may have been little selective pressure to eliminate nonfunctional DNA in order to permit rapid cellular replication.

Most Simple-Sequence DNAs Are Concentrated in Specific Chromosomal Locations

 Besides duplicated protein-coding genes and tandemly repeated genes, eukaryotic cells contain multiple copies of other DNA sequences, generally referred to as repetitious DNA (see Table 1). Of the two main types of repetitious DNA, the less prevalent is simple-sequence DNA, or satellite DNA. This type of DNA, which constitutes about 6 percent of the human genome, is composed of perfect or nearly perfect repeats of relatively short sequences. The second, more common type of repetitious DNA, collectively called interspersed repeats, is composed of much longer sequences.

Table1. Major Classes of Nuclear Eukaryotic DNA and Their Representation in the Human Genome

The length of each repeat in simple-sequence DNA can range from 1 to 500 base pairs. DNA sequences in which the repeats each contain 1–13 bp are often called micro satellites. Most microsatellite DNA has a repeat length of 1–4 bp, and the repeats usually occur in tandem sequences of 150 repeats or fewer. Microsatellites are thought to have originated by “backward slippage” of a daughter strand on its template strand during DNA replication so that the same short sequence was copied twice (Figure 2).

Fig2. Generation of microsatellite repeats by backward slippage of the nascent daughter strand during DNA replication. If, during replication (a), the nascent daughter strand “slips” back ward relative to the template strand by one repeat, one new copy of the repeat is added to the daughter strand when DNA replication continues (b). An extra copy of the repeat forms a single-stranded loop in the daughter strand of the daughter duplex DNA molecule. If this single-stranded loop is not removed by DNA repair proteins before the next round of DNA replication (c), the extra copy of the repeat is added to one of the double-stranded daughter DNA molecules.

Microsatellites occasionally occur within transcription units. Some individuals are born with a larger number of repeats in specific genes than are observed in the general population, presumably because of daughter-strand slippage during DNA replication in the germ cells from which they and their forebears developed. Such expanded microsatellites have been found to cause at least 14 different types of neuromuscular diseases, depending on the gene in which they occur. In some cases, expanded microsatellites behave like a recessive mutation because they interfere with just the function or expression of the gene in which they occur. But in the more common types of diseases associated with expanded microsatellites, the expanded microsatellites behave like dominant mutations. In some of these diseases, such as Huntington disease, triplet repeats occur within a coding region, resulting in the formation of long polymers of a single amino acid that may aggregate over time in long lived neuronal cells, eventually interfering with normal cellular function. For example, expansion of a CAG repeat in the first exon of the gene involved in Huntington disease leads to synthesis of long stretches of polyglutamine, which over several decades form toxic aggregates resulting in neuronal cell death in patients with the disease.

Pathogenic expanded repeats can also occur in the noncoding regions of some genes, where they are thought to function as dominant mutations because they interfere with the processing of a subset of mRNAs in the muscle cells and neurons where the affected genes are expressed. For example, in patients with myotonic dystrophy type 1, transcripts of the DMPK gene contain between 50 and 1500 repeats of the sequence CUG in the 3′ untranslated region, compared with 5–34 repeats in unaffected individuals. The extended stretch of CUG repeats in affected individuals is thought to form a long RNA hairpin, which binds and sequesters nuclear RNA-binding proteins that normally regulate alternative RNA splicing of a subset of pre-mRNAs essential for muscle and nerve cell function.

Most simple-sequence satellite DNA is composed of repeats of 14–500 bp in tandem arrays 20–100 kb long. In situ hybridization studies with metaphase chromosomes have localized this simple-sequence DNA to specific chromosomal regions. Much of this DNA lies near centromeres, the discrete chromosomal regions that attach to spindle micro tubules during mitosis and meiosis (Figure 3). Experiments in the fission yeast S. pombe indicate that these sequences are required to form a specialized chromatin structure called centromeric heterochromatin, necessary for the proper segregation of chromosomes to daughter cells during mitosis. Simple-sequence DNA is also found in long tandem repeats at the ends of chromosomes, the telomeres, where it functions to maintain those chromosome ends and prevent their joining to the ends of other DNA molecules, as discussed further in the last section of this chapter.

EXPERIMENTAL FIGURE 3. Simple-sequence DNA is localized at the centromere in mouse chromosomes. Purified simple-sequence DNA from mouse cells was copied in vitro using E. coli DNA polymerase I and fluorescently labeled dNTPs to generate a fluorescently labeled DNA “probe” for mouse simple-sequence DNA. Chromosomes from cultured mouse cells were fixed and denatured on a microscope slide, and the chromosomal DNA was then hybridized in situ to the labeled probe (light blue). The slide was also stained with DAPI, a DNA-binding dye, to visualize the full length of the chromosomes (dark blue). Fluorescence microscopy shows that the simple-sequence probe hybridizes primarily to one end of the telocentric mouse chromosomes (i.e., chromosomes in which the centromeres are located near one end). [Courtesy of Sabine Mal, Ph.D., Manitoba Institute of Cell Biology, Canada.]

DNA Fingerprinting Depends on Differences in Length of Simple-Sequence DNAs

Within a species, the nucleotide sequences of the repeat units composing a simple-sequence DNA tandem array are highly conserved among individuals. In contrast, the number of repeats, and thus the length of simple-sequence tandem arrays containing the same repeat unit, is quite variable among individuals. These differences in length are thought to result from unequal crossing over within regions of simple-sequence DNA during meiosis. As a consequence of this unequal crossing over, the lengths of some tandem arrays are unique in each individual.

In humans and other mammals, some simple-sequence DNA exists in relatively short 1–5-kb regions made up of 20–50 repeat units, each containing 14–100 bp. These regions are called minisatellites, in contrast to microsatellites made up of tandem repeats of 1–13 bp. Even slight differences in the total lengths of various minisatellites from different individuals can be detected by Southern blotting. This technique was exploited in the first application of DNA fingerprinting, which was developed to detect DNA polymorphisms (i.e., differences in sequence between individuals of the same species) (Figure 4). Today the far more sensitive polymerase chain reaction (PCR) technique is generally used in forensic genetic testing. Microsatellites consisting of tandem repeats of four bases in 30–50 copies are usually analyzed today. The exact number of repeats at a specific location in the genome generally varies between the two homologous chromosomes of an individual (one inherited from the mother and one from the father) and between the Y chromosomes of different males. A mixture of pairs of PCR primers that hybridize to unique sequences flanking 13 of these short tandem repeats and a Y-chromosome short tandem repeat are used to amplify DNA in a sample from one individual. The resulting mixture of PCR product lengths is unique in the human population, except for identical twins. The use of PCR allows analysis of minute amounts of DNA, and individuals can be distinguished more precisely and reliably than by conventional fingerprinting.

Fig4. Distinguishing individuals by DNA fingerprinting. (a) In this analysis of paternity, several minisatellite repeat lengths were determined by Southern blot analysis of restriction enzyme–digested genomic DNA and hybridization with a probe for a sequence shared by several minisatellite sequences. This method generated hypervariable multiband patterns for each individual called “DNA fingerprints.” Lane M shows the pattern of restriction fragment bands using the mother’s DNA; C, using the child’s DNA; and F1 and F2 using DNA from two potential fathers. The child has minisatellite repeat lengths inherited from either the mother or F1, demonstrating that F1 is the father. Arrows indicate restriction fragments from F1, but not F2, found in the child’s DNA. (b) In these “DNA fingerprints” of a specimen isolated from a rape victim and three men suspected of the crime, it is clear that minisatellite repeat lengths in the specimen match those of suspect 1. The victim’s DNA was included in the analysis to ensure that the specimen DNA was not contaminated with DNA from the victim.

Unclassified Intergenic DNA Occupies a Significant Portion of the Genome

About 45 percent of human DNA lies between transcription units. Much of this sequence is not repeated anywhere else in the genome. Enhancers on the order of 50–200 bp in length that help to regulate transcription at distant promoters occur in these long stretches of intergenic DNA, as well as in introns. These enhancers are often conserved during evolution, while the neighboring intergenic sequences are not conserved. Other conserved intergenic regions may perform significant functions that are not yet understood.

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إيذانًا ببدء فعاليات حفل التكليف المركزي.. انطلاق مسير التلميذات في مجمع أبي الفضل العباس (عليه السلام)

بدء توافد التلميذات المشاركات في فعاليات حفل التكليف الشرعي ضمن مشروع الورود الفاطمية

قسم الشؤون الفكرية يقدم محاضرة حول الأخلاق الإدارية في بيئة العمل لوفد من ذي قار

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طريقة جديدة لتجديد خلايا الدماغ

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7 أطعمة يومية "تسمم" جسمك بالبلاستيك دون أن تشعر

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بسبب الذكاء الاصطناعي.. هذه الوظائف "ستختفي" بعد أشهر قليلة ؟!

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مواضيع ذات صلة

RNA Interference Causes Gene Inactivation by Destroying the Corresponding mRNA

Linkage Studies Can Map Disease Genes with a Resolution of About 1 Centimorgan

DNA Polymorphisms Are Used as Markers for Linkage Mapping of Human Mutations

The Two- Hit Theory of Tumor Suppressor Gene Inactivation in Cancer

DNA Microarrays Can Be Used to Evaluate the Expression of Many Genes at One Time

Hybridization Techniques Permit Detection of Specific DNA Fragments and mRNAs

Cloned DNA Molecules Can Be Sequenced Rapidly by Methods Based on PCR

Cellular and Molecular Mechanisms in Development: Gene Regulation by Transcription Factors

Transcriptional Control of Megakaryocytopoiesis: GATA Family Transcription Factors

The Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture

Role of Developmentally Important Neutrophil-Specific Genes in Disease

Genes Can Be Identified by Their Map Position on the Chromosome