The Human Genome

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Chapter 74 The Human Genome

The Human Genome Project, culminating in the sequencing of the human genome, made it possible to study virtually any human gene and to explore the roles of genes in both rare and common disorders. It has also become apparent that the genome includes far more than a coded store of information to produce proteins.

The human genome has approximately 25,000 genes that encode the wide variety of proteins found in the human body. Reproductive or germline cells contain 1 copy (N) of this genetic complement and are haploid, whereas somatic (nongermline) cells contain 2 complete copies (2N) and are diploid. Genes are organized into long segments of DNA, which, during cell division, are compacted into intricate structures together with proteins to form chromosomes. Each somatic cell has 46 chromosomes: 22 pairs of autosomes, or nonsex chromosomes, and 1 pair of sex chromosomes (XY in a male, XX in a female). Germ cells (eggs, sperm) contain 22 autosomes and 1 sex chromosome, for a total of 23. At fertilization, the full diploid chromosome complement of 46 is again realized in the embryo.

Most of the genetic material is contained in the cell’s nucleus. The mitochondria (the cell’s energy-producing organelles) contain their own unique genome. The mitochondrial chromosome consists of a double-stranded circular piece of DNA, which contains 16,568 base pairs (bp) of DNA and is present in multiple copies within mitochondria per cell. The proteins that compose the mitochondria may either be produced in the mitochondria (from information contained in the mitochondrial genome) or from information contained in the nuclear genome and transported into the organelle. All mitochondria are maternally derived; sperm do not usually contribute mitochondria to the developing embryo. Hence, a child’s mitochondrial genetic makeup derives exclusively from his or her biological mother.

Fundamentals of Molecular Genetics

The central tenet of molecular genetics is that information encoded in DNA, predominantly located in the cell nucleus, is transcribed into messenger RNA (mRNA), which is then transported to the cytoplasm, where it is translated into protein. A gene is a unit that includes a regulatory region and a coding region that stores information corresponding to the sequence of amino acids in a specific protein.

DNA consists of a pair of chains of a sugar-phosphate backbone linked by pyrimidine and purine bases to form a double helix (Fig. 74-1). The sugar in DNA is deoxyribose. The pyrimidines are cytosine (C) and thymine (T); the purines are guanine (G) and adenine (A). The bases are linked by hydrogen bonds such that A always pairs with T and G with C. Each strand of the double helix has polarity, with a free phosphate at one end (5′) and an unbonded hydroxyl on the sugar at the other end (3′). The two strands are oriented in opposite polarity in the double helix.


Figure 74-1 DNA double helix, with sugar-phosphate backbone and nitrogenous bases.

(From Jorde LB, Carey JC, Bamshad MJ, et al, editors: Medical genetics, ed 2, St Louis, 1999, Mosby, p 8.)

The replication of DNA follows the paring of bases in the parent DNA strand. The original two strands unwind by breaking the hydrogen bonds between base pairs. Free nucleotides, consisting of a base attached to a sugar-phosphate, form new hydrogen bonds with their complementary bases on the parent strand; new phosphodiester bonds are created by the enzyme DNA polymerase. Replication of chromosomes begins simultaneously at multiple sites, forming replication bubbles that expand bidirectionally until the entire DNA molecule (chromosome) is replicated. Errors in DNA replication, or mutations induced by environmental mutagens such as irradiation or chemicals, are detected and potentially corrected by DNA repair systems.

A prototypical gene consists of a regulatory region, segments called exons that encode the amino acid sequence of a protein, and intervening segments called introns (Fig. 74-2). Transcription starts at the promoter region and continues through the entire length of the gene to form mRNA. The introns are removed and the exons spliced together to form a mature message, which is exported to the cytoplasm. There the mRNA is bound to ribosomes and translated into protein.

Transcription is initiated by attachment of RNA polymerase to the promoter site upstream of the beginning of the coding sequence. Specific proteins bind to the region to either repress or activate transcription by opening up the chromatin, which is a complex of DNA and histone proteins. It is the action of these regulatory proteins (transcription factors) that determines, in large part, when a gene is turned on or off. Some genes are also turned on and off by methylation of cytosine bases that are adjacent to guanines (CpG bases). Methylation is an example of an epigenetic change, meaning a change that can affect gene expression, and possibly the characteristics of a cell or organism, but that does not involve a change in the underlying genetic sequence. Gene regulation is flexible and responsive, with genes being turned on or off during development and in response to internal and external conditions and stimuli.

Transcription proceeds through the full length of the gene, synthesizing mRNA in a 5′ to 3′ direction. RNA, like DNA, is a sugar-phosphate chain with pyrimidines and purines. The sugar in this case is ribose; uracil replaces thymine in RNA. The RNA reads off one strand of DNA to copy a complementary RNA sequence. A “cap” consisting of 7-methylguanosine is added to the 5′ end of the RNA in a 5′-5′ bond and, for most transcripts, several hundred adenine bases are enzymatically added to the 3′ end after transcription.

mRNA processing occurs in the nucleus and consists of excision of the introns and splicing together of the exons. Specific sequences at the start and end of introns mark the sites where the splicing machinery will act on the transcript. In some cases, there may be tissue-specific patterns to splicing, so that the same primary transcript can produce multiple distinct proteins.

The processed transcript is next exported to the cytoplasm, where it binds to ribosomes, which are complexes of protein and RNA. The genetic code is then read in triplets of bases, each triplet corresponding with a specific amino acid or providing a signal that terminates translation. The triplet codons are recognized by transfer RNAs (tRNAs) that include complementary anticodons and bind the corresponding amino acid, delivering it to the growing peptide. A new amino acid is enzymatically attached to the peptide; each time an amino acid is added, the ribosome moves one triplet codon step along the mRNA. Eventually a stop codon is reached, at which point translation ends and the peptide is released. In some proteins, there are post-translational modifications such as attachment of sugars (glycosylation); the protein is then delivered to its destination within or outside the cell by trafficking mechanisms that recognize portions of the peptide.

An emerging layer of complexity and genetic regulation is that of noncoding RNAs. This refers to RNAs that are transcribed from DNA. However, they are not transported and translated into proteins. Instead, these RNAs are “noncoding” and serve diverse biologic functions often in complexes with different proteins. Traditionally, this has included RNAs that function in mediating splicing or processing of coding RNA or translation of coding RNAs in ribosomes. Small noncoding RNAs including microRNAs (miRNAs) are representative of a class of small RNAs (21-23 bp) that control gene expression in the cell by directly targeting specific sets of coding RNAs by direct RNA-RNA binding. This RNA-RNA interaction might lead to degradation of the target coding RNA or inhibition of translation of the protein specified by that coding RNA. miRNAs, in general, target and regulate several hundred mRNAs.

Genetic Variation

The process of producing protein from a gene is subject to disruption at multiple levels owing to alterations in the coding sequence (Fig. 74-3). Changes in the regulatory region can lead to altered gene expression, including increased or decreased rates of transcription, failure of gene activation, or activation of the gene at inappropriate times or in inappropriate cells. Changes in the coding sequence can lead to substitution of one amino acid for another (missense mutation or nonsynonymous) or creation of a stop codon in the place of an amino acid codon. Some single-base changes do not affect the amino acid (silent or wobble mutation or synonymous), because there may be several codons that correspond with a single amino acid. Amino acid substitutions can have a profound effect on protein function if the chemical properties of the substituted amino acid are markedly different from the usual one. Other substitutions can have a subtle or no effect on protein function, particularly if the substituted amino acid is chemically similar to the original one.

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