Biochemistry_Lippinco

Biochemistry_Lippinco. Cell-surface receptors: These receptors include those for insulin, epinephrine, and glucagon. Glucagon, for example, is a peptide hormone that binds its G protein–coupled plasma membrane receptor on glucagon-responsive cells. This extracellular signal is then transduced to intracellular cAMP, a second messenger (Fig. 33.11; also see Fig. 8.7 on p. 95), which can affect protein expression (and activity) through protein kinase A–mediated phosphorylation. In response to a rise in cAMP, a trans-acting factor (cAMP response element–binding [CREB] protein) is phosphorylated and activated. Active CREB protein binds via a leucine zipper motif to a cis-acting regulatory element, the cAMP response element (CRE), resulting in transcription of target genes with CRE in their promoters. [Note: The genes for phosphoenolpyruvate carboxykinase and glucose 6phosphatase, key enzymes of gluconeogenesis (see p. 122), are examples of genes upregulated by the cAMP/CRE/CREB system.]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Messenger RNA processing and use Eukaryotic mRNA undergoes several processing events before it is exported from the nucleus to the cytoplasm for use in protein synthesis. Capping at the 5′-end (see p. 441), polyadenylation at the 3′-end (see p. 442), and splicing (see p. 442) are essential for the production of a functional eukaryotic messenger from most pre-mRNA. Variations in splicing and polyadenylation can affect gene expression. In addition, messenger stability also affects gene expression. 1. Alternative splicing: Tissue-specific protein isoforms can be made from the same pre-mRNA through alternative splicing, which can involve exon skipping (loss), intron retention, and use of alternative splice-donor or -acceptor sites (Fig. 33.12). For example, the pre-mRNA for tropomyosin (TM) undergoes tissue-specific alternative splicing to yield a number of TM isoforms (see p. 443). [Note: Over 90% of all human genes undergo alternative splicing.] 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. Alternative polyadenylation: Some pre-mRNA transcripts have more than one site for cleavage and polyadenylation. Alternative polyadenylation (APA) generates mRNA with different 3′-ends, altering the untranslated region (UTR) or the coding (translated) sequence. [Note: APA is involved in the production of the membrane-bound and secreted forms of immunoglobulin M.] The use of alternative splicing and polyadenylation sites, as well as alternative transcription start sites explains, at least in part, how the ~20,000 to 25,000 genes in the human genome can give rise to well over 100,000 proteins. 3. Messenger RNA editing: Even after mRNA has been fully processed, it may undergo an additional posttranscriptional modification in which a base in the mRNA is altered. This is known as RNA editing. An important example in humans occurs with the transcript for apolipoprotein (apo) B, an essential component of chylomicrons (see p.


Biochemistry_Lippinco

Biochemistry_Lippinco. 228) and very-low-density lipoproteins ([VLDL] see p. 230). Apo B mRNA is made in the liver and the small intestine. However, in the intestine only, the cytosine (C) base in the CAA codon for glutamine is enzymatically deaminated to uracil (U), changing the sense codon to the nonsense or stop codon UAA, as shown in Figure 33.13. This results in a shorter protein (apo B-48, representing 48% of the message) being made in the intestine (and incorporated into chylomicrons) than is made in the liver (apo B-100, full-length, incorporated into VLDL). U = uracil. 4. Messenger RNA stability: How long an mRNA remains in the cytosol before it is degraded influences how much protein product can be produced from it. Regulation of iron metabolism and the gene-silencing process of RNA interference (RNAi) illustrate the importance of mRNA stability in the regulation of gene expression.


Biochemistry_Lippinco

Biochemistry_Lippinco. a. Iron metabolism: Transferrin (Tf) is a plasma protein that transports iron. Tf binds to cell-surface receptors (transferrin receptors [TfR]) that get internalized and provide cells, such as erythroblasts, with iron. The mRNA for the TfR has several cis-acting iron-responsive elements (IRE) in its 3′-UTR. IRE have a short stem-loop structure that can be bound by trans-acting iron regulatory proteins (IRP), as shown in Figure 33.14. When the iron concentration in the cell is low, the IRP bind to the 3′-IRE and stabilize the mRNA for TfR, allowing TfR synthesis. When intracellular iron levels are high, the IRP dissociate. The lack of IRP bound to the mRNA hastens its destruction, resulting in decreased TfR synthesis. [Note: The mRNA for ferritin, an intracellular protein of iron storage, has a single IRE in its 5′-UTR. When iron levels in the cell are low, IRP bind the 5′-IRE and prevent the use of the mRNA, and less ferritin is made. When iron accumulates in the cell, the IRP


Biochemistry_Lippinco

Biochemistry_Lippinco. has a single IRE in its 5′-UTR. When iron levels in the cell are low, IRP bind the 5′-IRE and prevent the use of the mRNA, and less ferritin is made. When iron accumulates in the cell, the IRP dissociate, allowing synthesis of ferritin molecules to store the excess iron. Aminolevulinic acid synthase 2, the regulated enzyme of heme synthesis (see p. 278) in erythroblasts, also contains a 5′-IRE.] (See Chapter 29 for a discussion of iron metabolism.) b. RNA interference: RNAi is a mechanism of gene silencing through decreased expression of mRNA, either by repression of translation or by increased degradation. It plays a key role in such fundamental processes as cell proliferation, differentiation, and apoptosis. RNAi is mediated by short (~22 nucleotides), noncoding RNA called microRNA (miRNA). The miRNA arise from far longer, genomically encoded nuclear transcripts, primary miRNA (pri-miRNA), that are partially processed in the nucleus to pre-miRNA by an endonuclease (Drosha) then


Biochemistry_Lippinco

Biochemistry_Lippinco. (miRNA). The miRNA arise from far longer, genomically encoded nuclear transcripts, primary miRNA (pri-miRNA), that are partially processed in the nucleus to pre-miRNA by an endonuclease (Drosha) then transported to the cytoplasm. There, an endonuclease (Dicer) completes the processing and generates short, double-stranded miRNA. A single strand (the guide or antisense strand) of the miRNA associates with a cytosolic protein complex known as the RNA-induced silencing complex (RISC). The guide strand hybridizes with a complementary sequence in the 3′-UTR of a full-length target mRNA, bringing RISC to the mRNA. This can result in repression of translation of the mRNA or its degradation by an endonuclease (Argonaute/Ago/Slicer) of the RISC. The extent of complementarity appears to be the determining factor (Fig. 33.15). RNAi can also be triggered by the introduction of exogenous double-stranded short interfering RNA (siRNA) into a cell, a process that has enormous therapeutic potential.


Biochemistry_Lippinco

Biochemistry_Lippinco. The first clinical trial of RNAi-based therapy involved the neovascular form of age-related macular degeneration (AMD), which is triggered by overproduction of vascular endothelial growth factor (VEGF), leading to the sprouting of excess blood vessels behind the retina. The vessels leak, clouding and often entirely destroying vision (therefore, neovascular AMD is also referred to as wet AMD). An siRNA was designed to target the mRNA of VEGF and promote its degradation. Although considerable effort and resources have been expended to develop RNAi-based therapeutics, especially for the treatment of cancer, no products have gone from trials to the market. The research applications of RNAi, however, have grown rapidly.


Biochemistry_Lippinco

Biochemistry_Lippinco. 5. Messenger RNA translation: Regulation of gene expression can also occur at the level of mRNA translation. One mechanism by which translation is regulated is through phosphorylation of the eukaryotic translation initiation factor, eIF-2 (Fig. 33.16). Phosphorylation of eIF-2 inhibits its function and so inhibits translation at the initiation step (see p. 459). [Note: Phosphorylation of eIF-2 prevents its reactivation by inhibiting GDP-GTP exchange.] Phosphorylation is catalyzed by kinases that are activated in response to environmental conditions, such as amino acid starvation, heme deficiency in erythroblasts, the presence of double-stranded RNA (signaling viral infection), and the accumulation of misfolded proteins in the rough endoplasmic reticulum (see p. 460). phosphate; = phosphate. C. Regulation through variations in DNA


Biochemistry_Lippinco

Biochemistry_Lippinco. phosphate; = phosphate. C. Regulation through variations in DNA Gene expression in eukaryotes is also influenced by the accessibility of DNA to the transcriptional apparatus, the amount of DNA, and the arrangement of DNA. [Note: Localized transitions between the B and Z forms of DNA (see p. 414) can also affect gene expression.] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Access to DNA: In eukaryotes, DNA is found complexed with histone and nonhistone proteins to form chromatin (see p. 425). Transcriptionally active, decondensed chromatin (euchromatin) differs from the more condensed, inactive form (heterochromatin) in a number of ways. Active chromatin contains histone proteins that have been covalently modified at their amino terminal ends by reversible methylation, acetylation, or phosphorylation (see p. 438 for a discussion of histone acetylation/deacetylation by histone acetyltransferase and histone deacetylase). Such modifications decrease the positive charge of these basic proteins, thereby decreasing the strength of their association with negatively charged DNA. This relaxes the nucleosome (see p. 425), allowing transcription factors access to specific regions on the DNA. Nucleosomes can also be repositioned, an ATP-requiring process that is part of chromatin remodeling. Another difference between transcriptionally active and inactive chromatin


Biochemistry_Lippinco

Biochemistry_Lippinco. regions on the DNA. Nucleosomes can also be repositioned, an ATP-requiring process that is part of chromatin remodeling. Another difference between transcriptionally active and inactive chromatin is the extent of methylation of cytosine bases in CG-rich regions (CpG islands) in the promoter region of many genes. Methylation is by methyltransferases that use S-adenosylmethionine as the methyl donor (Fig. 33.17). Transcriptionally active genes are less methylated (hypomethylated) than their inactive counterparts, suggesting that DNA hypermethylation silences gene expression. Modification of histones and methylation of DNA are epigenetic in that they are heritable changes in DNA that alter gene expression without altering the base sequence.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Amount of DNA: A change up or down in the number of copies of a gene can affect the amount of gene product produced. An increase in copy number (gene amplification) has contributed to increased genomic complexity and is still a normal developmental process in certain nonmammalian species. In mammals, however, gene amplification is seen with some diseases and in response to particular chemotherapeutic drugs such as methotrexate, an inhibitor of the enzyme dihydrofolate reductase (DHFR), required for the synthesis of thymidine triphosphate (TTP) in the pyrimidine biosynthetic pathway (see p. 303). TTP is essential for DNA synthesis. Gene amplification results in an increase in the number of DHFR genes and resistance to the drug, allowing TTP to be made. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Arrangement of DNA: The process by which immunoglobulins (antibodies) are produced by B lymphocytes involves permanent rearrangements of the DNA in these cells. The immunoglobulins (for example, IgG) consist of two light and two heavy chains, with each chain containing regions of variable and constant amino acid sequence. The variable region is the result of somatic recombination of segments within both the light-and the heavy-chain genes. During B-lymphocyte development, single variable (V), diversity (D), and joining (J) gene segments are brought together through gene rearrangement to form a unique variable region (Fig. 33.18). This process allows the generation of 109−1011 different immunoglobulins from a single gene, providing the diversity needed for the recognition of an enormous number of antigens. [Note: Pathologic DNA rearrangement is seen with translocation, a process by which two different chromosomes exchange DNA segments.] 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. Mobile DNA elements: Transposons (Tn) are mobile segments of DNA that move in an essentially random manner from one site to another on the same or a different chromosome. Movement is mediated by transposase, an enzyme encoded by the Tn itself. Movement can be direct, in which transposase cuts out and then inserts the Tn at a new site, or replicative, in which the Tn is copied and the copy inserted elsewhere while the original remains in place. In eukaryotes, including humans, replicative transposition frequently involves an RNA intermediate made by a reverse transcriptase (see p. 424), in which case the Tn is called a retrotransposon. Transposition has contributed to structural variation in the genome but also has the potential to alter gene expression and even to cause disease. Tn comprise ~50% of the human genome, with retrotransposons accounting for 90% of Tn. Although the vast majority of these retrotransposons have lost the ability to move, some are still active. Their


Biochemistry_Lippinco

Biochemistry_Lippinco. Tn comprise ~50% of the human genome, with retrotransposons accounting for 90% of Tn. Although the vast majority of these retrotransposons have lost the ability to move, some are still active. Their transposition is thought to be the basis for some rare cases of hemophilia A and Duchenne muscular dystrophy. [Note: The growing problem of antibiotic-resistant bacteria is a consequence, at least in part, of the exchange of plasmids among bacterial cells. If the plasmids contain Tn-carrying antibiotic resistance genes, the recipient bacteria gain resistance to one or more antimicrobial drugs.]


Biochemistry_Lippinco

V. CHAPTER SUMMARY

Biochemistry_Lippinco. V. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Gene expression results in the production of a functional gene product (either RNA or protein) through the processes of transcription and translation (Fig. 33.19). Genes can be either constitutive (always expressed, housekeeping genes) or regulated (expressed only under certain conditions in all cells or in a subset of cells). The ability to appropriately induce (positively regulate) or repress (negatively regulate) genes is essential in all organisms. Regulation of gene expression occurs primarily at transcription in both prokaryotes and eukaryotes and is mediated through trans-acting proteins binding to cis-acting regulatory DNA elements. In eukaryotes, regulation also occurs through DNA modifications and through posttranscriptional and posttranslational processing. In prokaryotes, such as Escherichia coli, the coordinate regulation of genes whose protein products are required for a particular process is achieved through operons (groups of genes sequentially arranged on the


Biochemistry_Lippinco

Biochemistry_Lippinco. such as Escherichia coli, the coordinate regulation of genes whose protein products are required for a particular process is achieved through operons (groups of genes sequentially arranged on the chromosome along with the regulatory elements that determine their transcription). The lac operon contains the Z, Y, and A structural genes, the protein products of which are needed for the catabolism of lactose. It is subject to negative and positive regulation. When glucose is available, the operon is repressed by the binding of the repressor protein (the product of the lacI gene) to the operator, thus preventing transcription. When only lactose is present, the operon is induced by an isomer of lactose (allolactose) that binds the repressor protein, preventing it from binding to the operator. In addition, cyclic adenosine monophosphate (cAMP) binds the catabolite activator protein (CAP), and the complex binds the DNA at the CAP site. This increases promoter efficiency and results in the


Biochemistry_Lippinco

Biochemistry_Lippinco. In addition, cyclic adenosine monophosphate (cAMP) binds the catabolite activator protein (CAP), and the complex binds the DNA at the CAP site. This increases promoter efficiency and results in the expression of the structural genes through the production of a polycistronic messenger RNA (mRNA). When both glucose and lactose are present, glucose prevents formation of cAMP, and transcription of these genes is negligible. The trp operon contains genes needed for the synthesis of tryptophan (Trp), and, like the lac operon, it is regulated by negative control. Unlike the lac operon, it is also regulated by attenuation, in which mRNA synthesis that escaped repression by Trp is terminated before completion. Transcription of ribosomal RNA and transfer RNA is selectively inhibited in prokaryotes by the stringent response to amino acid starvation. Translation is also a site of prokaryotic gene regulation: Excess ribosomal proteins bind the Shine-Dalgarno sequence on their own polycistronic


Biochemistry_Lippinco

Biochemistry_Lippinco. by the stringent response to amino acid starvation. Translation is also a site of prokaryotic gene regulation: Excess ribosomal proteins bind the Shine-Dalgarno sequence on their own polycistronic mRNA, preventing ribosomes from binding. Gene regulation is more complex in eukaryotes. Operons are not present, but coordinate regulation of the transcription of genes located on different chromosomes can be achieved through the binding of trans-acting proteins to cis-acting elements as seen in the galactose circuit in unicellular yeast. In multicellular organisms, hormones can cause coordinated regulation, either through the binding of the hormone receptor–hormone complex to the DNA (as with steroid hormones) or through the binding of a protein that is activated in response to a second messenger (as with glucagon). In each case, binding to DNA is mediated through structural motifs such as the zinc finger. Co-and posttranscriptional regulation is also seen in eukaryotes and includes


Biochemistry_Lippinco

Biochemistry_Lippinco. messenger (as with glucagon). In each case, binding to DNA is mediated through structural motifs such as the zinc finger. Co-and posttranscriptional regulation is also seen in eukaryotes and includes alternative mRNA splicing and polyadenylation, mRNA editing, and variations in mRNA stability as seen with transferrin receptor synthesis and with RNA interference. Regulation at the translational level can be caused by the phosphorylation and inhibition of eukaryotic initiation factor 2. Gene expression in eukaryotes is also influenced by accessibility of DNA to the transcriptional apparatus (as seen with epigenetic changes to histone proteins), the amount of DNA, and the arrangement of the DNA.


Biochemistry_Lippinco

Biochemistry_Lippinco. reticulum. Choose the ONE best answer. 3.1. Which of the following mutations is most likely to result in reduced expression of the lac operon? A. cya− (no adenylyl cyclase made) B. i− (no repressor protein made) C. Oc (operator cannot bind repressor protein) D. One resulting in impaired glucose uptake Correct answer = A. In the absence of glucose, adenylyl cyclase makes cyclic adenosine monophosphate (cAMP), which forms a complex with the catabolite activator protein (CAP). The cAMP–CAP complex binds the CAP site on the DNA, causing RNA polymerase to bind more efficiently to the lac operon promoter, thereby increasing expression of the operon. With cya− mutations, adenylyl cyclase is not made, and so the operon is unable to be maximally expressed even when glucose is absent and lactose is present. The absence of a repressor protein or decreased ability of the repressor to bind the operator results in constitutive (essentially constant) expression of the lac operon.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3.2. Which of the following is best described as cis-acting? A. Cyclic adenosine monophosphate response element–binding protein B. Operator C. Repressor protein D. Thyroid hormone nuclear receptor Correct answer = B. The operator is part of the DNA itself, and so is cis-acting. The cyclic adenosine monophosphate response element–binding protein, repressor protein, and thyroid hormone nuclear receptor protein are molecules that diffuse (transit) to the DNA, bind, and affect the expression of that DNA and so are trans-acting. 3.3. Which of the following is the basis for the intestine-specific expression of apolipoprotein B-48? A. DNA rearrangement and loss B. DNA transposition C. RNA alternative splicing D. RNA editing E. RNA interference


Biochemistry_Lippinco

Biochemistry_Lippinco. A. DNA rearrangement and loss B. DNA transposition C. RNA alternative splicing D. RNA editing E. RNA interference Correct answer = D. The production of apolipoprotein (apo) B-48 in the intestine and apo B-100 in liver is the result of RNA editing in the intestine, where a sense codon is changed to a nonsense codon by posttranscriptional deamination of cytosine to uracil. DNA rearrangement and transposition, as well as RNA interference and alternative splicing, do alter gene expression but are not the basis of apo B-48 tissue-specific production. 3.4. Which of the following is most likely to be true in hemochromatosis, a disease of iron accumulation? A. The messenger RNA for the transferrin receptor is stabilized by the binding of iron regulatory proteins to its 3′-iron-responsive elements. B. The messenger RNA for the transferrin receptor is not bound by iron regulatory proteins and is degraded.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. The messenger RNA for the transferrin receptor is not bound by iron regulatory proteins and is degraded. C. The messenger RNA for ferritin is not bound by iron regulatory proteins at its 5′-iron-responsive element and is translated. D. The messenger RNA for ferritin is bound by iron regulatory proteins and is not translated. E. Both B and C are correct. Correct answer = E. When iron levels in the body are high, as is seen with hemochromatosis, there is increased synthesis of the iron-storage molecule, ferritin, and decreased synthesis of the transferrin receptor (TfR) that mediates iron uptake by cells. These effects are the result of cis-acting iron-responsive elements not being bound by trans-acting iron regulatory proteins, resulting in degradation of the messenger RNA (mRNA) for TfR and increased translation of the mRNA for ferritin.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3.5. Patients with estrogen receptor–positive (hormone responsive) breast cancer may be treated with the drug tamoxifen, which binds the estrogen nuclear receptor without activating it. Which of the following is the most logical outcome of tamoxifen use? A. Increased acetylation of estrogen-responsive genes B. Increased growth of estrogen receptor–positive breast cancer cells C. Increased production of cyclic adenosine monophosphate D. Inhibition of the estrogen operon E. Inhibition of transcription of estrogen-responsive genes


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Increased production of cyclic adenosine monophosphate D. Inhibition of the estrogen operon E. Inhibition of transcription of estrogen-responsive genes Correct answer = E. Tamoxifen competes with estrogen for binding to the estrogen nuclear receptor. Tamoxifen fails to activate the receptor, preventing its binding to DNA sequences that upregulate expression of estrogen-responsive genes. Tamoxifen, then, blocks the growth-promoting effects of these genes and results in growth inhibition of estrogen-dependent breast cancer cells. Acetylation increases transcription by relaxing the nucleosome. Cyclic adenosine monophosphate is a regulatory signal mediated by cell-surface rather than nuclear receptors. Mammalian cells do not have operons. 3.6. The ZYA region of the lac operon will be maximally expressed if: A. cyclic adenosine monophosphate levels are low. B. glucose and lactose are both available. C. the attenuation stem-loop is able to form. D. the CAP site is occupied.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. cyclic adenosine monophosphate levels are low. B. glucose and lactose are both available. C. the attenuation stem-loop is able to form. D. the CAP site is occupied. Correct answer = D. It is only when glucose is gone, cyclic adenosine monophosphate (cAMP) levels are increased, the cAMP–catabolite activator protein (CAP) complex is bound to the CAP site, and lactose is available that the operon is maximally expressed (induced). If glucose is present, the operon is off as a result of catabolite repression. The lac operon is not regulated by attenuation, a mechanism for stopping transcription in some operons such as the trp operon. 3.7. X chromosome inactivation is a process by which one of two X chromosomes in mammalian females is condensed and inactivated to prevent overexpression of X-linked genes. What would most likely be true about the degree of DNA methylation and histone acetylation on the inactivated X chromosome?


Biochemistry_Lippinco

Biochemistry_Lippinco. Cytosines in CpG islands would be hypermethylated, and histone proteins would be deacetylated. Both conditions are associated with decreased gene expression, and both are important in maintaining X inactivation. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. In the past, efforts to understand genes and their expression have been confounded by the immense size and complexity of human deoxyribonucleic acid (DNA). The human genome contains ~3 billion (109) base pairs (bp) that encode 20,000–25,000 protein-coding genes located on 23 chromosomes in the haploid genome. It is now possible to determine the nucleotide sequence of long stretches of DNA, and the entire human genome has been sequenced. This effort (called the Human Genome Project and completed in 2003) was made possible by several tools that have already contributed to our understanding of many genetic diseases (Fig. 34.1). These include 1) the discovery of restriction endonucleases that permit the cleavage of huge DNA molecules into defined fragments, 2) the development of cloning techniques that provide a mechanism for amplification of specific nucleotide sequences, and 3) the ability to synthesize specific probes, which has allowed the identification and manipulation of nucleotide


Biochemistry_Lippinco

Biochemistry_Lippinco. that provide a mechanism for amplification of specific nucleotide sequences, and 3) the ability to synthesize specific probes, which has allowed the identification and manipulation of nucleotide sequences of interest. These and other experimental approaches have permitted the identification of both normal and mutant nucleotide sequences in DNA. This knowledge has led to the development of methods for the diagnosis of genetic diseases and some successes in the treatment of patients by gene therapy. [Note: The genomes of several viruses, prokaryotes, and nonhuman eukaryotes have also been sequenced.]


Biochemistry_Lippinco

Biochemistry_Lippinco. II. RESTRICTION ENDONUCLEASES One of the major obstacles to molecular analysis of genomic DNA is the immense size of the molecules involved. The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded DNA (dsDNA) into smaller, more manageable fragments, opened the way for DNA analysis. Because each enzyme cleaves dsDNA at a specific nucleotide sequence (restriction site), restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments. A. Specificity


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Specificity Restriction endonucleases recognize short stretches of dsDNA (4–8 bp) that contain specific nucleotide sequences. These sequences, which differ for each restriction enzyme, are palindromes, that is, they exhibit twofold rotational symmetry (Fig. 34.2). This means that, within a short region of the dsDNA, the nucleotide sequence on the two strands is identical if each is read in the 5′→3′ direction. Therefore, if you turn the page upside down (that is, rotate it 180° around its axis of symmetry) the sequence remains the same. In bacteria, restriction endonucleases limit (restrict) the expression of nonbacterial (foreign) DNA through cleavage. Bacterial DNA is protected from cleavage by methylation of adenine at the restriction site. B. Nomenclature


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Nomenclature A restriction enzyme is named according to the organism from which it was isolated. The first letter of the name is from the genus of the bacterium. The next two letters are from the name of the species. An additional letter indicates the type or strain (as needed), and a number (Roman numeral) is appended to indicate the order in which the enzyme was discovered in that particular organism. For example, HaeIII is the third restriction endonuclease isolated from the bacterium Haemophilus aegyptius. C. Sticky and blunt ends


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Sticky and blunt ends Restriction enzymes cleave dsDNA so as to produce a 3′-hydroxyl group on one end and a 5′-phosphate group on the other. Some restriction endonucleases, such as TaqI, form staggered cuts that produce sticky or cohesive ends (that is, the resulting DNA fragments have single-stranded regions that are complementary to each other), as shown in Figure 34.3. Other restriction endonucleases, such as HaeIII, produce fragments that have blunt ends that are entirely double stranded and, therefore, do not form hydrogen bonds with each other. Using the enzyme DNA ligase (see p. 418), sticky ends of a DNA fragment of interest can be covalently joined with other DNA fragments that have sticky ends produced by cleavage with the same restriction endonuclease (Fig. 34.4). [Note: A ligase encoded by bacteriophage T4 can covalently join blunt-ended fragments.] D. Restriction sites


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Restriction sites A DNA sequence that is recognized and cut by a restriction enzyme is called a restriction site. Restriction endonucleases cleave dsDNA into fragments of different sizes depending upon the size of the sequence recognized. For example, an enzyme that recognizes a specific 4-bp sequence produces many cuts in the DNA molecule, one every 44 bp. In contrast, an enzyme requiring a unique sequence of 6 bp produces fewer cuts (one every 46 bp) and, therefore, longer pieces. Hundreds of these enzymes, each having different cleavage specificities (varying in both nucleotide sequences and length of recognition sites), are commercially available. III. DNA CLONING


Biochemistry_Lippinco

Biochemistry_Lippinco. Introduction of a foreign DNA molecule into a replicating cell permits the cloning or, amplification (that is, the production of many identical copies) of that DNA. [Note: Human DNA for cloning can be obtained from blood, saliva, and solid tissue.] In some cases, a single DNA fragment can be isolated and purified prior to cloning. More commonly, to clone a nucleotide sequence of interest, the total cellular DNA is first cleaved with a specific restriction enzyme, creating hundreds of thousands of fragments. Each of the resulting DNA fragments is joined to a DNA vector molecule (referred to as a cloning vector) to form a hybrid, or recombinant, DNA molecule. Each recombinant molecule carries its inserted DNA fragment into a single host cell (for example, a bacterium), where it is replicated. [Note: The process of introducing foreign DNA into a cell is called transformation for bacteria and yeast and transfection for higher eukaryotes.] As the host cell multiplies, it forms a clone in


Biochemistry_Lippinco

Biochemistry_Lippinco. [Note: The process of introducing foreign DNA into a cell is called transformation for bacteria and yeast and transfection for higher eukaryotes.] As the host cell multiplies, it forms a clone in which every bacterium contains copies of the same inserted DNA fragment, hence the name “cloning.” The cloned DNA can be released from its vector by cleavage (using the appropriate restriction endonuclease) and isolated. By this mechanism, many identical copies of the DNA of interest can be produced. [Note: An alternative to amplification by biologic cloning, the polymerase chain reaction (PCR), is described on p. 495.]


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Vectors A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined. Essential properties of a vector include the 1) capacity for autonomous replication within a host cell, 2) presence of at least one specific nucleotide sequence recognized by a restriction endonuclease, and 3) presence of at least one gene (such as an antibiotic resistance gene) that confers the ability to select for the vector. Commonly used vectors include plasmids and viruses. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Prokaryotic plasmids: Prokaryotic organisms typically contain single, large, circular chromosomes. In addition, most species of bacteria also normally contain small, circular, extrachromosomal DNA molecules called plasmids (Fig. 34.5). Plasmid DNA undergoes replication that may or may not be synchronized to chromosomal division. Plasmids may carry genes that convey antibiotic resistance to the host bacterium and may facilitate the transfer of genetic information from one bacterium to another. They can be readily isolated from bacterial cells, their circular DNA cleaved at specific sites by restriction endonucleases, and up to 15 kb (kilobases) of foreign DNA (cut with the same restriction enzyme) inserted. The recombinant plasmid vector can be introduced into a bacterium, producing large numbers of copies of the plasmid. The bacteria are grown in the presence of antibiotics, thus selecting for cells containing the hybrid plasmids, which provide antibiotic resistance (Fig. 34.6).


Biochemistry_Lippinco

Biochemistry_Lippinco. numbers of copies of the plasmid. The bacteria are grown in the presence of antibiotics, thus selecting for cells containing the hybrid plasmids, which provide antibiotic resistance (Fig. 34.6). Artificial plasmids are routinely constructed. An example is the classic pBR322 (see Fig. 34.5), which contains an origin of replication, two antibiotic resistance genes, and >40 unique restriction sites. Use of plasmids is limited by the size of the DNA that can be inserted.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Other vectors: The development of improved vectors that can more efficiently accommodate larger DNA segments, or express the passenger genes in different cell types, has aided molecular genetics research and therapeutics. In addition to the prokaryotic plasmids described above, naturally occurring viruses that infect bacteria (bacteriophage λ, for example) or mammalian cells (retroviruses, for example), as well as artificial constructs such as cosmids and bacterial or yeast artificial chromosomes (BAC or YAC, respectively), are currently used as cloning vectors. [Note: BAC and YAC can accept DNA inserts of 100–300 kb and 250–1,000 kb, respectively.] B. DNA libraries


Biochemistry_Lippinco

Biochemistry_Lippinco. B. DNA libraries A DNA library is a collection of cloned restriction fragments of the DNA of an organism. Two kinds of libraries are commonly used: genomic libraries and complementary DNA (cDNA) libraries. Genomic libraries ideally contain a copy of every DNA nucleotide sequence in the genome. In contrast, cDNA libraries contain those DNA sequences that only appear as processed messenger RNA (mRNA) molecules, and these differ according to cell type and environmental conditions. [Note: cDNA lacks introns and the control regions of the genes, whereas these are present in genomic DNA.] 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. Genomic DNA libraries: A genomic library is created by digestion of the total DNA of an organism with a restriction endonuclease and subsequent ligation to an appropriate vector. The recombinant DNA molecules replicate within host bacteria. Thus, the amplified DNA fragments collectively represent the entire genome of the organism and are called a genomic library. Regardless of the restriction enzyme used, the chances are good that the gene of interest contains more than one restriction site recognized by that enzyme. If this is the case, and if the digestion is allowed to go to completion, the gene of interest is fragmented (that is, it is not contained in any one clone in the library). To avoid this usually undesirable result, a partial digestion is performed in which either the amount or the time of action of the enzyme is limited. This results in cleavage occurring at only a fraction of the restriction sites on any one DNA molecule, thus producing fragments of ~20 kb. Enzymes that


Biochemistry_Lippinco

Biochemistry_Lippinco. the time of action of the enzyme is limited. This results in cleavage occurring at only a fraction of the restriction sites on any one DNA molecule, thus producing fragments of ~20 kb. Enzymes that cut very frequently (that is, those that recognize 4-bp sequences) are generally used for this purpose so that the result is an almost random collection of fragments. This insures a high degree of probability that the gene of interest is contained, intact, in some fragment.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Complementary DNA libraries: If a protein-coding gene of interest is expressed at a high level in a particular tissue, the mRNA transcribed from that gene is likely also present at high concentrations in the cells of that tissue. For example, reticulocyte mRNA is composed largely of molecules that code for the α-globin and β-globin chains of hemoglobin


Biochemistry_Lippinco

Biochemistry_Lippinco. A (HbA). This mRNA can be used as a template to make a cDNA molecule using the enzyme reverse transcriptase (Fig. 34.7). Therefore, the resulting cDNA is a double-stranded copy of mRNA. [Note: The template mRNA is isolated from transfer RNA and ribosomal RNA by the presence of its poly-A tail.] cDNA can be amplified by biologic cloning or by PCR. It can be used as a probe to locate the gene that encodes the original mRNA (or fragments of the gene) in mixtures containing many unrelated DNA fragments. If the mRNA used as a template is a mixture of many different size species, the resulting cDNA is heterogeneous. These mixtures can be cloned to form a cDNA library. Because cDNA has no introns, it can be cloned into an expression vector for the synthesis of eukaryotic proteins by bacteria (Fig. 34.8). These special plasmids contain a bacterial promoter for transcription of the cDNA and a Shine-Dalgarno (SD) sequence (see p. 454) that allows the bacterial ribosome to initiate translation


Biochemistry_Lippinco

Biochemistry_Lippinco. 34.8). These special plasmids contain a bacterial promoter for transcription of the cDNA and a Shine-Dalgarno (SD) sequence (see p. 454) that allows the bacterial ribosome to initiate translation of the resulting mRNA molecule. The cDNA is inserted downstream of the promoter and within a gene for a protein that is expressed in the bacterium (for example, lacZ; see p. 466), such that the mRNA produced contains an SD sequence, a few codons for the bacterial protein, and all the codons for the eukaryotic protein. This allows for more efficient expression and results in the production of a fusion protein. [Note: Therapeutic human insulin is made in bacteria through this technology. However, the extensive co-and posttranslational modifications required for most other human proteins (for example, blood clotting factors) necessitates the use of eukaryotic, even mammalian, hosts.]


Biochemistry_Lippinco

C. Sequencing cloned DNA fragments

Biochemistry_Lippinco. C. Sequencing cloned DNA fragments


Biochemistry_Lippinco

Biochemistry_Lippinco. The base sequence of DNA fragments that have been cloned can be determined. The original procedure for this purpose was the Sanger dideoxy chain termination method illustrated in Figure 34.9. In this method, the single-stranded DNA (ssDNA) to be sequenced is used as the template for DNA synthesis by DNA polymerase (DNA pol). A radiolabeled primer complementary to the 3′-end of the target DNA is added, along with the four deoxyribonucleoside triphosphates (dNTP). The sample is divided into four reaction tubes, and a small amount of one of the four dideoxyribonucleoside triphosphates (ddNTP) is added to each tube. Because it contains no 3′-hydroxyl group, incorporation of a ddNMP terminates elongation at that point. The products of this reaction, then, consist of a mixture of DNA strands of different lengths, each terminating at a specific base. Separation of the various DNA products by size in an electric field using polyacrylamide gel electrophoresis, followed by autoradiography,


Biochemistry_Lippinco

Biochemistry_Lippinco. of different lengths, each terminating at a specific base. Separation of the various DNA products by size in an electric field using polyacrylamide gel electrophoresis, followed by autoradiography, yields a pattern of bands from which the DNA base sequence can be read. [Note: The shorter the fragment, the farther it travels on the gel, with the shortest fragment representing that which was made first (that is, the 5′-end).] In place of a labeled primer, a mixture of the four ddNTP linked to different fluorescent dyes and in a single reaction tube is now commonly used. The mixture is separated by capillary electrophoresis, the fluorescent labels are detected, and a color readout of the sequence is generated (Fig. 34.10). [Note: The Human Genome Project used variations of this technique to sequence the human genome.] Advances in sequencing technology, so-called next generation, or high-throughput sequencing, now allow the rapid sequencing of an entire genome with increased fidelity and


Biochemistry_Lippinco

Biochemistry_Lippinco. sequence the human genome.] Advances in sequencing technology, so-called next generation, or high-throughput sequencing, now allow the rapid sequencing of an entire genome with increased fidelity and decreased cost through the simultaneous (parallel) sequencing of many DNA pieces. [Note: Sequencing of the exome, that portion of the genome that encodes proteins, is now possible.]


Biochemistry_Lippinco

Biochemistry_Lippinco. IV. PROBES Cleavage of large DNA molecules by restriction enzymes produces an enormous array of fragments. How can the DNA sequence of interest be picked out of such a mixture? The answer lies in the use of a probe, a short piece of ssDNA or RNA, labeled with a radioisotope, such as 32P, or with a nonradioactive molecule, such as biotin or a fluorescent dye. The sequence of a probe is complementary to a sequence in the DNA of interest, called the target DNA. Probes are used to identify which band on a gel or which clone in a library contains the target DNA, a process called screening. A. Hybridization to DNA


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Hybridization to DNA The utility of probes hinges on the process of hybridization (or annealing) in which a probe containing a complementary sequence binds a single-stranded sequence of a target DNA. ssDNA, produced by alkaline denaturation of dsDNA, is first bound to a solid support, such as a nitrocellulose membrane. The immobilized DNA strands are prevented from self-annealing but are available for hybridization to the exogenous, radiolabeled, single-stranded probe. The extent of hybridization is measured by the retention of radioactivity on the membrane. Excess probe molecules that do not hybridize are removed by washing the membrane. B. Synthetic oligonucleotide probes


Biochemistry_Lippinco

Biochemistry_Lippinco. If the sequence of all or part of the target DNA is known, short, single-stranded oligonucleotide probes can be synthesized that are complementary to a small region of the gene of interest. If the sequence of the gene is unknown, the amino acid sequence of the protein, the final gene product, may be used to construct a nucleic acid probe using the genetic code as a guide. Because of the degeneracy of the genetic code (see p. 449), it is necessary to synthesize several oligonucleotides. [Note: Oligonucleotides can be used to detect single-base changes in the sequence to which they are complementary. In contrast, cDNA probes contain many thousands of bases, and their binding to a target DNA with a single-base change is unaffected.] 1. Detecting the βS-globin mutation: A synthetic allele-specific oligonucleotide (ASO) probe can be used to detect the presence of the sickle cell mutation in the β-globin gene (Fig. 34.11). DNA, isolated from white blood cells (WBC) and amplified, is


Biochemistry_Lippinco

Biochemistry_Lippinco. oligonucleotide (ASO) probe can be used to detect the presence of the sickle cell mutation in the β-globin gene (Fig. 34.11). DNA, isolated from white blood cells (WBC) and amplified, is denatured and applied to a membrane. A radiolabeled oligonucleotide probe, complementary to the point mutation (GAG → GTG, glutamate → valine) at codon 6 in patients with the βS gene, is applied to the membrane. DNA isolated from a heterozygous individual (sickle cell trait) or a homozygous patient (sickle cell anemia) contains a sequence that is complementary to the probe and a double-stranded hybrid form can be detected. In contrast, DNA obtained from normal individuals is not complementary at this position and, therefore, does not form a hybrid (see Fig. 34.11). Use of a pair of such ASO probes (one specific for the normal allele and one specific for the mutant allele) allows all three possible genotypes (homozygous normal, heterozygous, and homozygous mutant) to be distinguished (Fig. 34.12).


Biochemistry_Lippinco

Biochemistry_Lippinco. specific for the normal allele and one specific for the mutant allele) allows all three possible genotypes (homozygous normal, heterozygous, and homozygous mutant) to be distinguished (Fig. 34.12). [Note: ASO probes are useful only if the mutation and its location are known.]


Biochemistry_Lippinco

C. Biotinylated probes

Biochemistry_Lippinco. C. Biotinylated probes


Biochemistry_Lippinco

Biochemistry_Lippinco. Because the disposal of radioactive waste is becoming increasingly expensive, nonradiolabeled probes have been developed. One of the most successful is based on the vitamin biotin (see p. 385), which can be chemically linked to the nucleotides used to synthesize the probe. Biotin was chosen because it binds very tenaciously to avidin, a readily available protein contained in chicken egg whites. Avidin can be attached to a fluorescent dye detectable optically with great sensitivity. Thus, a DNA fragment (displayed, for example, by gel electrophoresis) that hybridizes with the biotinylated probe can be made visible by immersing the gel in a solution of dye-coupled avidin. After washing away the excess avidin, the DNA fragment that binds the probe is fluorescent. [Note: Labeled probes can allow detection and localization of DNA or RNA sequences in cell or tissue preparations, a process called in situ hybridization (ISH). If the probe is fluorescent (F), the technique is called FISH.]


Biochemistry_Lippinco

Biochemistry_Lippinco. D. Antibodies If no amino acid sequence information is available to guide the synthesis of a probe for direct detection of the DNA of interest, a gene can be identified indirectly by cloning cDNA in an expression vector that allows the cloned cDNA to be transcribed and translated. A labeled antibody is used to identify which bacterial colony produces the protein and, therefore, contains the cDNA of interest. V. SOUTHERN BLOTTING Southern blotting is a technique that combines the use of restriction enzymes, electrophoresis, and DNA probes to generate, separate, and detect pieces of DNA. A. Procedure


Biochemistry_Lippinco

Biochemistry_Lippinco. This method, named after its inventor, Edward Southern, involves the following steps (Fig. 34.13). First, DNA is extracted from cells, for example, a patient’s WBC. Second, the DNA is cleaved into many fragments using a restriction enzyme. Third, the resulting fragments (all of which are negatively charged) are separated on the basis of size by electrophoresis. [Note: Because the large fragments move more slowly than the smaller ones, the lengths of the fragments, usually expressed as the number of base pairs, can be calculated from comparison of the position of the band relative to standard fragments of known size.] The DNA fragments in the gel are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. If the original DNA represents the individual’s entire genome, the enzymic digest contains ≥106 fragments. The gene of interest is on only one (or a few if the gene itself was fragmented) of these pieces of DNA. If all the DNA fragments were visualized by a


Biochemistry_Lippinco

Biochemistry_Lippinco. the enzymic digest contains ≥106 fragments. The gene of interest is on only one (or a few if the gene itself was fragmented) of these pieces of DNA. If all the DNA fragments were visualized by a nonspecific technique, they would appear as an unresolved blur of overlapping bands. To avoid this, the last step in Southern blotting uses a probe to identify the DNA fragments of interest. The patterns observed on Southern blot analysis depend both on the specific restriction endonuclease and on the probe used to visualize the restriction fragments. [Note: Variants of the Southern blot have been facetiously named northern if RNA is being studied (see p. 499) and western if protein is being studied (see p. 500), neither of which relates to anyone’s name or to points of the compass.]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Mutation detection Southern blotting can detect DNA mutations such as large insertions or deletions, trinucleotide repeat expansions, and rearrangements of nucleotides. It can also detect point mutations (replacement of one nucleotide by another; see p. 449) that cause the loss or gain of restriction sites. Such mutations cause the pattern of bands to differ from those seen with a normal gene. Longer fragments are generated if a restriction site is lost. For example, in Figure 34.13, person 2 lacks a restriction site present in person 1. Alternatively, the point mutation may create a new cleavage site with the production of shorter fragments. [Note: Most sequence differences at restriction sites are harmless variations in the DNA.] VI. RESTRICTION FRAGMENT LENGTH POLYMORPHISM


Biochemistry_Lippinco

Biochemistry_Lippinco. It has been estimated that the genomes of any two unrelated people are 99.5% identical. With 6 billion bp in the diploid human genome, that represents variation in ~30 million bp. These genome variations are the result of mutations that lead to polymorphisms. A polymorphism is a change in genotype that can result in no change in phenotype or a change in phenotype that is harmless, causes increased susceptibility to a disease, or, rarely, causes the disease. It is traditionally defined as a sequence variation at a given locus (allele) in >1% of a population. Polymorphisms primarily occur in the 98% of the genome that does not encode proteins (that is, in introns and intergenic regions). A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed by cleaving the DNA into fragments (restriction fragments) with a restriction endonuclease. The length of the restriction fragments is altered if the variant alters the DNA so as to create or abolish a


Biochemistry_Lippinco

Biochemistry_Lippinco. cleaving the DNA into fragments (restriction fragments) with a restriction endonuclease. The length of the restriction fragments is altered if the variant alters the DNA so as to create or abolish a restriction site. RFLP can be used to detect human genetic variations, for example, in prospective parents or in fetal tissue.


Biochemistry_Lippinco

Biochemistry_Lippinco. A. DNA variations resulting in RFLP Two types of DNA variations commonly result in RFLP: single-base changes in the DNA sequence and tandem repeats of DNA sequences.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Single-base changes: About 90% of human genome variation comes in the form of single nucleotide polymorphisms (SNPs, pronounced “snips”), that is, variations that involve just one base (Fig. 34.14). The substitution of one nucleotide at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site can also be created by the same mechanism. In either case, cleavage with an endonuclease results in fragments of lengths that differ from the normal and can be detected by DNA hybridization (see Fig. 34.13). The altered restriction site can be either at the site of a disease-causing mutation (rare) or at a site some distance from the mutation. [Note: The HapMap, developed by The International Haplotype Map Project, is a catalog of common SNP in the human genome. The data are being used in genome-wide association studies (GWAS) to identify those alleles that affect health and disease.] 2. Tandem repeats: Polymorphisms in chromosomal


Biochemistry_Lippinco

Biochemistry_Lippinco. SNP in the human genome. The data are being used in genome-wide association studies (GWAS) to identify those alleles that affect health and disease.] 2. Tandem repeats: Polymorphisms in chromosomal DNA can also arise from the presence of a variable number of tandem repeats (VNTR), as shown in Figure 34.15. These are short sequences of DNA at scattered locations in the genome, repeated in tandem (one after another). The number of these repeat units varies from person to person but is unique for any given individual and, therefore, serves as a molecular “fingerprint.” Cleavage by restriction enzymes yields fragments that vary in length depending on how many repeated segments are contained in the fragment (see Fig. 34.15). Many different VNTR loci have been identified and are extremely useful for DNA fingerprint analysis, such as in forensic and paternity cases. It is important to emphasize that these polymorphisms, whether SNP or VNTR, are simply markers, which, in most cases, have no


Biochemistry_Lippinco

Biochemistry_Lippinco. for DNA fingerprint analysis, such as in forensic and paternity cases. It is important to emphasize that these polymorphisms, whether SNP or VNTR, are simply markers, which, in most cases, have no known effect on the structure, function, or rate of production of any particular protein.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Tracing chromosomes from parent to offspring If the DNA of an individual has gained a restriction site by base substitution, then enzymic cleavage yields at least one additional fragment. Conversely, if a mutation results in loss of a restriction site, fewer fragments are produced by enzymic cleavage. An individual who is heterozygous for a polymorphism has a sequence variation in the DNA of one chromosome and not in the homologous chromosome. In such individuals, each chromosome can be traced from parent to offspring by determining the presence or absence of the polymorphism. C. Prenatal diagnosis Families with a history of severe genetic disease, such as an affected previous child or near relative, may wish to determine the presence of the disorder in a developing fetus. Prenatal diagnosis, in association with genetic counseling, allows for an informed reproductive decision if the fetus is affected. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Methods available: The available diagnostic methods vary in sensitivity and specificity. Visualization of the fetus, for example, by ultrasound or fiberoptic devices (fetoscopy), is useful only if the genetic abnormality results in gross anatomic defects (for example, neural tube defects [NTD]). The chemical composition of the amniotic fluid can also provide diagnostic clues. For example, the presence of high levels of αfetoprotein is associated with NTD. Fetal cells obtained from amniotic fluid or from biopsy of the chorionic villi can be used for karyotyping, which assesses the morphology of metaphase chromosomes. Staining and cell sorting techniques permit the rapid identification of trisomies and translocations that produce an extra chromosome or chromosomes of abnormal lengths. However, molecular analysis of fetal DNA provides the most detailed genetic picture. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. DNA sources: DNA may be obtained from blood cells, amniotic fluid, or chorionic villi (Fig. 34.16). For amniotic fluid, it was formerly necessary to grow cells in culture for 2–3 weeks in order to have sufficient DNA for analysis. The ability to amplify DNA by PCR has dramatically shortened the time needed for a DNA analysis. 3. Direct diagnosis of sickle cell anemia using RFLP: The genetic disorders of Hb are the most common genetic diseases in humans. In the case of sickle cell anemia (Fig. 34.17), the point mutation that gives rise to the disease (see p. 35) is actually one and the same mutation that gives rise to the polymorphism. However, direct detection by RFLP of diseases that result from point mutations is limited to only a few genetic diseases. a. Early diagnostic efforts: In the past, prenatal diagnosis of sickle cell anemia involved the determination of the amount and kinds of Hb synthesized in the nucleated red cells obtained from fetal blood.


Biochemistry_Lippinco

Biochemistry_Lippinco. However, the invasive procedures to obtain fetal blood have a high mortality rate (~5%), and analysis cannot be carried out until late in the second trimester of pregnancy when HbA (and its HbS variant) begins to be produced.


Biochemistry_Lippinco

Biochemistry_Lippinco. b. RFLP analysis: In sickle cell anemia, the sequence alteration caused by the point mutation abolishes the recognition site of the restriction endonuclease MstII: CCTNAGG (where N is any nucleotide; see Fig. 34.17). Thus, the A-to-T mutation in codon 6 of the βS-globin gene eliminates a cleavage site for the enzyme. Normal DNA digested with MstII yields a 1.15-kb fragment, whereas a 1.35-kb fragment is generated from the βS gene as a result of the loss of one MstII cleavage site. Diagnostic techniques that allow analysis of fetal DNA from amniotic cells or chorionic villus sampling rather than fetal blood have proved valuable because they provide safe, early detection of sickle cell anemia as well as other genetic diseases. [Note: Genetic disorders caused by insertions or deletions between two restriction sites, rather than by the creation or loss of cleavage sites, will also display RFLP.] 4. Indirect diagnosis of phenylketonuria using RFLP: The gene for phenylalanine hydroxylase


Biochemistry_Lippinco

Biochemistry_Lippinco. two restriction sites, rather than by the creation or loss of cleavage sites, will also display RFLP.] 4. Indirect diagnosis of phenylketonuria using RFLP: The gene for phenylalanine hydroxylase (PAH), the enzyme deficient in phenylketonuria ([PKU] see p. 270), is located on chromosome 12. It spans ~90 kb of genomic DNA and contains 13 exons separated by introns (Fig. 34.18; see p. 442 for a description of exons and introns). Mutations in the PAH gene usually do not directly affect any restriction endonuclease recognition site. To establish a diagnostic protocol for PKU, DNA from family members of the affected individual must be analyzed. The goal is to identify genetic markers (RFLP) that are tightly linked to the disease trait. Once these markers are identified, RFLP analysis can be used to carry out prenatal diagnosis.


Biochemistry_Lippinco

a.

Biochemistry_Lippinco. a.


Biochemistry_Lippinco

Biochemistry_Lippinco. Mutant gene identification: Determining the presence of the mutant gene by identifying the polymorphism marker can be done if two conditions are satisfied. First, if the polymorphism is closely linked to a disease-producing mutation, the defective gene can be traced by detection of the RFLP. For example, if DNA from a family carrying a disease-causing gene is examined by restriction enzyme cleavage and Southern blotting, it is sometimes possible to find an RFLP that is consistently associated with that gene (that is, they show close linkage and are coinherited). It is then possible to trace the inheritance of the gene within a family without knowledge of the nature of the genetic defect or its precise location in the genome. [Note: The polymorphism may be known from the study of other families with the disorder or may be discovered to be unique in the family under investigation.] Second, for autosomal-recessive disorders, such as PKU, the presence of an affected individual in the


Biochemistry_Lippinco

Biochemistry_Lippinco. families with the disorder or may be discovered to be unique in the family under investigation.] Second, for autosomal-recessive disorders, such as PKU, the presence of an affected individual in the family would aid in the diagnosis. This individual would have the mutation present on both chromosomes, allowing identification of the RFLP associated with the genetic disorder.


Biochemistry_Lippinco

b.

Biochemistry_Lippinco. b.


Biochemistry_Lippinco

Biochemistry_Lippinco. RFLP analysis: The presence of abnormal genes for PAH can be shown using DNA polymorphisms as markers to distinguish between normal and mutant genes. For example, Figure 34.19 shows a typical pattern obtained when DNA from members of an affected family is cleaved with an appropriate restriction enzyme and subjected to electrophoresis. The vertical arrows represent the cleavage sites for the restriction enzyme used. The presence of a polymorphic site creates fragment “b” in the autoradiogram (after hybridization with a labeled PAH-cDNA probe), whereas the absence of this site yields only fragment “a.” Note that subject II-2 demonstrates that the polymorphism, as shown by the presence of fragment “b,” is associated with the mutant gene. Therefore, in this particular family, the appearance of fragment “b” corresponds to the presence of a polymorphic site that marks the abnormal gene for PAH. The absence of fragment “b” corresponds to having only the normal gene. In Figure 34.19,


Biochemistry_Lippinco

Biochemistry_Lippinco. of fragment “b” corresponds to the presence of a polymorphic site that marks the abnormal gene for PAH. The absence of fragment “b” corresponds to having only the normal gene. In Figure 34.19, examination of fetal DNA shows that the fetus inherited two abnormal genes from the parents and, therefore, has PKU.


Biochemistry_Lippinco

Biochemistry_Lippinco. c. Value of DNA testing: DNA-based testing is useful not only in determining if an unborn fetus is affected by PKU but also in detecting unaffected carriers of the mutated gene to aid in family planning. [Note: PKU is treatable by dietary restriction of phenylalanine. Early diagnosis and treatment are essential in preventing severe neurologic damage in affected individuals.] VII. POLYMERASE CHAIN REACTION PCR is an in vitro method for amplifying a selected DNA sequence that does not rely on the biologic (in vivo) cloning method described on p. 483. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source, including viral, bacterial, plant, or animal. The steps in PCR are summarized in Figures 34.20 and 34.21. A. Procedure


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Procedure PCR uses DNA pol to repetitively amplify targeted portions of genomic or cDNA. Each cycle of amplification doubles the amount of DNA in the sample, leading to an exponential increase (2n, where n = cycle number) in DNA with repeated cycles of amplification. The amplified DNA products can then be separated by gel electrophoresis, detected by Southern blotting and hybridization, and sequenced. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Constructing primer: It is not necessary to know the nucleotide sequence of the target DNA in the PCR method. However, it is necessary to know the nucleotide sequence of short segments on each side of the target DNA. These stretches, called flanking sequences, bracket the DNA sequence of interest. The nucleotide sequences of the flanking regions are used to construct two, single-stranded oligonucleotides, usually 20– 35 nucleotides long, which are complementary to the respective flanking sequences. The 3′-hydroxyl end of each oligonucleotide points toward the target sequence (see Fig. 34.20). These synthetic oligonucleotides function as primers in PCR. 2. Denaturing DNA: The target DNA to be amplified is heated to ~95°C to separate the dsDNA into single strands. 3. Annealing primers: The separated strands are cooled to ~50°C and the two primers (one for each strand) anneal to a complementary sequence on the ssDNA. 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. Extending primers: DNA pol and dNTP (in excess) are added to the mixture (~72°C) to initiate the synthesis of two new strands complementary to the original DNA strands. DNA pol adds nucleotides to the 3′-hydroxyl end of the primer, and strand growth extends in the 5′→3′ direction across the target DNA, making complementary copies of the target. [Note: PCR products can be several thousand base pairs long.] At the completion of one cycle of replication, the reaction mixture is heated again to separate the strands (of which there are now four). Each strand binds a complementary primer, and the step of primer extension is repeated. By using a heat-stable DNA pol (for example, Taq from the bacterium Thermus aquaticus that normally lives at high temperatures), the polymerase is not denatured and, therefore, does not have to be added at each successive cycle. However, Taq lacks proofreading activity. Typically, 20–30 cycles are run during this process, amplifying the DNA by a million-fold


Biochemistry_Lippinco

Biochemistry_Lippinco. and, therefore, does not have to be added at each successive cycle. However, Taq lacks proofreading activity. Typically, 20–30 cycles are run during this process, amplifying the DNA by a million-fold (220) to a billion-fold (230). [Note: Each extension product includes a sequence at its 5′-end that is complementary to the primer (see Fig. 34.20). Thus, each newly synthesized strand can act as a template for the successive cycles (see Fig. 34.21). This leads to an exponential increase in the amount of target DNA with each cycle, hence, the name “polymerase chain reaction.”] Probes can be made during PCR by adding labeled nucleotides to the last few cycles.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Advantages The major advantages of PCR over biologic cloning as a mechanism for amplifying a specific DNA sequence are sensitivity and speed. DNA sequences present in only trace amounts can be amplified to become the predominant sequence. PCR is so sensitive that DNA sequences present in an individual cell can be amplified and studied. Isolating and amplifying a specific DNA sequence by PCR is faster and less technically difficult than traditional cloning methods using recombinant DNA techniques. C. Applications PCR has become a very common tool in research, forensics, and clinical diagnostics. 1. Comparison of a normal gene to its mutant form: PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laborious biologic cloning of the DNA. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Comparison of a normal gene to its mutant form: PCR allows the synthesis of mutant DNA in sufficient quantities for a sequencing protocol without laborious biologic cloning of the DNA. 2. Forensic analysis of DNA samples: DNA fingerprinting by means of PCR has revolutionized the analysis of evidence from crime scenes. DNA isolated from a single human hair, a tiny spot of blood, or a sample of semen is sufficient to determine whether the sample comes from a specific individual. The DNA markers analyzed for such fingerprinting are most commonly a type of polymorphism known as short tandem repeats. These are very similar to the VNTR described previously (see p. 491) but are smaller in size. [Note: Paternity testing uses the same techniques.] 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 491) but are smaller in size. [Note: Paternity testing uses the same techniques.] 3. Detection of low-abundance nucleic acid sequences: Viruses that have a long latency period, such as human immunodeficiency virus (HIV), are difficult to detect at the early stage of infection using conventional methods. PCR offers a rapid and sensitive method for detecting viral DNA sequences even when only a small proportion of cells harbors the virus. [Note: Quantitative PCR (qPCR), also known as real-time PCR, allows quantification of the amount (copy number) of the target nucleic acid after each cycle of amplification (that is, in real time) rather than at the end and is useful in determining viral load (the amount of virus).] 4.


Biochemistry_Lippinco

Biochemistry_Lippinco. Prenatal diagnosis and carrier detection of cystic fibrosis: Cystic fibrosis is an autosomal-recessive genetic disease resulting from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common mutation is a three-base deletion that results in the loss of a phenylalanine residue from the CFTR protein (see p. 450). Because the mutant allele is three bases shorter than the normal allele, it is possible to distinguish them from each other by the size of the PCR products obtained by amplifying that portion of the DNA. Figure 34.22 illustrates how the results of such a PCR test can distinguish between homozygous normal, heterozygous (carriers), and homozygous mutant (affected) individuals. The simultaneous amplification of multiple regions of a target DNA using multiple primer pairs is known as multiplex PCR. It allows detection of the loss of ≥1 exons in a gene with many exons such as the gene for CFTR, which has 27 exons.


Biochemistry_Lippinco

Biochemistry_Lippinco. VIII. GENE EXPRESSION ANALYSIS The tools of biotechnology not only allow the study of gene structure, but also provide ways of analyzing the mRNA and protein products of gene expression. A. Determining messenger RNA levels mRNA levels are usually determined by the hybridization of labeled probes to either mRNA itself or to cDNA produced from mRNA. [Note: Amplification by PCR of cDNA made from mRNA by retroviral reverse transcriptase (RT) is referred to as RT-PCR.] 1. Northern blots Northern blots are similar to Southern blots (see Fig. 34.13), except that the sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized with a radiolabeled probe. The bands obtained by autoradiography give a measure of the amount and size of the mRNA molecules in the sample.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Microarrays: DNA microarrays contain thousands of immobilized ssDNA sequences organized in an area no larger than a microscope slide. These microarrays are used to analyze a sample for the presence of gene variations or mutations (genotyping) or to determine the patterns of mRNA production (gene expression analysis), analyzing thousands of genes at the same time. For genotyping analysis, the sample is from genomic DNA. For expression analysis, the population of mRNA molecules from a particular cell type is converted to cDNA and labeled with a fluorescent tag (Fig. 34.23). This mixture is then exposed to a gene (or, DNA) chip, which is a glass slide or membrane containing thousands of tiny spots of DNA, each corresponding to a different gene. The amount of fluorescence bound to each spot is a measure of the amount of that particular mRNA in the sample. DNA microarrays are used to determine the differing patterns of gene expression in two different types of cell (for example, normal


Biochemistry_Lippinco

Biochemistry_Lippinco. is a measure of the amount of that particular mRNA in the sample. DNA microarrays are used to determine the differing patterns of gene expression in two different types of cell (for example, normal and cancer cells; see Fig. 34.23). They can also be used to subclassify cancers, such as breast cancer, to optimize treatment. [Note: Microarrays involving proteins and the antibodies or other proteins that recognize them are being used to identify biomarkers to aid in the diagnosis, prognosis, and treatment of disease based on a patient’s protein expression profile. Protein (and DNA) microarrays are important tools in the development of personalized (precision) medicine in which the treatment and/or prevention strategies consider the genetic, environmental, and lifestyle variations among individuals.]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Protein analysis The kinds and amounts of proteins in cells do not always directly correspond to the amounts of mRNA present. Some mRNA are translated more efficiently than others, and some proteins undergo posttranslational modification. When analyzing the abundance and interactions of a large number of proteins, automated methods involving a variety of techniques, such as mass spectrometry and two-dimensional electrophoresis, are used. When investigating one, or a limited number of proteins, labeled antibodies (Ab) are used to detect and quantify specific proteins and to determine posttranslational modifications. 1.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. Enzyme-linked immunosorbent assays: These assays (known as ELISA) are performed in the wells of a microtiter dish. The antigen (protein) is bound to the plastic of the dish. The probe used consists of an Ab specific for the protein (such as troponin, see p. 66) to be measured. The Ab is covalently bound to an enzyme, which will produce a colored product when exposed to its substrate. The amount of color produced is proportional to the amount of Ab present and, indirectly, to the amount of protein in a test sample. 2. Western blots: Western blots (also called immunoblots) are similar to Southern blots, except that it is protein molecules in the sample that are separated by electrophoresis and blotted (transferred) to a membrane. The probe is a labeled Ab, which produces a band at the location of its antigen. 3.


Biochemistry_Lippinco

Biochemistry_Lippinco. 3. Detecting exposure to human immunodeficiency virus: ELISA and western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV Ab present in a patient’s blood sample. ELISA are used as the primary screening tool because they are very sensitive. Because these assays sometimes give false positives, however, western blots, which are more specific, are often used as a confirmatory test (Fig. 34.24). [Note: ELISA and western blots can only detect HIV exposure after anti-HIV Ab appear in the bloodstream. PCR based testing for HIV is more useful in the first few months after exposure.] C. Proteomics


Biochemistry_Lippinco

Biochemistry_Lippinco. C. Proteomics The study of the proteome, or all the proteins expressed by a genome, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other macromolecules, is known as proteomics. The 20,000–25,000 protein-coding genes of the human genome translate into well over 100,000 proteins when posttranscriptional and posttranslational modifications are considered. Although a genome remains essentially unchanged, the amounts and types of proteins in any particular cell change dramatically as genes are turned on and off. [Note: Proteomics (and genomics) required the parallel development of bioinformatics, the computer-based organization, storage, and analysis of biologic data.] Figure 34.25 compares some of the analytic techniques discussed in this chapter. IX. GENE THERAPY


Biochemistry_Lippinco

Biochemistry_Lippinco. The goal of gene therapy is to treat disease through delivery of the normal, cloned DNA for a gene into the somatic cells of a patient who has a defect in that gene as a result of a disease-causing mutation. Because somatic gene therapy changes only the targeted somatic cells, the change is not passed on to the next generation. [Note: In germline gene therapy, the germ cells are modified, and so the change is passed on. A long-standing moratorium on germline gene therapy is in effect worldwide.] There are two types of gene transfer: 1) ex vivo, in which cells from the patient are removed, transduced, and returned, and 2) in vivo, in which the cells are directly transduced. Both types require use of a viral vector to deliver the DNA. Challenges of gene therapy include development of vectors, achievement of long-lived expression, and prevention of side effects such as an immune response. The first successful gene therapy involved two patients with severe combined immunodeficiency


Biochemistry_Lippinco

Biochemistry_Lippinco. vectors, achievement of long-lived expression, and prevention of side effects such as an immune response. The first successful gene therapy involved two patients with severe combined immunodeficiency disease (SCID) caused by mutations to the gene for adenosine deaminase (ADA, see p. 301). It utilized mature T lymphocytes transduced ex vivo with a retroviral vector (Fig. 34.26). [Note: Human ADA cDNA is now used.] Since 1990, only a small number of patients (with a variety of disorders, such as hemophilia, cancers, and certain types of blindness) have been treated with gene therapy, with varying degrees of success.


Biochemistry_Lippinco

Biochemistry_Lippinco. Gene editing, as opposed to gene addition, allows a mutated gene to be repaired. Combinations of DNA-binding molecules (proteins or RNA) and endonucleases are used to identify and cleave the mutated sequence. Cleavage activates homologous recombination repair of dsDNA breaks (see p. 429) that integrates DNA containing the correct sequence into the gene. [Note: An endonuclease guided to a specific DNA sequence by a custom-designed RNA has been used in gene editing in human cell lines. The technique is based on (and named for) the prokaryotic CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats [CRISPR]-associated protein) system that identifies and cleaves foreign DNA in bacterial cells. CRISPR is currently used in the laboratory but not in the clinic.] X. TRANSGENIC ANIMALS


Biochemistry_Lippinco

Biochemistry_Lippinco. Transgenic animals can be produced by injecting a cloned foreign gene (a transgene) into a fertilized egg. If the gene randomly and stably integrates into a chromosome, it will be present in the germline of the resulting animal and can be passed from generation to generation. A giant mouse called “Supermouse” was produced in this way by injecting the gene for rat growth hormone into a fertilized mouse egg. [Note: Transgenic animals have been designed that produce therapeutic human proteins in their milk, a process called “pharming.” Antithrombin, an anticlotting protein (see online Chapter 35), was produced by transgenic goats and approved for clinical use in 2009.] If the functional transgene undergoes targeted (not random) insertion, a knockin (KI) mouse that expresses the gene is created. Targeted insertion of a nonfunctional version of the transgene creates a knockout (KO) mouse that does not express the gene. Such genetically engineered animals can serve as models for the study


Biochemistry_Lippinco

Biochemistry_Lippinco. Targeted insertion of a nonfunctional version of the transgene creates a knockout (KO) mouse that does not express the gene. Such genetically engineered animals can serve as models for the study of a corresponding human disease.


Biochemistry_Lippinco

XI. CHAPTER SUMMARY

Biochemistry_Lippinco. XI. CHAPTER SUMMARY


Biochemistry_Lippinco

Biochemistry_Lippinco. Restriction endonucleases are bacterial enzymes that cleave double-stranded DNA (dsDNA) into smaller fragments. Each enzyme cleaves at a specific 4–8 base-pair sequence (a restriction site), producing DNA segments called restriction fragments. The sequences that are recognized are palindromic. Restriction enzymes form either staggered cuts (sticky ends) or blunt-end cuts on the DNA. Bacterial DNA ligases can join two DNA fragments from different sources if they have been cut by the same restriction endonuclease. This hybrid combination of two fragments is called a recombinant DNA molecule. Introduction of a foreign DNA molecule into a replicating cell permits the amplification (production of many copies) of the DNA, a process called cloning. A vector is a molecule of DNA to which the fragment of DNA to be cloned is joined. Vectors must be capable of autonomous replication within the host cell, must contain at least one specific nucleotide sequence recognized by a restriction


Biochemistry_Lippinco

Biochemistry_Lippinco. the fragment of DNA to be cloned is joined. Vectors must be capable of autonomous replication within the host cell, must contain at least one specific nucleotide sequence recognized by a restriction endonuclease, and must carry at least one gene that confers the ability to select for the vector such as an antibiotic resistance gene. Prokaryotic organisms normally contain small, circular, extrachromosomal DNA molecules called plasmids that can serve as vectors. They can be readily isolated from the bacterium (or artificially constructed), joined with the DNA of interest, and reintroduced into the bacterium, which will replicate, thus making multiple copies of the hybrid plasmid. A DNA library is a collection of cloned restriction fragments of the DNA of an organism. A genomic library is a collection of fragments of dsDNA obtained by digestion of the total DNA of the organism with a restriction endonuclease and subsequent ligation to an appropriate vector. It ideally contains a copy of


Biochemistry_Lippinco

Biochemistry_Lippinco. collection of fragments of dsDNA obtained by digestion of the total DNA of the organism with a restriction endonuclease and subsequent ligation to an appropriate vector. It ideally contains a copy of every DNA nucleotide sequence in the genome. In contrast, complementary DNA (cDNA) libraries contain only those DNA sequences that are complementary to processed messenger RNA (mRNA) molecules present in a cell and differ according to cell type and environmental conditions. Because cDNA has no introns, it can be cloned into an expression vector for the synthesis of human proteins by bacteria or eukaryotes. Cloned, then purified, fragments of DNA can be sequenced, for example, using the Sanger dideoxy chain termination method. A probe is a small piece of RNA or single-stranded DNA (usually labeled with a radioisotope, such as 32P, or another identifiable compound, such as biotin or a fluorescent dye) that has a nucleotide sequence complementary to the DNA molecule of interest (target DNA).


Biochemistry_Lippinco

Biochemistry_Lippinco. with a radioisotope, such as 32P, or another identifiable compound, such as biotin or a fluorescent dye) that has a nucleotide sequence complementary to the DNA molecule of interest (target DNA). Probes can be used to identify which clone of a library or which band on a gel contains the target DNA. Southern blotting is a technique that can be used to detect specific sequences present in DNA. The DNA is cleaved using a restriction endonuclease, after which the pieces are separated by gel electrophoresis and are denatured and transferred (blotted) to a nitrocellulose membrane for analysis. The fragment of interest is detected using a probe. The human genome contains many thousands of polymorphisms (DNA sequence variations at a given locus). Polymorphisms can arise from single-base changes and from tandem repeats. A polymorphism can serve as a genetic marker that can be followed through families. A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed


Biochemistry_Lippinco

Biochemistry_Lippinco. from tandem repeats. A polymorphism can serve as a genetic marker that can be followed through families. A restriction fragment length polymorphism (RFLP) is a genetic variant that can be observed by cleaving the DNA into restriction fragments using a restriction enzyme. A base substitution in one or more nucleotides at a restriction site can render the site unrecognizable by a particular restriction endonuclease. A new restriction site also can be created by the same mechanism. In either case, cleavage with the endonuclease results in fragments of lengths differing from the normal that can be detected by hybridization with a probe. RFLP analysis can be used to diagnose genetic diseases early in the gestation of a fetus. The polymerase chain reaction (PCR), another method for amplifying a selected DNA sequence, does not rely on the biologic cloning method. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence,


Biochemistry_Lippinco

Biochemistry_Lippinco. a selected DNA sequence, does not rely on the biologic cloning method. PCR permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours. It can amplify the sequence, even when the targeted sequence makes up less than one part in a million of the total initial sample. The method can be used to amplify DNA sequences from any source. Applications of the PCR technique include 1) efficient comparison of a normal gene with a mutant form of the gene, 2) forensic analysis of DNA samples, 3) detection of low-abundance nucleic acid sequences, and 4) prenatal diagnosis and carrier detection (for example, of cystic fibrosis). The products of gene expression (mRNA and proteins) can be measured by techniques such as northern blots, which are like Southern blots except that the sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radiolabeled probe; microarrays are used to determine the differing patterns of gene


Biochemistry_Lippinco

Biochemistry_Lippinco. that the sample contains a mixture of mRNA molecules that are separated by electrophoresis, then hybridized to a radiolabeled probe; microarrays are used to determine the differing patterns of gene expression in two different types of cells (for example, normal and cancer cells); enzyme-linked immunosorbent assays (ELISA); and western blots (immunoblots) are used to detect specific proteins. Proteomics is the study of all the proteins expressed by a genome. The goal of gene therapy is the insertion of a normal cloned gene to replace a defective gene in a somatic cell, whereas the goal of gene editing is the repair of a mutated gene. Insertion of a foreign gene (transgene) into the germline of an animal creates a transgenic animal that can produce therapeutic proteins or serve as gene knockin or knockout models for human diseases.


Biochemistry_Lippinco

Biochemistry_Lippinco. Choose the ONE best answer. 4.1. HindIII is a restriction endonuclease. Which of the following is most likely to be the recognition sequence for this enzyme? A. AAGAAG B. AAGAGA C. AAGCTT D. AAGGAA E. AAGTTC Correct answer = C. The vast majority of restriction endonucleases recognize palindromes in double-stranded DNA, and AAGCTT is the only palindrome among the choices. Because the sequence of only one DNA strand is given, the base sequence of the complementary strand must be determined. To be a palindrome, both strands must have the same sequence when read in the 5′→3′ direction. Thus, the complement of 5′-AAGCTT-3′ is also 5′-AAGCTT-3′.


Biochemistry_Lippinco

Biochemistry_Lippinco. 4.2. An Ashkenazi Jewish couple has their 6-month-old son evaluated for listlessness, poor head control, and a fixed gaze. Tay-Sachs disease, an autosomal-recessive disease of lipid degradation, is diagnosed. The couple also has a daughter. The family’s pedigree is shown to the right, along with Southern blots of a restriction fragment length polymorphism very closely linked to the gene for hexosaminidase A, which is defective in Tay-Sachs disease. Which of the statements below is most accurate with respect to the daughter? A. She has a 25% chance of having Tay-Sachs disease. B. She has a 50% chance of having Tay-Sachs disease. C. She has Tay-Sachs disease. D. She is a carrier for Tay-Sachs disease. E. She is homozygous normal.


Biochemistry_Lippinco

Biochemistry_Lippinco. B. She has a 50% chance of having Tay-Sachs disease. C. She has Tay-Sachs disease. D. She is a carrier for Tay-Sachs disease. E. She is homozygous normal. Correct answer = E. Because they have an affected son, both the biological father and mother must be carriers for this disease. The affected son must have inherited a mutant allele from each parent. Because he shows only the 3kilobase (kb) band on the Southern blot, the mutant allele for this disease must be linked to the 3-kb band. The normal allele must be linked to the 4-kb band, and because the daughter inherited only the 4-kb band, she must be homozygous normal for the hexosaminidase A gene. 4.3. A physician would like to determine the global patterns of gene expression in two different types of tumor cells in order to develop the most appropriate form of chemotherapy for each patient. Which of the following techniques would be most appropriate for this purpose? A. Enzyme-linked immunosorbent assay B. Microarray


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Enzyme-linked immunosorbent assay B. Microarray C. Northern blot D. Southern blot E. Western blot Correct answer = B. Microarray analysis allows the determination of messenger RNA (mRNA) production (gene expression) from thousands of genes at once. A northern blot only measures mRNA production from one gene at a time. Western blots and enzyme-linked immunosorbent assay measure protein production (also gene expression) but only from one gene at a time. Southern blots are used to analyze DNA, not the products of DNA expression. 4.4. A 2-week-old infant is diagnosed with a urea cycle defect. Enzymic analysis showed no activity for ornithine transcarbamoylase (OTC), an enzyme of the cycle. Molecular analysis revealed that the messenger RNA (mRNA) product of the gene for OTC was identical to that of a control. Which of the techniques listed below was most likely used to analyze mRNA? A. Dideoxy chain termination B. Northern blot C. Polymerase chain reaction D. Southern blot


Biochemistry_Lippinco

Biochemistry_Lippinco. A. Dideoxy chain termination B. Northern blot C. Polymerase chain reaction D. Southern blot E. Western blot Correct answer = B. Northern blot allows analysis of the messenger RNA present (expressed) in a particular cell or tissue. Southern blot is used for DNA analysis, whereas western blot is used for protein analysis. Dideoxy chain termination is used to sequence DNA. Polymerase chain reaction is used to generate multiple, identical copies of a DNA sequence in vitro. 4.5. For the patient above, which phase of the central dogma was most likely affected? Correct answer = Translation. The gene is present and is able to be expressed as evidenced by normal production of messenger RNA. The lack of enzymic activity means that some aspect of protein synthesis is affected. For additional ancillary materials related to this chapter, please visit thePoint. I. OVERVIEW


Biochemistry_Lippinco

Biochemistry_Lippinco. Blood clotting (coagulation) is designed to rapidly stop bleeding from a damaged blood vessel in order to maintain a constant blood volume (hemostasis). Coagulation is accomplished through vasoconstriction and the formation of a clot (thrombus) that consists of a plug of platelets (primary hemostasis) and a meshwork of the protein fibrin (secondary hemostasis) that stabilizes the platelet plug. Clotting occurs in association with membranes on the surface of platelets and damaged blood vessels (Fig. 35.1). [Note: If clotting occurs within an intact vessel such that the lumen is occluded and blood flow is impeded, a condition known as thrombosis, serious tissue damage, and even death can occur. This is what happens, for example, during a myocardial infarction (MI).] Processes to limit clot formation to the area of damage and remove the clot once vessel repair is underway also play essential roles in hemostasis. [Note: Separate discussions of the formation of the platelet plug and the


Biochemistry_Lippinco

Biochemistry_Lippinco. formation to the area of damage and remove the clot once vessel repair is underway also play essential roles in hemostasis. [Note: Separate discussions of the formation of the platelet plug and the fibrin meshwork facilitate presentation of these multistep, multicomponent processes. However, the two work together to maintain hemostasis.]


Biochemistry_Lippinco

Biochemistry_Lippinco. II. FIBRIN MESHWORK FORMATION The formation of the fibrin meshwork involves two unique pathways that converge to form a common pathway (Fig. 35.2). In each pathway, the major components are proteins (called factors [F]) designated by Roman numerals. The factors are glycoproteins that are synthesized and secreted by the liver, primarily. [Note: Several factors are also denoted by alternative names. For example, factor X (FX), the point of pathway convergence, is also known as Stuart factor.] A. Proteolytic cascade


Biochemistry_Lippinco

Biochemistry_Lippinco. Within the pathways, a cascade is set up in which proteins are converted from an inactive form, or zymogen, to an active form by proteolytic cleavage in which the protein product of one activation reaction initiates another. The active form of a factor is denoted by a lowercase “a” after the numeral. The active proteins FIIa, FVIIa, FIXa, FXa, FXIa, and FXIIa are enzymes that function as serine proteases with trypsin-like specificity and, therefore, cleave a peptide bond on the carboxyl side of an arginine or lysine residue in a polypeptide. For example, FIX (Christmas factor) is activated through cleavage at arginine 145 and arginine 180 by FXIa (Fig. 35.3). The proteolytic cascade results in enormous rate acceleration, because one active protease can produce many molecules of active product each of which, in turn, can activate many molecules of the next protein in the cascade. In some cases, activation can be caused by a conformational change in the protein in the absence of


Biochemistry_Lippinco

Biochemistry_Lippinco. product each of which, in turn, can activate many molecules of the next protein in the cascade. In some cases, activation can be caused by a conformational change in the protein in the absence of proteolysis. [Note: Nonproteolytic proteins play a role as accessory proteins (cofactors) in the pathways. FIII, FV, and FVIII are the accessory proteins.]


Biochemistry_Lippinco

Biochemistry_Lippinco. B. Role of phosphatidylserine and calcium The presence of the negatively charged phospholipid phosphatidylserine (PS) and positively charged calcium ions (Ca2+) accelerates the rate of some steps in the clotting cascade. 1. Phosphatidylserine: PS is located primarily on the intracellular (cytosolic) face of the plasma membrane. [Note: Flippases create the asymmetry (see p. 205).] Its exposure signals injury to the endothelial cells that line blood vessels. PS is also exposed on the surface of activated platelets. 2.


Biochemistry_Lippinco

Biochemistry_Lippinco. 2. Calcium ions: Ca2+ binds the negatively charged γ-carboxyglutamate (Gla) residues present in four of the serine proteases of clotting (FII, FVII, FIX, and FX), facilitating the binding of these proteins to exposed phospholipids (Fig. 35.4). The Gla residues are good chelators of Ca2+ because of their two adjacent negatively charged carboxylate groups (Fig. 35.5). [Note: The use of chelating agents such as sodium citrate to bind Ca2+ in blood-collecting tubes or bags prevents the blood from clotting.] C. Formation of γ-carboxyglutamate residues γ-Carboxylation is a posttranslational modification in which 9–12 glutamate residues (at the amino [N]-terminus of the target protein) get carboxylated at the γ carbon, thereby forming Gla residues. The process occurs in the rough endoplasmic reticulum (RER) of the liver.


Biochemistry_Lippinco

Biochemistry_Lippinco. 1. γ-Carboxylation: This carboxylation reaction requires a protein substrate, oxygen (O2), carbon dioxide (CO2), γ-glutamyl carboxylase, and the hydroquinone form of vitamin K as a coenzyme (Fig. 35.6). In the reaction, the hydroquinone form of vitamin K gets oxidized to its epoxide form as O2 is reduced to water. [Note: Dietary vitamin K, a fat-soluble vitamin (see p. 393), is reduced from the quinone form to the hydroquinone coenzyme form by vitamin K reductase (Fig. 35.7).] 2. Inhibition by warfarin: The formation of Gla residues is sensitive to inhibition by warfarin, a synthetic analog of vitamin K that inhibits the enzyme vitamin K epoxide reductase (VKOR). The reductase, an integral protein of the RER membrane, is required to regenerate the functional hydroquinone form of vitamin K from the epoxide form generated in the γ-carboxylation reaction. Thus, warfarin is an anticoagulant that inhibits clotting by functioning as a vitamin K antagonist. Warfarin salts are used


Biochemistry_Lippinco

Biochemistry_Lippinco. vitamin K from the epoxide form generated in the γ-carboxylation reaction. Thus, warfarin is an anticoagulant that inhibits clotting by functioning as a vitamin K antagonist. Warfarin salts are used therapeutically to limit clot formation. [Note: Warfarin is used commercially as a pest control agent such as in rat poison. It was developed by the Wisconsin Alumni Research Foundation, hence the name.]