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Lecture Notes �APLAY MEDICAL USMLE™* Step 1 Biochemisty and Medical Genetics Lecture Notes BK4029J *USM LE™ is a joint program of the Federation of State Medical Boards of the United States and the National Board of Medical Examiners. ©2013 Kaplan, Inc. All rights reserved. No part of this book may be reproduced in any orm, by photostat, microilm, xerography or any other means, or incorporated into any inormation retrieval system, electronic or mechanical, without the written permission of Kaplan, Inc. Not or resale. BIOCHEMISTRY MEDICAL GENETICS Author Author Sam Turco, Ph.D. Vernon Reichenbecher, Ph.D. Professor, Department of Biochemistry Professor Emeritus, Department of University of Kentucky College of Medicine Biochemistry & Molecular Biology Lexington, KY Marshall University School of Medicine Huntington, V Contributors Roger Lane, Ph.D. Professor, Department of Biochemistry University of South Alabama College of Medicine Mobile, AL David Seastone, D.O., Ph.D. Department of Hematology/Oncology Cleveland Clinic - Taussig Cancer Institute Cleveland, OH Previous contributions by Barbara Hansen, Ph.D. and Lynn B. Jorde, Ph.D. Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Section I: Molecular Bioloy and Biochemistry Chapter 1: Nucleic Acid Structure and Organization Chapter 2: DNA Replication and Repair. . . . . . Chapter 3: Transcription and RNA Processing . . . . . . . . . . . . . . . . . . . . .. . . . . Chapter 4: The Genetic Code, Mutations, and Translation Chapter 5: Regulation of Eukaryotic Gene Expression Chapter 6: Recombinant DNA . . . .. . . . . . Chapter 7: Techniques of Genetic Analysis . . . . . . . . . . Chapter 8: Amino Acids, Proteins, and Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 . 17 . 33 49 73 83 101 117 Chapter 9: Hormones ........... ... .......... .. ............. 133 . Chapter 10: Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Chapter 11: Overview of Energy Metabolism ...... .............. 159 . Chapter 12: Glycolysis and Pyruvate Dehydrogenase .. ........... 169 . Chapter 13: Citric Acid Cycle and Oxidative Phosphorylation . . . . . . . . 187 Chapter 14: Glycogen, Gluconeogenesis, and the Hexose Monophosphate Shunt. ............................ 199 Chapter 15: Lipid Synthesis and Storage . . . . . . . . . . . . . . . . . . . . . . . .. 217 . � MEDICAL V Chapter 16: Lipid Mobilization and Catabolism Chapter 17: Amino Acid Metabolism . . . . . . . . . Chapter 18: Purine and yrimidine Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239 261 287 Section II. Medical Genetics Chapter 1: Single-Gene Disorders Chapter 2: Population Genetics Chapter 3: Cytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chapter 4: Genetics of Common Diseases Chapter 5: Gene Mapping . . . Chapter 6: Genetic Diagnosis Index Vi � MEDICAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 333 347 . 371 383 395 411 Preface hese 7 volumes of Lecture Notes represent the most-likely-to-be-tested material on the current USMLE Step 1 exam. Please note that these are Lecture Notes, not review books. The Notes were designed to be accompanied by aculty lectures live, on video, or on the web. Reading them without accessing the accompanying lectures is not an efective way to review or the USMLE. To maximize the efectiveness of these Notes, annotate them as you listen to lec tures. To acilitate this process, we've created wide, blank margins. While these margins are occasionally punctuated by aculty high-yield "margin notes;' they are, or the most part, let blank or your notations. Many students ind that previewing the Notes prior to the lecture is a very efec tive way to prepare or class. his allows you to anticipate the areas where you'll need to pay particular attention. It also afords you the opportunity to map out how the inormation is going to be presented and what sort of study aids (charts, diagrams, etc.) you might want to add. his strategy works regardless of whether you're attending a live lecture or watching one on video or the web. Finally, we want to hear what you think. What do you like about the Notes? What could be improved? Please share your feedback by e-mailing us at medeedback@ kaplan.com. hank you or joining Kaplan Medical, and best of luck on your Step 1 exam! Kaplan Medical � MEDICAL Vii SECTION Molecular B iolo gy and Biochemistry Nucleic Acid Structure and Organization 1 OVERVIEW: CENTAL DOGMA OF MOLECULAR BIOLOGY An organism must be able to store and preserve its genetic inormation, pass that inormation along to uture generations, and express that inormation as it carries out all the processes of life. he major steps involved in handling genetic inorma tion are illustrated by the central dogma of molecular biology (Figure I-1-1). Ge netic inormation is stored in the base sequence of DNA molecules. Ultimately, during the process of gene expression, this inormation is used to synthesize all the proteins made by an organism. Classically, a gene is a unit of the DNA that encodes a particular protein or RNA molecule. Although this deinition is now complicated by our increased appreciation of the ways in which genes may be expressed, it is still useul as a general, working deinition. Replication Transcription Translation � I Protein l -./ -.. Reverse transcription Figure 1-1-1. Central Dogma of Molecular Biology Gene xpression and DNA Replication Gene expression and DNA replication are compared in Table I-1-1. Transcrip tion, the irst stage in gene expression, involves transfer of inormation ound in a double-stranded DNA molecule to the base sequence of a single-stranded RNA molecule. If the RNA molecule is a messenger RNA, then the process known as translation converts the inormation in the RNA base sequence to the amino acid sequence of a protein. When cells divide, each daug hter cell must receive an accurate c opy of the genetic inormation. DNA replication is the process in which each chromosome is dupli cated beore cell division. � MEDICAL 3 Section I • Molecular Bioloy and Biochemistry Table 1-1-1. Comparison of Gene xpression and DNA Replication DNA Replication Gene Expression Produces all the proteins an organism Duplicates the chromosomes before requires cell division Transcription of DNA: RNA copy of DNA copy of entire chromosome a small section of a chromosome (average size of human gene, 104-1os (average size of human chromosome, 10s nucleotide pairs) Transcription occurs in the nucleus Occurs during S phase nucleotide pairs) throughout interphase Translation of RNA (protein synthesis) Replication in nucleus occurs in the cytoplasm throughout the cell cycle. The concept of the cell cycle (Figure I-1-2) can be used to describe the timing of some of these events in a eukaryotic cell. The M phase (mitosis) is the time in which the cell divides to orm two daughter cells. Interphase is the term used to describe the time between two cell divisions or mitoses. Gene expression occurs throughout all stages of interphase. Interphase is subdivided as ollows: • G1 phase (gap 1) is a period of cellular growth preceding DNA synthesis. Cells that have stopped cycling, such as muscle and nerve cells, are said to be in a special state called G0. • S phase (DNA synthesis) is the period of time during which DNA repli cation occurs. At the end of S phase, each chromosome has doubled its DNA content and is composed of two identical sister chromatids linked Note at the centromere. Many chemotherapeutic agents function by targeting speciic phases of the cell cycle. This is a frequently tested area on the USMLE. Below are • G2 phase (gap 2) is a period of cellular growth ater DNA synthesis but preceding mitosis. Replicated DNA is checked or any errors beore cell division. some of the commonly tested agents with the appropriate phase of the cell cycle they target: • S-phase: methotrexate, 5-flurouracil, M hydroxyurea • G2 phase: bleomycin • M phase: paclitaxel, vincristine, vinblastine • Non cell-cycle speciic: cyclophosphamide, cisplatin Figure 1-1-2. The Eukaryotic Cell Cycle 4 � MEDICAL Chapter 1 • Nucleic Acid Structure and Organization Reverse transcription, which produces DNA copies of an RNA, is more com monly associated with life cycles of retroviruses, which replicate and express their genome through a DNA intermediate (an integrated provirus). Reverse tran scription also occurs to a limited extent in human cells, where it plays a role in ampliying certain highly repetitive sequences in the DNA (Chapter 7). NUCLEOTIDE STRUCTURE AND NOMENCLATURE Nucleic acids (DNA and RNA) are assembled rom nucleotides, which consist of three components: a nitrogenous base, a ive-carbon sugar (pentose), and phosphate. Five-Carbon Sugars Nucleic acids (as well as nucleosides and nucleotides) are classiied according to the pentose they contain. If the pentose is ribose, the nucleic acid is RNA (ribo nucleic acid); if the pentose is deoxyribose, the nucleic acid is DNA (deoxyribo nucleic acid). Bases here are two tpes of nitrogen-containing bases commonly ound in nucleo tides: purines and pyrimidines (Figure I-1-3): Pyrimidines 0 N H� olNJ H J � l) HN 0 Uracil H Thymine Figure 1-1-3. Bases Commonly Found in Nucleic Acids • Purines contain two rings in their structure. The two purines com monly ound in nucleic acids are adenine (A) and guanine (G); both are ound in DNA and RNA. Other purine metabolites, not usually ound in nucleic acids, include xanthine, hypoxanthine, and uric acid. • Pyrimidines have only one ring. Ctosine ( C) is present in both DNA and RNA. Thymine (T) is usually ound only in DNA, whereas uracil (U) is ound only in RNA. Nucleosides and Nucleotides Nucleosides are ormed by covalently linking a base to the number 1 carbon of a sugar (Figure I-1-4). he numbers identiying the carbons of the sugar are labeled with "primes" in nucleosides and nucleotides to distinguish them rom the car bons of the purine or pyrimidine base. � MEDICAL 5 Section I • Molecular Bioloy and Biochemisty � Deoxythymidine Adenosine >I NH2 N N� � N N 5' CH20H I 0 1' 4' I 1' 1 _j OH Figure 1-1-4. Examples of Nucleosides Nucleotides are ormed when one or more phosphate groups is attached to the 5' carbon of a nucleoside (Figure 1-1-5). Nucleoside di- and triphosphates are high energy compounds because of the hydrolytic energy associated with the acid an hydride bonds (Figure 1-1-6). Uridine Monophosphate (UMP) ATP NH2 High-energy bonds N�J(> �N o II { \i o I� o II I I I N I OP-0-P-0-P-0-CH2 0 _ o- o- 1 Deoxyguanosine Monophosphate (dGMP) I I 0 - 11 0 5' o- oOH Figure 1-1-6. High-Energy Bonds in 5' 0- P-O-CH2 I OHOH 11 _ 0- P-O-CH2 I 1 I 0 o- Figure 1-1-5. Examples of Nucleotides a Nucleoside Triphosphate he nomenclature or the commonly ound bases, nucleosides, and nucleotides is shown in Table 1-1-2. Note that the "deoxy" part of the names deoxythymidine, dTMP, etc., is sometimes understood, and not expressly stated, because thymine is almost always ound attached to deoxyribose. 6 � MEDICAL Chapter . • Nucleic Acid Structure and Organization Table 1-1-2. Nomenclature of Important Bases, Nucleosides, and Nucleotides Base Nucleoside Nucleotides Adenine Adenosine AMP (dAMP) ADP (dADP) ATP (dATP) GMP (dGMP) GDP (dGDP) GTP (dGTP) CMP (dCMP) CDP (dCDP) CTP (dCTP) UMP (dUMP) UDP (dUDP) UTP (dUTP) (dTMP) (dTDP) (dTTP) (Deoxyadenosine) Guanine Guanosine (Deoxyguanosine) Cytosine Cytidine (Deoxyc ytidine) Uridine Uracil (Deoxyuridine) Thymine (Deoxythymidine) Names of nucleosides and nucleotides attached to deoxyribose are shown in parentheses. NUCLEIC ACIDS In a Nutshell Nucleic acids are polymers of nucleotides joined by 3', 5'-phosphodiester bonds; Nucleic Acids that is, a phosphate group links the 3' carbon of a sugar to the 5' carbon of the next sugar in the chain. Each strand has a distinct 5' end and 3' end, and thus has • polarity. A phosphate group is oten ound at the 5' end, and a hydroxyl group is oten ound at the 3' end. • on the let in Figure I-1-7 must be written Have distinct 3' and 5' ends, thus polarity The base sequence of a nucleic acid strand is written by convention, in the 5' �3' direction (let to right). According to this convention, the sequence of the strand Nucleotides linked by 3', 5' phosphodiester bonds • Sequence is always speciied as 5'-)3' 5'-TCAG-3' or TCAG: • If written backward, the ends must be labeled: 3' -GACT-5' • The positions of phosphates may be shown: pTpCpApG • In DNA, a "d" (deoxy) may be included: dTdCdAdG In eukaryotes, DNA is generally double-stranded (dsDNA) and RNA is gener ally single-stranded (ssRNA). Exceptions occur in certain viruses, some of which have ssDNA genomes and some of which have dsRNA genomes. � MEDICAL 7 Section I • Molecular Bioloy and Biochemistry 5'- Phosphate o- 1 0-P=O H 6 3 C 0- M sdH, N v� I ----------- H H - I N - H ·----------N O N HI ( A �N OH N 0 3' 5'C H2 0 I H - O-P = O I Hydroxyl 3'- A--- - - - - J H, v 0 I -o-P= O I 0 I 5'CH2 N -O -P=O I I 0 I 5'CH2 H- \� H " N N N- H - - --- { �N--- -------A N/ ( � �H ( N N N N G -H 0 � � H3 0 H- N � 0 / O------------·H - N N I 0 HJ � -�) G � ---- -- -- H O 0 0 N N - H ---------------O I I -o-P=O N N-H------------ 0 - I 0 I -o-P=O I 0 5'CH2 I 0 H 0 � -------- N 5'CH2 I -O -P=O I N 0 I H 5'CH2 OH 3' I 9 -o-P=O 3' - I o- Hydroxyl 5 '- Figure 1-1-7. 8 5' � MEDICAL Hydrogen-Bonded Base Pairs in DNA Phosphate Chapter 1 • Nucleic Acid Structure and Organization DNA Structure Note Figure I-1-8 shows an example of a double-stranded DNA molecule. Some of the Using Charga's Rules features of double-stranded DNA include: In dsDNA (or dsRNA) • The two strands are an tiparallel (opposite in direction). • The two strands are complementary. A always pairs with T (two hydrogen bonds), and G always pairs with C (three hydrogen bonds). Thus, the base sequence on one strand deines the base sequence on the other strand. • (ds = double-stranded) %A=% T % ( U) %G=%C Because of the speciic base pairing, the amount of A equals the amount of T, and the amount of G equals the amount of C. Thus, total purines equals total pyrimidines. These properties are known as Charga's rules. With minor modiication (substitution ofU or T) these rules also apply to dsRNA. % purines=% pyrimidines A sample of DNA has 10% G; what is the% T? 10% G Most DNA occurs in nature as a right-handed double-helical molecule known as Watson-Crick DNA or B-DNA (Figure I-1-8). he hydrophilic sugar-phosphate backbone of each strand is on the outside of the double helix. The hydrogenbonded base pairs are stacked in the center of the molecule. here are about 10 base pairs per complete turn of the helix. A rare let-handed double-helical orm + 10% C = 20% therefore,%A+%T must total 80% 40%A and 40% T Ans: 40% T of DNA that occurs in G-C-rich sequences is known as Z-DNA. he biologic unction of Z- DNA is unknown, but may be related to gene regulation. Bridge to Pharmacology Daunorubicin and doxorubicin are antitumor drugs that are used in the treatment of leukemias. They exert their efects by intercalating between the bases of DNA, thereby interfering with the activity of topoisomerase II and preventing proper replication of the DNA. Minor Groove Other drugs, such as cisplatin, which is used in the treatment of bladder and lung tumors, bind tightly to the DNA, causing structural distortion and malfunction. Figure 1-1-8. The B-DNA Double Helix � MEDICAL 9 Section I • Molecular Bioloy and Biochemistry Denaturation and Renaturation of DNA Double-helical DNA can be denatured by conditions that disrupt hydrogen bonding and base stacking, resulting in the "melting" of the double helix into two single strands that separate rom each other. No covalent bonds are broken in this process. Heat, alkaline pH, and chemicals such as ormamide and urea are com monly used to denature DNA. Double-stranded DNA l Denaturation (heat) Denatured single-stranded DNA can be renatured (annealed) if the denaturing condition is slowly removed. For example, if a solution containing heat-dena tured DNA is slowly cooled, the two complementary strands can become base paired again (Figure I-1-9). Such renaturation or annealing of complementary DNA strands is an important step in probing a Southern blot and in perorming the polymerase chain reaction (reviewed in Chapter Single-stranded DNA l Renaturation (cooling) 7). In these techniques, a well-characterized probe DNA is added to a mixture of target DNA molecules. he mixed sample is denatured and then renatured. When probe DNA binds to target DNA sequences of suicient complementarity, the process is called hybridization. ORGANIZATION OF DNA Double-stranded DNA Denaturation and Renaturation of DNA Large DNA molecules must be packaged in such a way that they can it inside the cell and still be unctional. Figure 1-1-9. Supercoiling Mitochondrial DNA and the DNA of most prokaryotes are closed circular struc tures. hese molecules may eist as relaxed circles or as supercoiled structures in which the helix is twisted around itself in three-dimensional space. Supercoiling re sults rom strain on the molecule caused by under- or overwinding the double helix: • Negatively supercoiled DNA is ormed if the DNA is wound more loosely than in Watson-Crick DNA. This orm is required or most biologic reactions. • Positively supercoiled DNA is ormed if the DNA is wound more tightly than in Watson-Crick DNA. • Topoisomerases are enzymes that can change the amount of supercoiling in DNA molecules. T hey make transient breaks in DNA strands by alter nately breaking and resealing the sugar-phosphate backbone. For example, in Escherichia coli, DNA gyrase (DNA topoisomerase negative supercoiling into DNA. 10 � MEDICAL II) can introduce Chapter 1 • Nucleic Acid Structure and Organization Nucleosomes and Chromatin Without +HI H1 Expanded view of a nucleosome Expanded view Figure 1-1-10. Nucleosome and Nucleofilament Structure in Eukaryotic DNA Nuclear DNA in eukaryotes is ound in chromatin associated with histones and nonhistone proteins. he basic packaging unit of chromatin is the nucleosome (Figure I-1-10): • • • • • Histones are rich in lysine and arginine, which confer a positive charge on the proteins. Two copies each of histones the histone octamer. H2A, H2B, H3, and H4 aggregate to orm DNA is wound around the outside of this octamer to orm a nucleo some (a series of nucleosomes is sometimes called "beads on a string", but is more properly referred to as a lOnm chromatin iber). Histone Hl is associated with the linker DNA ound between nucleo somes to help package them into a solenoid-like structure, which is a thick 30-nm iber. Further condensation occurs to eventually orm the chromosome. Each eukaryotic chromosome in Go or G 1 contains one linear molecule of double-stranded DNA. Cells in interphase contain two types of chromatin: euchromatin (more opened and available or gene expression) and heterochromatin (much more highly con densed and associated with areas of the chromosomes that are not expressed.) (Figure I-1-11). � MEDICAL 11 Section I j • Molecular Biology and Biochemistry Less active More active DNA double helix 10 nm chromatin 30 nm chromatin 30 nm fiber forms loops attached Higher order to scaffolding proteins packaging Heterochromatin Euchromatin Figure 1-1-11. DNA Packaging in Eukaryotic Cell Euchromatin generally corresponds to the nucleosomes (10-nm ibers) loosely as sociated with each other (looped 30-nm ibers). Heterochromatin is more highly condensed, producing interphase heterochromatin as well as chromatin charac teristic of mitotic chromosomes. Figure 1-1-12 shows an electron micrograph of an interphase nucleus containing euchromatin, heterochromatin, and a nucleolus. he nucleolus is a nuclear region specialized or ribosome assembly (discussed in Chapter 3). Figure 1-1-12. An lnterphase Nucleus During mitosis, all the DNA is highly condensed to allow separation of the sister chromatids. his is the only time in the cell cycle when the chromosome struc ture is visible. Chromosome abnormalities may be assessed on mitotic chromo somes by karyotype analysis (metaphase chromosomes) and by banding tech niques (prophase or prometaphase), which identiy aneuploidy, translocations, deletions, inversions, and duplications. 12 � MEDICAL Chapter 1 • Nucleic Acid Structure and Organization Chapter Summary • Nucleic acids: - RNA and DNA - Nucleotides (nucleoside monophosphates) linked by phosphodiester bonds • - Have polarity (3' end versus 5' end) - Sequence always speciied 5'-to-3' (let to right on page) Double-stranded nucleic acids: - Two strands associate by hydrogen bonding - • Sequences are complementary and antiparallel Eukaryotic DNA in the nucleus: - Packaged with histones (H2a, H2b, H3, H4)2 to form nucleosomes (10-nm iber) - 10-nm iber associates with Hl (30-nm iber). - 10-nm iber and 30-nm iber comprise euchromatin (active gene expression). - Higher-order packaging forms heterochromatin (no gene expression). - Mitotic DNA most condensed (no gene expression) � MEDICAL 13 Section I • Molecular Bioloy and Biochemisty Review Questions Select the ONE best answer. l. A double-stranded RNA genome isolated rom a virus in the stool of a child with gastroenteritis was ound to contain 15% uracil. hat is the percent age of guanine in this genome? 2. A. 15 B. 25 c. 35 D. 75 E. 85 hat is the structure indicated below? N� � N 5' CH20H 0 1' 4' OH 3. A. Purine nucleotide B. Purine c. Pyrimidine nucleoside D. Purine nucleoside E. Deoyadenosine Endonuclease activation and chromatin ragmentation are characteristic features of eukaryotic cell death by apoptosis. Which of the ollowing chro mosome structures would most likely be degraded irst in an apoptotic cell? 14 � MEDICAL A. Barr body B. 10-nm iber c. 30-nm iber D. Centromere E. Heterochromatin