Principles of surgery

PART I
Basic Considerations

CHAPTER 15
Molecular Biology

BASIC SCIENCE QUESTIONS

1. Functional genomics is a term used to describe which of the following?

A. Transcription of DNA

B. Translation of RNA

C. Proteomics

D. All of the above

Answer: D

Functional genomics seeks to assign a biochemical, physiologic, cell biologic, and/or developmental function to each predicted gene. An ever-increasing arsenal of approaches, including transgenic animals, RNA interference (RNAi), and various systematic mutational strategies, will allow dissection of functions associated with newly discovered genes. (See Schwartz 9th ed., p 385, and Fig. 15-1.)

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FIG. 15-1. The flow of genetic information from DNA to protein to cell functions. The process of transmission of genetic information from DNA to RNA is called transcription, and the process of transmission from RNA to protein is called translation. Proteins are the essential controlling components for cell structure, cell signaling, and metabolism. Genomics and proteomics are the study of the genetic composition of a living organism at the DNA and protein level, respectively. The study of the relationship between genes and their cellular functions is called functional genomics.

2. An intron is

A. A segment of DNA removed prior to transcription

B. The remaining (functional) segment of DNA after removal of nonfunctional DNA

C. A segment of mRNA removed prior to translation

D. The remaining (functional) segment of mRNA after removal of nonfunctional RNA

Answer: A

Living cells have the necessary machinery to enzymatically transcribe DNA into RNA and translate the mRNA into protein. This machinery accomplishes the two major steps required for gene expression in all organisms: transcription and translation. However, gene regulation is far more complex, particularly in eukaryotic organisms. For example, many gene transcripts must be spliced to remove the intervening sequences. The sequences that are spliced off are called introns, which appear to be useless, but in fact may carry some regulatory information. The sequences that are joined together, and are eventually translated into protein, are called exons. Additional regulation of gene expression includes modification of mRNA, control of mRNA stability, and its nuclear export into cytoplasm (where it is assembled into ribosomes for translation). After mRNA is translated into protein, the levels and functions of the proteins can be further regulated posttranslationally. (See Schwartz 9th ed., p 382.)

3. Translation of mRNA into proteins occurs in the

A. Mitochondria

B. Ribosomes

C. Cytoplasm

D. Cell membrane

Answer: B

DNA directs the synthesis of RNA; RNA in turn directs the synthesis of proteins. Proteins are variable-length polypeptide polymers composed of various combinations of 20 different amino acids and are the working molecules of the cell. The process of decoding information on mRNA to synthesize proteins is called translation. Translation takes place in ribosomes composed of rRNA and ribosomal proteins. (See Schwartz 9th ed., p 383, and Fig. 15-2.)

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FIG. 15-2. Four major steps in the control of eukaryotic gene expression. Transcriptional and posttranscriptional control determine the level of messenger RNA (mRNA) that is available to make a protein, while translational and posttranslational control determine the final outcome of functional proteins. Note that posttranscriptional and posttranslational controls consist of several steps.

4. Approximately how many genes are present in the human genome?

A. 25,000

B. 100,000

C. 250,000

D. 750,000

Answer: A

Genome is a collective term for all genes present in one organism. The human genome contains DNA sequences of 3 billion base-pairs, carried by 23 pairs of chromosomes. The human genome has an estimated 25,000 to 30,000 genes, and overall it is 99.9% identical in all people. Approximately 3 million locations where single-base DNA differences exist have been identified and termed single nucleotide polymorphisms. Single nucleotide polymorphisms may be critical determinants of human variation in disease susceptibility and responses to environmental factors. (See Schwartz 9th ed., p 385.)

5. DNA replication occurs in which phase of the cell cycle?

A. G1

B. S

C. G2

D. M

Answer: B

Many cells grow, while some cells such as nerve cells and striated muscle cells do not. All growing cells have the ability to duplicate their genomic DNA and pass along identical copies of this genetic information to every daughter cell. Thus, the cell cycle is the fundamental mechanism to maintain tissue homeostasis. A cell cycle comprises four periods: G1 (first gap phase before DNA synthesis), S (synthesis phase when DNA replication occurs), G2 (the gap phase before mitosis), and M (mitosis, the phase when two daughter cells with identical DNA are generated) (Fig. 15-3). After a full cycle, the daughter cells enter G1 again, and when they receive appropriate signals, undergo another cycle, and so on. The machinery that drives cell cycle progression is made up of a group of enzymes calledcyclin-dependent kinases (CDK). Cyclin expression fluctuates during the cell cycle, and cyclins are essential for CDK activities and form complexes with CDK. The cyclin A/CDK1 and cyclin B/CDK1 drive the progression for the M phase, while cyclin A/CDK2 is the primary S phase complex. Early G1 cyclin D/CDK4/6 or late G1 cyclin E/CDK2 controls the G1-S transition. There also are negative regulators for CDK termed CDK inhibitors, which inhibit the assembly or activity of the cyclin-CDK complex. Expression of cyclins and CDK inhibitors often are regulated by developmental and environmental factors. (See Schwartz 9th ed., pp 385-386.)

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FIG. 15-3. The cell cycle and its control system. M is the mitosis phase, when the nucleus and the cytoplasm divide; S is the phase when DNA is duplicated; G1 is the gap between M and S; G2 is the gap between S and M. A complex of cyclin and cyclindependent kinase (CDK) controls specific events of each phase. Without cyclin, CDK is inactive. Different cyclin/CDK complexes are shown around the cell cycle. A, B, D, and E stand for cyclin A, cyclin B, cyclin D, and cyclin E, respectively.

6. Apoptosis is accomplished by activation of

A. Capsases

B. Metalloproteases

C. Complement

D. Heat shock protein

Answer: A

Normal tissues undergo proper apoptosis to remove unwanted cells, those that have completed their jobs or have been damaged or improperly proliferated. Apoptosis can be activated by many physiologic stimuli such as death receptor signals (e.g., Fas or cytokine tumor necrosis factor), growth factor deprivation, DNA damage, and stress signals. Two major pathways control the biochemical mechanisms governing apoptosis: the death receptor and mitochondrial. However, recent advances in apoptosis research suggest an interconnection of the two pathways. What is central to the apoptotic machinery is the activation of a cascade of proteinases called caspases. (See Schwartz 9th ed., pp 385-386, and Fig. 15-4.)

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FIG. 15-4. A simplified view of the apoptosis pathways. Extracellular death receptor pathways include the activation of Fas and tumor necrosis factor (TNF) receptors, and consequent activation of the caspase pathway. Intracellular death pathway indicates the release of cytochrome c from mitochondria, which also triggers the activation of the caspase cascade. During apoptosis, cells undergo DNA fragmentation, nuclear and cell membrane breakdown, and are eventually digested by other cells.

7. Cells sense changes in their environment which then subsequently affects gene expression in the cell. These changes are transmitted to the cell by “ligands,” substances that interact with receptors on or in the cell. Ligands are

A. Peptides

B. Dissolved gases

C. Retinoids

D. All of the above

Answer: D

Gene expression in a genome is controlled in a temporal and spatial manner, at least in part by signaling pathways. A signaling pathway generally begins at the cell surface and, after a signaling relay by a cascade of intracellular effectors, ends up in the nucleus (Fig. 15-5). All cells have the ability to sense changes in their external environment. The bioactive substances to which cells can respond are many and include proteins, short peptides, amino acids, nucleotides/nucleosides, steroids, retinoids, fatty acids, and dissolved gases. Some of these substances are lipophilic and thereby can cross the plasma membrane by diffusion to bind to a specific target protein within the cytoplasm (intracellular receptor). Other substances bind directly with a transmembrane protein (cell-surface receptor). Binding of ligand to receptor initiates a series of biochemical reactions (signal transduction) typically involving protein-protein interactions and the transfer of high-energy phosphate groups, leading to various cellular end responses. (See Schwartz 9th ed., p 386.)

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FIG. 15-5. Cell-surface and intracellular receptor pathways. Extracellular signaling pathway: Most growth factors and other hydrophilic signaling molecules are unable to move across the plasma membrane and directly activate cell-surface receptors such as G-protein coupled receptors and enzyme-linked receptors. The receptor serves as the receiver, and in turn activates the downstream signals in the cell. Intracellular signaling pathway: Hormones or other diffusible molecules enter the cell and bind to the intracellular receptor in the cytoplasm or in the nucleus. Either extracellular or intracellular signals often reach the nucleus to control gene expression.

8. Identification of a specific DNA segment can be accomplished by

A. Southern blotting

B. Northern blotting

C. Western blotting

D. Eastern blotting

Answer: A

Southern blotting refers to the technique of transferring DNA fragments from an electrophoresis gel to a membrane support, and the subsequent analysis of the fragments by hybridization with a radioactively labeled probe (Fig. 15-6). Southern blotting is named after E. M. Southern, who in 1975 first described the technique of DNA analysis. It enables reliable and efficient analysis of size-fractionated DNA fragments in an immobilized membrane support. (See Schwartz 9th ed., p 392.)

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FIG. 15-6. Southern blotting. Restriction enzymatic fragments of DNA are separated by agarose gel electrophoresis, transferred to a membrane filter, and then hybridized to a radioactive probe.

Northern blotting refers to the technique of size fractionation of RNA in a gel and the transferring of an RNA sample to a solid support (membrane) in such a manner that the relative positions of the RNA molecules are maintained. The resulting membrane then is hybridized with a labeled probe complementary to the mRNA of interest. Signals generated from detection of the membrane can be used to determine the size and abundance of the target RNA. In principle, Northern blot hybridization is similar to Southern blot hybridization (and hence its name), with the exception that RNA, not DNA, is on the membrane.

Analyses of proteins are primarily carried out by antibody-directed immunologic techniques. For example, Western blotting, also called immunoblotting, is performed to detect protein levels in a population of cells or tissues, whereas immunoprecipitation is used to concentrate proteins from a larger pool.

There is no technique known as Eastern blotting.

9. DNA microarrays allow identification of gene mutations using

A. Polymerase chain reaction

B. Hybridization

C. Western blotting

D. Molecular cloning

Answer: B

DNA microarray, also called gene chipDNA chip, and gene array, refers to large sets of probes of known sequences orderly arranged on a small chip, enabling many hybridization reactions to be carried out in parallel in a small device (Fig. 15-7). Like Southern and Northern hybridization, the underlying principle of this technology is the remarkable ability of nucleic acids to form a duplex between two strands with complementary base sequences. DNA microarray provides a medium for matching known and unknown DNA samples based on base-pairing rules, and automating the process of identifying the unknowns. Microarrays require specialized robotics and imaging equipment that spot the samples on a glass or nylon substrate, carry out the hybridization, and analyze the data generated. DNA microarrays containing different sets of genes from a variety of organisms are now commercially available, allowing biologists to simply purchase the chips and perform hybridization and data collection.

Polymerase chain reaction (PCR) is an in vitro method for the polymerase-directed amplification of specific DNA sequences using two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in the target DNA.

Western blotting, also called immunoblotting, is performed to detect protein levels in a population of cells.

Molecular cloning refers to the process of cloning a DNA fragment of interest into a DNA vector that ultimately is delivered into bacterial or mammalian cells or tissues. (See Schwartz 9th ed., p 394.)

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FIG. 15-7. DNA microarrays. DNA microarrays, also referred to as gene chips, have arrayed oligonucleotides or complementary DNAs (cDNAs) corresponding to tens or hundreds of distinct genes. DNA microarray is used to comparatively analyze gene expression in different cells or tissues. Messenger RNAs (mRNAs) extracted from different sources are converted into cDNAs, which are then labeled with different fluorescent dyes. The two fluorescent cDNA probes are mixed and hybridized to the same DNA microarrays. The ratio of dark brown to light brown fluorescence at each spot on the chip represents the relative expression of levels of that gene between two different cells. In the example shown in the figure, cDNA from cell #1 is labeled with dark brown fluorescence and the cell #2 light brown fluorescence. On the microarray, dark brown spots demonstrate that the gene in cell sample #1 is expressed at a higher level than the corresponding gene in cell sample #2. The light brown spots indicate that the gene in cell sample #1 also is expressed at a higher level than the corresponding gene in cell sample #2. Beige spots represent equal expression of the gene in both cell samples.

CLINICAL QUESTIONS

1. Trastuzumab is a monoclonal antibody which targets which of the following cell receptors in susceptible patients with breast cancer?

A. BRAC-1

B. BRAC-2

C. HER2

D. CAD-1

Answer: C

One of the most exciting applications of immunotherapy has come from the identification of certain tumor targets called antigens and the aiming of an antibody at these targets. This was first used as a means of localizing tumors in the body for diagnosis, and was more recently used to attack cancer cells. Trastuzumab (Herceptin) is an example of such a drug. Trastuzumab is a monoclonal antibody that neutralizes the mitogenic activity of cell-surface growth factor receptor HER2. Approximately 25% of breast cancers overexpress HER2. These tumors tend to grow faster and generally are more likely to recur than tumors that do not overproduce HER2. Trastuzumab is designed to attack cancer cells that overexpress HER2. Trastuzumab slows or stops the growth of these cells and increases the survival of HER2-positive breast cancer patients. (See Schwartz 9th ed., p 390.)

2. STI157, also known as Gleevec, is a molecularly targeted therapy for

A. Acute lymphocytic leukemia

B. Acute myeloid leukemia

C. Chronic lymphocytic leukemia

D. Chronic myeloid leukemia

Answer: D

The primary function of anticancer chemicals is to block different steps involved in cell growth and replication. These chemicals often block a critical chemical reaction in a signal transduction pathway or during DNA replication or gene expression. For example, STI571, also known as Gleevec, is one of the first molecularly targeted drugs based on the changes that cancer causes in cells. STI571 offers promise for the treatment of chronic myeloid leukemia (CML) and may soon surpass interferon-γ as the standard treatment for the disease. In CML, STI571 is targeted at the Bcr-Abl kinase, an activated oncogene product in CML (Fig. 15-8). Bcr-Abl is an overly activated protein kinase resulting from a specific genetic abnormality generated by chromosomal translocation that is found in the cells of patients with CML. STI571-mediated inhibition of Bcr-Abl-kinase activity not only prevents cell growth of Bcr-Abl–transformed leukemic cells, but also induces apoptosis. Clinically, the drug quickly corrects the blood cell abnormalities caused by the leukemia in a majority of patients, achieving a complete disappearance of the leukemic blood cells and the return of normal blood cells. (See Schwartz 9th ed., p 390.)

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FIG. 15-8. Mechanism of STI571 as a molecular drug. Bcr-Abl is an overly activated oncogene product resulting from a specific genetic abnormality generated by chromosomal translocation that is found in cells of patients with chronic myeloid leukemia. Bcr-Abl is an activated protein kinase and thus requires adenosine triphosphate (ATP) to phosphorylate substrates, which in turn promote cell proliferation. STI571 is a small molecule that competes with the ATP-binding site and thus blocks the transfer of phosphoryl group to substrate. PO4 = phosphate; Tyr = tyrosine.