AAOS Comprehensive Orthopaedic Review
Section 1 - Basic Science
Chapter 1. Cellular and Molecular Biology, Immunology, and Genetics
I. Terminology and Definitions
A. Nuclear structure of cells
1. DNA-related terms
a. DNA—A double-stranded deoxyribose that contains pairs of nucleotides connected by hydrogen bonds. Four nitrogenous bases (adenine, guanine, cytosine, thymine) are found in DNA. DNA contains biologic information for replication and regulation of gene expression. The nucleotide sequence in DNA determines the specific information.
b. Genome—Complete complement of genetic information of an organism.
c. Chromosome—Nuclear structure containing a linear strand of DNA; humans have 46 chromosomes (23 pairs).
d. Solenoid—Three-dimensional shape found in cells that resembles an electromagnetic coil wrapped around a core.
e. Nucleosome—DNA/histone complex consisting of DNA wrapped around four pairs of proteins called histones (
f. Gene—Specific segment of DNA that contains all the information required for synthesis of a protein, including both coding and noncoding sequences.
g. Recombinant DNA—DNA artificially made by recombinant technique (manipulation of a DNA segment).
h. Exon—Portion of a gene that codes for messenger RNA (mRNA).
i. Intron—Portion of a gene that does not code for mRNA.
j. Gene promoter—Regulatory portion of DNA that controls initiation of transcription adjacent to the transcription start site of a gene.
k. Promoter DNA sequences—DNA sequences that are upstream of the coding sequences that are necessary for binding of transcription factors. Each transcription factor has a strong affinity for specific sequences (
l. Gene enhancer—Regions of a gene that positively regulate rates of transcription.
m. Transgene—A gene that is artificially placed into a single-celled embryo and is present in all cells of that organism.
2. RNA-related terms
a. RNA—Nucleic acid composed of ribonucleotide monomers, each of which contains ribose, a phosphate group, and a purine or pyrimidine.
b. Messenger RNA (mRNA)—Translates and transmits DNA information into protein synthesis machinery.
c. Small interfering RNA (siRNA)—Short double-stranded RNA that interferes with the expression of a specific gene.
[Figure 1. DNA wraps around histone octamers to create a histone/DNA complex called a nucleosome, which is wound further into a solenoid form. The solenoid form is packed into the chromosome.]
[Figure 2. The promoter region includes the binding site for various transcriptional factors, including the TATA box, and a binding site for RNA polymerase II. TBP = TATA-binding protein.]
d. Ribosomal RNA (rRNA)—Major constituent of the ribosome, which is the cell machinery for synthesizing proteins.
e. Transfer RNA (tRNA)—Transfers a specific amino acid to mRNA.
f. RNA polymerase RNAP I (Pol I)—Transcribes the rRNA genes.
g. RNAP II (Pol II)—Transcribes protein-encoding genes into mRNA.
h. RNAP III (Pol III)—Transcribes all the tRNA genes.
B. Gene expression (transcription; DNA mRNA) (
1. Transcription—Process of reading DNA information by RNA polymerase to make specific mRNA.
2. Translation—Building a protein from amino acids by specific mRNA. tRNA interprets the code on the mRNA and delivers the amino acids.
3. Transformation—Inserting a plasmid into a bacterium with added recombinant DNA.
4. Splicing—Removal of intronic sequences from newly transcribed RNA, resulting in the production of mRNA.
5. Transcription factor—Protein that can initiate transcription.
C. Protein expression (translation; mRNA peptides)—tRNA interprets the code on the mRNA and delivers amino acids to the peptide chain, which is mediated by the ribosome machinery.
D. Cell division and cell cycle
1. Haploid—Amount of DNA in a human egg or sperm cell, or half the DNA in a normal cell.
2. Diploid—Twice haploid; the amount of DNA in a normal resting human cell (the G0/G1 phase of the cell cycle).
3. Tetraploid—Four times haploid, or twice the amount of DNA in a resting cell; the amount of DNA in a cell in the G2 phase of the cell cycle.
4. Point mutation—An alteration in the genomic DNA at a single nucleotide.
E. Extracellular matrix
1. Extracellular matrix (ECM)—The noncellular portion of a tissue that provides structural support and affects the development and biochemical functions of cells (
2. Collagen—Triple-helix proteins that form most of the fibrils in the ECM (Table 1). Consists of various combinations of α1, α2, and α3 chains.
3. Glycosaminoglycans (GAGs)—Structural polysaccharides in the ECM. A GAG is composed of a repeating disaccharide. These include hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparan sulfate, and keratan sulfate. Most
[Figure 3. The general flow of gene expression (from DNA to RNA to protein).]
GAGs attach covalently to a protein core to become a proteoglycan. Hyaluronic acid, a nonsulfated GAG, does not attach to proteins, but proteoglycans are linked to hyaluronic acid to form giant molecules that act as excellent lubricators or shock absorbers.
4. Fibronectin—The role of fibronectin is to attach cells to various fibrous collagens (type I, II, III, V) in the ECM. Fibronectin is composed of several functional units that contain binding sites for ECM molecules such as heparin, collagen, and fibrin. Fibronectin also contains cell-binding domains, which have RGD (Arg-Gly-Asp) integrin-binding sequences. Fibronectin has been known to regulate cell migration and differentiation.
5. Laminin—An important component of the basal lamina. Laminin and type IV collagen form a network for the basement membrane scaffold.
[Table 1. Collagen Types and Representative Tissues]
Table 2. Genetic Defects Associated With Skeletal Dysplasias]
II. Basic Genetics
A. Genomic DNA
1. Human chromosomes contain 6 billion base pairs, which encode approximately 50,000 to 100,000 individual genes. All the genetic information present in a single haploid set of chromosomes constitutes the genome for a human being. A variety of orthopaedic disorders are secondary to genetic mutation (Tables 2 through
2. Only 5% to 10% of genomic DNA in humans is transcribed. Genes in DNA are organized into introns, or noncoding sequences, and exons, which contain the code for the mRNA to produce the proteins as a gene product.
3. The noncoding sequences contain promoter regions, regulatory elements, and enhancers. About half of the coding genes in human genomic DNA are solitary genes, and their sequences occur only once in the haploid genome.
4. Directionality—Single-strand nucleic acid is synthesized
Table 3. Genetic Defects Associated with Metabolic Bone Diseases and Connective Tissue Disorders]
Table 4. Genetic Defects Associated With Musculoskeletal Tumors]
[Table 5. Genetic Defects Associated With Other Musculoskeletal Disorders]
in vivo in a 5´ to 3´ direction, meaning from the fifth to the third carbon in the nucleotide sugar ring (Figure 3).
5. Simple-sequence repeated DNA in long tandem array is located in centromeres, telomeres, and specific locations within the arms of particular chromosomes. Because a particular simple-sequence tandem array is variable between individuals, these differences form the basis for DNA fingerprinting for identifying individuals.
6. Mitochondrial DNA (mtDNA) encodes rRNA, tRNA, and proteins for electron transport and adenosine triphosphate (ATP) synthesis. mtDNA originates from egg cells. Mutations in mtDNA can cause neuromuscular disorders.
B. Control of gene expression
1. Transcription—Transcriptional control is the primary step for gene regulation (Figure 2). A transcription process means the synthesis of complementary RNA by RNA polymerase from a strand of DNA molecule. Transcription is controlled by a regulatory sequence in DNA called the cis-acting sequence, which includes enhancer and promoter sites. Trans-acting factors bind to the cis-acting sequence and regulate gene expression. The trans-acting factor is usually a protein such as a transcriptional factor. Promoter regions include the binding site for various transcriptional factors, including the TATA box, and a binding site for RNA polymerase II. The TATA-binding protein, together with other transcriptional proteins, initiates transcription, followed by binding of RNA polymerase. RNA polymerase II generates mRNA template.
2. Translation (Figure 3)—The ribosome binds to the translation start sites of the mRNA and initiates protein synthesis. tRNA interprets the code on the mRNA and delivers amino acids to the peptide chain. Each amino acid is encoded by a 3-nucleotide sequence (codon). For example, UUC is a codon for lysine, and GGG or GGU are codons for glycine. UGA, UUA, and UAG are codons that stop translation.
C. Inheritance patterns of genetic disease
1. Autosomal mutation—A gene mutation that is located on a chromosome other than the X or Y chromosome.
2. Sex-linked mutation—A gene mutation that is located on the X or Y chromosome.
3. Dominant mutation—A mutation of one allele is sufficient to cause an abnormal phenotype.
4. Recessive mutation—A mutation of both alleles is necessary to cause an abnormal phenotype.
D. Musculoskeletal genetic disorders are listed in Tables 2 through 5.
III. Extracellular Signaling (cell-to-cell interaction)
A. Signaling by secretary molecules
1. Endocrine signaling—Hormones secreted from distant endocrine cells are carried by the blood to its target cells. Some lipophilic hormones (steroid hormone, thyroxine, and retinoids) can diffuse across the cell membrane and bind to specific receptors in the cytosole or nucleus. The hormone-receptor complex affects DNA by altering the transcription of specific genes.
2. Paracrine signaling—Water-soluble hormones (peptide hormones such as insulin and glucagons, catecholamines) and some lipophilic hormones (prostaglandins) bind to cell-surface receptors of neighboring cells to affect their growth and proliferation.
3. Autocrine signaling—Some kinds of growth factors are secreted from cells to stimulate their own growth and proliferation.
B. Signaling by membrane-bound protein—Certain membrane-bound proteins on cells can bind directly to specific receptors on adjacent cells to transfer signals.
IV. Intracellular Signaling
A. Cell response—Cells express specific genes in response to extracellular influences such as mechanical forces; extracellular matrices; and contact with other cells, hormones, and cytokines. External influences are used by cells to coordinate intracellular signaling events and regulate the synthesis of specific genes that impact cell proliferation, differentiation, and paracrine function (
Figures 4 and
B. Signal transduction—The process of converting extracellular signals to cell response.
1. Initiation of signal transduction—Binding of the ligand to a specific receptor initiates signal transduction in various ways, depending on the type of receptor.
a. G protein-coupled receptors—Binding the ligand to the receptor activates a G protein. The G protein modulates a specific second messenger or an ion channel.
b. Ion-channel receptors—Ligand binding alters the conformation of a specific ion channel. The resultant ion movement across the cell membrane activates a specific intracellular molecule.
c. Tyrosine kinase-linked receptors—Binding the ligand activates cytosolic protein-tyrosine kinase.
d. Receptors with intrinsic enzyme activity—Some receptors have intrinsic catalytic activity. Some have guanine cyclase activity to convert guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). The others have tyrosine kinase activity to phosphorylate various protein substrates (referred to as receptor tyrosine kinase).
2. Secondary messengers—Intracellular signaling molecules, the concentration of which is controlled by binding the ligand to a membrane receptor. The elevated concentration of secondary messengers activates other signaling molecules. These include cyclic adenosine monophosphate (cAMP), cGMP, diacylglycerol (DAG), IP3, phosphoinositides, and Ca2+.
[Figure 4. The potential differentiation fates of mesenchymal stem cells.]
[Figure 5. The differentiation schema of bone marrow precursor cells into osteoclasts. Cortical cytokine regulators are indicated. M-CSF = macrophage colony-stimulating ligand; RANKL = receptor activator for nuclear factor κB ligand; OPG = osteoprotegerin.]
3. Other intracellular signaling proteins—In addition to secondary messengers, GTP-binding proteins such as Ras and protein kinases can accomplish the signal transduction without secondary messengers, through kinase cascades.
4. Activation and translocation of a protein kinase to the nucleus activates transcription factors, which regulate gene expression.
A. Innate and adaptive immunity—Defense against foreign pathogens is mediated early by innate immunity and later by adaptive immunity.
1. Innate immunity, which provides the early defense line, is stimulated by a certain structure shared by a group of microbes. It responds rapidly to infection, and will respond in the same way to repeated infections. Physical barriers: epidermis, dermis, mucosa; cellular barriers: phagocytotic cells and natural killer (NK) cells; chemical barriers: antimicrobial substances, blood proteins (complement system), and cytokines.
2. Adaptive immunity memorizes the specific antigens of foreign pathogens. It is able to recognize diverse and specific antigens. Successive exposure to antigens increases the magnitude of the immune reaction. The two types of adaptive immune responses are humoral immunity and cell-mediated immunity.
a. Humoral immunity—Mediated by antibodies produced by B lymphocytes.
b. Cell-mediated immunity—Mediated by T lymphocytes (T cells). T cells can activate macrophages to kill phagocytosed antigens or can destroy infected cells directly. Example: An
Figure 6. The general sequence of events for cloning a DNA fragment: Transformation means the introduction of recombinant DNA into host cells.]
individual who has had chicken pox has immunity against chicken pox.
B. Immune mediators and regulation of bone mass
1. Inflammatory bone destruction or osteolysis is seen clinically in rheumatoid arthritis, chronic inflammatory disease, periodontitis, and wear particle-induced osteolysis. Osteoblasts and osteoclasts communicate in the regulation of bone mass.
2. Inflammatory stimuli may stimulate osteoblasts to express receptor activator of nuclear factor κB ligand (RANKL), a member of the tumor necrosis factor (TNF) superfamily of proteins. RANKL is the key molecule that induces osteoclastogenesis.
3. Anabolic factors such as transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) may stimulate osteoblatic precursors to differentiate into osteoblasts.
VI. Experimental Methods
A. Recombinant technology (Figure 6)
1. Definition—Manipulation of DNA or RNA segments, including production of specific desired DNA, RNA, or amino acids
2. Recombinant protein—A desired protein can be made by introducing the genetic sequence coding the specific protein into the genome of an organism.
3. Recombinant DNA—A DNA fragment that is removed from its original genome and ligated into the other genome.
4. Manipulation of DNA (cutting, pasting, copying)
a. Restriction digestion (cutting DNA)—Restriction enzyme is used to cut double-stranded DNA at a specific sequence of DNA.
b. Ligation (pasting a DNA fragment)—Can be accomplished using enzymes called ligases, which make complete covalent phosphate bonds between nucleotides.
c. Hybridization techniques—Each base of DNA or RNA pairs with a complementary base by hydrogen bonding. A probe (labeled segment of DNA or RNA) enables detection of complementary sequences of either DNA or RNA.
5. Southern blotting—DNA is subjected to agarose gel electrophoresis to identify specific DNA fragments by the size of base pairs.
6. Northern blotting—Used to identify and quantify specific RNA. As with Southern blotting, RNA is subjected to agarose gel electrophoresis and separated by size.
7. DNA sequencing—Several methods have been developed for detection of the nucleotide sequences of DNA. The Sanger method uses dideoxynucleotides (a, c, g, t instead of A, C, G, T) during DNA polymerization. Because dideoxynucleotides do not have a 3´ OH group to link the next nucleotide, they stop DNA synthesis. One dideoxynucleotide (eg, a instead of A) is mixed with the other three deoxynucleotides (C, G, T) in the DNA synthesis; this reaction will stop at the a-site, indicating that there is A at this location.
8. Polymerase chain reaction (PCR)—Method of amplifying a sequence of DNA to a significantly detectable level by repeating a thermal cycle. A DNA template with two primers (forward and reverse) complementary to the target sequence is incubated with nucleotides and polymerase.
B. Methods of protein detection
1. Western blotting—Proteins are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) according to molecular weight, confirmation, and charge. A specific antibody is used to recognize the protein of interest.
2. Immunohistochemistry—Method to detect and localize a target protein in the cells or tissue using a specific antibody for the protein of interest.
3. ELISA (enzyme-linked immunosorbent assay)—Method to detect and quantify a specific soluble protein.
C. Modulation of gene expression
1. Transfection—Method to introduce a foreign DNA into a cell or organism using vectors such as plasmid DNA, retroviruses, and adenovirouses, where a specific gene is incorporated.
2. Antisense strategies—Method to introduce an RNA or DNA complementary to an mRNA of interest. The antisense sequence will bind to a specific mRNA and inhibit its translation. Referred to as "gene knockdown" because it reduces the expression level of the target gene.
3. Transgenics—Technique to generate a transgenic animal by introducing a cloned gene into a fertilized ovum. Accomplished by microinjection or by transfection of embryonic stem cells in an embryo.
VII. Pharmaceutical Interventions
A. Anti-TNF—TNF inhibitors reportedly reduce erosive damage and disability in patients with rheumatoid arthritis (RA). Three anti-TNFs have been approved for the treatment of RA:
1. Infliximab and adalimumab—Monoclonal anti-TNF-α antibodies with high a affinity for TNF-α; they prevent TNF-α from binding to its receptors.
2. Etanercept—A fusion protein that binds to TNF-α and prevents it from interacting with its receptors.
B. Osteoclast inhibitors—Control excessive osteoclastogenesis
1. DNA vaccination against RANKL has been tried on an experimental basis in animals.
2. Anti-RANKL antibody has been tried clinically for treatment of osteoporosis.
3. Osteoprotegerin (OPG)—OPG binds to RANKL, preventing its binding to receptor activator of nuclear factor κB (RANK), a receptor for osteoclast differentiation. Direct OPG injection and modulation of OPG expression in cells are being considered as therapeutic strategies.
4. Other inhibitors of osteoclast—Cathepsin K inhibitors, ανβ3 integrin receptor blockers, and an osteoclast-selective H1-ATPase inhibitor could potentially be used to block bone resorption.
Top Testing Facts
1. DNA is a double-stranded deoxyribose. An exon is a portion of a gene that codes for mRNA.
2. mRNA translates and transfers DNA information into protein synthesis machinery. tRNA transfers amino acid to mRNA.
3. Transcription: DNA mRNA; translation: mRNA protein.
4. Achondroplasia is related to a defect in FGF receptor 3.
5. Signal transduction is the process of converting extracellular signals to cell response.
6. Inflammatory stimuli may stimulate osteoblasts to express RANKL, a key molecule of osteoclastogenesis.
7. Recombinant technology is manipulation of DNA or RNA segments to produce specific desired DNA, RNA, or amino acids.
8. Infliximab is a monoclonal antibody for TNF-α; it prevents TNF-α from binding to its receptors.
9. Etanercept is a competitive inhibitor of TNF-α signaling; it is a fusion protein that combines the ligand-binding domain of the TNF-α receptor.
10. OPG, anti-TNFs, and anti-RANKLs can control excessive osteoclastogenesis.
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