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Figure 4-1: Formation of polynucleotides from nucleotide precursors. Nucleotides are joined together by a phosphodiester linkage to form a nucleic acid. Arrows indicate the carbon atoms of deoxyribose that are joined by phosphodiester bonds to form polynucleotides. Note that the bases are attached to 1' carbon position of the sugar molecule and face the interior of the molecule. The backbone is formed by the sugar linked by phosphate groups binding to 5' and 3' carbons of the sugar. (From Mares A Jr, Towbin J, Bies RG, Roberts R. Molecular biology for the cardiologist. Curr Probi Cardiol 1992; 17:9-72. Reproduced with permission from the publisher and authors.) Figure 4-2: The common purine and pyrimidine bases found in DNA. Uracil is substituted for thymine in RNA. (From Mares A Jr, Towbin J, Bies RG, Roberts R. Molecular biology for the cardiologist. Curr Probi Cardiol 1992; 17:9-72. Reproduced with permission from the publisher and authors.)

Figure 4-3: DNA replication conserves the nucleotide sequence. DNA is a double-stranded helical molecule bound together by the nucleotide bases contained on each individual strand. During cell division, two identical copies of the original parental strand are made by unwinding the DNA and then synthesis of a complementary second strand to make two identical new daughter strands. Figure 4-4: Central dogma of molecular biology.

Figure 4-5: Schematic localization of the processes of transcription and translation. Figure 4-6: Illustration of how RNA polymerase II interacts with DNA and the promoter to generate a single-stranded mRNA. RNA polymerase II attaches to the initiation site promoted by the 5' promoter sequence. mRNA is synthesized in the 5' to 3' direction from just one strand, the antisense strand. The specificity of base pairing between mRNA and the antisense strand provides for an mRNA with sequences complementary to that of the antisense strand and identical to that of the sense strand.

Figure 4-7: Transcription. Transcription occurs in the nucleus, producing mRNA that is processed into mature mRNA and transported to the cytoplasm. In the cytoplasm, translation occurs, with the mRNA coding for specific amino acids that are linked together to form a polypeptide and ultimately to form a mature protein. (From Mares A Jr, Towbin J, Bies RG, Roberts R. Molecular biology for the cardiologist. Curr Probi Cardiol 1992; 17:9-72. Reproduced with permission from the publisher and authors.) Figure 4-8: A summary of the multiple steps involved in gene expression from the genomic DNA to the protein showing how the protein destined for secretion follows a systematic path different from proteins destined to remain in the cytoplasm. (From Campbell PN, Smith AD. Nucleic acids and protein biosynthesis. In: Campbell PN, Smith AD, eds. Biochemistry Illustrated, 2d ed. New York: Churchill Livingstone; 1988:111. Reproduced with permission from the publisher.)

' Figure 4-9: Structure of a gene. These small functional units within the nucleus contain the coding information for the synthesis of a polypeptide and on their 5' ends have regulatory sequences that include silencers, enhancers, and promoters. The coding region consisting of exons (code for protein) as well as intervening noncoding sequences (introns) is followed by a 3' noncoding region that is translated into the mRNA. The 3' end appears important for exit of the mRNA from the nucleus and its stability in the cytoplasm but does not code for protein. The TATA is the initiation site for polymerase and is present in most eukaryotes at about 10 to 30 base pairs 5' from the start codon (TAC) of the coding region. The AATAA will become the recognition site on the mRNA to which attaches an enzyme that cleaves the 3' region and replaces the distal portion with a poly(A) tail. (From Mares A Jr, Towbin J, Bies RG, Roberts R. Molecular biology for the cardiologist. Curr Probl Cardiol 1992; 17:9-72. Reproduced with permission from the publisher and authors.)

' Figure 4-10: Types of transcription factors that affect gene activation. Schematic representation of the shapes of four types of protein transcription factors that bond to DNA and influence gene activation. Helix-turn-helix is a protein with two d-helices separated by a P-turn. Leucine zippers are protein dimers with entering leucine amino acids. Zinc fingers have a peptide loop connected at the base by a zinc ion tetrahedran between cysteine and/or histidine in amino acids. The helix-loop-helix consists of Gt-helix but uses leucine zippers and has a loop between the Qf-helices. The darkened areas are believed to be the regions of the protein that interact with the DNA to modulate transcription.

' Figure 4-11: Generation of a complementary DNA (cDNA). Taking advantage of the enzyme reverse transcriptase, mRNA is converted to DNA, referred to as complementary DNA (cDNA). The DNA is single-stranded and complementary to the sequence of RNA, except thymine now replaces uracil. Using DNA polymerase, one can then make the single-stranded DNA into double-stranded cDNA. The cDNA can be used as a probe to identify specific sequences or genes of the genomic DNA, or it can be inserted into vectors to be cloned or expressed in a variety of hosts.

' Figure 4-12: Southern blotting technique. The DNA is cleaved with an appropriately selected restriction endonuclease. The digested fragments are separated by electrophoresis on agarose gel, and the fragments of gene A are located at positions 1, 2, and 3 but cannot be seen against the background of many other randomly occurring DNA fragments. The DNA is denatured and transferred to a membrane in an identical pattern to what it was on the agarose gel. It is difficult to manipulate anything on a soft gel or to remove it. Once transferred to the membrane (filter), a solid support system, the DNA is much easier to handle. A DNA probe (cDNA) that has been labeled with 32P is hybridized to its cDNA and visualized after exposure of the nylon membrane to an autoradiograph. The transfer of the DNA from the gel to the membrane developed by Southern was a major innovation illustrated in the next figure. (From Mares A Jr, Towbin J, Bies RG, Roberts R. Molecular biology for the cardiologist. Curr Probl Cardiol 1992; 17:9-72. Reproduced with permission from the publisher and authors.)

s Figure 4-13: A typical Southern blot with distinct bands. Each vertical lane consists of DNA from a separate individual. All the individual DNAs were digested with the same restriction endonuclease. Following separation on electrophoresis and transfer to a nylon membrane, hybridization was performed with the selected radioactive probe, and thus only those fragments complementary to the probe are visualized. This is an analysis of a family with hypertrophic cardiomyopathy, and the different patterns reflect restriction fragment length polymorphisms (RFLPs) characteristic of the marker locus, which is linked to the disease locus. (From Mares A Jr, Towbin J, Bies RG, Roberts R. Molecular biology for the cardiologist. Curr Probl Cardiol 1992; 17:9-72. Reproduced with permission from the publisher and authors.)

' Figure 4-14: Restriction endonucleases recognize specific sequences and cut in a specific manner. The sequences recognized may be anywhere from 3 to 8 base pairs long and may cut to give a blunt end or a staggered end (EcoR1). Enzymes that provide staggered ends (cohesive or sticky ends) have unpaired bases that are easy to ligate together because they are complementary to each other, as shown in this illustration. This feature is exploited in cloning or in the formation of any recombinant DNA molecule. For cloning purposes, the fragment of DNA to be inserted is digested with the same restriction enzyme as is used to digest the DNA of the vector into which it will be inserted. Thus the sticky ends of the DNA insert and the vector will be complementary and easy to ligate together in the presence of the enzyme DNA ligase, as illustrated in Fig. 4-15.

' Figure 4-15: DNA cloning. The basic objective of cloning is to provide multiple copies of a DNA fragment of interest. The fundamental principles for in vitro cloning of specific DNA fragments are as follows: (1) The human genome DNA of interest is isolated after digested by a restriction endonuclease, which is often referred to as the DNA insert. (2) A DNA vector is selected (shown on the right); the vector is a plasmid that has circular DNA and contains the necessary replication site and the reporter gene (drug resistance gene) to subsequently recognize which host has the insert. The vector and the DNA fragment to be inserted are digested with the same restriction endonuclease so that the ends are complementary for ligation. (3) DNA ligase ligates compatible insert and vector ends together. (4) Finally, host cells are transformed by incorporating vectors containing insert fragments and are identified by characteristics encoded by resident genes on the vector. Some of the clones will be viable and others not. (From Mares A Jr, Towbin J, Bies RG, Roberts R. Molecular biology for the cardiologist. Curr Probl Cardiol 1992; 17:9-72. Reproduced with permission from the publisher and authors.)

s Figure 4-16: Relationship of sarcomere length and tension generated during isometric contraction of striated muscle. Maximum tension is generated at sarcomere lengths that allow maximum interaction of myosin heads and actin filaments (positions 2 and 3). If the sarcomere length is too short (positions 4 and 5), actin filaments overlap one another and prevent optimal interaction with myosin heads. (From Darnell J, Lodish H, Baltimore D, eds. Molecular Cell Biology. New York: Scientific American Books, W. H. Freeman; 1990. Reproduced with permission from the publisher.)

s Figure 4-17: Sarcomere ultrastructure.

' Figure 4-18: Molecular basis of myocardial contraction. (Adapted with permission from Alberts B, Bray A, Lewis J, et al, eds. Molecular Biology ofthe Cell, 2d ed. New York: Garland; 1991:621.)

' Figure 4-19: The cell cycle in a mammalian cell having a generation time of 16 h. The three phases spanning the first 15 h or so-the G1 (first gap) phase, and S (synthetic) phase, and the G2 (second gap) phase-make up the interphase, during which DNA and other cellular macromolecules are synthesized. The remaining hour is the M (mitotic) phase, during which the cell actually divides.

= Figure 4-20: Illustration of many proteins with varied functions for which oncogenes are known to encode. It is clear from this diagram that oncogenes encode proteins that function as growth factors, receptors, coupling proteins, signaling proteins, and transcription factors.

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