Student Learning Outcomes
At completion of this exercise, the student will be able to:
1. Name major contributors in the history of DNA.
2. Describe the composition of nucleotides.
3. Compare and contrast DNA and RNA.
4. Explain the molecular con figuration of DNA.
5. Describe the make-up of chromosomes.
6. Trace the basic process of replication.
7. Describe the basic process of protein synthesis.
8. Relate the cause and impact of mutations
9. Describe mitochondrial and chloroplast DNA.
10. Explain the role of DNA in heredity, medicine, forensics, and evolution.
11. Trace the process of spooling DNA.
OVERVIEWIt is hard to believe that the humble beginnings of our knowledge of DNA can be traced back to an obscure physician studying the chemical composition of pus-soaked rags and sperm of salmon .During his studies in the 1870s , the Swiss physician Johann Miescher (1844-1895) described a mysterious substance that he coined nuclein.
Eventually, nuclein would be known as deoxyribonucleic acid, or DNA.Scientists studying nuclein described this as a nucleic acid. Albert Kossel (1853-1927 ) labeled two distinct types of nucleic acids:
1. Thymus nucleic acid (DNA), and
2. Yeast nucleic acid (RNA)
Early scientists thought that DNA perhaps was involved in heredity and RNA as an energy source for cellular metabolism. Nucleic acids are macromolecules composed of repeating units called nucleotides. Each nucleotide consists of a pentose sugar (5 carbon), a phosphate group, and a nitrogenous base (Fig. 16.1). The nucleotides in DNA contain deoxyribose sugar and in RNA the nucleotides contain ribose sugar. In addition, the nitrogenous bases are different in DNA and RNA. Nuleotides are composed of five different bases. The bases are further divided into either double-ringed purines-adenine and guanine or they are single-ringed pyramidines-thymine , cytosine, and uracil. In RNA uracil replaces thymine.
As a result of the contributions of Erwin Chargaff (1905-2002) and others, it was determined that in DNA, the purine adenine (A) is hydrogen-bonded to the pyrimidine thymine (T) and the purine guanine (G) is paired to the pyramidine cytosine (C) by hydrogen bonds. In RNA, thymine is replaced by uracil (U).
By 1950s, DNA was widely accepted as the heredity molecule, but its molecular configuration remained a mystery. As a result of the x-ray diffraction studies of Rosalind Franklin (1920-1958) and Maurice Wilkins (1916-2004), it was suspected that a molecule of DNA was shaped like a helix. Using the work of Chargaff, Franklin, Wilkins and others James Watson (1928-present) and Francis Crick (1916-2004) developed the double helix model of DNA ( Fig. 16.2 ). Watson and Crick described DNA as a double-stranded helical structure with a backbone of deoxyribose sugar and phosphate and rungs of complementary base pairs (ATCG ) held together by hydrogen bonds. As a result of bonding, the two strands of the DNA molecule run in opposite directions and accordingly are antiparallel.
In eukaryotic cells, a chromosomes consists of a continuous molecule of DNA and several types of associated proteins. Humans have 46 chromosomes per cell. Genes are units of heredity located on specific chromosomes. For example, the cystic fibrosis transmembranal regulatory gene exists at a specific residence on chromosome 7.
The collective genes that comprise an organism are called the genome. The Human genome consists of fewer than 30,000 genes. In a chromosome, DNA is tightly coiled around proteins termed histones, which resemble a bead-like structure, forming a nucleosome. The nucleosome is composed of units of eight histone proteins capped by another histone known as a linker. The framework of DNA is maintained by specialized scaffold proteins. Collectively Chromosomes make up chromatin, which consists of 60% DNA, 30% histones, 30% other proteins, and 10% RNA. One of the most interesting characteristics of DNA is its ability to undergo replication, allowing DNA to make copies of itself. Usually, this action occurs with extreme fidelity and mistakes are minimal. DNA demonstrates semiconservative replication. One strand serves as a direct template for the new strand, and the other strand is pieced together. In 1958, Matthew Meselson and Franklin Stahl, while working with bacteria, proved that DNA was semiconservative.
STEPS IN DNA REPLICATIONA human chromosome replicates at several hundred points along its length. In eukaryotes, from 500 to 5000 base pairs are assembled per minute in up to 50,000 origins of replication.
1. Replication begins at a point called the origin of replication, or the initiation site, on the parental strands of DNA (fig. 16.3). Here, an enzyme called helicase facilitates unwinding. Another enzyme, gyrase, prevents the strands of DNA from tangling. This unwinding results in a replication fork; gyrase forms a nick in the DNA that will repair later. The enzyme that repairs this nick is ligase.
2. One strand of the replication fork is called the leading strand (continuous) (3’ to 5’), continuing in one direction. The other strand, the lagging strand (discontinuous) (5’to 3’), continues in the opposite direction. Then, at the start of each DNA segment to be replicated, an enzyme called RNA primase builds a short piece of RNA called an RNA primer.
3. The RNA primer attracts an enzyme called DNA polymerase, which attracts the proper nucleotides to the template. The new strand grows as hydrogen bonds are formed. Copying occurs in two directions: In the 5’ to 3’ direction, Okazaki fragments are formed because of the nature of the molecules. The ends of these fragments are joined by ligase (fig. 16.4).
4. Enzymes called proofreading enzymes are responsible for ensuring the fidelity of replication. DNA replication is accurate; only 1 in 10,000 base pairs are incorrect.
5. Repair enzymes also ensure fidelity. Excision and post-replication enzymes are common in the repair process. Unfortunately, repair systems can be damaged by prolonged exposure to sunlight and can bring about skin cancer. Examples of two genetic disorders that involve repair enzymes are xeroderma pigmentosum and ataxia telangiectasia.
RNA is the best known as the chemical “ancestor” of DNA. Evolutionarily, RNA appeared before DNA. RNA plays a key role in building proteins. Five fundamental differences between DNA and RNA are the following.
1. DNA has deoxyribose sugar, and RNA has ribose sugar. Ribose has a hydroxyl instead of a hydrogen attached to the 2’carbon.
2. DNA has base pairs consisting of A-T-C-G, while RNA has A-Uracil, C-G.
3. DNA is a double-strand while RNA is a single strand.
4. DNA usually is longer than RNA
5. DNA is more stable than RNA.
The three types of RNA are the following:
1. Ribosomal RNA: forms ribosomes. Ribosomal RNA is made inside of the nucleolus, whereas other RNA is made in the nucleus. Ribosomal RNA is 100-30,000 nucleotides long. Ribosomal RNA has two sub-units: The small subunit binds the mRNA to the ribosome, and the large subunit attaches to the tRNA and helps bind it to the protein. (figure. 16.5)
2. Messenger RNA: a single strand of nucleotides whose bases are complementary to those of the template DNA to which the RNA was transcribed. Most mRNAs consists of 500-1000 bases. Working in groups of three, or triplets, the mRNA forms codons that specify specific amino acids in protein synthesis (fig. 16.6)
3. Transfer RNA: connectors linking an mRNA codon to a specific amino acid. These consist of 75-80 nucleotide base pairs. A loop of tRNA has three bases called anticodons, which are complimentary to the codon. The end opposite to the anticodon covalently bonds to a specific amino acid. tRNA always attaches to specific amino acids (fig. 16.7).
Protein synthesis is the process of building proteins from amino acids. This process is directed and coordinated by DNA. Protein synthesis consists of two major steps: transcription and translation.
TRANSCRIPTIONThe process by which chemical information encoded in DNA is copied into RNA is called transcription. Generally, only one strand of the double helix of DNA is transcribed and is called the sense strand. The noncoding portion is called the nonsense strand. Most genes are composed of a coding region that is transcribed into RNA, and a regulatory region that oversees transcription in the coding portion. The promoter is a specific part of the regulatory region that serves as the starting point of transcription.
Building of the complementary strand of RNA is completed by large molecules of RNA polymerase. Beginning, at the promoter, RNA polymerase unwinds the DNA, breaking the hydrogen bonds. Transcription ceases at a transcription termination signal on the DNA. Once RNA for a specific region is made, the DNA quickly re-forms and the RNA segment is displaced. The process can be prolific. This new RNA is called messenger RNA.
Messenger RNA now exits the nucleus and enters the cytoplasm via nuclear pores. Once in the cytoplasm, the mRNA attaches to a ribosome. The messenger RNA works in units of three called triplets, which serve as code word called codons. Codons specify which one of 20 standard amino acids to pick up. For example, the codon GAG specifies glutamic acid. There are 64 different codons. One codon, AUG, encodes for methionine and serves as a start signal for building a protein. UAA, UAG, and UGA represents stop codons, ending the formation of proteins, these codons are responsible for the genetic code that, for the most part, is universal.
The genetic code is degenerate; that is, more than one codon can encode for a specific amino acid. These codons are synonymous codons. Glycine is coded by the codons GGU, GCA, and GGG. The first two nucleotides are the same in each codon. This phenomenon, described by Francis Crick, is known as the wobble hypothesis.
The nest step, translation, cannot happen without post-transcriptional modifications. The beginning of the RNA sequence is called a leader, and the end part is called the trailer. The DNA sequence contains coding portions called exons and noncoding portions called introns. Before translation, introns are removed by protein complexes called spiceosomes. Introns range from 65 to 100,000 bases, and exons range from 100 to 300 bases. Many genes are riddled with introns. Many scientists consider the introns to be the “genetic junk”, such as old genes, which may be slices of viral material or could be the basis of future genes. Introns, it is thought, may even regulate other gene activity.
TRANSLATIONTranslation is the actual process of expressing the genetic code and building a protein. The codons transcribe a complementary anticodon loop that can be found at the opposite end of protein attachment in the newly made transfer RNA. Transfer RNA will seek a specific amino acid in the cytoplasm as dictated by the codon. Translation is divided into three parts:
1. Initiation, the start of protein synthesis, begins when mRNA associates with a small ribosomal subunit. If all goes well, the AUG codon will pick up methionine to serve as the initiation point. The other codons then are read three at a time in the next step.
2. Elongation is the process by which all of the amino acids are joined by peptide bonds.
3. Termination is the point at which the stop sequence appears.
Protein folding and the final touches to the proteins occur after termination. The process of protein synthesis is accurate, but mistakes called mutations can happen. Protein synthesis is economical, as cells can produce large amount of a protein from just one or two copies of a gene (fig. 16.9). For example, a plasma cell in human immune system can produce more than 2,000 identical antibodies.
BEYOND THE LAB – VISITING HISTORYDescribe the contributions of the following in establishing our understanding of DNA:
1. Frederick Griffith: ______
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2. Archibald Garrod: ______
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3. Oswald Avery, Colin MacLeod, and Maclyn McCarty: ______
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4. Alfred Hershey and Martha Chase: ______
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5. George Beadle and Edward Tatum: ______
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6. Matthew Meselson and Franklin Stahl: ______
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7. Linus Pauling and Vernon Ingram: ______
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8. Severo Ochoa: ______
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9. Kary Mullis: ______
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10. Alec Jefferys: ______
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STUDENT ACTIVITY -SPOOLING DNASpooling DNA
In this activity you will isolate DNA from your cheek cells: This two-step process, requiring in the first step the removal of the cell and nuclear membranes, and in the second step isolating the DNA.
Materials:
· 8 ounces of clear Gatorade or a 0.9% salt water solution ( ½ teaspoon salt added to 8 ounces water)
· 25% soap solution ( 5 ml dish liquid soap and 15 ml water )
· 15 ml of ice cold 95% ethanol (keep in the freezer or on ice until use )
· 6 –ounces plastic cup
· 30-35 ml glass test tube
· 25 ml graduated cylinder
· Glass stirring rod
· Stop clock or timer
· Test tube rack
Procedure 16.1 DNA
1. Obtain materials from the lab instructor.
2. Using the graduated cylinder, pour 10 ml of 0.9% salt water solution into a plastic cup.
3. Swirl the contents of the cup in your mouth vigorously for 30 seconds. The vigorous swirling will allow you to slough off a large number of cheek cells.
4. Carefully spit the contents from your mouth into the cup.
5. Add 5 ml of the soap solution to a glass test tube.
6. Pour the contents from the cup into the test tube the containing soap solution.
7. Using a glass stirring rod, stir the contents for 3 minutes. Use a gentle motion to avoid forming bubbles. (In this step of the activity, the soap solution is used to break down the cell membranes.)
8. Remove the glass stirring rod, and carefully tilt the test tube at a 45-degree angle. Add 15 ml of the ice cold ethanol slowly down the side of the test tube. Do not shake or mix the ethanol with the contents of the test tube. The alcohol will form a layer on top of the solution.