Lecture Outline - Chapter 25

25.1. DNA Structure and Replication (p. 476)

1. Genes were known to be on chromosomes in nucleus of cell in mid-1900s.
2. Hereditary material was suspected to be either DNA or protein component of chromosomes.
3. Hereditary material was proved to be DNA. (Fig. 25.1)
   • a. T₂ viruses were labeled with ³⁵S in their protein outer coat; ³²P-labeled DNA on inside.
   • b. When viruses attached to new bacteria, scientists found only DNA entered cells and produced more viral particles.
   • c. Therefore only DNA was needed to reproduce these viruses -- DNA was the genetic material.
4. DNA Structure
   • a. Major discovery by American biologist James Watson and English physicist Francis Crick at University of Cambridge, England, 1951.
   • b. Worked on puzzle made from pieces of data available from other researchers.
     • i. DNA is composed of four nucleotides, each nucleotide consists of a phosphate group, sugar (deoxyribose), and a nitrogen base.
     • ii. The four nitrogen bases are: adenine (A) and guanine (G), both purines with double rings; and thymine (T) and cytosine (C), pyrimidines with a single ring.
     • iii. Chemist Erwin Chargaff discovered "Chargaff's rules" in late 1940s: number of purines in DNA equals the number of pyrimidines, amount of T equals the amount of A, and amount of G equals the amount of C.
     • iv. Rosalind Franklin and Maurice Wilkins at King's College, London, prepared X-ray diffraction photograph suggesting DNA was double helix with bases of constant diameter and stacked. (477)
   • c. Watson and Crick Model of DNA
     • i. DNA consists of two strands of nucleotides, twisted together.
     • ii. Sugar-phosphate molecules make up sides of ladder, bases make up rungs.
     • iii. The base pairs are complimentary: A pairs with T; G pairs with C.
     • iv. The hydrogen bonding of A=T and G=C provide rungs of constant width.
     • v. Watson and Crick built DNA model of wire and tin. (477)
     • vi. Their historic paper describing structure pointed out "possible copying mechanism for the genetic material."
5. Complimentary Base Pairing
   • a. Purine is always bonded to a pyrimidine. (Fig. 25.3)
   • b. Bases can be in any order, providing overwhelming variability; a chromosome can have 140 million base pairs with total number of possible nucleotide sequences in the chromosome of 4^140,000,000.
6. DNA Replication: Unzipping and Molding (p. 479)
   • a. In double-stranded DNA, replication is possible because each strand serves as template for complementary strand.
   • b. First step of replication involves DNA strands unwinding and unzipping; hydrogen bonds between bases broken by enzyme called helicase.
   • c. New complementary nucleotides move into place by complementary base pairing opposite each original template strand.
   • d. Complementary nucleotides join together by enzyme DNA polymerase complex.
   • e. DNA again is double stranded with two DNA molecules identical to original molecule.
   • f. Semiconservative replication means each new double helix has one parental strand conserved, and one new strand.
7. Cancer involves rapidly dividing cells; chemotherapeutic drugs stop replication by providing analogs of the four nucleotides, thus stopping synthesis of effective DNA.

1. DNA serves in nucleus as template for RNA formation (transcription) which in turn is used to synthesize proteins (translation) in ribosomes of cell cytoplasm.
2. Structure of RNA (ribonucleic acid) (Table 25.1)
   • a. RNA is polynucleotide like DNA but has sugar ribose, not deoxyribose. (Fig. 25.5)
   • b. Pyrimidine thymine of DNA is replaced by pyrimidine uracil in RNA; therefore in RNA, adenine pairs with uracil.
   • c. RNA is single stranded unless it doubles back on itself.
3. Three types of RNA involved in protein synthesis:
   • a. Messenger RNA (mRNA) carries coded sequence of bases from nucleus to ribosomes.
   • b. Ribosomal RNA (rRNA) is found in ribosomes.
   • c. Transfer RNA (tRNA) carries amino acids to ribosomes.
4. DNA Controls the Cell
   • a. Occurrence of inherited metabolic disorders pointed to genes controlling cell metabolism.
     • i. In phenylketonuria (PKU), mental retardation is due to inability to convert phenylalanine to tyrosine.
     • ii. In albinism, tyrosine cannot be converted to melanin skin pigment.
   • b. Evolution of gene -- product concepts:
     • i. Early experiments with bread mold Neurospora led to "one gene -- one enzyme" hypothesis.
     • ii. This was broadened to one gene -- one protein since not all proteins are enzymes; for example, actin and myosin.
     • iii. When it was discovered some proteins have more than one polypeptide, hypothesis became one gene -- one polypeptide.
5. Overview of Protein Synthesis
   • a. DNA both codes for its replication and is template for RNA formation.
   • b. Transcription makes an RNA molecule complementary to a portion of DNA.
   • c. Micrographs using labeled RNA show it moving through pores of nucleus to cytoplasm.
   • d. Messenger RNA (mRNA) carries information for synthesis of polypeptides.
   • e. Translation uses this information to sequence amino acids of polypeptide.
6. DNA Base Sequence is Coded
   • a. Order of bases in DNA codes for order of amino acids in polypeptides.
   • b. A triplet code of four bases would supply 64 different triplets, far more than needed to code for the 20 essential and different amino acids; a doublet code would only provide 16 combinations.
   • c. A codon is a "three letter" unit of three nucleotides in messenger RNA (mRNA).
   • d. All 64 mRNA codons have been determined. (Fig. 25.7)
   • e. Sixty-one codons correspond to a particular amino acid; three are stop codons.
   • f. One codon for methionine is also a start codon initiating polypeptide formation.
7. Genetic code is essentially universal.
   • a. Codons stand for same amino acids in most bacteria, plants, and animals.
   • b. Uniformity suggests all living things have common evolutionary ancestor.
8. Transcription (p. 482)
   • a. During transcription, portion of DNA helix unwinds and unzips.
   • b. Complementary RNA nucleotides pair with DNA nucleotides of one strand.
   • c. RNA nucleotides are joined together by RNA polymerase.
   • d. On RNA, uracil substitutes for thymine; where A, T, G, or C is present in DNA template, U, A, C, or G is incorporated into mRNA.
9. Processing Strips Introns from mRNA
   • a. Exons are portions of DNA that are ultimately expressed by producing proteins.
   • b. Introns are segments of DNA between genes and are not expressed (intragene segments).
   • c. Transcription produces mRNA complementary to both exons and introns.
   • d. Nucleotides complementary to introns are processed or enzymatically removed before mRNA exits from nucleus.
   • e. Ribozymes are enzymes that remove introns; therefore, not all enzymes are proteins.
   • f. In eukaryotes, after processing occurs in nucleus, it passes into cytoplasm to ribosomes.
10. Translation (p. 484)
    • a. During translation, sequence of codons in mRNA determines order of amino acids in polypeptide.
    • b. Term translation is because sequence of DNA bases is finally translated into sequence of amino acids.
    • c. Requires several enzymes, transfer RNA, and ribosomal RNA.
11. Transfer RNA (tRNA)
    • a. Transfer RNA molecules bring amino acids to ribosomes in cytoplasm.
    • b. Single-stranded nuclei acid doubles back on itself with hydrogen bonds across complementary bases.
    • c. An amino acid binds at one end of tRNA molecule; requires ATP.
    • d. Research underway on how correct amino acid joins correct tRNA; tRNA synthetase recognizes correct amino acid.
    • e. Opposite end has specific anticodon which binds complementarily to mRNA codon. (Fig. 25.9b)
    • f. Example: Codon ACC has anticodon UGG to produce amino acid threonine.
12. Ribosomal RNA
    • a. Called structural RNA; found in ribosome.
    • b. Produced in nucleolus inside nucleus.
    • c. Joins with proteins migrated in from cytoplasm.
    • d. Ribosomal subunits then migrate to cytoplasm.
    • e. Subunits join as protein synthesis begins; small subunit has one rRNA and proteins; large subunit has two rRNA plus proteins and subunits are joined by peptide bond.
13. Translation in Three Steps (p. 484)
    • a. Initiation: mRNA binds to smaller, then larger of the two ribosomal subunits.
    • b. Elongation:
      • i. Polypeptide chain lengthens one amino acid at a time.
      • ii. Ribosome has sites to accommodate two tRNA molecules, an incoming tRNA-amino acid complex and outgoing tRNA.
      • iii. Ribosome moves laterally so that next mRNA codon is available to receive incoming tRNA_amino acid complex.
    • c. Termination:
      • i. Occurs at stop codon on mRNA.
      • ii. The two ribosome subunits dissociate from mRNA.
    • d. Polyribosomes are several ribosomes that move along one mRNA at a time. (Fig. 25.11)

1. In eukaryotic cells, there are four levels of control of gene activity. (Fig. 25.13)
   • a. Transcriptional control:
     • i. Involves mechanisms controlling which genes are transcribed and/or rate at which transcription occurs.
     • ii. Includes organization of chromatin.
     • iii. Includes use of transcription factors.
   • b. Posttranscriptional control:
     • i. Occurs in nucleus after DNA is transcribed and preliminary mRNA is formed.
     • ii. Involves differential processing of mRNA before it leaves the nucleus.
     • iii. Involves speed with which mature mRNA leaves the nucleus.
   • c. Translational control:
     • i. How long mRNA exists in cytoplasm, or can bind ribosomes, varies.
     • ii. Some mRNAs may need changes before they are translated.
   • d. Posttranslational control:
     • i. Occurs in cytoplasm after protein synthesis.
     • ii. Polypeptide product may have to undergo additional changes before biologically functional.
     • iii. Functional enzyme is subject to feedback control; binding of end product may change shape of enzyme and inactivate it.
2. Transcriptional Control in Prokaryotes (p. 488)
   • a. Operon model for transcriptional control in prokaryotes includes several elements:
     • i. Regulator gene codes for repressor protein; repressor protein binds to operator, prevents RNA polymerase from binding to promoter.
     • ii. Promoter is short sequence of DNA where RNA polymerase first attaches when a gene is to be transcribed.
     • iii. Operator is short sequence of DNA where repressor binds, preventing RNA polymerase from attaching to promoter; often called on/off switch of transcription.
     • iv. Structural genes are one to several genes of a metabolic pathway that are transcribed as a unit; note that regulator genes regulate activity of structural genes.
3. Lac Operon (Fig. 25.14)
   • a. Was first operon discovered; used the bacterium Escherichia coli.
   • b. Bacterium uses glucose energy source; if denied glucose and given lactose (milk sugar), it begins to make three enzymes needed to metabolize lactose. (Fig. 25.14)
   • c. Structural genes in this operon are normally not transcribed; regulator genes code for active repressor protein that attaches to operator, preventing transcription.
   • d. Operon becomes active when repressor joins with inducer (lactose) to form inactive repressor unable to bind to operator.
4. Transcriptional Control in Eukaryotes (p. 489)
   • a. Involves organization of chromatin and regulatory proteins called transcription factors.
   • b. Activated Chromatin
     • i. Genes are ordinarily inactive; must be turned on.
     • ii. Lampbrush chromosomes are easily seen in developing vertebrate egg cells. (Fig. 25.15).
     • iii. Many loops synthesize large quantities of mRNA to provide for rapid protein synthesis after fertilization.
     • iv. Larval fly salivary glands have chromosomes that duplicate many times without cell division; resultant sister chromatids synapse together to form giant polytene chromosomes.
     • v. Chromosome puffs are regions of polytene chromosomes that bulge out to allow DNA to be actively transcribed; tested by radioactive-labeled uridine.
5. Transcription Factors
   • a. In eukaryotic cells, transcription is controlled by DNA-binding proteins called transcription factors.
   • b. Every cell contains many different transcription factors; specific combination regulates gene activity.
   • c. After correct transcription factors combine to DNA, RNA polymerase attaches to DNA and begins transcription.
   • d. Cells specialize as they mature; signals received from inside and outside cell could turn on or off genes that code for certain transcription factors.

1. A gene mutation is a change in nucleotide sequence of a gene.
2. Frameshift Mutations Are Drastic
   • a. Reading frame is sequence of codons read from starting point.
   • b. Frameshift mutation involves either addition or deletion of a nucleotide; for example, removing the C below changes:
     THE CAT ATE THE RAT to THE ATA TET HER AT

     c. Usually results in completely nonfunctional protein produced.
3. Point Mutations Can Be Drastic
   • a. Point mutations change one specific codon by changing a single nucleotide.
   • b. When UAC is changed to UAU, has no effect since amino acid remains same; is called a silent mutation.
   • c. When UAC is changed to UAG, UAG is stop codon and results in short protein unable to function.
   • d. When glutamic acid is replaced by valine, it produces sickle-cell hemoglobin. (Fig. 25.16)
   • e. Missense mutation, as in UAC to CAC, may have no effect if change occurs in noncritical area, or if two amino aids have same chemical properties.
4. Cause and Repair of Mutations
   • a. Mutations from DNA replication errors are rare; one in 10⁵ to 10⁸ divisions.
   • b. DNA polymerase carries out replication and proofreads new strand; detects mismatched pairs, corrects them.
   • c. Mutagens are environmental factors that cause mutations.
     • i. Radiation includes X rays, ultraviolet light, rays from radioactive ores.
     • ii. Chemical mutagens include some pesticides, cigarette smoke, etc.
     • iii. Mutation in gametes may affect traits of offspring.
     • iv. Mutation in body cells may cause cancer.
5. Transposons: Jumping Genes
   • a. Are specific DNA sequences that have ability to move within and between chromosomes.
   • b. Movement to a new location may alter neighboring genes, increasing or decreasing expression.
   • c. First discovered in corn 40 years ago, only recently recognized as significant; may occur in all organisms.

1. Cancer cell have severe failure in control of gene expression.
2. Cancer cells lack differentiation; the abnormal cells are nonspecialized and fail to play original role in body functions.
3. Normal cells only divide about 50 times; cancer cells are "immortal" and die only if they run out of nutrients or killed by own waste products.
4. Abnormal nuclei of cancer cells may be enlarged, have mutated chromosomes, possess duplications and deletions, and have gene amplification more than normal cells.
5. Cancer cells form tumors because they lack contact inhibition and pile up on tissue culture plates; normal cells stop dividing once they make contact with nearby cells and form single layer.
6. Growth factors are hormones needed by normal cells to grow; cancer cells have less need for growth factors.
7. Tumor is abnormal mass of cells that invades and destroys neighboring tissue; termed neoplasia.
8. Benign tumor is disorganized and does not invade adjacent tissue.
9. Cancer in situ is still growing in place of origin; has not invaded normal tissues.
10. A malignancy is establishment of new tumors distant from primary tumor.
11. Metastasis is migration across basement membranes into blood or lymph vessels.
12. Cancer cells travel in blood or lymph and start tumors in lung, liver, elsewhere.
13. Progression of cancer involves: tumor invading surrounding tissues, dispersal into lymph nodes, establishment of metastatic tumors in other organs (most serious).
14. What Causes Cancer (p. 494)
    • a. Carcinogens
      • i. Are environmental agents that cause mutations leading to cancer.
      • ii. Activate oncogenes (cancer-causing genes) or inactivate tumor-suppressor genes.
    • b. Oncogenes
      • i. Cell growth, differentiation, and division are regulated
      • ii. A regulatory pathway controls cell division and involves:
        - extracellular growth factors.
        - plasma membrane growth factor receptors.
        - proteins within cytoplasm.
        - genes within nucleus.

        iii. Proto-oncogenes are genes where a single mutation can cause them to become an oncogene; about 100 identified so far.

        iv. Oncogenes can produce abnormal protein product or abnormally high levels of abnormal product; both can cause uncontrolled growth.

        v. Alteration of one nucleotide in ras proto-oncogene produces human oncogene; rasK oncogene in many lung, colon, and pancreatic cancers, rasN oncogene in blood cell leukemias, lymphomas often have both.

        vi. Researchers are probing regulation and inactivation of ras protein.
15. Tumor-Suppressor Genes Stop Cancer (p. 496)
    • a. Researchers have identified half dozen tumor-suppressor genes.
    • b. When tumor-suppressor genes malfunction, tumor results.
    • c. RB tumor-suppressor
      • i. Discovered by Alfred Knudson in research on inherited retinoblastoma, a cancer of retina.
      • ii. If child receives one normal RB tumor-suppressor gene and it mutates, eye tumors develop by age three.
      • iii. RB tumor-suppressor gene malfunctions incriminated in cancers of breast, prostate, bladder, etc.
      • iv. Active RB protein turns off expression of proto-oncogene.
    • d. Another major tumor-suppressor gene is p53; more frequently mutated in human cancers than any other known gene; currently under research.