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Download Lectures prepared by Christine L. Case Chapter 8 Microbial Genetics
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Chapter 8 Microbial Genetics © 2013 Pearson Education, Inc. Copyright © 2013 Pearson Education, Inc. Lectures prepared by Christine L. Case Lectures prepared by Christine L. Case Structure and Function of the Genetic Material Genetics (遺傳學): The study of what genes are how they carry information how information is expressed how genes are replicated Gene: A segment of DNA that encodes a functional product, usually a protein. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Genome (基因組): The genetic information in a cell A cell’s genome includes its chromosome and plasmid Chromosome (染色體): Structures containing DNA that physically carry hereditary information The chromosomes contain the genes Gene (基因): The segment of DNA that code for functional product Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Genotype and phenotype Genomics (基因體學): The molecular study of genomes Genotype (基因型): The genes of an organism Phenotype (外表型): Expression of the genes In microbes, most proteins are either enzymatic or structural. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings DNA and Chromosomes DNA in cells exists as a double-stranded helix associated with various proteins that regulate genetic activity the two strands are held together by hydrogen bonds between specific nitrogenous base pairs (complementary) : A=T C = G. bacterial DNA is circular Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Chromosome of E. coli contains about 4.6 million base pairs 1 mm takes up only about 10% of the cell’s volume because the DNA is supercoiled Disrupted E. coli cell Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.1a The location of genes on a bacterial chromosome was determined by experiments on the transfer of genes from one cell to another. The bacterial map is marked in minutes. KEY Amino acid metabolism Carbohydrate metabolism DNA replication and repair Membrane synthesis Lipid metabolism Genetic map of the chromosome of E. coli Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.1b The entire genome does not consist of back-to-back genes. Noncoding regions called short tandem repeats (STRs). Open-reading frame (ORF, 開放譯讀區): region of DNA that are likely to encode a protein. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Flow of Genetic Information Parent cell DNA 重組 基因表現 expression recombination Genetic information is used within a cell to produce the proteins needed for the cell to function. Genetic information can be Insert Fig 8.2 transferred between cells of the same generation. 複製 replication Genetic information can be transferred between generations of cells. New combinations of genes Transcription 轉錄 Translation 轉譯 Cell metabolizes and grows Recombinant cell Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Daughter cells Figure 8.2 Transcription genetic information in DNA is copied into a complementary base sequence of RNA Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.2 Translation mRNA is translated in codons (three nucleotides) Translation of mRNA begins at the start codon: AUG Translation ends at a stop codon: UAA, UAG, UGA Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.2 DNA replication DNA Polymer of nucleotides: Adenine (腺嘌呤), thymine (胸腺嘧啶), cytosine (胞嘧啶), and guanine (鳥糞嘌呤) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.3b Double helix associated with proteins "Backbone" is deoxyribosephosphate Strands are held together by hydrogen bonds between AT and CG. Strands are antiparallel. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.3a Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.4 DNA is copied by DNA polymerase In the 5' 3' direction Initiated by an RNA primer Leading strand is synthesized continuously Lagging strand is synthesized discontinuously Okazaki fragments RNA primers are removed and Okazaki fragments joined by a DNA polymerase and DNA ligase Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings REPLICATION Proteins stabilize the unwound parental DNA. The leading strand is synthesized continuously by DNA polymerase. 3' DNA polymerase 5' Enzymes unwind the 1 parental double helix. Replication fork RNA primer Insert Fig 8.5 Primase 5' DNA polymerase 3' Parental strand Okazaki fragment DNA polymerase The lagging strand is synthesized discontinuously. Primase, an RNA polymerase, synthesizes a short RNA primer, which is then extended by DNA polymerase. DNA polymerase digests RNA primer and replaces it with DNA. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings DNA ligase 3' 5' DNA ligase joins the discontinuous fragments of the lagging strand. Figure 8.5 Important Enzymes Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings DNA replication is semiconservative (半保留). Replication forks REPLICATION An E. coli1chromosome in the process of replicating Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.6a An E. coli 1chromosome in the process of replicating Origin of replication Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.6b The Meselson and Stahl experiment: evidence demonstrating semiconservative replication Semiconservative replication Because each double-stranded DNA molecule contains one original and one new strand, the replication process is called semiconservative replication DNA polymerase proofreads new molecules of DNA and removes mismatched bases before continuing DNA synthesis. Errors only occur ~1 time for every 1010 bases added. Each daughter bacterium receives a chromosome identical to the parent's. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings RNA and protein synthesis Transcription DNA is transcribed to make RNA (mRNA, tRNA, and rRNA). Transcription begins when RNA polymerase binds to the promoter (啟動子) sequence Transcription proceeds in the 5' 3' direction Transcription stops when it reaches the terminator sequence Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.7 (1 of 2) 啟動子 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.7 (2 of 2) Translation Degeneracy (退化性) Sense codons and nonsense codons Anticodon Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.8 Translation steps: Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.9, step 1-2 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.9, step 3-4 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.9, step 5-6 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.9, step 7-8 RNA polymerase Met In prokaryotic cells, the translation of mRNA into protein can begin even before transcription is complete. Met RNA Ribosome Peptide 1 Insert Fig 8.10 Met Met Met Met Direction of transcription RNA polymerase DNA 5′ Peptide Polyribosome Ribosome Direction of translation Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings mRNA Figure 8.10 RNA processing in Eukaryotes Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.11 The Regulation of Bacterial Gene Expression Constitutive enzymes, perhaps 60-80%, are expressed at a fixed rate. Other enzymes are expressed only as needed. Repressible enzymes Inducible enzymes Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Pre-transcriptional control Two genetic control mechanisms regulate the transcription of mRNA: Repression (抑制作用) To inhibit gene expression and decrease the synthesis of enzymes 抑制物 To be mediated by regulatory proteins, repressors To block the ability of RNA polymerase to initiate transcription Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Induction (誘導作用) To turn on the transcription of gene or genes Inducer, to act to induce transcription Inducible enzyme: enzymes that are synthesized in the presence of inducers (enzyme induction, βgalactosidase) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The operon model of gene expression Operon: a unit of prokarytoic gene expression and regulation which typically includes: 1. Structural genes for enzymes in a specific biosynthetic pathway whose expression is coordinately controlled. 2. Control elements, such as operator and promoter. 3. Regulator gene(s) whose products recognize the control elements. Regulator gene(s) sometimes are encoded by the gene under the control of a different promoter. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Operon lac operon Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings trp operon Figure 8.12, 8.13 An inducible operon Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.12, step 2-3 A repressible operon Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.13, step 2-3 Positive regulation Bacteria growing on glucose as the sole carbon source grow faster than on lactose. Figure 8.14 Bacteria growing in a medium containing glucose and lactose first consume the glucose and then, after a short lag time, the lactose. During the lag time, intracellular cAMP increases, the lac operon is transcribed, more lactose is transported into the cell, and β-galactosidase is synthesized to break down lactose. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings CAP: catabolic activator protein Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings cyclic AMP (cAMP) Figure 8.15 Epigenetic control (表觀調控) Eukaryotic and bacterial cells can turn genes off by methylating certain nucleotides. can pass to offspring isn’t permanent Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Post-transcriptional control Some regulatory mechanisms stop protein synthesis after transcription has occurred: DNA Transcription of miRNA occurs. miRNA miRNA binds to target mRNA that has at least six complementary bases. Ex: microRNAs (miRNAs) mRNA mRNA is degraded. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.16 Mutation: Change in the Genetic material A change in the genetic material Mutations may be neutral, beneficial, or harmful. Mutagen (致突變原): Agent that causes mutations Spontaneous mutations (自發性突變): Occur in the absence of a mutagen Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Types of mutations Base substitution (point mutation) Silent mutation Missense mutation Nonsense mutation Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.17 Base substitution Silent mutation Result in no change in amino acid Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.8 Base substitution Missense mutation Result in change in amino acid Figure 8.18a–b Ex: Sickle-cell disease – A single missense mutation, a change from an A to a T at a specific site, results in the change from glutamic acid to valine in the protein of hemoglobin. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Base substitution Nonsense mutation Results in a nonsense codon Figure 8.18a, c Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Frameshift mutation Insertion or deletion of one or more nucleotide pairs Figure 8.18a, d Huntington’s disease – A progressive neurological disorder caused by extra bases inserted into a particular gene. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Spontaneous mutations occur without the presence of a mutagen. The spontaneous mutation rate varies from genome to genome. Copying errors in the genetic material during cell division Induced mutations occur as a result of applying mutagens. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Mutagens Mutagens (致突變原) are agent in environment that brings about DNA mutation. Usually chemically or physically interact with DNA to cause change. Once mistake is fixed into the DNA the change is permanent. Chemical mutagens Radiation Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Chemical mutagens Nitrous acid: converts A so it pairs with C instead of T 亞硝酸 Adenosine Figure 8.19 thymine or uracil Adenosine Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings cytosine Figure 8.19 Nucleoside analogs: have chemical structure similar to a base but do not base pair correctly 2-aminopurine and 5-bromouracil incorporated into DNA in place of A and T but base pairs with C Advantage: antiviral and antitumor drugs. Ex: AZT for HIV Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.20 苯芘 Benzpyrene (cigarette smoke): causes frameshift mutations binds between bases and offsets the double helix strands Another ex: aflatoxin, produced by Aspergillus flavus Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Radiation x-rays and -rays: create ions and free radicals that break molecular bonds UV causes crosslinking of T bases (Thymine dimer) which can prevent unwinding for replication or transcription. Cells have light repair enzymes called photolyases which cut out damaged Ts and replace them. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Nucleotide excision repair = enzymes that can cut out and replace DNA damage Figure 8.21 Nucleotide excision repairs mutations damaged parts are removed leaving gap in strand gap is filled by complementary base pairing from other strand often repair restores correct sequence sometimes errors are made during repair: once nucleotide excision repair mechanisms seal the DNA, mutation is permanent Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Damage on one strand Damage on both strands ATGCTAGGCTATTATCG TACGATCCGATAATAGC ATGCTAGGCTATTATCG TACGATCCGATAATAGC ATGCT GCTATTATCG TACGAT GATAATAGC ATGCTA?GCTATTATCG TACGAT?CGATAATAGC Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The frequency of mutation Mutation rate: probability that gene will mutate when cell divides Spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated genes If harmful, organism dies. If beneficial, organism thrives and passes mutation to offspring (drives adaptation and evolution). Mutagens increase to 10–5 or 10–3 per replicated gene. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Identifying mutation Mutants can be detected by selecting or testing for an altered phenotype Positive (direct) selection detects mutant cells because they grow or appear different. Ex: penicillin resistant Negative (indirect) selection detects mutant cells because they do not grow. Technique: replica plating very effective the mutant is known as an auxotroph (營養需求突變株) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Replica Plating (複製平板法) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.22 Identifying chemical carcinogens Carcinogen (致癌物): substances that cause cancer in animal. The Ames test (安氏試驗) is a relatively inexpensive and rapid test for identifying possible chemical carcinogens. The test assumes that a mutant cell can revert to a normal cell in the presence of mutagen, and that many mutagens are carcinogens. Histidine auxotrophs (組蛋白營養需求突變株) of Samonella are exposed to an enzymatically treated potential carcinogen, and reversions to the nonmutant state are selected. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The Ames Test for Chemical Carcinogens Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.22 Genetic Transfer and Recombination Genetic recombination refer to the exchange of genes between two DNA molecules to form new combinations of genes on chromosome involves crossing over Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.24 Genetic recombination contributes to population diversity: recombination more likely than mutation to provide beneficial change since it tends not to destroy gene function. Eukaryotes: recombination during meiosis for sexual reproduction. creates diversity in offspring but parent remains unchanged vertical gene transfer = genes passed from organism to offspring Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Prokaryotes: recombination via gene transfer between cells or within cell by transformation (轉型), conjugation (接合), or transduction (轉導) original cell is altered horizontal gene transfer = genes passed to neighboring microbes of same generation transfer involves donor cell that gives portion of DNA to recipient cell when donor DNA incorporated into recipient, recipient now called recombinant cell if recombinant cell acquired new function/characteristic as result of new DNA, cell has been transformed Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Generation of recombinant cells is very low frequency event (less than 1%): very few cells in population are capable of exchanging and incorporating DNA. Three methods of prokaryotic gene transfer: Transformation (轉型) Conjugation (接合) Transduction (轉導) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Transformation in bacteria Genes are transferred as “naked” DNA in solution. It can occur between unrelated genus/species. It was discovered by F. Griffith 1928 who studied Streptococcus pneumoniae virulent strain had capsule non-virulent stain did not in mouse, dead virulent strain could pass “virulence factor” to live nonvirulent strain Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Griffith’s experiment Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.25 Competent cells (勝任細胞) can pick up DNA from dead cells and incorporate it into genome by recombination (e.g. antibiotic resistance). Transformed cell than passes genetic recombination to progeny. Transformation works best when donor and recipient are related but they do not have to be. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings competent = permeable to DNA: alterations in cell wall that allow large molecule like DNA to get through (in lab we use chemical agents to poke holes) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.26 Conjugation in bacteria Genes are transferred between two live cells via sex pilus (Gram -) or surface adhesion molecules (Gram +). Transfer is mediated by a plasmid: small circle of DNA separate from genome that is self replicating but contains no essential genes. Plasmid has genes for its own transfer. Gram negative plasmids have genes for pilus. Gram positive plasmids have genes for surface adhesion molecules. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Conjugation requires cell to cell contact between two cells of opposite mating type, usually the same species, must be same genus. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.27 During conjugation plasmid is replicated and single stranded copy is transferred to recipient. Recipient synthesizes complementary strand to complete plasmid. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.28a Plasmid can remain as separate circle or plasmid can be integrated into host cell genome resulting in permanent chromosomal changes. 高頻重 組細胞 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.28b Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.28c Transduction in bacteria DNA from a donor is carried by a virus to a recipient cell. Bacteriophage (Phage) is a kind of virus that infects bacterial cells Each phage is species specific (donor and recipient are the same species). Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Transduction mechanism: 1. Phage attaches to donor cell and injects phage DNA. 2. Phage DNA directs donor cell to synthesize phage proteins and DNA, phage enzymes digest the bacterial chromosome. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.29, step 1-2 3. New phages are assembled: phage DNA is packaged into capsids. Occasionally bacterial DNA is packaged by mistake. 4. Capsid containing bacterial DNA “infects” new host recipient cell by injecting the DNA. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.29, step 3-4 5. Donor DNA does not direct viral replication (not viral DNA): instead integrates into recipient genome. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.29, step 5 Plasmids and Transposons Plasmids Plasmids are self-replicating circle of DNA containing “extra” genes. 1. Conjugative plasmid: used in bacterial conjugation. Carries genes for sex pili and transfer of the plasmid. 2. Dissimilation plasmids (異化質體): Encode enzymes for catabolism of unusual compounds. 3. Pathogenicity plasmids: carry genes that code for virulence traits. Ex: capsules, toxins, adhesion molecules, bacteriocins. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 4. Resistance factor (R factor): carry genes for resistance to antibiotic and toxins. R factor has two parts: Resistance transfer factor (RTF) R-determinant 抗汞 抗磺胺劑 抗鏈黴素 抗 性 移 轉 因 子 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings r決 定 體 四環素 抗氯黴素 Figure 8.30 Plasmids can be transferred between species: allows spread of antibiotic resistance between different pathogens wide use of antibiotics has put selective pressure on microbes to “develop” and “share” resistance genes Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Transposons (轉位子) Transposons are small segments of DNA that can move independently from one region of DNA to another. discovered 1950s by McClintock: mosaic pattern in indian corn (Nobel Prize 1983) transposons pop out and randomly insert at rate of 10-5 to 10-7 per generation integration is random: can disrupt genes at minimum transposons carry genetic info to carry out own transposition, may also carry other genes Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Simplest transposon = insertion sequence (IS) gene for transposase (轉位酶) (enzyme that cuts DNA at recognition sites and religates it elsewhere in genome) two recognition sites called inverted repeats, mark ends of transposon, recognized by transposase Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.31a Complex transposons have inverted repeats outside other genes. Genes will get carried with transposon when it moves. Depending on where it inserts and what genes it carries, it can mediate good or bad genetic changes. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.31b Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.31c