* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Microbial Genetics - MyCourses
Bisulfite sequencing wikipedia , lookup
History of RNA biology wikipedia , lookup
Messenger RNA wikipedia , lookup
Genome (book) wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Genomic library wikipedia , lookup
Frameshift mutation wikipedia , lookup
United Kingdom National DNA Database wikipedia , lookup
Genealogical DNA test wikipedia , lookup
Non-coding RNA wikipedia , lookup
Genetic code wikipedia , lookup
Epigenetics of human development wikipedia , lookup
DNA polymerase wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Epitranscriptome wikipedia , lookup
DNA damage theory of aging wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Genetic engineering wikipedia , lookup
DNA vaccination wikipedia , lookup
Molecular cloning wikipedia , lookup
Epigenomics wikipedia , lookup
Designer baby wikipedia , lookup
Nucleic acid double helix wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Genome editing wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
DNA supercoil wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Non-coding DNA wikipedia , lookup
Extrachromosomal DNA wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Microevolution wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Point mutation wikipedia , lookup
Helitron (biology) wikipedia , lookup
History of genetic engineering wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
11/21/2016 PowerPoint® Lecture Presentations prepared by Bradley W. Christian, McLennan Community College CHAPTER 8 Microbial Genetics © 2016 Pearson Education, Ltd. © 2016 Pearson Education, Ltd. 1 11/21/2016 Big Picture: Genetics • • • • The science of heredity Central dogma of molecular biology Mutations Gene expression controlled by operons © 2016 Pearson Education, Ltd. Big Picture pg. 228 (2 of 9). © 2016 Pearson Education, Ltd. 2 11/21/2016 Big Picture pg. 228 (6 of 9). An inducible operon includes genes that are in the "off" mode with the repressor bound to the DNA, and is turned "on" by the environmental inducer. Active repressor "OFF" (gene not expressed) DNA Inducer Inactive repressor "ON" (gene expressed) DNA A repressible operon includes genes that are in the "on" mode, without the repressor bound to the DNA, and is turned "off" by the environmental corepressor and repressor. Inactive repressor "ON" (gene expressed) DNA Corepressor "OFF" (gene not expressed) Active repressor DNA © 2016 Pearson Education, Ltd. Big Picture: Genetics • Alteration of bacterial genes and gene expression • Cause of disease • Prevent disease treatment • Manipulated for human benefit © 2016 Pearson Education, Ltd. 3 11/21/2016 Big Picture pg. 229 (2 of 6). © 2016 Pearson Education, Ltd. Structure and Function of the Genetic Material Learning Objectives 8-1 Define genetics, genome, chromosome, gene, genetic code, genotype, phenotype, and genomics. 8-2 Describe how DNA serves as genetic information. 8-3 Describe the process of DNA replication. 8-4 Describe protein synthesis, including transcription, RNA processing, and translation. 8-5 Compare protein synthesis in prokaryotes and eukaryotes. © 2016 Pearson Education, Ltd. 4 11/21/2016 Structure and Function of the Genetic Material • Genetics: the study of genes, how they carry information, how information is expressed, and how genes are replicated • Chromosomes: structures containing DNA that physically carry hereditary information; the chromosomes contain genes • Genes: segments of DNA that encode functional products, usually proteins • Genome: all the genetic information in a cell © 2016 Pearson Education, Ltd. Structure and Function of the Genetic Material • The genetic code is a set of rules that determines how a nucleotide sequence is converted to an amino acid sequence of a protein • Central dogma: © 2016 Pearson Education, Ltd. 5 11/21/2016 Genotype and Phenotype • Genotype: the genetic makeup of an organism • Phenotype: expression of the genes © 2016 Pearson Education, Ltd. DNA and Chromosomes • Bacteria usually have a single circular chromosome made of DNA and associated proteins • Short tandem repeats (STRs): repeating sequences of noncoding DNA © 2016 Pearson Education, Ltd. 6 11/21/2016 Figure 8.1 A prokaryotic chromosome. Chromosome Chromosome © 2016 Pearson Education, Ltd. The Flow of Genetic Information • Vertical gene transfer: flow of genetic information from one generation to the next © 2016 Pearson Education, Ltd. 7 11/21/2016 Figure 8.2 The Flow of Genetic Information. Parent cell DNA Genetic information is used within a cell to produce the proteins needed for the cell to function. Transcription Genetic information can be transferred horizontally between cells of the same generation. Genetic information can be transferred vertically to the next generation of cells. New combinations of genes Translation Cell metabolizes and grows Recombinant cell Offspring cells © 2016 Pearson Education, Ltd. Check Your Understanding ü Give a clinical application of genomics. 8-1 ü Why is the base pairing in DNA important? 8-2 © 2016 Pearson Education, Ltd. 8 11/21/2016 DNA Replication • DNA forms a double helix • "Backbone" consists of deoxyribose-phosphate • Two strands of nucleotides are held together by hydrogen bonds between A-T and C-G • Strands are antiparallel • Order of the nitrogen-containing bases forms the genetic instructions of the organism © 2016 Pearson Education, Ltd. Figure 8.3b DNA replication. © 2016 Pearson Education, Ltd. 9 11/21/2016 DNA Replication • One strand serves as a template for the production of a second strand • Topoisomerase and gyrase relax the strands • Helicase separates the strands • A replication fork is created © 2016 Pearson Education, Ltd. Figure 8.3a DNA replication. © 2016 Pearson Education, Ltd. 10 11/21/2016 DNA Replication • DNA polymerase adds nucleotides to the growing DNA strand In the 5' ® 3' direction Initiated by an RNA primer Leading strand is synthesized continuously Lagging strand is synthesized discontinuously, creating Okazaki fragments • DNA polymerase removes RNA primers; Okazaki fragments are joined by the DNA polymerase and DNA ligase • • • • © 2016 Pearson Education, Ltd. Table 8.1 Important Enzymes in DNA Replication, Expression, and Repair © 2016 Pearson Education, Ltd. 11 11/21/2016 Figure 8.5 A summary of events at the DNA replication fork. REPLICATION Proteins stabilize the unwound parental DNA. The leading strand is synthesized continuously by DNA polymerase. 3' 5' DNA polymerase Enzymes unwind the parental double helix. Replication fork RNA primer Primase 5' DNA polymerase 3' Parental strand Okazaki fragment DNA polymerase DNA ligase 3' 5' 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. DNA ligase joins the discontinuous fragments of the lagging strand. © 2016 Pearson Education, Ltd. DNA Replication • Energy for replication is supplied by nucleotides • Hydrolysis of two phosphate groups on ATP provides energy © 2016 Pearson Education, Ltd. 12 11/21/2016 Figure 8.4 Adding a nucleotide to DNA. New Strand Template Strand Sugar Phosphate When a nucleoside triphosphate bonds to the sugar, it loses two phosphates. Hydrolysis of the phosphate bonds provides the energy for the reaction. © 2016 Pearson Education, Ltd. DNA Replication • Most bacterial DNA replication is bidirectional • Each offspring cell receives one copy of the DNA molecule • Replication is highly accurate due to the proofreading capability of DNA polymerase © 2016 Pearson Education, Ltd. 13 11/21/2016 Figure 8.6 Replication of bacterial DNA. Replication fork An E. coli chromosome in the process of replicating REPLICATION Origin of replication Parental strand Daughter strands Replication fork Replication fork Replication fork Termination of replication Bidirectional replication of a circular bacterial DNA molecule © 2016 Pearson Education, Ltd. RNA and Protein Synthesis • Ribonucleic acid • Single-stranded nucleotide • 5-carbon ribose sugar • Contains uracil (U) instead of thymine (T) © 2016 Pearson Education, Ltd. 14 11/21/2016 RNA and Protein Synthesis • Ribosomal RNA (rRNA): integral part of ribosomes (ribosomaalinen RNA) • Transfer RNA (tRNA): transports amino acids during protein synthesis (siirtäjä-RNA) • Messenger RNA (mRNA): carries coded information from DNA to ribosomes (lähetti-RNA) © 2016 Pearson Education, Ltd. Transcription in Prokaryotes • Synthesis of a complementary mRNA strand from a DNA template • Transcription begins when RNA polymerase binds to the promoter sequence on DNA • Transcription proceeds in the 5' ® 3' direction; only one of the two DNA strands is transcribed • Transcription stops when it reaches the terminator sequence on DNA © 2016 Pearson Education, Ltd. 15 11/21/2016 Figure 8.7 The process of transcription. TRANSCRIPTION RNA polymerase DNA RNA polymerase mRNA RNA nucleotides Protein DNA RNA polymerase bound to DNA RNA Template strand of DNA Promoter (gene begins) RNA synthesis RNA polymerase RNA RNA polymerase binds to the promoter, and DNA unwinds at the beginning of a gene. RNA is synthesized by complementary base pairing of free nucleotides with the nucleotide bases on the template strand of DNA. The site of synthesis moves along DNA; DNA that has been transcribed rewinds. Terminator (gene ends) Transcription reaches the terminator. Complete RNA strand RNA and RNA polymerase are released, and the DNA helix re-forms. © 2016 Pearson Education, Ltd. Translation • mRNA is translated into the "language" of proteins • Codons are groups of three mRNA nucleotides that code for a particular amino acid • 61 sense codons encode the 20 amino acids • The genetic code involves degeneracy, meaning each amino acid is coded by several codons © 2016 Pearson Education, Ltd. 16 11/21/2016 Figure 8.8 The genetic code. Second position Phe Tyr Cys Ser Leu His Pro Arg Gln Asn Ser Lys Arg Ile Third position First position Leu Thr Asp Val Ala Gly Glu © 2016 Pearson Education, Ltd. Translation • Translation of mRNA begins at the start codon: AUG • Translation ends at nonsense codons: UAA, UAG, UGA • Codons of mRNA are "read" sequentially © 2016 Pearson Education, Ltd. 17 11/21/2016 Translation • tRNA molecules transport the required amino acids to the ribosome • tRNA molecules also have an anticodon that base-pairs with the codon • Amino acids are joined by peptide bonds © 2016 Pearson Education, Ltd. Figure 8.9 The process of translation (1 of 4). Ribosome P Site Ribosomal subunit tRNA Anticodon Ribosomal subunit Components needed to begin translation come together. mRNA Start codon Second codon mRNA On the assembled ribosome, a tRNA carrying the first amino acid is paired with the start codon on the mRNA. The place where this first tRNA sits is called the P site. A tRNA carrying the second amino acid approaches. © 2016 Pearson Education, Ltd. 18 11/21/2016 Figure 8.9 The process of translation (2 of 4). Peptide bond forms A site E site mRNA mRNA Ribosome moves along mRNA The second codon of the mRNA pairs with a tRNA carrying the second amino acid at the A site. The first amino acid joins to the second by a peptide bond. This attaches the polypeptide to the tRNA in the P site. The ribosome moves along the mRNA until the second tRNA is in the P site. The next codon to be translated is brought into the A site. The first tRNA now occupies the E site. © 2016 Pearson Education, Ltd. Figure 8.9 The process of translation (3 of 4). Growing polypeptide chain tRNA released mRNA mRNA The second amino acid joins to the third by another peptide bond, and the first tRNA is released from the E site. The ribosome continues to move along the mRNA, and new amino acids are added to the polypeptide. © 2016 Pearson Education, Ltd. 19 11/21/2016 Figure 8.9 The process of translation (4 of 4). Polypeptide released mRNA New protein mRNA Stop codon When the ribosome reaches a stop codon, the polypeptide is released. Finally, the last tRNA is released, and the ribosome comes apart. The released polypeptide forms a new protein. © 2016 Pearson Education, Ltd. Translation • In bacteria, translation can begin before transcription is complete (why?) © 2016 Pearson Education, Ltd. 20 11/21/2016 Figure 8.10 Simultaneous transcription and translation in bacteria. TRANSLATION RNA polymerase Met DNA Met DNA mRNA Protein DNA RNA Direction of transcription polymerase 5' Peptide Polyribosome Ribosome mRNA Direction of translation © 2016 Pearson Education, Ltd. Transcription in Eukaryotes • In eukaryotes, transcription occurs in the nucleus, whereas translation occurs in the cytoplasm • Exons are regions of DNA that code for proteins • Introns are regions of DNA that do not code for proteins • Small nuclear ribonucleoproteins (snRNPs) remove introns and splice exons together © 2016 Pearson Education, Ltd. 21 11/21/2016 Figure 8.11 RNA processing in eukaryotic cells. © 2016 Pearson Education, Ltd. Splicing: recognition sites (ei kirjassa, not in the book) • Specific recognition sites © 2016 Pearson Education, Ltd. 22 11/21/2016 Check Your Understanding ü What is the role of the promoter, terminator, and mRNA in transcription? 8-4 ü How does mRNA production in eukaryotes differ from the process in prokaryotes? 8-5 © 2016 Pearson Education, Ltd. The Regulation of Bacterial Gene Expression Learning Objectives 8-6 Define operon. 8-7 Explain pre-transcriptional regulation of gene expression in bacteria. 8-8 Explain post-transcriptional regulation of gene expression. © 2016 Pearson Education, Ltd. 23 11/21/2016 The Regulation of Bacterial Gene Expression • Constitutive genes are expressed at a fixed rate • Other genes are expressed only as needed • Inducible genes • Repressible genes • Catabolite repression © 2016 Pearson Education, Ltd. Pre-transcriptional Control • Repression inhibits gene expression and decreases enzyme synthesis • Mediated by repressors, proteins that block transcription • Default position of a repressible gene is on • Induction turns on gene expression • Initiated by an inducer • Default position of an inducible gene is off © 2016 Pearson Education, Ltd. 24 11/21/2016 PLAY Animation: Operons: Induction © 2016 Pearson Education, Ltd. PLAY Animation: Operons: Repression © 2016 Pearson Education, Ltd. 25 11/21/2016 The Operon Model of Gene Expression • Promoter: segment of DNA where RNA polymerase initiates transcription of structural genes • Operator: segment of DNA that controls transcription of structural genes • Operon: set of operator and promoter sites and the structural genes they control © 2016 Pearson Education, Ltd. The Operon Model of Gene Expression • In an inducible operon, structural genes are not transcribed unless an inducer is present • In the absence of lactose, the repressor binds to the operator, preventing transcription • In the presence of lactose, lactose (inducer) binds to the repressor; the repressor cannot bind to the operator and transcription occurs © 2016 Pearson Education, Ltd. 26 11/21/2016 Figure 8.12 An inducible operon (1 of 3). Operon Control region Structural genes I P O Z Y A DNA Regulatory gene Promoter Operator Structure of the operon. The operon consists of the promoter (P) and operator (O) sites and structural genes that code for the protein. The operon is regulated by the product of the regulatory gene (I) © 2016 Pearson Education, Ltd. Figure 8.12 An inducible operon (2 of 3). RNA polymerase I P Z Y A Transcription Repressor mRNA Active repressor protein Translation Repressor active, operon off. The repressor protein binds with the operator, preventing transcription from the operon. © 2016 Pearson Education, Ltd. 27 11/21/2016 Figure 8.12 An inducible operon (3 of 3). I P O Z Y A Transcription Operon mRNA Translation Allolactose (inducer) Inactive repressor protein Transacetylase Permease β-Galactosidase Repressor inactive, operon on. When the inducer allolactose binds to the repressor protein, the inactivated repressor can no longer block transcription. The structural genes are transcribed, ultimately resulting in the production of the enzymes needed for lactose catabolism. © 2016 Pearson Education, Ltd. The Operon Model of Gene Expression • In repressible operons, structural genes are transcribed until they are turned off • Excess tryptophan is a corepressor that binds and activates the repressor to bind to the operator, stopping tryptophan synthesis © 2016 Pearson Education, Ltd. 28 11/21/2016 Figure 8.13 A repressible operon (1 of 3). Operon Control region Structural genes I O P D E C B A DNA Regulatory gene Promoter Operator Structure of the operon. The operon consists of the promoter (P) and operator (O) sites and structural genes that code for the protein. The operon is regulated by the product of the regulatory gene (I) © 2016 Pearson Education, Ltd. Figure 8.13 A repressible operon (2 of 3). RNA polymerase I P O E D C B A Transcription Repressor mRNA Operon mRNA Translation Inactive repressor protein Polypeptides comprising the enzymes for tryptophan synthesis Repressor inactive, operon on. The repressor is inactive, and transcription and translation proceed, leading to the synthesis of tryptophan. © 2016 Pearson Education, Ltd. 29 11/21/2016 Figure 8.13 A repressible operon (3 of 3). I P E D C B A Active repressor protein Tryptophan (corepressor) Repressor active, operon off. When the corepressor tryptophan binds to the repressor protein, the activated repressor binds with the operator, preventing transcription from the operon. © 2016 Pearson Education, Ltd. Positive Regulation • Catabolite repression inhibits cells from using carbon sources other than glucose • Cyclic AMP (cAMP) builds up in a cell when glucose is not available • cAMP binds to the lac promoter, initiating transcription and allowing the cell to use lactose © 2016 Pearson Education, Ltd. 30 11/21/2016 Figure 8.14 The growth rate of E. coli on glucose and lactose. Bacteria growing on glucose as the sole carbon source grow faster than on lactose. Glucose Lactose All glucose consumed Glucose used Lactose used Lag time 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. © 2016 Pearson Education, Ltd. Figure 8.15 Positive regulation of the lac operon. Promoter lacl lacZ DNA Operator RNA polymerase can bind and transcribe CAP-binding site cAMP Active CAP Inactive lac repressor Inactive CAP Lactose present, glucose scarce (cAMP level high). If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA for lactose digestion. Promoter lacl lacZ DNA Operator CAP-binding site RNA polymerase can't bind Inactive lac repressor Inactive CAP Lactose present, glucose present (cAMP level low). When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription. © 2016 Pearson Education, Ltd. 31 11/21/2016 Epigenetic Control • Methylating nucleotides turns genes off • Methylated (off) genes can be passed to offspring cells • Not permanent © 2016 Pearson Education, Ltd. Post-Transcriptional Control • microRNAs (miRNAs) base pair with mRNA to make it double-stranded • Double-stranded RNA is enzymatically destroyed, preventing production of a protein © 2016 Pearson Education, Ltd. 32 11/21/2016 Figure 8.16 MicroRNAs control a wide range of activities in cells. DNA Transcription of miRNA occurs. miRNA miRNA binds to target mRNA that has at least six complementary bases. mRNA mRNA is degraded. © 2016 Pearson Education, Ltd. Check Your Understanding ü What is the role of cAMP in regulating gene expression? 8-7 ü How does miRNA stop protein synthesis? 8-8 © 2016 Pearson Education, Ltd. 33 11/21/2016 Changes in the Genetic Material Learning Objectives 8-9 Classify mutations by type. 8-10 Describe two ways mutations can be repaired. 8-11 Describe the effect of mutagens on the mutation rate. 8-12 Outline the methods of direct and indirect selection of mutants. 8-13 Identify the purpose of and outline the procedure for the Ames test. © 2016 Pearson Education, Ltd. Changes in Genetic Material • Mutation: a permanent change in the base sequence of DNA • Mutations may be neutral, beneficial, or harmful • Mutagens: agents that cause mutations • Spontaneous mutations: occur in the absence of a mutagen © 2016 Pearson Education, Ltd. 34 11/21/2016 Big Picture pg. 228 (3 of 9). © 2016 Pearson Education, Ltd. Types of Mutations • Base substitution (point mutation) • Change in one base in DNA © 2016 Pearson Education, Ltd. 35 11/21/2016 Figure 8.17 Base substitutions. Parental DNA Replication During DNA replication, a thymine is incorporated opposite guanine by mistake. Daughter DNA Daughter DNA Daughter DNA If not corrected, in the next round of replication, adenine pairs with the new thymine, yielding an AT pair in place of the original GC pair. Replication Granddaughter DNA Transcription When mRNA is transcribed from the DNA containing this substitution, a codon is produced that, during translation, encodes a different amino acid: tyrosine instead of cysteine. mRNA Translation Amino acids Cysteine Tyrosine Cysteine Cysteine © 2016 Pearson Education, Ltd. Types of Mutations • Missense mutation • Base substitution results in change in an amino acid © 2016 Pearson Education, Ltd. 36 11/21/2016 Figure 8.18a-b Types of mutations and their effects on the amino acid sequences of proteins. DNA (template strand) Transcription mRNA Translation Amino acid sequence Met Lys Phe Gly Stop Lys Phe Ser Stop Normal DNA molecule DNA (template strand) mRNA Amino acid sequence Met Missense mutation © 2016 Pearson Education, Ltd. Types of Mutations • Nonsense mutation • Base substitution results in a nonsense (stop) codon © 2016 Pearson Education, Ltd. 37 11/21/2016 Figure 8.18a-c Types of mutations and their effects on the amino acid sequences of proteins. DNA (template strand) Transcription mRNA Translation Amino acid sequence Met Lys Phe Gly Stop Normal DNA molecule Met Stop Nonsense mutation © 2016 Pearson Education, Ltd. Types of Mutations • Frameshift mutation • Insertion or deletion of one or more nucleotide pairs • Shifts the translational "reading frame" © 2016 Pearson Education, Ltd. 38 11/21/2016 Figure 8.18a-d Types of mutations and their effects on the amino acid sequences of proteins. DNA (template strand) Transcription mRNA Translation Met Amino acid sequence Lys Phe Gly Stop Normal DNA molecule Met Lys Leu Ala Frameshift mutation © 2016 Pearson Education, Ltd. Check Your Understanding ü How can a mutation be beneficial? 8-9 © 2016 Pearson Education, Ltd. 39 11/21/2016 Chemical Mutagens • Nitrous acid: causes adenine to bind with cytosine instead of thymine • Nucleoside analog: incorporates into DNA in place of a normal base; causes mistakes in base pairing © 2016 Pearson Education, Ltd. Radiation • Ionizing radiation (X rays and gamma rays) causes the formation of ions that can oxidize nucleotides and break the deoxyribose-phosphate backbone • UV radiation causes thymine dimers © 2016 Pearson Education, Ltd. 40 11/21/2016 Radiation • Photolyases separate thymine dimers • Nucleotide excision repair: Enzymes cut out incorrect bases and fill in correct bases © 2016 Pearson Education, Ltd. Figure 8.21 The creation and repair of a thymine dimer caused by ultraviolet light. Ultraviolet light Thymine dimer Exposure to ultraviolet light causes adjacent thymines to become cross-linked, forming a thymine dimer and disrupting their normal base pairing. An endonuclease cuts the DNA, and an exonuclease removes the damaged DNA. New DNA DNA polymerase fills the gap by synthesizing new DNA, using the intact strand as a template. DNA ligase seals the remaining gap by joining the old and new DNA. © 2016 Pearson Education, Ltd. 41 11/21/2016 The Frequency of Mutation • Spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated genes • Mutagens increase the mutation rate to 10–5 or 10–3 per replicated gene © 2016 Pearson Education, Ltd. Check Your Understanding ü How can mutations be repaired? 8-10 ü How do mutagens affect the mutation rate? 8-11 © 2016 Pearson Education, Ltd. 42 11/21/2016 Identifying Mutants • Positive (direct) selection detects mutant cells because they grow or appear different than unmutated cells • Negative (indirect) selection detects mutant cells that cannot grow or perform a certain function • Auxtotroph: mutant that has a nutritional requirement absent in the parent • Use of replica plating © 2016 Pearson Education, Ltd. Figure 8.22 Replica plating. Handle Sterile velvet is pressed on the grown colonies on the master plate. Velvet surface (sterilized) Master plate with medium containing histidine Cells from each colony are transferred from the velvet to new plates. Petri plate with medium containing Plates are incubated. histidine Auxotrophic mutant Petri plate with medium lacking histidine Colony missing Growth on plates is compared. A colony that grows on the medium with histidine but could not grow on the medium without histidine is auxotrophic (histidine-requiring mutant). © 2016 Pearson Education, Ltd. 43 11/21/2016 Identifying Chemical Carcinogens • The Ames test exposes mutant bacteria to mutagenic substances to measure the rate of reversal of the mutation • Indicates degree to which a substance is mutagenic © 2016 Pearson Education, Ltd. Figure 8.23 The Ames reverse gene mutation test. Experimental sample Suspected mutagen Rat liver extract Experimental plate Incubation Cultures of histidine-dependent Salmonella Control (no suspected mutagen added) Colonies of revertant bacteria Media lacking histidine Rat liver extract Incubation Control plate Two cultures are prepared of Salmonella bacteria that have lost the ability to synthesize histidine (histidinedependent). The suspected mutagen is added to the experimental sample only; rat liver extract (an activator) is added to both samples. Each sample is poured onto a plate of medium lacking histidine. The plates are then incubated at 37°C for two days. Only bacteria whose histidine-dependent phenotype has mutated back (reverted) to histidinesynthesizing will grow into colonies. The numbers of colonies on the experimental and control plates are compared. The control plate may show a few spontaneous histidine-synthesizing revertants. The test plates will show an increase in the number of histidine-synthesizing revertants if the test chemical is indeed a mutagen and potential carcinogen. The higher the concentration of mutagen used, the more revertant colonies will result. © 2016 Pearson Education, Ltd. 44 11/21/2016 Check Your Understanding ü How would you isolate an antibiotic-resistant bacterium? An antibiotic-sensitive bacterium? 8-12 ü What is the principle behind the Ames test? 8-13 © 2016 Pearson Education, Ltd. Genetic Transfer and Recombination Learning Objectives 8-14 Differentiate horizontal and vertical gene transfer. 8-15 Compare the mechanisms of genetic recombination in bacteria. 8-16 Describe the functions of plasmids and transposons. © 2016 Pearson Education, Ltd. 45 11/21/2016 Genetic Transfer and Recombination • Genetic recombination: exchange of genes between two DNA molecules; creates genetic diversity • Crossing over: Two chromosomes break and rejoin, resulting in the insertion of foreign DNA into the chromosome © 2016 Pearson Education, Ltd. Figure 8.24 Genetic recombination by crossing over. DNA from one cell aligns with DNA in the recipient cell. Notice that there is a nick in the donor DNA. Donor DNA Recipient chromosome DNA from the donor aligns with complementary base pairs in the recipient's chromosome. This can involve thousands of base pairs. RecA protein catalyzes the joining of the two RecA protein strands. The result is that the recipient's chromosome contains new DNA. Complementary base pairs between the two strands will be resolved by DNA polymerase and ligase. The donor DNA will be destroyed. The recipient may now have one or more new genes. © 2016 Pearson Education, Ltd. 46 11/21/2016 Genetic Transfer and Recombination • Vertical gene transfer: transfer of genes from an organism to its offspring • Horizontal gene transfer: transfer of genes between cells of the same generation © 2016 Pearson Education, Ltd. Transformation in Bacteria • Transformation: genes transferred from one bacterium to another as "naked" DNA © 2016 Pearson Education, Ltd. 47 11/21/2016 Figure 8.26 The mechanism of genetic transformation in bacteria. Recipient cell a b c d D A B DNA fragments from donor cells C Chromosomal DNA Recipient cell takes up donor DNA. 5' 3' a b c d 5' b c d D A C B Donor DNA aligns with complementary bases. 3' D A B C Recombination occurs between donor DNA and recipient DNA. Degraded unrecombined DNA a D B C Genetically transformed cell © 2016 Pearson Education, Ltd. Conjugation in Bacteria • Conjugation: plasmids transferred from one bacterium to another • Requires cell-to-cell contact via sex pili © 2016 Pearson Education, Ltd. 48 11/21/2016 Figure 8.27 Bacterial conjugation. Mating bridge Sex pilus F– cell F+ cell Sex pilus Mating bridge © 2016 Pearson Education, Ltd. Conjugation in Bacteria • Donor cells carry the plasmid (F factor) and are called F+ cells • Hfr cells contain the F factor on the chromosome © 2016 Pearson Education, Ltd. 49 11/21/2016 Figure 8.28a Conjugation in E. coli. RECOMBINATION Bacterial chromosome Mating bridge Replication and transfer of F factor F factor F+ cell F– cell F+ cell F+ cell When an F factor (a plasmid) is transferred from a donor (F+) to a recipient (F–), the F– cell is converted to an F+ cell. © 2016 Pearson Education, Ltd. Figure 8.28b Conjugation in E. coli. RECOMBINATION Recombination between F factor and chromosome, occurring at a specific site on each Insertion of F factor into chromosome Integrated F factor F+ cell Hfr cell When an F factor becomes integrated into the chromosome of an F+ cell, it makes the cell a high frequency of recombination (Hfr) cell. © 2016 Pearson Education, Ltd. 50 11/21/2016 Figure 8.28c Conjugation in E. coli. © 2016 Pearson Education, Ltd. Transduction in Bacteria • DNA is transferred from a donor cell to a recipient via a bacteriophage • Generalized transduction: Random bacterial DNA is packaged inside a phage and transferred to a recipient cell • Specialized transduction: Specific bacterial genes are packaged inside a phage and transferred to a recipient cell © 2016 Pearson Education, Ltd. 51 11/21/2016 Figure 8.30 Transduction by a bacteriophage. RECOMBINATION Phage protein coat Phage DNA Bacterial chromosome Donor cell A phage infects the donor bacterial cell. Phage DNA and proteins are made, and the bacterial chromosome is broken into pieces. Occasionally during phage assembly, pieces of bacterial DNA are packaged in a phage capsid. Then the donor cell lyses and releases phage particles containing bacterial DNA. Phage DNA Bacterial DNA A phage carrying bacterial DNA infects a new host cell, the recipient cell. Recipient cell Donor bacterial DNA Recipient bacterial DNA Recombinant cell reproduces normally Recombination can occur, producing a recombinant cell with a genotype different from both the donor and recipient cells. Many cell divisions © 2016 Pearson Education, Ltd. Plasmids • Plasmids are self-replicating circular pieces of DNA • 1 to 5% the size of a bacterial chromosome • Often code for proteins that enhance the pathogenicity of a bacterium © 2016 Pearson Education, Ltd. 52 11/21/2016 Figure 8.31 R factor, a type of plasmid. Origin of replication Mercury resistance Sulfonamide resistance Pilus and conjugation proteins Origin of transfer RTF Streptomycin resistance Chloramphenicol resistance Tetracycline resistance © 2016 Pearson Education, Ltd. Plasmids • Conjugative plasmid: carries genes for sex pili and transfer of the plasmid • Dissimilation plasmids: encode enzymes for the catabolism of unusual compounds • Resistance factors (R factors): encode antibiotic resistance © 2016 Pearson Education, Ltd. 53 11/21/2016 Transposons • Transposons are segments of DNA that can move from one region of DNA to another • Contain insertion sequences (IS) that code for transposase that cuts and reseals DNA • Complex transposons carry other genes (e.g, in antibiotic resistance) © 2016 Pearson Education, Ltd. Figure 8.32a Transposons and insertion. IS1 Transposase gene Inverted repeat Inverted repeat An insertion sequence (IS), the simplest transposon, contains a gene for transposase, the enzyme that catalyzes transposition. The tranposase gene is bounded at each end by inverted repeat sequences that function as recognition sites for the transposon. IS1 is one example of an insertion sequence, shown here with simplified IR sequences. © 2016 Pearson Education, Ltd. 54 11/21/2016 Figure 8.32b-c Transposons and insertion. Transposase gene Transposase cuts DNA, leaving sticky ends. Tn5 Kanamycin resistance IS1 IS1 Complex transposons carry other genetic material in addition to transposase genes. The example shown here, Tn5, carries the gene for kanamycin resistance and has complete copies of the insertion sequence IS1 at each end. IS1 IS1 Sticky ends of transposon and target DNA anneal. Insertion of the transposon Tn5 into R100 plasmid © 2016 Pearson Education, Ltd. Check Your Understanding ü What types of genes do plasmids carry? 8-16 © 2016 Pearson Education, Ltd. 55 11/21/2016 Genes and Evolution Learning Objective 8-17 Discuss how genetic mutation and recombination provide material for natural selection to act upon. © 2016 Pearson Education, Ltd. Genes and Evolution • Mutations and recombination create cell diversity • Diversity is the raw material for evolution • Natural selection acts on populations of organisms to ensure the survival of organisms fit for a particular environment © 2016 Pearson Education, Ltd. 56