Ch 8. Microbial Genetics

Transcription

1 Ch 8 Microbial Genetics

2 SLOs Define the terms genome and gene, and differentiate between genotype and phenotype. Draw a detailed segment of DNA. Summarize the steps of bacterial DNA replication, and identify the enzymes used in this process. Compare and contrast the synthesis of leading and lagging strands. Provide an overview of the relationship among DNA, RNA, and proteins. Identify structural and functional differences between RNA and DNA. Outline the process of transcription. List the three types of RNA directly involved in translation. Define the terms codon and anticodon, and recite the start codon. Outline the process of translation. Indicate how eukaryotic transcription and translation differ from these processes in bacteria. Define operon, and explain one advantage it provides to a bacterial cell. Highlight the main parts of the lac operon Explain the defining characteristics of a recombinant organism. Describe the three forms of horizontal gene transfer used in bacteria. Define the term mutation and distinguish between the different types. Explain the importance of restriction endonucleases to genetic engineering. Describe in detail how to clone a gene into a bacterium and gain a desired protein 2004 by Jones product and Bartlett Publishers

3 Vocabulary Genetics Genome of cells vs. genome of viruses Genes, 3 categories of genes - Structural genes: - Genes that code for - Regulatory genes: control gene expression Chromosome Haploid vs. diploid Base pairs Genetic code Genotype vs. Phenotype

4 Fig 8.1

5 Unit molecule:, composed of: DNA Code Unit molecules covalently linked to form a sugarphosphate backbone Phosphates linked to number 5 (five prime) carbon of sugar and to number 3 (three prime carbon) Compare to Fig 8.3

6 DNA Code cont. DNA is double helix associated with proteins Strands are held together by bonds between and Strands are antiparallel

7 The Bacterial DNA Mostly single circular chromosome Attached to plasma membrane Chromosome length 1mm (Cell length? ) DNA is supercoiled Number of genes in E. coli Extra-chromosomal bacterial DNA: (1-5% of chromosome size)

8 Flow of Genetic Information

9 DNA Replication Collaboration of ~ 30 enzymes. DNA polymerase initiated by RNA primer bidirectional leading strand: continuous DNA synthesis lagging strand: discontinuous DNA synthesis Okazaki fragments semiconservative 2

10 Elongation and Termination of the Daughter Molecules Speed can be 750 bases per second DNA pol I removes RNA primers and replaces them with DNA. Ligases move along the lagging strand to.. Mistakes in DNA replication happen ~ every 10 8 to 10 9 bases, but most corrected by DNA polymerase III.

11 Replication fork Replication in 5' 3' direction

12 Compare to Fig 8.4

13 A wide variety of RNAs are used to regulate gene function Transcription produces 3 types of RNA (?) Enzyme necessary? Promoters and terminators Protein Synthesis Translation produces the protein Sense codons vs. nonsense codons Anticodons Exceptions to this pattern: - RNA viruses convert RNA to other RNA - Retroviruses convert RNA to DNA

14 Fig. 8.5 Also: Primer RNAs in both bacterial and eukaryotic cells Ribozymes: remove unneeded sequences from other RNAs

15 Genetic code: universal and degenerate/redundant Codons: groups of three nucleotides determining the amino acid Advantage of redundancy and wobble position?

16 More Details on Transcription RNA polymerase binds to promotor sequence proceeds in 5' 3' direction stops when it reaches terminator sequence Fig 8.7 Fig 8.7

17 After Transcription: Translation Ribosomes of prokaryotes and eukaryotes differ in size - Bacteria: 70S (50S and 30S subunits) - Eukaryotes: 80S (60S and 40S subunits) Small subunit binds to 5 end of mrna. Large subunit supplies enzymes for making peptide bonds.

18 More Details on Translation Nucleotide sequence of mrna is translated into amino acid sequence of protein using three letter words = codons Translation of mrna begins at the start codon: Translation ends at a stop codon: UAA, UAG, UGA Requires various accessory molecules and 3 major components:? In Prokaryotes: Simultaneous transcription and translation Polyribosomes

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20 The Translation Process in Protein Synthesis

21 Simultaneous Transcription and Translation in Prokaryotes

22 Differences Between Eukaryotic and Bacterial Transcription and Translation Characteristic Bacteria Eukaryotes Start codon Always AUG AUG, but codes for a different form of methionine mrna Transcription and translation: Genes Can code for several genes in a series Occur simultaneously in the cytoplasm Exist as an uninterrupted set of triplets coding for a protein Only codes for one protein Transcription occurs in the nucleus, translation occurs in the cytoplasm Contain introns that do not code for proteins and exons that do code for proteins. Introns must be edited out.

23 Genetic Regulation of Protein Synthesis Genes are only expressed when needed Example of regulation of gene expression in bacteria: Operons: Set of genes regulated as a single unit Inducible operons for catabolic enzymes. Induced by the substrate of the enzyme(s) for which the structural genes code Repressible operons for anabolic enzymes. Operon turned off by product. Lac Operon

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25 DNA Recombination Bacteria have no sexual reproduction. Horizontal gene transfer allows for DNA recombination Recombinant: Any organism containing genes that originated in another organism Allows for rapid spreading of genes for drug resistance and exotoxins...

26 Flow of Genetic Information Horizontal Gene Transfer: Any DNA transfer that results in organisms acquiring new genes that did not come from parent organisms.

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28 Conjugation Plasmid and chromosomal DNA transfer via direct cell to cell contact Gram-negative conjugation: F + = donor cell. Contains F plasmid (factor) and produces conjugation (F) pilus (aka sex pilus ) Recipient cell (F ) becomes F + In some cells F factor integrates into chromosome Hfr cell E.g.: R plasmids Fig 8.11

29 Gram-positive conjugation: Opening created between two adjacent cells Replicated DNA passes from one cell to another Fig 8.11 Conjugation is a conservative process. I.e.: Donor bacterium retains (conserves) copy of genetic material being transferred.

30 Transformation - Capturing DNA from Solution - DNA released by lysed cell breaks into fragments accepted by recipient cell Facilitated by DNA-binding proteins on cell wall Competent cells are capable of accepting DNA Adapted for use in recombinant DNA technology

31 Transduction DNA Transfer from donor to recipient cell with help of bacteriophage (= transducing phage) Generalized vs. specialized transduction Many exotoxins Compare to Fig 8.12

32 Mutations: Changes in the Genetic Code Driving force of evolution May be neutral, beneficial, or harmful Wild type vs. mutant Spontaneous mutations: Occur in the absence of a mutagen Mutagen: Physical or chemical agents inducing mutations. (E.g.: UV light, X rays, nitrous acid) Types of Mutations: 1. Point mutation = base substitution (silent, missense, nonsense, readthrough) 2. Frameshift mutation = Insertion or deletion of one or more nucleotide pairs

33 What type of mutation? 1. Nonsense mutation 2. Missense mutation 3. Silent mutation 4. Point mutation 5. Frameshift mutation

34 Various Point Mutations Missense Compare to Table 8.8 Silent Nonsense TAA

35 Radiation as a Mutagen 1. Ionizing radiation ( and ) Formation of highly reactive radicals and ions that damage nucleotides mutations. Ds breaks of covalant bonds in backbone deletion mutations 2. UV rays lead to

36 Chemical Mutagens examples: 1. Nucleoside (base) analogs have altered basepairing properties. They can be randomly incorporated into growing cells (cancer drugs) only used by viral enzymes (e.g. AZT) 2. Frameshift mutagens such as intercalating agents (e.g.:, aflatoxin, ethidium bromide)

37 Intercalation Distortion due to intercalating agent will lead to one or more base-pairs inserted or deleted during replication. Potent carcinogens!

38 Repair of Mutations DNA Pol has proofreading capacity DNA repair of replication mistakes The cell has additional systems for finding and repairing DNA that has been damaged. Photolyases for UV damage repair. Light repair enzymes separate thymine dimers using energy from visible light Nucleotide excision repair repairs all mutations Compare to Fig 8.15

39 Genetic Engineering Manipulation and change of the genome using biotechnology Restriction Endonucleases (REs) = Molecular scissors Recognize and clip at palindromes specific cuts! Bunt ends vs. sticky ends Destroy bacteriophage DNA in bacterial cells

40 Restriction Endonuclease, aka Restriction Enzymes Staggered symmetrical cuts leave short tails called sticky ends Site of cleavage EcoRI Adhesive tails will basepair with complementary tails on other DNA fragments or plasmids Restriction fragments: pieces of DNA produced by restriction endonucleases

41 Role of Restriction Enzymes in Making Recombinant DNA Molecules Compare to Fig 8.16

42 Additional Important Enzymes Ligase, for Genetic Engineering seals (ligates) sticky ends together used in final step of splicing genes into plasmids and chromosomes Reverse transcriptase - Role in nature? - Converts RNA into DNA to make cdna cdna - Made from mrna, trna, and rrna - Used to synthesize eukaryotic genes from mrna transcripts. Advantage vs. using DNA directly?

43 Recombinant DNA Technology Intent to remove DNA from one organism and combine it with that of a different organism Bacteria are genetically engineered to mass produce: Hormones Enzymes Vaccines

44 Vectors Also known as cloning vectors. Must be Small and easy to manipulate. & serve as vectors. self-replicating large quantities When they carry insert : = Recombinant DNA molecules Introduce foreign DNA (desired gene) into host cells Shuttle vectors can exist in several different species.

45 ... One of most commonly used vectors: Compare to Fig 8.18 Gene Cloning Requires 2 Main Ingredients how do you get this?

46 Various Ways of Obtaining Gene of Interest 1. DNA removed from cells and separated into fragments by REs 2. Gene synthesized from isolated mrna transcripts using RT 3. Gene amplified using PCR (See lab) Recombinant vector is then inserted into host cell

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48 Blue and White Screening Method for Selecting a Clone (or Recombinant DNA Molecule) Direct selection of engineered vector via antibiotic-resistance markers (ampr) on plasmid vectors. Vector also contains -galactosidase gene for blue-white screening Desired gene is inserted into the -galactosidase gene site gene inactivated Not in book

49 Three possible outcomes: 1. Bacteria lack vector 2. Bacterial clones contain vector without the new gene colony type? 3. Bacterial clones contain recombinant vector resistant to Ampicillin and unable to hydrolyze X-gal colony type? 1) Plasmid cloning

50 2) Selecting Recombinant Bacteria

51 Which type of colonies do you want? a)white b)blue c) I don t want any

52 Making a Gene Product E. coli: prokaryotic workhorse of biotech. Easily grown and genomics well understood. Disadvantage: Cells must be lysed to get product release of Yeast: Saccharomyces cerevisiae is eukaryotic workhorse of biotechnology. Advantage: Continuous secretion of gene product. Mammalian cells: May express eukaryotic genes easily. Disadvantage: Harder to grow. Plant cells: Easy to grow. May express eukaryotic genes easily.

53 Some Therapeutic Applications of Recombinant DNA Technology 1. Pharmaceutical applications, e.g.: Insulin production 2. Subunit vaccines 3. DNA vaccines 4. Gene therapy to replace defective or missing genes

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55 PCR and Gel Electrophoresis covered in lab Who will present? Case File: A Body Attacking Itself Inside the Clinic: Using Recombinant DNA to Produce Insulin (covered by teacher)