Ch 8

Microbial SLOs • Define the terms and , and differentiate between genotype and phenotype. • Draw a detailed segment of DNA. • Summarize the steps of bacterial DNA replication, and identify the used in this process. • Compare and contrast the synthesis of leading and lagging strands. • Provide an overview of the relationship among DNA, RNA, and . • 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 . • Define , 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 and distinguish between the different types. • Explain the importance of restriction endonucleases to . • Describe in detail how to clone a gene into a bacterium and gain a desired © 2004 by Jones product and Bartlett Publishers

Vocabulary Genetics Genome of cells vs. genome of , 3 categories of genes - Structural genes: ______- Genes that code for ______- Regulatory genes: control Haploid vs. diploid Base pairs  Genotype vs. Phenotype Fig 8.1 Unit molecule: DNA Code ______, composed of:

Unit molecules covalently linked to form a sugar- phosphate backbone

Phosphates linked to number 5’ (five prime) carbon of sugar and to number 3’ (three prime carbon) Compare to Fig 8.3

DNA Code cont.

DNA is double helix associated with proteins

Strands are held together by ___ bonds between ____ and ____

Strands are antiparallel

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) Flow of Genetic Information DNA Replication Collaboration of ~ 30 enzymes. DNA polymerase initiated by RNA primer bidirectional leading strand: continuous DNA synthesis lagging strand: 2 discontinuous DNA synthesis  Okazaki fragments semiconservative 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 108 to 109 bases, but most corrected by DNA polymerase III.

Replication fork Replication in 5'  3' direction Compare to Fig 8.4 Protein Synthesis Transcription  produces 3 types of RNA (?)  necessary ?  Promoters and terminators 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 A wide variety of are used to regulate gene function Fig. 8.5

Also: Primer RNAs in both bacterial and eukaryotic cells

Ribozymes: remove unneeded sequences from other RNAs Genetic code: universal and degenerate/redundant Codons: groups of three determining the amino acid Advantage of redundancy and wobble position?

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 After Transcription: Translation of 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.

More Details on Translation

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

The Translation Process in Protein Synthesis Simultaneous Transcription and Translation in Prokaryotes 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 Can code for several Only codes for one genes in a series protein Transcription and Occur simultaneously Transcription occurs in translation: in the cytoplasm the nucleus, translation occurs in the cytoplasm Genes Exist as an Contain introns that uninterrupted set of do not code for triplets coding for a proteins and exons protein that do code for proteins. Introns must be edited out. Genetic Regulation of Protein Synthesis • Genes are only expressed when needed Example of regulation of gene expression in bacteria: • : 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

DNA Recombination – Bacteria have no sexual . – 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.... Flow of Genetic Information

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

Conjugation 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

Fig 8.11 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. 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

Transduction

DNA Transfer from donor to recipient cell with help of (= transducing phage) Generalized vs. specialized Many exotoxins Compare to Fig 8.12 : 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 What type of mutation?

1. Nonsense mutation 2. Missense mutation 3. Silent mutation 4. Point mutation 5. Frameshift mutation Various Point Mutations

Compare to Table 8.8 Missense

Nonsense TAA Silent 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 ______

Chemical Mutagens examples: 1. Nucleoside (base) analogs have altered base- pairing 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) Intercalation

Distortion due to intercalating agent will lead to one or more base-pairs inserted or deleted during replication.

Potent carcinogens! 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 Genetic Engineering Manipulation and change of the genome using

Restriction Endonucleases (REs) = Molecular scissors  Recognize and clip at palindromes  specific cuts!  Bunt ends vs. sticky ends

 Destroy bacteriophage DNA in bacterial cells

Restriction Endonuclease, aka Restriction Enzymes Site of cleavage Staggered symmetrical EcoRI cuts leave short tails called “sticky ends” Adhesive tails will base- pair with complementary tails on other DNA fragments or plasmids

Restriction fragments: pieces of DNA produced by restriction endonucleases Role of Restriction Enzymes in Making Recombinant DNA Molecules

Compare to Fig 8.16 Additional Important Enzymes for Genetic Engineering • Ligase, – seals (ligates) sticky ends together – used in final step of splicing genes into plasmids and • 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?

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 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.

... One of most commonly used vectors:

Gene Cloning Requires 2 Main Ingredients 1. ______Compare to Fig 8.18 2. ______how do you get this? 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

Blue and White Screening Method Not in book 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 Three possible outcomes: 1) Plasmid cloning 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? ______2) Selecting Recombinant Bacteria Which type of colonies do you want? a)White b)Blue c)I don’t want any Making a Gene Product E. coli: prokaryotic workhorse of biotech. Easily grown and well understood. Disadvantage: Cells must be lysed to get product  release of ______

Yeast: 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. Some Therapeutic Applications of Recombinant DNA Technology

1. Pharmaceutical applications, e.g.: production 2. Subunit vaccines 3. DNA vaccines 4. Gene therapy to replace defective or missing genes

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)