Microbial Genetics Nestor T. Hilvano, M.D., M.P.H.

Learning Objectives You should be able to: 1.Compare and contrast the genomes of prokaryotes and eukaryotes. 2.Describe the structure of DNA. 3.Describe the structure and function of plasmids. 4.State the central dogma of genetics and explain the roles of DNA and RNA in polypeptide synthesis. 5.Compare and contrast the steps in RNA transcription and the translation of polypeptides. 6.Describe three types of point mutations and their effects. 7.Describe types of mutagens and their effects. 8.Discuss DNA repair. 9.Define genetic recombination. 10.Contrast vertical gene transfer with horizontal gene transfer. 11.Define biotechnology and recombinant DNA technology, mention its applications. 12.Discuss the safety and ethics of recombinant DNA technology.

Structure and Replication of Genomes Genes – specific sequences of nucleotides that code for polypeptides Genomes - sum of all genetic material in a cell or virus Prokaryotic and eukaryotic cells – use DNA as genetic materials Viruses – use either DNA or RNA

Structure of Nucleic Acids DNA and RNA Bases 1.Purine = Adenine (A); Guanine (G) 2.Pyrimidine = Cytosine (C); Thymine (T); Uracil (U) Complimentary bases: A-T and G- C for DNA; A-U and G-C for RNA Chromosomes – double stranded; anti-parallel (5’-3’ and 3’-5’) histones not present in bacteria

Prokaryotic Genomes Single copies of 1 or 2 chromosomes - circular molecules of DNA - localized in region of cytoplasm called ____. a. nucleus b. nucleoid c. both d. neither Contain plasmids w/c function to - regulate bacterial conjugation; resistance to antimicrobial drugs, heavy metals, or toxins; destruction of competing bacteria; and pathogenicity

Eukaryotic Genomes Nuclear chromosomes in linear pair/s - contain proteins called histones - arranged as nucleosomes - form chromatin fibers Also contain extrachromosomal DNA in mitochondria, chloroplasts, and plasmids

Central Dogma of Genetics DNA Replication- Semiconservative; DNA polymerase and DNA ligase enzymes Transcription- Synthesis of mRNA (codons) from DNA template ; RNA polymerase; mRNA- Introns (nonsense codons) & exons (sense codons); Splicing of exons (mRNA-carrier of genetic information to manufacture polypeptide) Translation- interpretation of mRNA into the amino acid language of proteins

Translation mRNA to tRNA (polypeptides with specific amino acid sequences) rRNA (has A site; P site) on ribosomes, where A.A. are assembled into protein tRNA (anticodon)- chain of A.A.(polypeptide forming protein)

Mutations of Genes Change in the nucleotide sequence Point mutations: 1. Substitutions 2. Frameshift Insertion or Frameshift Deletion Effects of Substitution: a.) silent – no change b.) missense – different A.A. sequence c.) nonsense – polypeptide synthesis stops Effects of frameshift insertion and frameshift deletion – major difference in A.A. sequence

Mutagens Cancer causing agents other than viruses: Radiation – ionizing radiation (X-rays); nonionizing radiation (UV light) Nucleotide analogs - 2 amino adenine; 5 bromo- uracil; azidothymine (AZT) Aflatoxins (Aspergilus flavus) - causing missense mutation and liver CA Acridine orange in dye - frameshift (base insertion) Nitrous acid – base substitution

Genetic Recombination Exchange of nucleotide sequences Vertical gene transfer – transmission of genes from parents to offspring Horizontal gene transfer – DNA from donor cell is transmitted to a recipient cell 1. transformation = competent recipient cell takes up DNA from the environment (in lab) 2. transduction = virus (bacteriophage) 3. conjugation = bacterial sex; gram – (pili); gram + (direct contact)

Recombinant DNA Technology and Biotechnology Recombinant DNA technology – process of modifying genomes of organisms ex. insulin production of E. coli 3 main goals of Recombinant DNA technology 1.To eliminate undesirable phenotypic traits 2.To combine beneficial traits of organisms to create valuable new organisms 3.To create organisms that synthesize products that human need

Medical Applications Protein synthesis – inserted genes for insulin synthesis Vaccines – hepatitis B vaccine Genetic screening - for inherited forms of breast CA DNA fingerprinting – use gel electrophoresis and southern blotting to identify unique DNA sequences of individuals or organisms Gene therapy – missing or defective genes are replaced with normal genes (in SCID- Severe Combined Immunodeficiency) Medical diagnosis – use PCR, fluorescent genetic probes, and DNA microassays in diagnostic applications Cloning – nuclear transfer Stem cell research – adult; embryonic

Agricultural Applications Herbicide resistance Salt tolerance Freeze resistance Pest resistance Improvements in nutritional value and yield

Ethics and Safety of Recombinant DNA Technology Concerns over accidental release of altered organisms into the environment Ethics of altering animals for human use Potential for creating genetically modified biological weapons Unknown long-term effects and unforeseen problems Other ethical issues – rights to screen people for genetic disease; rights to privacy and confidentiality of genetic data

Homework 1.Define terms – frameshift mutation, transcription, translation, genes, genome, plasmid, semiconservative, codon, anticodon, mutagens, recombinant DNA technology 2.Describe the central dogma of genetics. 3.List 3 medical applications and 2 agricultural applications of recombinant DNA technology. 4.List the uses of DNA fingerprinting. 5.Differentiate vertical gene transfer from horizontal gen transfer. 6.List the 3 main goals of recombinant DNA technology.