Channarong Rodkhum D.V.M. (Hons), Ph.D. Department of Veterinary Microbiology Faculty of Veterinary Science Chulalongkorn University

Outline A Brief History of Bacterial Genetics Chromosome Structure, Replication, and Segregation Gene Expression and It s regulation DNA repair and Mutagenesis Plasmids Transposon Recombinant DNA Technology Sequences Alignment and Phylogenetic Analysis

A Brief PART History 1 of Bacterial Genetics

Bacterial Genetics Bacteria are haploid : They have only one copy or allele of each gene. Short generation times Asexual reproduction Genetic exchange by transformation, conjugation and transduction

Phenotypic properties of Bacteria and Archaea compared with Eukarya. Property Biological Domain Eukarya Bacteria Archaea Cell configuration eukaryotic procaryotic procaryotic Nuclear membrane present absent absent Number of chromosomes >1 1 1 Chromosome topology linear circular circular Murein in cell wall - + - Cell membrane lipids ester-linked glycerides; unbranched; polyunsaturated ester-linked glycerides; unbranched; saturated or monounsaturated ether-linked branched; saturated Cell membrane sterols present absent absent Organelles (mitochondria and chloroplasts) present absent absent Ribosome size 80S (cytoplasmic) 70S 70S Cytoplasmic streaming + - - Meiosis and mitosis present absent absent Transcription and translation coupled - + + Amino acid initiating protein synthesis methionine N-formyl methionine methionine Protein synthesis inhibited by streptomycin and chloramphenicol - + - Protein synthesis inhibited by diphtheria toxin + - +

Transformation Recipient cell uptake of free DNA released into the environment when a bacterial cell dies and undergoes lysis. Certain bacteria are able to take up this free DNA, that is, are able to undergoes transformation. Such Bacteria are called competent cell.

Conjugation This process occurs between two living cells. Intercellular conatact by Sex Pilus (not in all bacteria) Transfer plasmid, transposable elements and chromosome.

Transduction This process is mediated by viruses that infect bacteria (bacteriophage) These viruses integrate their DNA into the bacterial chromosome. Viruses replicate within the bacterial cells. The viruses are released when the infected bacterial cell lyses and then infect new bacterial cell.

Transduction

PART 1 Chromosome Structure, Replication, and segregation

Bacterial Gene and Chromosome Gene is a DNA sequence that encodes for a specific product (RNA or Protein). Thounsands of genes taken together within and organism comprise that organism s genome. The genome is organized into discrete elements know as chromosome

Nucleic acids (I) DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid) DNA is unbranched DNA consist of four subunits (Deoxyribonucleotides) Adenine (A) Cytosine (C) Guanine (G) Thymine (T) [ for RNA = Uracil (U)] Sununits consist of : Base (A,C,G,T) + deoxyribose + phosphate Linked by covalent phosphodiester bonds between 5 C One deoxyribose and 3 C of other deoxyribose.

Nucleic acids (II) DNA has a free 3 end and free 5 end. DNA is helical, double stranded molecule 1953, Watson & Click proposed the double helix model Sugar phosphate backbone outside, bases inside Bases of the 2 strands linked together with hydrogen bonds A+T : 2 hydrogen bonds G+C : 3 hydrogen bonds Gene are made of DNA

DNA Structure

DNA Replication ( I ) DNA replicates each cell division Two strands dissociate and act as template for new strands Two duplexs with one new and one old strand Semiconservative replication DNA replication fork, Okazaki fragments Energy provided by release of pyrophosphate 500 nucleotides/sec in bacteria, 50 nucleotides/sec in mammals

DNA Replication ( II ) Replication starts with polymerization of short RNA primers Helicases open up the DNA double helix Single-strand-binding protein (SSB) proteins keep the DNA single stranded Mismatch repair or mismatch proofreading Recognition base on methylation in prokaryotes Replication start at replication origin Raplication initiation is major point of control

DNA Replication ( III ) Replication is very accurate One mistake every 10 9 basepairs Change in the DNA sequence is called mutation DNA sequence determines the amino acid sequence of a protein Two duplexs with one new and one old strand DNA is very stable and inert DNA is transcriped into RNA

Okazaki fragments DNA can t start polymerisation from scratch. It needs a free 3 OH group to start with. Pimase enzyme is used to synthesize a short RNA strand which serves as a primer for DNA synthesis. The leading strand only needs one RNA primer, however the lagging strand which is synthesized discontiuously needs new primers all the time. A relatively short fragments (Okazaki fragments) are created on the lagging strand which later in the process are ligated together by DNA ligase.

Replication of Bacterial Chromosome and Cell Division

Replication Errors Replication is very accurate One mistake every 10 9 basepairs Change in the DNA sequence is called mutation

Supercoiling Condensed bacterial DNA is called the bacterial nucleoid. DNA supercoiling in the nucleoid. Positively supercoiled and Negatively supercoiled. Enzymes involved in this process is Topoisomerases.

Bacterial nucleoid

PART 1 Gene expression and it s regulation

Central Dogma

Transcription One strand of the DNA double helix is used as a template RNA strand released Not all genes are express all the time Depends on gene regulatory proteins RNA polymerase copies DNA into RNA (Transcription) RNA polymerase is a large subunit protein : Binds randomly to DNA Binds strongly to a promoter sequence

Transcription Promoter contains starts signal TATAAT Box (-10) TTGACA Box (-35) RNA polymerase stops at termination or stop signal RNA polymerase polymerizes from 5 to 3

Transcription

Differences between initiation of transcription in procaryotes and eucaryotes Procaryote 1 RNA polymerase Eucaryote 3 RNA polymerase (I,II and III) RNA polymerase can initiate transcription by itself RNA polymerase needs a large number of general transcription factors genes often have a single regulatory sequence genes can be regulated by DNA sequences thousands of basepairs away

RNA (Ribonucleic acid) Four bases [Thymine (T) replaced by Uracil (U)] Ribose phosphate backbone Single stranded Three Types of RNA: Messenger RNA (mrna) : information Ribosonal RNA (rrna) : amino acids and specificity Transfer RNA (trna) : machinery and catalytic activity Released after transcription

Procaryotic mrna compared with eucaryotic mrna

trna

mrna binding site at ribosome

Translation ( I ) RNA is translated in groups of three nucleoties (codons) Reading frame is determined by beginning of sequence mrna is translated by trna trna contains an anti-codon that basepairs with a codon Ribosome consists of 50+ different proteins and rrnas Finds a start site on the mrna Moves along the mrna and translates the protein

Translation ( II ) trna molecules are adaptors. Each amino acid has at least one trna trna is specific for codon and brings correct amino acid to correct codon Aminoacyl-tRNA synthetases charge specific trnas with specific amino acid Aminoacyl trnas Protein production is catalysed on ribosomes.

Translation

Protein structure

The operon model Operon is a cluster of genes interacted together. Clusters of genes transcribed as a single mrna molecule are common in bacteria Many operons in bacteria Lac operon is a prototype of bacterial operon Regulatory sequence called the operator

PART 1 DNA repair and Mutagenesis

DNA repair ( I ) Accurate DNA replication, repair of DNA damage DNA damage : Chemical changed bases Reaction with mutagens/carcinogens Mismatches DNA damage : Problem during transcription Possible mutation Problem during transcription Wrong RNA Mutation rate : Extremaly low 1 mutation every 10 9 basepairs Average gene : 1 mutation every 10 6 cell generation

DNA repair ( II ) Rapaired by DNA repair systems (Mismatch repair system) Basic pathway : Recognition and removal Filling in Ligation of nick Two major pathways : Base excision repair Nucleotide excision repair DNA is easily repaired Extensive proofreading by DNA polymerase 3 5 exonuclease removes mismatch

Missense mutation

Nonesense mutation

Frameshift mutation

Plasmid and PART Transposable 1 elements

Plasmid : Extrachromosomal DNA Miniature chromosome Double-stranded, close, circilar structures with size ranges from 1-2 Kbp to 1 Mbp or more. The number of plasmids per bacterial cell varies extensively. Each plasmid is composed of several genes.

Transposable elements : Pieces of DNA that move from one genetic element to another, from plasmid to chromosome or vice versa. Unlike plasmids, they do not exist as separate entities Within the bacterial cell. They must either be incorporated into a plasmid or the chromosome. The two types of transposable elements are : - Insertion sequences (IS) - Transposon Insertion sequences contain genes that simply encode for information required for movement among plasmid and Chromosome. Transposons contain genes for moement as well as genes that encode for other features such as drug resistance.

Transposon Jumping genes Transposition is the process by which transposon excise from One genomic location and insert into another.

PART 1 Recombinat DNA Technology

Recombinant DNA technology comprises a large number of techniques. Most of this techniques are based On either one of the following basic techniques : Restriction/cutting/cleaving of DNA by restriction nuclease Sequencing of DNA fragments Nucleic acid hybridization DNA Cloning DNA engineering

Cleaving of DNA by Restriction endonuclease

Separation and detection of DNA molecules by size using gel electrophoresis

Sequencing ( I )

Sequencing ( II )

Nucleic acid hybridization

Polymerase Chain Reaction (PCR) [ I ]

Polymerase Chain Reaction (PCR) [ II ]

DNA Cloning ( I ) The formation of recombinant DNA molecules In vitro

DNA Cloning ( II )

DNA Cloning ( III )

Nucleotide Sequences and amino acid Sequences Alignment Alignment Multiple alignment Basic local alignment search tool : Computer software : - ClustalW algorithm - Genetyxt -etc.

Phylogenetic Analysis Clustering the sequences Computer program : - Mega4 - Bioedit/ClustalX