Chapter 9 Microbial Genetics Genetics and Genes Genetics

Chapter 9 Microbial Genetics

Genetics and Genes §Genetics §The – the study of heredity science of genetics explores: § transmission of biological traits from parent to offspring § expression and variation of those traits § structure and function of genetic material § how this material changes § the study of genetics takes place on several levels 2

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Levels of Structure and Function of the Genome §Genome – sum total of genetic material of an organism (chromosomes + mitochondria/chloroplasts and/or plasmids) § genome of cells – DNA § genome of viruses – DNA or RNA §DNA complexed with protein constitutes the genetic material as chromosomes §Bacterial chromosomes are a single circular loop §Eucaryotic chromosomes are multiple and linear 4

The Genetic Material §Chromosome is subdivided into genes, the fundamental unit of heredity responsible for a given trait § site on the chromosome that provides information for a certain cell function § segment of DNA that contains the necessary code to make a protein or RNA molecule §Three basic categories of genes: § genes that code for proteins - structural genes § genes that code for RNA § genes that control gene expression - regulatory genes 5

The Genetic Material §All types of genes constitute the genetic makeup – genotype §The expression of the genotype creates observable traits – phenotype 6

Genomes Vary in Size §Smallest virus – 4 -5 genes §E. coli – single chromosome containing 4, 288 genes; 1 mm; 1, 000 X longer than cell §Human cell – 46 chromosomes containing 31, 000 genes; 6 feet; 180, 000 X longer than cell 7

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DNA §Two strands twisted into a helix §Basic unit of DNA structure is a nucleotide §Each nucleotide consists of 3 parts: a 5 carbon sugar - deoxyribose § a phosphate group § a nitrogenous base – adenine, guanine, thymine, cytosine § §Nucleotides covalently bond to form a sugar-phosphate linkage – the backbone § each sugar attaches to two phosphates – § 5′ carbon and 3′ carbon 10

DNA §Nitrogenous bases covalently bond to the 1′ carbon of each sugar and span the center of the molecule to pair with an appropriate complementary base on the other strand § adenine binds to thymine with 2 hydrogen bonds § guanine binds to cytosine with 3 hydrogen bonds §Antiparallel strands 3′ to 5′ and 5′ to 3′ §Each strand provides a template for the exact copying of a new strand §Order of bases constitutes the DNA code 11

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Significance of DNA Structure §Maintenance of code during reproduction - constancy of base pairing guarantees that the code will be retained §Providing variety - order of bases responsible for unique qualities of each organism 13

DNA Replication §Making an exact duplicate of the DNA involves 30 different enzymes §Begins at an origin of replication §Helicase unwinds and unzips the DNA double helix §An RNA primer is synthesized by primase §DNA polymerase III adds nucleotides in a 5′ to 3′ direction leading strand – synthesized continuously in 5′ to 3′ direction § lagging strand – synthesized 5′ to 3′ in short segments; overall direction is 3′ to 5′ § 14

§DNA polymerase I removes the RNA primers and replaces them with DNA §When replication forks meet, ligases link the DNA fragments along the lagging strand to complete the synthesis §Separation of the daughter molecules is complete 15

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§DNA replication is semiconservative because each chromosome ends up with one new strand of DNA and one old strand 19

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Applications of the DNA code §Information stored on the DNA molecule is conveyed to RNA molecules through the process of transcription §The information contained in the RNA molecule is then used to produce proteins in the process of translation 21

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Gene-Protein Connection §Each triplet of nucleotides on the RNA specifies a particular amino acid §A protein’s primary structure determines its shape and function §Proteins determine phenotype. Living things are what their proteins make them §DNA is mainly a blueprint that tells the cell which kinds of proteins to make and how to make them 23

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RNAs §Single-stranded molecule made of nucleotides § 5 carbon sugar is ribose § 4 nitrogen bases – adenine, uracil, guanine, cytosine § phosphate 25

RNAs § 3 types of RNA: § messenger RNA (m. RNA) – carries DNA message through complementary copy; message is in triplets called codons § transfer RNA (t. RNA) – made from DNA; secondary structure creates loops; bottom loop exposes a triplet of nucleotides called anticodon which designates specificity and complements m. RNA; carries specific amino acids to ribosomes § ribosomal RNA (r. RNA) – component of ribosomes where protein synthesis occurs 26

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Transcription §RNA polymerase binds to promoter region upstream of the gene §RNA polymerase adds nucleotides complementary to the template strand of a segment of DNA in the 5′ to 3′ direction §Uracil is placed as adenine’s complement §At termination, RNA polymerase recognizes signals and releases the transcript § 100 -1, 200 bases long 29

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Translation §Ribosomes assemble on the 5′ end of a m. RNA transcript §Ribosome scans the m. RNA until it reaches the start codon, usually AUG §A t. RNA molecule with the complementary anticodon and methionine amino acid enters the P site of the ribosome and binds to the m. RNA 31

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Translation Elongation §A second t. RNA with the complementary anticodon fills the A site §A peptide bond is formed §The first t. RNA is released and the ribosome slides down to the next codon §Another §This t. RNA fills the A site and a peptide bond is formed process continues until a stop codon is encountered 33

Translation Termination §Termination codons – UAA, UAG, and UGA – are codons for which there is no corresponding t. RNA §When this codon is reached, the ribosome falls off and the last t. RNA is removed from the polypeptide 34

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The Master Genetic Code §Represented by the m. RNA codons and the amino acids they specify §Code is universal – prokaryotes, eukaryotes or viruses §Code is redundant – a particular amino acid can be coded for by more than a single codon. So one cannot predict (backwards) from protein structure what the exact m. RNA codons are 36

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Polyribosomal complex allows for the synthesis of many protein molecules simultaneously from the same m. RNA molecule 39

Eucaryotic Transcription and Translation §Do not occur simultaneously – transcription occurs in the nucleus and translation occurs in the cytoplasm §Eucaryotic start codon is AUG, but it does not use formyl- methionine §Eucaryotic m. RNA encodes a single protein, unlike bacterial m. RNA which encodes many §Eucaryotic DNA contains introns – intervening sequences of noncoding DNA- which have to be spliced out of the final m. RNA transcript 40

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Genetics of Animal Viruses §Viral genome - one or more pieces of DNA or RNA; contains only genes needed for production of new viruses §Requires access to host cell’s genetics and metabolic machinery to instruct the host cell to synthesize new viral particles 42

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Regulation of Protein Synthesis and Metabolism §Genes are regulated to be active only when their products are required §In procaryotes this regulation is coordinated by operons, a set of genes, all of which are regulated as a single unit 45

Operons § 2 types of operons: § inducible – operon is turned ON by substrate: catabolic operonsenzymes needed to metabolize a nutrient are produced when needed § repressible – genes in a series are turned OFF by the product synthesized; anabolic operon – enzymes used to synthesize an amino acid stop being produced when they are not needed 46

Lactose Operon: Inducible Operon §Made of 3 segments: §Regulator§Control gene that codes for repressor locus- composed of promoter and operator §Structural locus- made of 3 genes each coding for an enzyme needed to catabolize lactose – § § § β-galactosidase – hydolyzes lactose permease - brings lactose across cell membrane β -galactosidase transacetylase – uncertain function 47

Lac Operon §Normally § In the absence of lactose, the repressor binds with the operator locus and blocks transcription of downstream structural genes §Lactose § off turns the operon on Binding of lactose to the repressor protein changes its shape and causes it to fall off the operator. RNA polymerase can bind to the promoter. Structural genes are transcribed 48

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Arginine Operon: Repressible §Normally on and will be turned off when nutrient is no longer needed §When excess arginine is present, it binds to the repressor and changes it. Then the repressor binds to the operator and blocks arginine synthesis 50

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Antibiotics That Affect Transcription and Translation §Rifamycin – binds to RNA polymerase §Actinomycin D - binds to DNA and halts m. RNA chain elongation §Erythromycin and spectinomycin – interfere with attachment of m. RNA to ribosomes §Chloramphenicol, linomycin and ribosome and block elongation §Streptomycin tetracycline-bind to – inhibits peptide initiation and elongation 52

Mutations: Changes in the Genetic Code §A change in phenotype due to a change in genotype (nitrogen base sequence of DNA) is called a mutation §A natural, nonmutated characteristic is known as a wild type (wild strain) §An organism that has a mutation is a mutant strain, showing variance in morphology, nutritional characteristics, genetic control mechanisms, resistance to chemicals, etc. 53

Causes of Mutations §Spontaneous mutations– random change in the DNA due to errors in replication that occur without known cause §Induced mutations – result from exposure to known mutagens, physical (primarily radiation) or chemical agents that interact with DNA in a disruptive manner 54

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Categories of Mutations §Point mutation – addition, deletion or substitution of a few bases §Missense mutation – causes change in a single amino acid §Nonsense mutation – changes a normal codon into a stop codon §Silent mutation – alters a base but does not change the amino acid §Back-mutation – when a mutated gene reverses to its original base composition §Frameshift mutation – when the reading frame of the m. RNA is altered by the addition or deletion of nucleotides in a newly synthesized DNA 56

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Repair of Mutations §Since mutations can be potentially fatal, the cell has several enzymatic repair mechanisms in place to find and repair damaged DNA § DNA polymerase – proofreads nucleotides during DNA replication § mismatch repair – locates and repairs mismatched nitrogen bases that were not repaired by DNA polymerase § light repair – for UV light damage § excision repair – locates and repairs incorrect sequence by removing a segment of the DNA and then adding the correct nucleotides 58

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The Ames Test §Any compound known to be mutagenic is considered to be carcinogenic §Agricultural, industrial, and medicinal compounds are screened using the Ames test §Indicator organism is a mutant strain of Salmonella typhimurium that has lost the ability to synthesize histidine §This mutation is highly susceptible to back-mutation 60

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Positive and Negative Effects Of Mutations §Mutations leading to nonfunctional proteins are harmful, possibly fatal §Organisms with mutations that are beneficial in their environment can readily adapt, survive, and reproduce – these mutations are the basis of change in populations §Any change that confers an advantage during selection pressure will be retained by the population 62

DNA Recombination Events §Genetic recombination – occurs when an organism acquires and expresses genes that originated in another organism § 3 means for genetic recombination in bacteria: § conjugation § transformation § transduction 63

Conjugation §Conjugation – transfer of a plasmid or chromosomal fragment from a donor cell to a recipient cell via a direct connection § Gram-negative cell donor has a fertility plasmid (F plasmid, F′ factor) that allows the synthesis of a conjugation (sex) pilus § recipient cell is a related species or genus without a fertility plasmid § donor transfers fertility plasmid to recipient through pilus §High-frequency recombination – donor’s fertility plasmid has been integrated into the bacterial chromosome §When conjugation occurs, a portion of the chromosome and a portion of the fertility plasmid are transferred to the recipient 64

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Transformation §Transformation – chromosome fragments from a lysed cell are accepted by a recipient cell; the genetic code of the DNA fragment is acquired by the recipient §donor and recipient cells can be unrelated §useful tool in recombinant DNA technology 67

Insert figure 9. 23 transformation 68

Transduction §Transduction – bacteriophage serves as a carrier of DNA from a donor cell to a recipient cell §Two types: § generalized transduction – random fragments of disintegrating host DNA are picked up by the phage during assembly; any gene can be transmitted this way § specialized transduction – a highly specific part of the host genome is regularly incorporated into the virus 69

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Transposons §Special DNA segments that have the capability of moving from one location in the genome to another – “jumping genes” §Cause rearrangement of the genetic material §Can move from one chromosome site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome §May be beneficial or harmful 73

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