Microbial genetics - Quia

Microbial genetics - Quia

Bacterial Genetics Overview Two general mechanisms of genetic change in bacteria: Mutation - alteration in existing DNA sequence Spontaneous Induced (caused by mutagens) DNA transfer - acquisition of DNA from another source

Overview Two general mechanisms of genetic change in bacteria: Mutation - alteration in existing DNA sequence Spontaneous Induced (caused by mutagens) DNA transfer - acquisition of DNA from another source Why study bacterial genetics? Model system Spontaneous mutations occur in all cells at a very low frequency (one

per billion nucleotides) Bacteria quickly grow to high concentrations (109/ml) in culture, making it possible to study rare occurrences Test chemicals for potential carcinogens Understand bacterial adaptation Resistance to antimicrobial drugs Agency Urges Change in Antibiotics for Gonorrhea

By LAWRENCE K. ALTMAN Published: NY Times April 13, 2007 The rates of drug-resistant gonorrhea in the United States have increased so greatly in the last five years that doctors should now treat the infection with a different class of antibiotics, the last line of defense for the sexually transmitted disease, officials said yesterday.. No new antibiotics for gonorrhea are in the pipeline, officials of the

centers told reporters by telephone. Now we are down to one class of drugs, said Dr. Gail Bolan, an expert in sexually transmitted diseases at the California Department of Health Services. Thats a very perilous situation to be in. Overview Two general mechanisms of genetic change in bacteria: Mutation - alteration in existing DNA sequence

Spontaneous Induced (caused by mutagens) DNA transfer - acquisition of DNA from another source Why study bacterial genetics? Model system Spontaneous mutations occur in all cells at a very low frequency (one per billion nucleotides) Bacteria quickly grow to high concentrations (109/ml) in culture, making it possible to study rare occurrences

Some mutagens are carcinogens Understand bacterial adaptation Resistance to antimicrobial drugs Acquisition of disease-causing traits Terms Phenotype Genotype

Terms Phenotype - the observable characteristics of an organism Genotype - the sequence of nucleotides in the DNA of an organism Wild type - characteristics similar to the organism as it occurs in nature. Prototroph - requires the same nutrients as the wild type. Auxotroph - a strain that has lost the ability to synthesize a specific compound; as a consequence, that compound must be supplied as a nutrient in the growth medium.

Prototroph disrupt gene required for histidine synthesis When studying mutations, you only see what you look for His- auxotroph

Part I Mutation How mutations occur, and their consequences How cells can repair errors/damage How we can select (and therefore, study) mutants Spontaneous Mutation Mistakes during replication

Base substitution TGT cysteine Silent mutation Missense mutation Nonsense mutation

TGC cysteine TGG tryptophan TGA Stop codon

No consequence Consequence varies Truncated protein; generally non-functional Spontaneous Mutation

Mistakes during replication Base substitution Removal or addition of nucleotides TGTTTGACCTAGGT Spontaneous Mutation Mistakes during replication Base substitution Removal or addition of nucleotides

TGTTTGACCTAGGT TGT TTG ACC TAG GT TGTTGACCTAGGT TGT TGA CCT AGG T Frameshift mutation Generates an entirely different set of triplets

Often, a stop codon is generated Spontaneous Mutation Mistakes during replication Base substitution Removal or addition of nucleotides Spontaneous Mutation Transposons jumping genes

Insertional inactivation of the gene in which the transposon lands A transposon can insert elsewhere in the same DNA molecule, or into an entirely different DNA molecule Some transposons simply hop; others replicate then hop Summary Mutations spontaneous

mistakes during replication base substitution addition/removal of nucleotides transposable elements induced Induced Mutation Chemical mutagens (potential carcinogens) Chemicals that modify purines and pyrimidines

Alter the base-pairing properties Induced Mutation Chemical mutagens Chemicals that modify purines and pyrimidines Alter the base-pairing properties Example: nitrous acid strips the amino group from nucleotides :G

:A Induced Mutation Chemical mutagens Chemicals that modify purines and pyrimidines Alter the base-pairing properties Example: nitrous acid strips the amino group from nucleotides Base analogs

Resemble nucleotide bases; erroneously incorporated into DNA Analog base-pairs with a different nucleotide T C Induced Mutation Chemical mutagens

Chemicals that modify purines and pyrimidines Alter the base-pairing properties Example: nitrous acid strips the amino group from nucleotides Base analogs Resemble nucleotide bases; erroneously incorporated into DNA Analog base-pairs with a different nucleotide Intercalating agents Insert between base-pairs, pushing nucleotides apart; extra nucleotide

may then be erroneously added during replication Induced Mutation Transposons Intentional use of an agent that naturally creates spontaneous mutations Induced Mutation

Radiation Ultraviolet irradiation Causes formation of covalent bonds (thymine dimers) between adjacent thymine bases Distorted DNA can be repaired, but the process (SOS repair) may introduce errors High doses are used to sterilize

surfaces, lower doses to introduce mutations X rays Causes double- and single-stranded breaks in DNA Summary Mutations spontaneous

mistakes during replication base substitution removal or addition of nucleotides transposable elements induced chemical mutagens radiation transposons

DNA Repair Repair of errors in base incorporation DNA polymerase proofreading Mismatch repair excision/replacement Repair of thymine dimmers

DNA Repair Repair of errors in base incorporation DNA polymerase proofreading Mismatch repair excision/replacement Repair of thymine dimmers Light reactivation (photorepair)

DNA Repair Repair of errors in base incorporation DNA polymerase proofreading Mismatch repair excision/replacement Repair of thymine dimmers

Light reactivation (photorepair) Excision repair (dark repair; lightindependent repair) DNA Repair Repair of errors in base incorporation DNA polymerase proofreading Mismatch repair excision/replacement

Repair of thymine dimers Light reactivation (photorepair) Excision repair (dark repair; lightindependent repair) Repair of Modified Bases Glycosylase removes oxidized guanine SOS repair

Induction of SOS system New polymerase (tolerates slop) Mutant Selection Direct selection Obtain resistant mutants (ex. antibiotic resistant) Obtain prototrophs that have

reverted from auxotrophs Prototroph (revertant) Auxotrophs Application of direct selection Ames Test - screens for mutagens (used to narrow down list of

possible carcinogens) Minimal medium (glucose-salts) Enriched complex medium The Ames Test

Also do expt. with liver extract added Mutant Selection Indirect selection (replica plating) Obtain auxotrophs 106 prototrophs

1 auxotroph Indirect selection (replica plating) Obtain auxotrophs Indirect selection (replica plating) Obtain auxotrophs Joshua and Esther Lederberg

Summary Mutations spontaneous mistakes during replication transposons induced chemical mutagens radiation transposons

Repair repair of errors in base incorporation repair of thymine dimmers SOS repair Selecting mutants direct - obtain antibiotic resistant mutants, Ames test indirect - obtain prototrophs Part II DNA Transfer

Donor Recipient Horizontal (lateral) transfer DNA Transfer 1920s; Frederick Griffithstrains of Streptococcus pneumoniae that produce capsules kill mice

transforming principle (DNA) DNA Transfer 1/109 =10-9 10-9 x 10-9 = 10-18 DNA Transfer

Donor Recipient To be stably maintained, transferred DNA must either be a plasmid (has an origin of replication), or integrate into the host cells genome DNA Transfer Donor

Recipient To be stably maintained, transferred DNA must either be a plasmid (has an origin of replication), or integrate into the host cells genome DNA Transfer Donor

Recipient Integrate into host genome by Homologous recombination (site-specific recombination) DNA Transfer Donor Recipient

Integrate into host chromosome by Homologous recombination (site-specific recombination) DNA Transfer Donor Recipient

Integrate into host chromosome by Homologous recombination (site-specific recombination) DNA Transfer Donor Recipient Integrate into host chromosome by

Homologous recombination (site-specific recombination) heteroduplex replication DNA Transfer Donor Recipient

Integrate into host chromosome by Homologous recombination (site-specific recombination) DNA Transfer Donor Recipient

Horizontal (lateral) gene transfer A+, B- A- , B + B- AA+, B+

A-, BA+, B- DNA Transfer Donor DNA-mediated transformation Transduction Conjugation

Recipient DNA-Mediated Transformation Uptake of naked DNA Process is sensitive to the addition of DNAse DNA-Mediated Transformation Uptake of naked DNA Process is sensitive to the addition of DNAse

Recipient cell must be competent Natural competence Observed in only certain species Example - Streptococcus pneumoniae (GPC) Becomes competent in late log phase Competent cell binds ds DNA Enzymes cut DNA into smaller fragments (5 - 15 kb) Single strand is taken up by cell Example - Haemophilus influenzae (GNR)

Cell binds DNA only from related species Takes up ds DNA Artificial competence In the laboratory, treat cells with specific chemicals (plasmids taken up) Conjugation Requires cell-to-cell contact Involves a conjugative plasmid F plasmid (fertility plasmid) serves as a model

Three types of donors: F+ Hfr F Conjugation: F+ donor male female

Conjugation: F+ donor Conjugation: F+ donor Conjugation: F+ donor In donor cell, replication replaces strand thats being transferred

In recipient cell, complement to transferred strand is synthesized Conjugation: F+ donor Note: some R plasmids (encode resistance to one or more antibiotics) are conjugative F+ + F- F+ + F+

In donor cell, replication replaces strand thats being transferred In recipient cell, complement to transferred strand is synthesized Conjugation Formation of an Hfr cell Figure 8.25

Hfr = High-frequency recombination Conjugation: Hfr donor Conjugation: Hfr donor Some F plasmid DNA is transferred first, followed by chromosomal DNA

In donor cell, replication replaces strand thats being transferred In recipient cell, complement to transferred strand is synthesized Cells inevitably separate before entire chromosome is transferred Conjugation: Hfr donor

Some F plasmid DNA is transferred first, followed by chromosomal DNA In donor cell, replication replaces strand thats being transferred In recipient cell, complement to transferred strand is synthesized Cells inevitably separate before entire chromosome is transferred

Conjugation: Hfr donor Hfr + F- Hfr + F- Significance of Hfr strains: Chromosomal transfer Allowed mapping of E. coli chromosome

Recombinant DNA and Biotechnology DNA RNA protein Preview

Fundamental tools of biotechnology Molecular Cloning PCR Fundamental Tools Used in Biotechnology Restriction Enzymes - used to cut DNA at specific sequences Gel Electrophoresis - used to separate nucleotide (or protein) fragments DNA Probes - used to find specific nucleotide sequences Primers - used to initiate DNA synthesis at a specific location

Restriction Enzymes - cut DNA 1 reflects name of org. from which enz. was first isolated palindrome

Restriction Enzymes - cut DNA Restriction Enzymes - cut DNA Gel Electrophoresis - separates fragments Note: millions of each player Gel Electrophoresis - separates fragments

DNA Probes - find sequences Primers - dictate sites of synthesis initiation Techniques Used in Genetic Engineering DNA

Techniques Used in Genetic Engineering DNA Techniques Used in Genetic Engineering Self-replicating DNA

(ex. plasmid) insert vector recombinant molecule Cloning Overview

Cut out the gene of interest from donor Put the gene into a vector Transfer the vector into a recipient Select for the recipient from a mixed

population Cloning Vectors

1) Plasmids 2) Bacteriophage lambda 3) P1 Phage 4) Cosmids 5) Yeast artificial chromosomes (YAC) Characteristics of cloning vectors

1) Should have its own replicon i.e., be capable of autonomous replication in the host cell 2) Should carry one or more selectable markers that permit identification of parent

and recombinant vectors 3) Restriction sites in non-essential regions of DNA into which foreign DNA can be inserted Molecular Cloning Genetically Engineering Bacterial Cells

Note: millions of each player Applications of molecular cloning Medical application gene therapy production of drugs (insulin, antibiotics, hormones) Agricultural application Nutrients enriched food

Nitrogen fixing plants Scientific research (PCR) Polymerase chain reaction Amplifies target sequence ds DNA containing the target Taq polymerase (Thermus aquaticus)

nucleotides primers thermocycler PCR Medical application: Genetic diseases Infectious disease

Forensic science: Identify criminals parental identification Research application DDC: DNA Test Sets Inmate Free After 18 Years Forensic Resources

DNA Diagnostics Centers Forensics Division provided the DNA testing that eventually resulted in the release of inmate Robert McClendon. McClendon had spent 18 years in prison, convicted of a child rape that he has always maintained he did not commit. McClendons is 1 of 30 cases in Ohio that were identified to have legitimate claims of innocence in an investigation conducted by The Columbus Dispatch together with the Ohio Innocence Project (OIP). DDC

has volunteered to provide DNA testing for the OIP's post-conviction cases free of charge.

Recently Viewed Presentations

  • The SKILLS exam

    The SKILLS exam

    These are some of the skills we have already covered at least once in class: You will also have covered many of these skills in GCSE Maths and Geography Annotating diagrams, sketches, photos, graphs etc. Overlays . Literacy and numeracy...
  • Dia 1 - Kennisnet

    Dia 1 - Kennisnet

    3.1 STILLEVEN IN SOORTEN EN MATEN. De werkelijkheid nagebootst. Als de werkelijkheid nauwkeurig is weergegeven , zeggen we dat het er . realistisch . uitziet. Door goed te kijken naar vorm en oppervlak van de voorwerpen is het mogelijk deze...
  • Chapter 2: Population

    Chapter 2: Population

    Varna. Brahmins are holy men, priests with the responsibility for performing rituals and teaching others the meaning of the holy books.Nowadays, many Indian professionals are brahmins. Kshatriyas . were the kings and warriors who were charged with the responsibility of...
  • Credit Risk - University of Belgrade

    Credit Risk - University of Belgrade

    Credit Ratings. In the S&P rating system, AAA is the best rating. After that comes AA, A, BBB, BB, B, and CCC. The corresponding Moody's ratings are Aaa, Aa, A, Baa, Ba, B, and Caa. Bonds with ratings of BBB...
  • Advisory Class

    Advisory Class

    IB MISSION STATEMENT. International baccalaureate® aims to develop inquiring, knowledgeable and caring young people who help to create a better and more peaceful world through intercultural understanding and respect.. These programmes encourage students across the world to become active, compassionate...
  • Physical Geography of Europe

    Physical Geography of Europe

    The Apennine or Italian Peninsula is a long, thin, boot-shaped piece of land on which the country of Italy lies. The Apennines mountain range, which includes the active volcano Mount Vesuvius, extends down the center of the peninsula. Only about...
  • A Tipsy Idol, Lowing Cows, and Golden Rats

    A Tipsy Idol, Lowing Cows, and Golden Rats

    The Philistines had two main temples in honor of Dagon-one at Ashdod and one at Gaza, with smaller temples elsewhere. More Facts, Please! Lowing cows-In Bible times the terms used in regard to cattle were the same as now: a...
  • P r i m e r o n

    P r i m e r o n

    Rule 2: Purpose specification. Are we clear about the purpose (or purposes) for which we keep personal information? Are the individuals on our database also clear about this purpose? If we are required to register with NPC, does our register...