Exam 3 - Lecture Notes Review Microbiology 2421 (Chp. 8-9)

Genetics

Science of heredity

• Central dogma of molecular biology

• Mutations

• Gene expression controlled by operons

Typical chain of events described by central dogma

DNA → mRNA → Protein → Function

How mutations alters a genome

Mutated DNA → Altered mRNA → Altered protein → Altered function

Genetics

Study of genes, how they carry information, how information is expressed, and how genes are replicated

Chromosomes

Structures containing DNA that physically carry hereditary information; the chromosomes contain genes

Genes

Segments of DNA that encode functional products, usually proteins

Genome

All the genetic information in a cell

Genetic code

Set of rules that determines how a nucleotide sequence is converted to an amino acid sequence of a protein

Central dogma

Theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein

Genotype

Genetic makeup of an organism

Phenotype

Expression of the genes

Bacteria usually has a ______ circular _____

single, chromosome made of DNA and associated proteins

Short tandem repeats (STRs)

Repeating sequence of noncoding DNA

Vertical gene transfer

Flow of genetic information from one generation to the next

DNA forms a double helix, indicating

• Backbone (consist of deoxyribose-phosphate)

• Two strands of nucleotide are held together by hydrogen bonds between A-T and C-G

• Strands are antiparallel

DNA Replication (structure)

One strand serves as a template for the production of a second strand

• Topoisomerase and gyrase relax the strands

• Helicase separates the strands

• A replication fork is created

DNA polymerase adds _________ to the __________

nucleotides; growing DNA strand

• 5→3’ direction

  • Initated by an RNA primer

Leading strands is synthesized _______, while lagging strands is synthesized _______, creating Okazaki fragments

continuously; discontinuously

What removes RNA primers?

DNA polymerae; Okazaki fragments are joined by the DNA polymerase and DNA ligase

DNA replication: Energy needs:

• Nucleotides (Energy for replication is supplied by nucleotides)

• Hydrolysis of two phosphate groups on ATP provides energy

DNA replication

• Most bacterial DNA replication is bidirectional

• Each offspring cell receives one copy of the DNA molecule

  • Replication is highly accurate due to the proofreading capability of DNA polymerase

Ribonucleic acid

• Single-stranded nucleotide

• 5 - carbon ribose sugar

  • Contains uracil (U) instead of thymine (T)

Ribosomal RNA (rRNA)

Integral part of ribosomes

Transfer RNA (tRNA)

Transports amino acids during protein synthesis

Messenger RNA (mRNA)

Carries coded information from DNA to ribosomes

Transcription in Prokaryotes

Synthesis of a complementary mRNA strand from a DNA template

Transcription begins when ____________ and stops when ______________

RNA polymerase binds to the promoter sequence on DNA; it reaches the terminator sequence on DNA

mRNA is ____

Translated into the “language” of proteins

Translation: Codons

Groups of three mRNA nucleotide that code for a particular amino acid

• 61 sense condons encode the 20 amino acids

• Genetic code involves degeneracy, meaning each amino acid is coded by several condons

Translation of mRNA

Beings at the start codon (AUG), ends at nonsense codons (UAA, UAG, UGA)

• Codons of mRNA are “read” sequentially

Translation of tRNA

tRNA molecules transport the required amino acids to the ribosomes

• Have anticodons that base-pairs with the codon (amino acids are joined by peptide bonds)

In bacteria, translation

Translation can begin before transcription is complete

Transcription in Eukaryotes

Transcription occurs in the nucleus, whereas translation occurs in the cytoplasm

Transcription in Eukaryotes: Exons

Regions of DNA that code for protein

Transcription in Eukaryotes: Introns

Regions of DNA that do not code for proteins

Transcription in Eukaryotes: Small nuclear ribonucleoproteins (snRNPs)

Remove introns and splice exons together

The Regulation of Bacterial Gene Expression

Constitutive genes are expressed at a fixed rate

• Other genes are expressed only as needed

  • Inducible gene

  • Repressible genes

  • Catabolite repression

Pre-Transcriptional Control: Repression

Inhibits gene expression and decreases enzyme synthesis​

• Mediated by repressors, proteins that block transcription​

• Default position of a repressible gene is on

Pre-Transcriptional Control: Induction

Turns on gene expression

• Initiated by an inducer

• Default position of an inducible gene if off

Promoter

Segment of DNA where RNA polymerase initiates transcription of structural genes

Operator

Segment of DNA that controls transcription of structural genes

Operon

Set of operator and promoter sites and the structural genes they control

Inducible operon

In an inducible operon, structural genes are not transcribed unless an inducer is present​

• In the absence of lactose, the repressor binds to the operator, preventing transcription​

• In the presence of lactose, metabolite of lactose-allolactose (inducer)-binds to the repressor; the repressor cannot bind to the operator and transcription occurs

Repressible operaons

Structural genes are transcribed until they are turned off​

• Excess tryptophan is a corepressor that binds and activates the repressor to bind to the operator, stopping tryptophan synthesis

Positive Regulations: Catabolite repression

Inhibits cells from using carbon sources other than glucose

Positive Regulations: Cyclic AMP (cAMP)

Builds up in a cell when glucose is not available

• cAMP binds to the catabolic activator protein (CAP) that in turn binds the lac promoter, initiating transcription and allowing the cell to use lactose

Epigenetic Control

• Methylating nucleotides turn genes off​

• Methylated (off) genes can be passed to offspring cells​

• Not permanent

Post-Transcriptional Control: Riboswitch

Is a part of an mRNA molecule that binds to a substrate and changes the mRNA structure.​

• translation is initiated or stopped

Post-Transcriptional Control: microRNAs (miRNAs)

Base pair with mRNA to make it double-stranded​

• Double-stranded RNA is enzymatically destroyed, preventing production of a protein

Mutation

Permanent change in the base sequence of DNA

• may be neutral, beneficial, or harmful

Mutagens

Agents that cause mutations

Spontaneous mutations

Occur in the absence of a mutagen

Mutation: Base substitution (point mutation)

Change in one base in DNA (C-G → A-T)

Missense mutation

Base substitution results in change in an amino acid (before stop)

Nonsense mutation

Base substitution results in a nonsense (stop) codon

Freameshift mutation

• Insertion or deletion of one or more nucleotide pairs​

• Shifts the translational “reading frame”

Chemical mutagens: Nitrous acid

Causes adenine to bind with cytosine instead of thymine

Chemical mutagens: Nucleoside analog

Incorporates into DNA in place of a normal base; causes mistakes in base pairing

Radiation

• Ionizing radiation (X - ray and gamma rays) causes the formation of ions that can oxidize nucleotides and break the deoxyribose-phosphate backbone​

• U V radiation causes thymine dimers

Radiation: Photolyases

Seperate thymine dimers

Radiation: Nucleotide excision repair

Enzymes cut out incorrect bases and fill in correct bases

Spontaneous mutation rate is ___________ while mutagen increase the mutation rate to _____________

1 10^9 replicated in base pairs or 1 10^6 replicated genes; 10^-5 or 10^-3 per replicated gene

Positive (direct) selection

Detects mutant cells because they grow or appear different than unmutated cells

Negative (indirect) selection

Detects mutant cells that cannot grow or perform a certain function

Autotroph

Mutant that has a nutritional requirement absent in the parent​

• Use of replica plating

Identifying Chemical Carcinogens: Ames test

The Ames test exposes mutant bacteria to mutagenic substances to measure the rate of reversal of the mutation​

• Indicates degree to which a substance is mutagenic

Genetic recombination:

Exchange of genes between two DNA molecules; creates genetic diversity

Crossing over:

Two chromosomes break and rejoin, resulting in the insertion of foreign DNA into the chromosome

Vertical gene transfer

Transfer of genes from an organism to its offspring

Horizontal gene transfer

Transfer of genes between cells of the same generation

Plasmid and Transposons

• Mobile genetic elements​

  • Move from one chromosome to another or from one cell to another​

• Occur in prokaryotic and eukaryotic organisms​

Plasmids

Are self-replicating circular pieces of DNA​

• 1 to 5% the size of a bacterial chromosome​

• Often code for proteins that enhance the pathogenicity of a bacterium

Conjugative plasmids

​​Carries genes for sex pili and transfer of the plasmid

Dissimilation plasmids

Encode enzymes for the catabolism of unusual compounds

Resistance factors (R factors)

Encode antibiotic resistance

Transposons

Are segments of DNA that can move from one region of DNA to another​

• Contain insertion sequences (IS) that code for transposase that cuts and reseals DNA​

• Complex transposons carry other genes (gram ., in antibiotic resistance)

Transformation in Bacteria

Genes transferred from one bacterium to another as “naked” DNA

Conjugation in Bacteria

Plasmids transferred from one bacterium to another​

• Requires cell-to-cell contact via sex pili

• Donor cell carry the plasmid (F factor) and are called cells

• Hfr cells contain the F factor on the chromosome

• Can be used to map the location of genes on a chromosome

Bacteriophage

DNA is transferred from a donor cell to a recipient

Generalized transduction

Random bacterial DNA is packaged inside a phage and transferred to a recipient cell

Specialized trasnduction

Specific bacterial genes are packaged inside a phage and transferred to a recipient cell

Genes and Evolution

• Mutations and recombination create cell diversity​

• Diversity is the raw material for evolution​

• Natural selection acts on populations of organisms to ensure the survival of organisms fit for a particular environment

Biotechnology

The use of microorganisms, cells, or cell components to make a product​

• Foods, antibiotics, vitamins, enzymes

Recombinant DNA (rDNA) technology

The insertion or modification of genes to produce desired proteins

Vector

Self-replicating DNA molecule used to transport foreign DNA into a cell

Clone

Population of genetically identical cells arising from one cell; each carries the vector

Selection

Selecting for a naturally occurring microbe that produces a desired product

Mutations

Mutagens cause mutations that might result in a microbe with a desirable trait

Site-directed mutagenesis

A targeted and specific change in a gene

Restriction enzymes

Cut specific sequences of DNA

Restriction Enzymes: Bacterial cells

Destroy bacteriophage

Restriction enzyme: Methylated cytosines in bacteria

Protect their own DNA from digestion

Restriction Enzymes: Sticky ends

Create blunt ends or staggered cuts

Vectors

Carry new DNA to desired cells and must be able to self-replicate

Plasmids and viruses can be used as

Vectors

Vectors: Shuttle vectors

Exist in several different species and can move cloned sequences among various organisms

Polymerase Chain Reaction

• Process of increasing small quantities (amplifying) of ​DNA for analysis​

• Used for diagnostic tests for genetic diseases and detecting pathogens​

• Reverse-transcription PCR uses mRNA as template

DNA can be inserted into a cell by

Transformation, Electroporation, and Protoplast fusion

Transformation

Cells take up DNA from the surrounding environment

Electroporation

Electrical current forms pores in cell membranes