MCB2000 Exam 2

Microbiology Exam

Central Dogma

The flow of genetic information. Typical chain of events are DNA> mRNA> Protein> Function (Pleated sheets or Alpha Helix).

How mutations alter a genome

Mutated DNA> Mutated mRNA> Altered Protein> Altered Function

Big Picture-Genetics

Genetics is the science of heredity. It includes the study of genes: how they are replicated, expressed, and passed on from one generation to another.

Chromosome

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

RNA

Ribonucleic Acid. SS, 5 carbon ribose sugar, contains uracil instead of thymine

rRNA

Ribosomal RNA, Integral part of ribosomes

tRNA

Transfer RNA. Transports amino acids during protein synthesis. Contains the anticodon.

mRNA

Messenger RNA. Carries coded information from DNA to ribosomes. Contains the codon.

Describe the process of replication, transcription, and translation.

Expression

Genetic info is used within a cell to produce the proteins needed for the cell to function

Recombination

Genetic info can be transferred horizontally between cells of the same generation

Replication

Genetic info can be transferred vertically to the next generation of cells

Product of Replication

Forms a double helix

Product of Transcription

Forms RNA from DNA

Product of Translation

Forms proteins

Reverse Transcriptase

Catalyzes the synthesis of double-stranded DNA from an RNA template. A difficulty with this method is that long molecules of mRNA may not be completely reverse-transcribed into DNA; the reverse transcription often aborts, forming only parts of the desired gene.

DNA Polymerase vs. RNA Polymerase

DNA polymerase synthesizes DNA while RNA polymerase synthesizes RNA from a DNA template.

Complementary Strands

The base pairs always occur in a specific way: adenine always pairs with thymine, and cytosine always pairs with guanine. Because of this specific base pairing, the base sequence of one DNA strand determines the base sequence of the other strand.

Anti-Parallel

The paired DNA strands are oriented in opposite directions relative to each other. The carbon atoms of the sugar component of each nucleotide are numbered 1' (pronounced “one prime”) to 5'. For the paired bases to be next to each other, the sugar components in one strand are upside down relative to the other.

Semiconservative replication

The process of DNA replication in which each double-stranded DNA molecule contains one original strand and one new strand.

Double Helix

Backbone consists of deoxyribose-phosphate. Two strands of nucleotides are held together by hydrogen bonds and form a helix shape.

DNA Ligase

Makes covalent bonds to join DNA strands; Okazaki fragments, and new segments in excision repair

DNA Polymerase

Synthesizes DNA; proofreads and repairs DNA

Helicase

Unwinds double-stranded DNA

RNA Polymerase

Copies RNA from a DNA template

Topoisomerase

Relaxes supercoiling ahead of the replication fork; separates DNA circles at the end of DNA replication

Post-Transcriptional Control

Some regulatory mechanisms stop protein synthesis after transcription has occurred. A part of an mRNA molecule, called a riboswitch, that binds to a substrate can change the mRNA structure. Depending on the type of change, translation can be initiated or stopped. Both eukaryotes and prokaryotes use riboswitches to control expression of some genes.

microRNAs (miRNAs)

Inhibit protein production in eukaryotic cells. In humans, it is produced during development to allow different cells to produce different proteins. It base-pairs with a complementary mRNA, forming a double-stranded RNA. This double-stranded RNA is enzymatically destroyed so that the mRNA-encoded protein is not made.

Genetic Material of Viruses

Contain DNA or RNA; SS or DS

Where in eukaryotes is DNA?

DNA is found in linear chromosomes in nucleus, mitochondria and chloroplast in plants

Where in prokaryotes is DNA?

DNA is found in circular or linear chromosomes in supercoil in nucleoid, plasmid

Pre-transcriptional control

Two genetic control mechanisms known as repression and induction regulate the transcription of mRNA and, consequently, the synthesis of enzymes from them. These mechanisms control the formation and amounts of enzymes in the cell, not the activities of the enzymes.

Operons

System for highly regulated gene expression. Region of DNA that controls several genes. There are two types that they code for:

* Inducible genes: Catabolic processes (ex: Lactose operon)
* Repressible genes: Anabolic processes (ex: Tryptophan operon)

Catabolic Repression

Inhibition of the metabolism of alternative carbon sources by glucose. Can override operon. It only affects inducible operons (breakdown).

Why do cells prefer glucose over lactose?

When there is lactose and glucose together, the lactose operon is off. Glucose is used up and then during lag time you turn on lactose operon.

What are ribosomes made of?

rRNA and Proteins

Difference between prokaryotic and eukaryotic ribosomes

Prokaryotic ribosomes are a target for antibiotics, eukaryotes have bigger and denser ribosomes.

Recombinant DNA technology

The insertion or modification of genes to produce desired proteins

Describe the use of Recombinant DNA in producing Insulin

A gene from one organism can be inserted into the DNA of a bacterium or yeast. In many cases, the recipient can then be made to express the gene, which may code for a commercially useful product. Thus, bacteria with genes for human insulin are now being used to produce insulin for treating diabetes, and a vaccine for hep B is being made by yeast carrying a gene for part of the hepatitis virus (the yeast produces a viral coat protein).

Purpose of cloning

To have identical cells arising from one cell. The main goals are to eliminate undesirable phenotypes, combine beneficial traits, and create organisms that can produce human product (insulin)

Explain how RFLP analyzes DNA

Restriction Fragment Length Polymorphisms are different lengths of DNA restriction fragments from different individuals of same species becase of the deletion or addition of DNA between sites where restriction enzyme cuts

What are RFLPs used for

Used to determine ancestry of individual and identify DNA from specific individual, determine the location

of genes causing genetic diseases and identifies new inserted genes or DNA sequences

Agricultural applications of recombinant DNA

Bt toxin: Plants have toxin-producing gene, toxins kill insects that eat the plants (Bt cotton, Bt corn).

* Herbicide resistance
* Suppression of genes-antisense DNA (improve shelf life)
* Nutrition-human proteins

RoundUp (glyphosphate): plants have a bacterial gene; allows use of herbicides on weeds without damaging crops

Scientific applications of recombinant DNA

* Genetic screening
* DNA fingerprinting
* Gene therapy
* Replace defective and missing genes with normal ones

Restriction enzymes

* Cut specific sequences of DNA


* Destroy bacteriophage DNA in bacterial cells


* Methylated cytosines in bacteria protect their own DNA from digestion
* Create blunt ends or staggered cuts, aka, sticky ends

Vectors

Carry new DNA to desired cells (plasmids, viruses)

Gene library

* Collections of clones containing different DNA fragments
* The collection of cells (usually bacterial cells)
* An organism’s DNA is digested and spliced into plasmid or phage vectors and are introduced in bacteria
* At least one clone exists for every gene in the organism

The Polymerase Chain Reaction

* Process of increasing small quantities (amplifying) of DNA analysis
* Used for diagnostic tests for genetic diseases detecting pathogens
* Reverse-transcriptase PCR uses mRNA as template

Inserting Foreign DNA into Cells

Options include:

* Transformation
* Electroporation
* Protoplast fusion
* Microinjection
* Gene Gun

Transformation

Cells take up DNA from the surrounding environment

Electroporation

Electrical current forms pores in cell membrane

Protoplast Fusion

Removing cell walls from two bacteria allows them to fuse

Microinjection

A technique for introducing a solution of DNA into a cell using a fine micro-capillary pipette. Gene Gun: A way of introducing foreign DNA into plants cells.

Gene Gun

Can be used to insert DNA-coated “bullets” into a cell

How can one amplify DNA fragments in laboratory

The polymerase chain reaction: the process of increasing small quantities (amplifying) of DNA for analysis. Reverse-transcription PCR uses mRNA as template

Synthetic DNA

Building genes using a DNA synthesis machine

Therapeutic applications of recombinant DNA technology

* Human enzymes and other proteins such as insulin
* Subunit Vaccines: made from pathogen proteins in genetically modified yeasts
* Nonpathogenic viruses carrying genes for pathogen’s antigens as DNA vaccines
* Gene therapy to replace defective or missing genes
* Cervical cancer vaccine
* Colony-Stimulating Factor (CSF)
* Interferon
* Hep B Vaccine

Cervical Cancer Vaccine

Consists of viral proteins; produced by Saccharomyces cerevisiae or by insect cells

Colony-Stimulating Factor (CSF)

Counteracts effects of chemotherapy; improves resistance to infectious disease such as AIDS; produced by Escherichia coli and S. cerevisiae

Hep B Vaccine

S. cerevisiae

Human Insulin

Produced by E. coli

Human Genome Project

* aimed at deciphering the chemical makeup of the entire human genetic code (i.e., the genome). The primary work of the project is to develop three research tools that will allow scientists to identify genes involved in both rare and common diseases.


* Sequenced the entire human gene

Nanotechnology

the branch of engineering that deals with things smaller than 100 nanometers (especially with the manipulation of individual molecules)

The use of human microbiome in crime scene investigation

Forensic Microbiology

* DNA Fingerprinting: used to identify pathogens
* RFLP:
* PCR Microarrays and DNA chips can search for multiple pathogens
* Differs from medicine b/c it requires
* Proper collection of evidence
* Establishing a chain of custody

Important agricultural products produced using recombinant DNA tech

* Button mushroom: Delete gene for polyphenyl oxidase (causes browning)
* Bt Cotton/Corn: Bt is a toxin that kills insects that eat plants
* Genetically modified tomatoes/raspberries: antisense genes block pectin degradation for long shelf life
* Pseudomonas syringae, ice-minus bacterium: Lacks protein that causes ice formation on plants
* RoundUp resistant crops: bacterial gene allows the use of herbicide on weeds without damaging crops

Spontaneous mutation

Occur in the absence of a mutagen. Chemicals & radiation can lead to this.

Base Analogs, how do they induce mutation

are molecules that can substitute for normal bases in nucleic acids. Usually, substitution of a base analogue will result in altered base pairings and structural changes that affect DNA replication and transcription of genes

Nucleoside analog

incorporates into DNA in place of normal base, causes mistakes in base pairing

Types of Radiation

* Ionizing radiation (xrays, gamma rays) causes the formation of ions that can oxidize nucleotides and break the deoxyribose-phosphate backbone.
* UV radiation causes thymine dimers (fusing two T’s)
* Repair damage by photolyases (separate thymine dimers) and nucleotide excision repair (enzymes cutout incorrect base pairs and fill with correct bases)

Frameshift mutation

Insertion or deletion of one or more nucleotide pairs; shifts the translational “reading frame”

Missense mutation

Base substitution results in change in an amino acid

Nonsense mutation

Base substitution results in a nonsense (stop) codon

Chemical agents that can cause cancer (mutagens)

* Nitrous acid: causes adenine to bind with cystosine instead of thymine
* Nucleoside analog incorporate into DNA in place of normal base; causes mistakes in base pairing

Difference in Radiation types

Ionizing radiation> more serious mutation. DNA is actually broken here. You cannot repair broken DNA UV radiation> fuses two T together (thymine dimers). You can fix the thymine dimer.

Vertical gene transfer

Transfer of genes from an organism to its offspring (parent to two daughter cells)

Horizontal gene transfer

Transfer of genes between cells of the same generation (sideways from one bacteria to another)

Connection between horizontal gene transfer and antibiotic resistance genes

Plasmids carry genes that cause diseases and antibiotic resistance. Through horizontal gene transfer, bacteria can transfer plasmids with these problematic genes to one another. Plasmids are self-replicating circular pieces of DNA carry genes that can cause diseases and antibiotic resistant genes. They may code for proteins that enhance the pathogenicity of bacterium

Yeast (Saccharomyces cerevisiae) cells in making gene products

* Easily grown and has a longer genome than bacteria
* Expresses eukaryotic genes easily

Plant cells in making gene products

* Express eukaryotic genes easily
* Plants are easily grown, large-scale, and low cost

Mammalian cells in making gene products

* Express eukaryotic genes easily
* Can make products for medical use
* Harder to grow (Hard to work with)

Therapeutic applications of genetic engineering

* Human enzymes and other proteins such as insulin
* Subunit vaccines: made from pathogen proteins in genetically modified yeasts
* Nonpathogenic viruses carrying genes for pathogen’s antigens as DNA vaccines
* Gene therapy to replace defective or missing genes

Significance of Ti Plasmid

used as a vector for genetic modification in plants

General features of archaea

* prokaryotic
* contains no peptidoglycan
* no antibiotic sensitivity
* Lacks rRNA
* Grow in extreme conditions and are very beneficial to the planet

General features of bacteria

* prokaryotic
* contains peptidoglycan
* sensitive to antibiotics
* has rRNA loop

General features of eukarya

* Eukaryotic
* cellulose, chitin, and carbohydrates within cell wall
* no sensitivity to antibiotics
* Lacks rRNA

Protozoa

A catchall kingdom for a variety of organisms; autotrophic and heterotrophic (clades based on rRNA)

Fungi

chemoheterotrophic; unicellular or multicellular; cell walls of chitin; develop from spores (decomposers)

Plantae

Multicellular; cellulose well walls

Viruses

Submicroscopic, obligate intracellular parasites with host specificity. They can infect plants, fungi, bacteria, and archae and are acellular

Eukaryotic vs Prokaryotic

Eukaryotic is

* Linear DNA
* 80S Ribosomes
* Growth through Mitosis

Prokaryotic is

* One circular, some two circular; some linear DNA
* 70S Ribosomes
* Growth through Binary Fission

What is the importance of Cyanobacteria/Algae

* They are primary producers
* Fix CO2 through photosynthesis
* Produce O2

Features of Chlamydia

* Obligate intracellular bacteria
* No peptidoglycan in cell wall; grow intracellularly
* Chlamydia and chamydophilia
* Form an elementary body that is infective
* Chlamydia trachomatis causes trachoma and urethritis
* Chlamydophila psittaci causes respiratory psittacosis

Spirochetes

* Coiled and move via axial filaments
* Treponema
* T. pallidum causes syphilis
* Borrelia
* Causes relapsing fever and lyme disease

Mycoplasma

* No cell wall; pleomorphic
* Mycoplasmatales
* M. pneumonie causes mild pneumonia

Archaea types

* Extremophiles
* Halophiles (salt aboce 25%)
* Thermophiles (growth temp above 80C)
* Methanogens
* Anaerobic and produce methane

Fungal types

* Chemoheterotrophs
* Aerobic vs Facultative anaerobic
* Sexual asexual w/ spores
* Yeast vs. Mold
* Grow best at pH 5

Yeast

* Nonfilamentous and unicellular
* Budding yeasts divide unevenly, fission yeasts don’t
* Dimorphic Fungi
* Yeast like at 37C and mold like at 25C

Economic Effects of Fungi

* Trichoderma: cellulase
* Entomophaga: biocontrol
* Coniothyrum minitans: kills fungi on crops
* Paecilomyces: kills termites
* Saccharomyces: cerevisiae bread, wine, hep B vaccine
* Taxomyces: Taxol