microbio exam 2 (chs. 6 and 7)

microbial genetics and viruses

What are genomes and what makes them up?

the sum of ALL the genetic material of an organism, typically in the form of chromosome(s) BUT bacteria have plasmids and eukaryotes have mitochondrial and/or chloroplast DNA

What is a chromosome and its form in prokaryotes/eukaryotes?

defined as a distinct structure of DNA in an organized molecule

• eukaryotic: in nucleus, tightly bound to histone proteins, linear appearance, few-100s

• prokaryotic: condense into single circular shape

What is true of ALL DNA function and form?

all of it is complementary and double-stranded, as well as double-helixed. stores code to make all of the proteins (hereditary info) in an organism

What is a gene?

a specific place/location/spot of DNA on a chromosome that codes for a specific protein with a specific trait/characteristic

What is the difference between the genotype and phenotype?

genotype refers to the genetic makeup for traits, while phenotype is the actual physical manifestation of them

What is included in a nucleotide (basic unit of DNA)?

• deoxyribose sugar

• phosphate group

• nitrogenous base

What are the nitrogenous bases?

• purines: adenine and guanine

• pyrimidines: thymine, cytosine, and uracil (mRNA only)

What are the 5’ and 3’ ends of DNA?

• 5’ - has phosphate group sticking out from C

• 3’ - has -OH bound from third C

What is replication?

when the cell makes copies of ALL of the chromosomes (DNA) it contains

What does it mean that replication is semiconservative?

each original DNA strand is a template for the “new” strand

WHEN does replication occur in eukaryotes and prokaryotes?

• binary fission for prokaryotes

• “S” phase of mitosis for eukaryotes

What is step 1 in replication with the correct enzymes?

• DNA gyrase untwists DNA to prevent supercoiling

• DNA helicase breaks/unzips H bonds between complementary nucleotide bases

What is step 2 of replication with the enzyme?

SSBP (single-stranded binding proteins) keep DNA strands opened so that the H bonds do not reform

What is step 3 of replication with correct enzyme?

Primase: synthesizes a short strand of RNA primer in the 5’ to 3’ direction

What is step 4 of replication with correct enzyme?

DNA pol III finds/recognizes DNA primer and begins adding complementary nucleotides from there in 5’ to 3’ direction - other end proofreads

What is step 5 of replication with correct enzyme?

DNA pol I locates RNA primer, digests it, and replaces it with DNA

What are the steps involved with replication of the lagging strand?

• all same beginning steps

• primase builds primer AT replication fork b/c moves in 5’ to 3’ direction

• DNA pol III finds primer and synthesizes Okazaki fragments

• DNA pol I replaces primer with DNA

• DNA ligase forms the final bond between Okazaki fragments

What are the leading and lagging strands in replication?

• leading strand is built continuously in the 5’ to 3’ direction with a SINGLE primer

• lagging strand is built in opposite direction with multiple primers and Okazaki fragments

What is transcription?

the process a cell uses to copy a piece of DNA (gene) in a single strand to RNA

What is initiation in transcription?

the first beginning step in which RNA pol is directed to the promotor sequence of DNA, called the TATAAA box. RNA pol binds, breaks bonds, forms transcription bubble

What is elongation in transcription?

second step: when RNA pol adds RNA bases (this means U instead of T) in the 5’ to 3’ direction by sliding along the DNA strand

What is termination in transcription?

third/final step: RNA pol reaches terminator region that ends transcription

What is important about mRNA?

• single-stranded with 600-900 nucleotides forming 200-300 proteins

• copy of structural genes in DNA = carries DNA’s message

• 3 base codons (a triplet) create 1 amino acid!

• The Genetic Code chart shows what codon/triplet corresponds to what amino acid

What is important about tRNA?

• in a 3D cloverleaf shape

• contains an anticodon that is complementary to a mRNA codon

• acts as translator = knows both codon and amino acid languages

What is important about ribosomal RNA?

• some are ds, some are ss

• physically inside of ribosome

• 3 major types lead to our 3 different Domains!

What is translation?

the process by which cells use mRNA as a template to build amino acids

What is the first step of translation?

• mRNA slides into ribosome and binds to the small subunit

• a tRNA molecule with the complementary anticodon binds to the mRNA in the ribosome at the P site

What are the E, P, and A sites of the ribosome?

• A site: “available” spot where new tRNA binds

• P site: “peptide” spot where a protein is being built

• E site: “exit” spot where tRNA leaves the ribosome

What is step 2 of translation?

the next tRNA molecule enters the A site and binds to next mRNA codon there

What is step 3 of translation?

a peptide bond is formed between the amino acids in the A and P sites

What is step 4 of translation?

the ribosome moves over by 1 codon so that the empty/uncharged tRNA is in the E site, the growing protein in the P site, and next available amino acid is in the A site

What is step 5 of translation?

go back to step 2 and continue to repeat until a STOP codon is reached - the protein is then released by the ribosome

How is eukaryotic transcription/translation different?

• extra bases called introns do not code for proteins

• spliceosomes are needed to get rid of them

What are posttranslational modifications?

extra structures or tweaks to the translated protein once it leaves the ribosome, such as enzymes or cofactors

What is co-transcriptional translation?

the fact that archaea and bacteria carry out transcription and translation at the same time

What is a polyribosomal complex?

the chain of ribosomes upon which a single long mRNA strand passes through = many proteins made from same mRNA

What is an operon in bacteria?

a set of genes acting as a unit for a specific function

What are the types of operons in bacteria?

• inducible: are off most of the time except for when needed

• repressible: are ON most of time

What is the lac operon in bacteria?

an inducible operon used to regulate lactose metabolism - turned ON when glucose is low, lactose is available, cyclic AMP is low

What are the main areas of the lac operon?

1. regulator: has genes that code for the repressor

2. promoter: a segment recognized by RNA pol for starting transcription

3. operator: initiation site of structural gene transcription

How is the lac operon maintained in “off” mode?

the repressor is allosteric (2 binding sites), but usually completely blocks the operator in the absence of lactose

What happens to the repressor when lactose is present?

it binds to lactose, which frees open the operator for the RNA pol

What is different about a repressible operon?

a repressor is typically inactive/non-functioning, but with the help of a co-repressor (molecule in excess amounts) can bind to the operator and stop transcription

What is phase variation?

turning a gene set on/off that has phenotypic changes

What is horizontal gene transfer?

when DNA moves from one cell to another

What is conjugation in bacteria?

• 1 bacteria that has a plasmid, called F+, makes a pilus that attaches to a F- cell of the SAME species

• DNA from plasmid crosses through and makes the F- cell become F+

What is transformation in bacteria?

“naked” DNA from dead cells is accepted from the environment/solution by competent cells

How was transformation discovered?

in 1928, Griffith injected mice with smooth and rough Streptococcus pneumoniae samples. combining the heat-killed smooth with live rough cells caused the mouse to die and all appeared to be smooth - DNA from the dead smooth cells was taken in by the live rough ones

What are transposons?

• “jumping genes” in bacteria that move from one location to another

• they have enzymes that allow them to move in and out of the plasmid or chromosome

What is transduction?

when a bacteriophage (virus) accidentally packages host DNA into another bacteria’s genome

• phage releases DNA into host cell

• an enzyme from the phage breaks the host DNA into small fragments

• phage mRNA and structures form to make original viruses, but a new virus with some host DNA also is formed

• the altered (defective) phage injects DNA from the bacteria into a new cell

What is a mutation?

ANY change to the nucleotides/bases of DNA

*the spontaneous rate is 1 in 1 mil bases = 1 in 1,000 genes

What is a point substitution?

only 1 single base is changed, such as a A for a T

What is a silent mutation?

type of point mutation where a nucleotide is changed, so is the mRNA codon, but the same amino acid is produced

What is a missense mutation?

type of point mutation where the DNA nucleotide is changed, mRNA codon is changed, and a different amino acid is coded (ex. sickle cell disease)

What is a nonsense mutation?

type of point mutation where a DNA nucleotide is changed, mRNA codon is changed for a STOP codon - protein usually stopped too soon

What is a frameshift mutation?

1 or more nucleotides are added or deleted, so ALL the mRNA codons code for a different amino acid (unless in a group of 3)

What is a mutagen?

a physical force (radiation from UV, X-ray, gamma rays) or chemical that increases the spontaneous mutagen rate

How is toxicology tested?

through the Ames Test: checks if a substance in a product is mutagenic

• Sal his- is added to control and Sal his- is added with Chemical X to the experimental = both put on media that LACKS histidine (only mutagenic survives)

• if the experimental media has many colonies, it is designated a mutagen b/c it increases back-mutation of Sal his- into Sal his+

Are mutations all bad?

NO, they are drivers of population variation and therefore evolution. they can be beneficial if they stand the test of time through natural selection

What is important about the size of viruses?

generally 250 to 22 nanometers - WAY SMALLER than bacteria and require an electron microscope

What is the proper way to describe a virus?

obligate intracellular parasite: they require hosts to be “active” and are NOT living things

What kinds of nucleic acid may a virus contain?

ss DNA, ds DNA, ds RNA, ss RNA (positive or negative sense), multiple RNA strands

**always only has ONE TYPE

What structure do all viruses have besides nucleic acid?

They all contains a capsid made of a capsomere protein coat that surrounds the genetic info - may have envelopes or spike proteins

How are viruses classified based on traits?

• type of nucleic acid: ds DNA is primary

• enveloped vs non-enveloped

• size in nanometers

How is a viral envelope built?

comes from a virus exiting a cell and taking a piece of its plasma membrane on the way - mixes with viral proteins to become viral envelope

What are positive and negative sense RNA?

• positive sense: single stranded RNA ready for immediate protein translation

• negative: must be converted to proper form before translation

What other substances can be inside a virus?

enzymes for specific operations inside host: polymerases to synthesize DNA/RNA and replicases that copy RNA. reverse transcriptase in HIV makes DNA from RNA template

How are viruses named?

• order: -virales

• family: -viridae

• genus: -virus

What are the phases of animal virus multiplication?

• adsorption

• penetration

• uncoating

• synthesis

• assembly

• release

What occurs during animal adsorption?

proteins on the surface of the viral envelope bind to receptor on host OR by fusing with cell membrane bilayer directly

What are viruses specific to?

their HOST CELLS - explains why viruses typically don’t affect multiple species, why they are associated with one area of the body

What happens during penetration?

virus enters the cell via endocytosis - absorbed inside

What happens during uncoating?

the host cell enzymes digest the viral envelope and/or capsid

What ALWAYS occurs during virus synthesis?

• the viral genome must be copied MULTIPLE times

• viral proteins must be made, but using HOST machinery (organelles, ATP, etc)

*if integrated into the host genome, tumors or cancer may develop

What happens during viral assembly?

the new viruses are formed from their proteins and structures - can be 1 or 2, or up to 50 depending on the type

What happens during viral release?

the newly-formed virus leaves the cell through lysis (cell death) or budding (exocytosis) - bacteriophages only do lysis

What are CPEs?

cytopathic effects = the viral damage to the host cell, which is observed microscopically

What is syncytium?

a CPE in which a giant cell is formed from neighboring cells fusing - signaling is destroyed - cell dies

What are the main CPEs of host cells?

• syncytium

• inclusion bodies: virions remain inside cell

• lysis: lysosomes destroyed and cell dies

• transformation: changing DNA of host cell through shape, inhibiting/nonstop mitosis - can lead to cancer

What are bacteriophages?

viruses that target bacteria

What occurs in the lytic phase of a T-even phage?

1. adsorption to specific receptors

2. penetration of nucleic acid from phage tube - head stays outside, lysozyme (enzyme) softens the peptidoglycan cell wall

3. biosynthesis: viral DNA is replicated and viral proteins synthesized using host cell resources

4. assembly: parts become whole phage

5. release: cell lyses from being overcrowded - plaques in media are counted

What are the stages of a lysogenic (lambda) phage?

the viral DNA integrates to the host chromosome, but does NOT immediately kill it - becomes a prophage. viral DNA is copied and retained during cell division, but the virus is not assembled yet. during induction, the prophage is activated from some type of cell “stress” and begins the normal lytic cycle to form the virus, with some parts of bacterial DNA in viral genome

What are advantages of prophages in bacterial hosts?

• resistant to superinfection

• gain genes for toxins, antibiotic resistance, enzymes

• gain genes from other host cells by specialized transduction

How are bacteriophages cultivated?

the bacteria is placed in media and the plaque-forming units (PFUs) are counted

What are the methods of cultivating an animal virus?

• in vivo: embryos or live animals

• in vitro: from cell or tissue cultures

• embryonated chicken eggs

How does animal viral multiplication differ in DNA vs RNA viruses?

• DNA: viral DNA enters nucleus, RNA synthesized on ribosomes, proteins come back to nucleus

• RNA: viral RNA may be replicated in the cytoplasm or nucleus

Animal vs bacterial virus multiplication?

• animal: ENTIRE virus enters, has extra uncoating step, DNA viruses do most work in the nucleus, can be released by budding or lysis

• bacteria: only NUCLEIC ACID enters, no extra uncoating step, biosynthesis occurs in cytoplasm b/c no nucleus, released by lysis only