quiz 2 - final lectures 10,11,12,13 Flashcards
describe a virus
non living
not able of reproducing without host cell
small 20-200nm
genetic info like DNA or RNA contained in capsid (sometimes surrounded by additional membranous envelope)
Class IV, V and VI animal viruses
how many classes of viruses are there
6
describe class I
adenovirus & HPV
ds DNA
special enzymes/proteins
describe class II
parvovirus
ssDNA
special enzymes/proteins
describe class III
reovirus
ds DNA
special enzymes/proteins
describe class IV
coronavirus
rhinovirus
ssRNA, positive strand
RNA is mRNA
special enzymes/proteins
describe class V
influenza
ebola
ssRNA negative (antisense) strand
RNA is template for mRNA
special enzymes/proteins
describe class VI
HIV
ssRNA, retro
RNA is template for dsDNA
reverse transcriptase - special enzymes/proteins
what do all 3 classes of ssRNA viruses have
special enzymes/proteins (glycoproteins in envelope)
definition of contagion
highly contagious airborne virus
more likely to result in epidemic
definition of incubation time
time it takes to produce symptoms of disease
longer incubation time = more spread
definition of virulence of virus
ability to cause disease and kill
rapid killing lowers spread
describe structure of class IV, V & VI (diagram)
single stranded mRNA
capsid proteins
membrane envelope - phospholipid bilayer
spike proteins - membrane/surface glycoproteins
describe HPV (class I) steps of replication (5 steps)
1- entry and uncoating/disassembly
2 - replication
3 - transcription and manufacture of capsid proteins
4 - self assembly of new virus particles and exit from cell
5 - host cell death ->disease
viral gene transcription and translation are… (HPV)
dependent on host enzymes
RNA polymerase
host ribosomes
viral DNA replication is … (HPV)
also generally dependent on
host enzymes
DNA polymerase
describe steps of class VI-corona and class v -influenza (general)
1 goes in
binding to receptor
fusion of viral and plasma membrane (entry)
uncoating/disassembly
RNA replication
Viral RNA
self asembly
1000s exit
host cell death and disease
whats the main difference between class IV and class V
class IV - ssRNA is mRNA
class V - ssRNA to mRNA (transcription)
describe retroviruses - HIV class VI
STDs, blood, injections
no universal vaccine
genetic info as 2 ssRNA molecules
reverse transcriptase, integrase, protease
coat proteins
membranous envelope with gp120
infects - CD4+ immune cells, lymphocytes and macrophages
how many steps of HIV replication
7
describe AIDS symptoms (appear when? and why?)
10 years after infection (latent phase)
BECAUSE
the provirus is not active and viral proteins are not expressed
the immune system of host keeps viral replication under control
describe anti HIV drugs (3 things)
cocktail of antiviral drugs
1 - CCR5 blockers inhibit a non essential cellular protein CCR5 - prevents entry
2 - reverse transcriptase inhibits it’s activity AZT mimics nucleotides (competitive inhibition)
3 - viral proteases that cleave viral poly protein to individual and functional polypeptide chains the drugs inactivate the viral proteases so functional proteins are not made (prevent assembly and exit)
what are bacteriophages
bacterial viruses
reproduce according to lytic and or lysogenic cycle
what are virulent phages
T4
always kills the host (bacteria)
lytic cycle
what are temperate phages
lambda
doesn’t always kill the infected host
lysogenic and lytic cycles
describe lytic cycle
early genes - nucleases to destroy host DNA
middle genes - capsid proteins, head, tail
late genes - protease (lysozyme) to lyse host (bacteria) by damaging cell wall
describe lysogenic cycle
default state of temperate phage only
involves incorporation of phage DNA into bacterial chromosome called prophage (replication occurs together with bacterial DNA replication during cell division)
bacteria are not lysed
phage and bacteria co exist
describe main steps of lytic cycle (5)
attachment
entry of phage DNA and degradation of host DNA
(early, middle)
synthesis of viral genomes and proteins
assembly
(late)
release / exit
generally describe bacterial genetics (prokaryotes)
singular circular chromosome (+/- plasmid)
binary fission (can’t sexually reproduce)
mechanisms to increase genetic variation
genetic variation = diversity
increase chances of survival in ever changing environment
has steps (a,b,c,d)
in the case of pathogenic bacteria - changing environment = host and immune system - solutions?
acquiring antibiotic resistance through mutations or other means
developing better virulence genes for attacking host
changing antigenic makeup to avoid immune system of host (similar to RNA viruses)
what is vertical gene transfer
within species
what is horizontal gene transfer
between species
describe mutations (a)
random errors in DNA
can be beneficial - selection
can be harmful - elimination
describe transformation (b)
acquisition of foreign DNA from the environment or surrounding
may involve plasmids or chromosomal DNA
most common way of acquiring antibiotic resistance
(Griffith experiment and e. coli lab 7)
describe transduction (c)
transfer of DNA between donor and recipient bacteria using phase vector (transmission agent)
bacteriophages acquire random genes (from donor to recipient)
virulent or temperate pages during lytic cycle
describe conjugation
and how many types are there
like sexual reproduction
divert transfer of DNA between bacteria using conjugation bridge
2 types
describe type I (conjugation)
involves transfers of DNA from F+ donor to F- recipient
info transferred is the F plasmid
at end - both cells are F+
(R plasmids can be transferred this way too)
describe type II (conjugation)
involved an Hfr donor bacterium
F plasmid has integrated into bacterial chromosome
transfers parts of F factor along with bacterial genes of the chromosome to F- recipient bacterium (acquires bacterial genes that become incorporated into its own chromosome by recombination)
F- recipient doesn’t become F+
what are plasmids
circular pieces of DNA
may contain genes that help
relaxed plasmid replication is independent of bacterial chromosome (opposite of stringent replication)
how are plasmids transferred
by transformation or conjugation type I
what are F plasmids
contain info for formation of sex pili and conjugation
what are R plasmids
F plasmids that contain in addition info for antibiotic resistance
what do plasmids used for cloning usually have
origin of replication
multiple cloning site (MCS)
antibiotic resistance gene
what is cloning
insertion of a gene or any foreign DNA into a bacterial plasmid making 1000s of copies of genes of interest
why do we do cloning (3 reasons)
study gene structure (gene and promoter sequence)
produce recombinant protein in bacteria - then be used for medical or industrial purposes
manipulate gene of interest by introducing mutations, introducing the DNA into organism of interest (GMOS)
how many steps of cloning are there
5
describe generally (diagram) of steps of cloning
1- bacteria plasmid DNA
2 - restriction digestion
3 - ligation (used ligaments seals plasmid and DNA)
4 - transformation (competency and heat shock)
5 - cell division and selection on plating
describe step 1 of cloning
isolation or purification of DNA genomic or cDNA from source (organism of interest)
isolation of plasmid DNA from bacteria
describe step 2 of cloning
restriction digestion of DNA and bacterial plasmid DNA with same restriction endonucleases
describe restriction enzymes (step 2 of cloning)
cut within DNA
cleave ssDNA at recognition sites (palindromic)
hydrolyze phosphodiesther bonds on both strands
blunt ends and sticky ends
describe function of restriction enzymes (step 2 of cloning)
have evolved to digest foreign DNA (phage)
prevent its insertion into bacterial chromosome
prevent phage replication
host DNA is methylated (not digested bc of this)
describe step 3 of cloning
ligation of foreign DNA into bacterial plasmid (ligase + ATP) anneal compatible
sticky ends
not all plasmid DNA will incorporate foreign DNA or DNA of interest
describe diagram of step 3 of cloning (plasmid DNA used as vector)
plasmid used for cloning
MCS - multiple cloning site, insertion of foreign DNA (multiple restriction sites)
AMPR, Bla, beta lactamase - destroys ampicillin (allows bacterial growth in the presence of antibiotic ampicillin)
origin of replication
describe step 4 of cloning
transformation of recombinant plasmid into bacteria by heat shock
describe bacteria before transformation in step 4 of cloning
AMP S - un transformed
describe bacteria after transformation in step 4 of cloning
AMP R - resistant
describe step 5 of cloning
plating and selection
antibiotic resistance - selection for bacterial with plasmid on medium
each col out has identical bacteria carrying same plasmid
each colony carries different plasmid
colonies can be screened (colony hybridization)
radioactive probe = bait, fluorescent, ssDNA sequence complementary to gene of interest
describe cloning using PCR plasmid (steps)
1 - isolate source DNA, plasmid DNA
2 - PCR with specific primers that anneal to ends of gene of interest
3 - PCR products and plasmid
4 - ligation
5 - transformation
6 - plating and selection on ampicillin medium
what is a library
pool of recombinant clones
storage of genes from organism/cell of interest
library can be screened at a later date for gene of interest
what is cDNA
complementary DNA
made from mRNA of cell of interest that has been reverse transcribed using reverse transcriptase
what are the advantages of cDNA
better source of genetic info (bacterial expression plasmid)
no eukaryotic promoter sequence
no intron sequences
bacteria can therefore express foreign protein (eukaryotic intron and promoter won’t be recognized by bacteria)
describe steps of cDNA library (6 steps)
1 - mRNA - reverse transcriptase
2 - cDNA - restriction digestion
3 - cDNA fragments - ligation into plasmid
4 - library of cDNA plasmids - transformation
5 - library of bacterial cDNA clones
6 - plating and selection
what is genomic DNA
isolated from chromosomes of cells
what are the advantages of genomic DNA
uncertain about expression of gene of interest (low levels mRNA made - may be hard to clone as cDNA)
interest in studying gene sequences and regulatory sequences (splicing has removed introns in mRNA and in cDNA, mRNA doesn’t contain promoter and upstream regulatory sequences)
what is polymerase chain reaction
amplification of DNA sequence of interest
describe PCR
used in forensic sciences, paternity testing and cloning, hiv, covid tests
synthesis reaction test tube in vitro reaction
(also entails cutting amplified with restriction enzyme, ligation into plasmid, transforming bacteria, selecting recombinant plasmid of interest
name and describe PCR ingredients (5)
DNA source - 2 strands template
heat tolerant DNA polymerase (TAQ polymerase)
specific primers to delimit region of interest to be amplified (2 F & R DNA primers)
dNTPs
thermocycler = adjusts temp rapidly automatically for each step
describe 3 steps of PCR
repetition 20-30x
1 - denauturation at 94c
2 - annealing/hybridization with specific F & R primers 45-65c
3 - elongation/extension (complementary strand to region of interest is synthesized with TAQ polymerase at 72c
what are RFLPs
restriction fragment length polymorphisms
describe RFLPs
polymorphisms are regions of genome that are variable between individuals
distinguished based on patterns of DNA fragment sizes follow restriction digestion of DNA
describe 3 steps of RFLPs
DNA isolation
restriction digestion
AGE - agarose gel electrophoresis
describe RFLPs and alleles (genes)
sickle cell anemia - recessive aa, mutation in B globin gene - defective hemoglobin and defective RBC - anemia
aa = recessive
AA = dominant
Aa = heterozygous carrier
what is DNA sequencing
method to sternum if cloned DNA is the one of interest if there is a polymorphism (single gene mutations associated with inherited diseases)
synthesis reaction in vitro reaction
name and describe ingredients of DNA sequencing (5)
DNA of interest - one template strand
DNA polymerase
primer for specific DNA (one primer, provided 3’ OH)
dNTPs
ddNTPs - fluorescently labelled (ddGTP - green…)
what are dideoxyribonucleotides
stop elongation/extension reaction whenever they get incorporated into the daughter strand synthesized
produce DNA strands or fragments of different sizes lengths and colours
whats the difference between dNTP (deoxyribonucleotides) and ddNTP (dideoxyribonucleotides)
dNTP - OH anchor for synthesis
ddNTP - H synthesis stops
name the 2 types of immunity
innate
adaptive - acquired
describe innate immunity
rapid response against a broad range of microbes (non specific)
cells - macrophages, neutrophils, natural killer cells