Genetics (4)
science of heredity and gene fxn
- study of what genes are
- how they carry info
- how they are replicated and pass to generations
- how gene expression determines the particular characteristics of an organism
what organisms have RNA genes?
viruses and bacteriophages
What is a bacteriophage?
A virus that infects bacteria
What does the cell's genome include?
chromosomes and plasmids
Ribozymes are
RNAs that process catalytic activity
genome
all the genetic info in a cell
name 6 RNA products expressed in either prok or euk cells
1. mRNA- (not expressed)
2. tRNA-
3. rRNA-
4. miRNA-
5. sRNA
6. snRNA-
7. snoRNA- in nucleolus
Chromosome features (in bacteria-4)
- one, circular double-stranded DNA or linear
- size ranges 500-6,000kb
- lots of DNA that is not useful (humans)
- looped, folded, and attached at one or many points on plasma membrane
T/F: human cell have lots of more DNA that is not converted in mRNA?
T
plasmid features
- autonomously replicating extrachromosomal DNA
- range sizes 1.5-300
- contains genes that are conditional
(not necessary for day to day survival but required under certain conditions)
how is DNA compacted in bacteria?
-supercoiling
how is DNA compacted in archaea and euk
-histones
What are chromosomes? (what do they contain)
DNA that physically carry hereditary info
is a bacteria chromosomes= bacterial genome?
genome is all genetic info and chromosome is not because chromosome has a plasmid too
What is a gene?
A segment of DNA or RNA that encodes for a polypeptide or RNA chain that has a fxn
how are plasmids classified?
by function
types of plasmids
1. conjugative plasmid
2. dissimilation plasmid
3. R factors/plasmids
what is conjugative plasmid?
carries genes needed for transfer of the plasmid to another cell
what is dissimilation plasmid?
encodes enzymes for catabolism of unusual compounds
- common in pseudomonas (toluene, camphor, and petroleum hydrocarbs)
what is R factors/plasmids?
encode resistance to abx or heavy metals and often
- carry multiple resistance genes (r-determinants) problem when transfer abx resistance (transfers all)
- are conjugative (resistance transfer factor RTF)- easier to transfer
understand figure R factor
know diff components
- origin of replication
- origin or transfer
- origin of replication- by bidirectional
- origin or transfer- during conjugation
- *
encode gram - cell goes to pillis and attach to recipient cell and encode other genes for conjugation
*
what are other fxnal plasmid groups?
- virulence plasmid-
- bacteriocin plasmid
what are virulence plasmids?
encode for proteins that enhance the pathogenicity of the bacteria
- host attachment
- toxins
what are bacteriocin plasmids?
encode for bacteriocins
bacteriocins...
ribosome synthesized peptides that kill closely related bacterial species or even diff strains of the same species
difference btw bacteriocin and abx?
- bacteriocin- active against closely related species
are peptides
- abx- broad and affect wide variety
*
some abx are peptide but most derived from a.a
* not translated
araD139 (triangle)(araA-leu)
what is the D and triangle meaning?
D- sugar
triangle- deletion btw araganose and leu are deleted
What does a nucleotide consist of?
- nucleobase
- deoxyribose
- phosphate group
Two DNA strands are held together by...
hydrogen bonds
how do base pairs occur? and how many H-bonds?
A-T (2 H-bonds)
C-G (3- H-bonds)
structure of DNA helps explain two primary features...
1. linear sequence of bases provides actual info
2. complimentary structure allows for precise duplication of DNA during cell division
how would you modify DNA backbone to be RNA?
change thymine to uracil and use ribose sugar (OH on 2nd C)
What did Meselson and Stahl discover?
DNA replication is semi-conservative
before semiconservative was discovered how was it thought to be?
- conservative
- dispersive
genetic code...
a set of rules that determines how a nucleotide sequence is converted to an amino acid sequence of a protein
what gene products are used in protein synthesis?
mRNA, tRNA, miRNA
genotype is...
genetic makeup of an organism (characteristic)
Genetic Nomenclature for bacteria/archaea (4)
- 3 letters depending on pathway, fxn, cell structure
- lower case/italicized
- diff genes affecting the same pathway are distinguished by capital letters
- each diff mutants is assigned a unique allele #
phenotype is...
external looks of an organism genotype
what does the following mean?
bio-
arg-
met-
requires biotin, arginine, and methionine added as a supplement to minimal medium
what does the following mean
lac-
gal-
cannot utilize lactose and galactose as a carbon source
what does the following mean?
str^r
str^s
r- resistance/ s-sensitive
to abx streptomycin
how long is genetic map of chromosomes?
100 mins now before used to be 90 mins to transfer
What are ORFs?
Open reading frames- part of DNA btw starts and end codon that can be translated into proteins
what are short tandem repeats?
non-coding short DNA sequences (2-5 bases) that repeat in introns
what are the polymorphisms in STRs due to?
# of copies of the repeat element that can occur in a population
In microbes, most proteins are either...
enzymatic or structural
Bacteria DNA...
single circular chromosome
why does the chromosome only take up about 10% of cells volume (when there are many base pairs)?
DNA is twisted or supercoiled
noncoding regions are called
short tandem repeats (STRs)
genomics...
the molecular study of genomes
Flow of genetic information
-
expression
- used w/in cell to produce proteins needed for cell fnx
-
recombination
- transferred btw cells of same generation
-
replication
- transferred btw generations of cells
when replication begins what protein is used to relax supercoiling?
topoisomerase or gyrase
what protein helps unwound (separate) the parental DNA?
helicase
what happens when bases are improperly base-paired?
they are removed and replaced by replication enzymes
the newly added nucleotide is joined to growing DNA by what enzyme?
DNA polymerase
the point at which replication occurs
replication fork
How is DNA read?
5' to 3'
what initiates DNA synthesis?
RNA primer
how is leading and lagging strand synthesized?
leading- continuous
lagging- discontinuously (Okazaki fragments)
how are RNA primers removed and Okazaki fragments joined?
DNA polymerase and DNA ligase
semiconservative replication
original strand (conserved) and newly synthesized daughter strain rewind
what proteins prevent DNA to rewind after is unwinded?
single stranded binding proteins
hydroxyl attached to the...
3' end of DNA strand
phosphate attached to...
5' end
DNA polymerase adds new nucleotides (dNTPs deoxyribonucleotide triphosphate) to what end?
3' end only
the two new strands must grow in what direction?
opposite (different)
DNA replication requires a great deal of...
ENERGY
where does the energy come from to make DNA replication?
supplied from nucleoside triphosphate bonded to sugar loses 2 Pi when they bind to correct base
what the difference btw ATP and adenine nucleotide in DNA?
sugar component in ATP is ribose
what is deoxyribose used for?
to synthesize DNA
what are nucleoside triphosphate w/ ribose used for?
synthesize RNA
how do you add nucleotide to growing strand of DNA? what type of E?
-by removed 2 phosphate groups
- hydrolysis of nucleoside is exergonic and provides E to make new bonds in DNA strand
since bacteria DNA is circular what happens at end of replication?
replication fork eventually meet when replication is complete (full circle)
at what rate are mistakes made in DNA replication?
1 in every 10^10
whats the major factor for the low rate of mistakes?
proofreading of DNA polymerase
proofreading function
corrects DNA mismatch to allow each daughter chromosomes to be identical to parent DNA
how are DNA gyrase and topoisomerase similar/different?
- they both relax DNA to unwind
- but gyrase uses E to supercoil
which enzymes are euk specific? and fxn
- DNA ligase- joins okaz and new segments
- DNA polymerase- syn DNA, proofreads, and repaids
- endonucleases- cut DNA backbone help repair/insertions
- exonucleases- cut DNA from an exposed end of DNA, help repair
- snRNP- protein comples removed introns
E. coli DNA replication is...
bidirectional
how is it possible that it takes 40 mins to replicate E. coli chromo yet doubling time of organism can be as fast as 20 mins?
when 1/2 done replication starts again dividing so when your done you still have a replication happening...
bacteria transcription:
transcription begins when ____ binds to ____
transcription begins when
RNA poly
binds to
promoter
sequence
when does bacterial transcription stop?
when reaches
terminator
sequence
process of transcription in prok?
1. RNA poly binds promoter and DNA unwinds
2. mRNA is synth by complementary bases
3. site of synth moves along DNA, DNA transcribes rewinds
4. transcription reaches terminator
5. RNA and RNA poly are release and DNA helix re-forms
how many RNA poly do euk have?
3; other prok have 5 from mito and chloroplast
mRNA transcription and processing in euk?
1. in nucleus, gene (exons+introns) transcribed to mRNA
2. involved snRNPs in nucleus to remove introns and splice together exons
3. further modification ==> cytoplasm for protein synthesis
4. add poly A tail at 3' and 5' cap for stability
RNA is terminated in bacteria through
1. road dependent rho- binds DNA and cause polymerase to fall off
2. road independent rho- RNA made, there is a stretch of UUUUU forms upside down U (stem loop) and at the end there it terminates
mRNA is translated in...
codons
start codon:
nonsense codons:
AUG
UAG, UGA, UAA
how many sense codons?
how many total codons?
61
64
genetic code is degenerate; what does this mean? and is this good or bad? and why?
- that many codons can code for the same a.a
- good bc enables silent mutations
tRNA carries...
complementary anticodon
what is the start codon in bacteria?
N-formylmethionine- only at start codon bc there it replaces NH3 (eventually cleaved off)
what is codon bias?
there are many codons that may code for
same
a.a and the fact that some are more common even though they code same a.a is codon bias
is the genetic code universal?
NO- mito has its own
how is the correct AUG start codon recognized in euk and prok?
euk- scan the 1st AUG
prok- recognize shine-dalgarno sequence ~8 nucleotides away from correct AUG
what is an operon?
Group of genes under control of a single operator. Multiple genes on a single mRNA transcript...
process of translation
1. EPA sites anticodon- AUG comes to P site
2. next anticodon enters A site
3. btw a.a form a peptide bond
4. anticodon goes to E site to exit
5. ribosomes moves along mRNA 5' to 3'
6. stop codon ends translation
7. polypeptide is release==> protein
T/F: in prok transcription and translation work independently?
F: they are coupled together (happens at same time)
why is transcription and translation not coupled in euk?
bc transcription occurs in nucleus and has to be proper before leaves and starts translation in cytoplasm
what are constitutive genes?
60-80% expressed at a fixed rate
repressible genes...
transcription rate
decrease
by regulatory protein
repressor
default position is
on
inducible gene...
transcription rate
increased
by a regulatory protein
inducer
default position is
off
catabolite repression: (subclass of repressible gene)
inhibition of the synth of alternative catabolic enzymes by preferred carbon source (glucose)
[when glu present--> inhibits the enzymes to break down lactose]
catabolic repression (graph)
glucose present shut off all catabolic genes that degrades other cmpds (don't need to make those enzymes if not needed)
- no more glucose then lactose is used
negative regulation of the lac operon:
1. RNA poly binds promoter
2. active repressor protein binds operator and
stops
progression of polymerase
3.
repressor active and operon off
operon includes....
control region ( P & O )
structural genes (Z, Y, A)
what is allolactose?
lactose is converted to allolactose (diff bond) still 2 sugars
what breaks down lactose?
B-galactosidase
what does the permease do?
transports lactose into cell
positive regulation of lac operon:
1. allolactose (inducer) binds repressor to remove repressor protein
2. RNA poly runs through sequence and translate mRNA
3.
repressor inactive and operon
still off
**
what makes up galactose?
glucose + galactose
positive regulation
1. lactose + glucose (genes not expressed)
2. lactose, NO glucose
1. CAP binding site is inactive and RNA poly
cannot
bind
2. cAMP binds inactive CAP then binds promoter to recruit RNA polymerase (inducer) --> transcribe
how are cAMP levels controlled?
-cAMP levels are high when NO glucose is present.
-enzyme adenylyl cyclase converts AMP--> cAMP (when low levels of glu)
only way to turn on lactose lac operon...
no glucose and yes lactose
4 scenarios of glucose + lactose
Trp Operon (biosynthetic operon)
1.
inactive repressor protein
=
operon on
(does not bind sequence and RNA poly goes through)
**
no inducer
**
Trp Operon (negative)
1.
repressor is active
blocking RNA poly from going through =
operon OFF
how can bacterial transcriptional regulator proteins recognize same sequence but activate one gene and repress another?
- depends where they bind on the DNA
- block upstream of promoter- blocks transcription
epigenetic control
- heritable changes in gene expression that occur w/o changes in DNA sequence
change in phenotype w/o change in genotype
which nucleotides in euk are methylated?
C*G (silence genes passed to offspring cells but can be reversed)
which nucleotides are methylated in prok?
GA*TC (important for restriction modification and replication fidelity)
- may also influence gene expression
restriction modification ? 1:32
one enzyme modify at specific point and at another is restricted (cut) if not methylated ?
If DNA poly makes mistakes and does not know where to modify will modify the new nucleotide
where else can you regulate gene expression in prok?
1. mRNA stability (short poly A tails)
2. translation
3. protein stability
miRNAs are nucleotide single stranded RNAs incorporated where?
RISC
what do RISC do?
bind mRNA and degrade
or bind and inhibit translation or stability
bacterial small regulatory RNAs are:
non coding RNAs 1. bind protein and alter fxn or 2. bind RNA and regulate translation and stability
mutations
a change in the base sequence of DNA bc of DNA poly
mutagen
agent that causes mutations
spontaneous mutations
occur in absence of mutagen
different types of mutations and fxn?
1. base substitution
2. additions and deletions
3. inversion: deletion and reassertion in opposite orientation
different types of base substitution and fxn?
1. transitions- change purine for purine
2. transversions- change purine for pyrimidine
point mutation (similar to base sub)
change w/in a gene in which one base pair in the DNA sequence is altered
which mutations is most easily reverted spontaneously?
base substitution (either transition or transversion)
which mutations can impact protein translation? (4 & fxn)
1. missense mutants- change of a.a
2. nonsense mutants- creates stop codon
3. silent mutant- does not alter a.a of protein
4. frame shift mutants- insertion or deletion of one or a few bases ==> alters reading frame
why do frame shift mutations generally lead to premature termination?
bc shifting can make a stop codon more frequent since theres about 3/20 a.a (avg gene 1000s)
do nucleotide changes outside ORFs impact gene expression?
yes bc mutation can be in promotor or region where inducer or repressor binds
what are temp sensitive mutants and how are they related to conditional lethal mutants?
- temp sensitive- protein in gene is unstable at high temps
- temp sens are class of conditional mutants (fxnal at specific conditions)
mutations rates defines as
1. mutation
per base pair
per generations/cell division
2.
per gene
per generation/cell division
3.
per genome
per generation/cell division
mutagens increase mutation rate 10 to...
1000x
are mutations generally beneficial, neutral or detrimental?
either neutral or detrimental
types of mutagenic agents (5)
1. base modifiers
2. base analogs
3. radiation
4. frame shift mutagens
5. mobile genetic elements
base modifiers: nitrous acid causes...
deaminates adenines and cytosines
so they base pair C-A
base analogs
polymerase puts them into DNA and changes what they bind too
1.
2 aminopurine
binds
thymine
(gets protonated) and then changes to bind to
cytosine
2.
5 bromouracil
(anticancer drug) normally pairs
adenine
if turns to enol pairs
guanine
types of radiation
ionizing radiation ionize H2O to hydroxyls and they can oxidize bases and alter binding properties
1. direct action-
2. indirect action-
direct action (radiation)
X rays and gamma rays physically break one or both of sugar phosphate backbones, break the H-bond or damage the bases of DNA
indirect action (radiation)
cause the formation of ions that can oxidize bases resulting in errors in replication and repair
UV radiation causes
thymine dimers
repair mechanisms for thymine dimers
1. photolyases- separate thymine dimers
2. nucleotide excision repair- cut both sides of damage, remove it and polymerase repairs it
can nucleotide excision repair deletions?
no
In gram - bacteria, how are incorrect bases that are undamaged distinguished from correct bases?
cells assume methylated strand is correct and changes the complement
frame shift mutagens and examples?
insert btw bases and causes kinks
-
benzopyrene
: in smoke and soot
-
aflatoxin
: produced by variety of molds that grow on peanuts and grain
-
acridine orange
- fluorescent cationic dye
Azidothymidine (AZT) is used as an antiretroviral drug to help?
treat AIDS
mechanism of action of AZT
terminates growing chains by blocking replication bc N=N=N is on 5' where OH normal binds
why is the retrovirus impaired more than the host?
1. viral RT has a 100 fold greater affinity for AZT than cellular DNA poly
2. cells can quickly repair their DNA chain if broken by AZT whereas virus cannot
is mitochondrial RNA poly sensitive to AZT?
yes, it is more sensitive than cellular DNA poly
2 strategies to detect mutants?
1. positive (direct)- detects mutant cells that
grow under conditions their unmutated parents won't
2. negative (indirect)- detects mutants cells bc
they do not grow
(replica plating)
or...3. screen identifies mutant cells that appear diff
why is it useful to generate mutants?
- study particular gene (if mutant present) study fxn
- find mutant that tolerate high temp or that make higher protein
why are bacteria useful for generating mutants?
reproduce quickly and are haploid (1 set of X (chromo))
direct selection process
- grow in media supporting mutant but not wild type
- only rare mutants will survive
indirect selection process
(replica plating)
- used when there is NO medium on which the mutant can grow and the parent cell cannot
- mutants that require growth factor not normally present
how would replica plating be modified to identify temperature sensitive mutants?
use two rich plates one at low temp and one at high temp
- low temp see growths
- colonies missing at high temps
what advantage does direct selection have over indirect selection?
- want to find mutant direct is easier bc looking for growth on plate can look at 10 billion cells all at once
- indirect is restricted to see a specific # of colony (can see few) need many plates
phenotypic screening (more indirect)
recognizable differences in colony morphology (not selection)
- pH indicators
- differential breakdown of materials in media
what is the Ames test used for?
to identify chemical carcinogen by using bacteria as carcinogen indicators
procedure for Ames test:
experimental sample (w/ suspect mutagen) and control (no mutagen added)
mutagen- histidine dependent salmonella
1. both are added rat liver extract (activator) (enzymes in body, liver, converts cmpds into mutagenic)
2. put on media lacking histidine
3. in
are mutagens also carcinogens?
yes most are
vertical gene transfer
occurs
during reproduction
btw generations of cells
horizontal gene transfer
transfer of genes
btw cells of the same generation
3 mechanism of gene transfer in bacteria
1. transformation
2. conjugation
3. transduction
DNA needs to go into cell and replicate by...
homologous recombination
homologous recombination process
1. exchange of genes btw two DNA molecules
2. cross over occurs when 2 chromosomes break and rejoin
3. recA protein cataluzes joining of two strands (donor DNA+ recipient chromo)
4. recipient chromo has 1 or more new genes and donor is destroyed
who discovered an experiment with rats to demonstrate genetic transformation?
frederick griffiths
what was fredericks griffiths experiment?
- living encapsuled bacteria injected in mouse= died
- living nonencapsulated bacteria inject into mouse= healthy
-heated encapsulated= healthy
- nonencapsulated+ heated killed encapsulated= dead mouse
*
proved transforming factor to be DNA
*
transformation
direct uptake of DNA by recipient cells
natural transformation
active process demanding specific enzymes produced by the recipient cell
why is transformation useful?
extremely useful for experimentally for introducing DNA into bacterial cells
what is meant by cell competence?
ability of cells to take up DNA from environment
bacterial conjugation
transfer of a plasmid from one cell to another (cell to cell contact)
- plasmid is replicated during transfer of a single-stranded copy of plasmid DNA to the recipient, where the complementary strand is synthesized
how does conjugation differ from transformation
1. conjugation requires cell to cell contact
2. conjugation cells must be generally be of opposite mating type
(
donor cells must carry plasmids
)
conjugation in gram - bacteria
- plasmid carries genes that code for synthesis of sex pili
- projections from donors cells surface makes contact with recipient bringing two cells into direct contact forming
cytoplasmic bridge
(cytoplasmic bridge not true anymore)
conjugation in gram + bacteria
- cells produce sticky surface molecules==> causing contact
- NO sex pili
which was the first plasmid observed to be transferred btw cells during conjugation?
F factor F+ (fertility factor)
a cell w/ an integrated F is called a
Hfr cell
Hfr (high frequency recombination)
F+==> Hfr- replicate and parental strand of chromosome is transferred to recipient
formation of Hfr
a+ goes into F- at 5 mins
b+ goes into F- at 8 mins
c+ goes into F- at 17 mins
d+ does into F- at 30 mins
takes about 90 mins to transfer the chromosome
what sequence do F plasmids and bacterial chromosomes have in common that permit integration via homologous recombination?
insertions elements and transposons
following conjugation btw Hfr and F- does F- cells become Hfr?
no bc it did not recieve a complete F factor
is integration of the F plasmid into the bacterial chromosome random?
no bc it goes where the insertion elements are
F' bullshit...
excision involve a different bacterial IS element than was originally used for integration, excised F factor will contain segment of bacterial DNA
generation of F' lac
1. insertion-
2. excision-
transduction
bacterial DNA is transferred from a donor cell -->recipient cell inside a bacteriphage
two major kinds of transduction
1. generalized trans-
2. specialized trans
generalized transduction
a random fragment of bacterial DNA is accidentally encapsulated in a phage protein coat in place of the phage DNA
specialized transduction
- only those genes located on the bacterial genome in close proximity to the prophage insertion site can be transduced
- specialized transduction particle contains a
hybrid phage
and a
bacterial DNA
molecule which integrates into phage genome as the phage
why is the frequency of transducing a specific gene from a donor cell to recipient cell low? (4)
1. encapsulation of bacteri DNA is low 1/1000
2. probability that the transducing particle carries the gene of interest is low
3. successful infection of the P1 transducing particle is required
4. transduced gene must recombine w/ homologous portion of ch
what is cotransduction frequency?
if 2 mins apart genes then cotrans frequency= 0
*
closer they are higher cotransduction
*
transposable elements
self-mobilizable genetic element or "jumping genes" that can be transferred from place to place on the chromosome and into and out of plasmids
replicative and non-replicative transposons
a replicative- leaves a copy to itself at original location
non-replicative- does NOT leave a copy
are target site preferences stringent and long?
yes, thus suitable insertions locations for many ISs
are insertions random?
NO
3 major kinds of transposable elements
1. insertion sequence- contains genes for transposons (milk)
2. transposons-
3. certain phages (no need to know)
what is a transposons and subtypes?
- resistance gene captured by 2 insertion sequences
1. composite
2. Tn3- resolvable diff mechanism needs other gene and has transposases--> have same sort of resistance gene
T/F: most resistance gene transfer is plasmid mediated?
T- but plasmid mediated traits can interchange w/ chromosomal elements
T/F: virtually all resistance determinants genes on R plasmids are NOT present as transposons
F- they are present
toxin of E.coli
shiga- leading cause of diarrhea
how did E. coli acquire this toxin gene?
sequence pathogen shiga toxin carried bacteriophage which picked up specialized transduction
what are the benefits of E. coli
suppresses growth of harmful bacterial species and synthesizes vitamins