bacterial genetics Flashcards
polymorphism
cite in a gene sequence displaying variation in a population
hox genes
group of related genes controlling body plan of an embryo along a head-tail axis
scabrous
ethanol reward as memory aid insignalling pathway of long-term memory
hangover
ethanol tolerance aid as well as response to heat and oxidative stress
syntaxin
mutations affect ethanol tolerance to sedation, encoding synaptic vesicle release protein
simple/ single gene defect disease
Duchenne MD
huntington’s
complex/multi gene defect disease
Cancer
diabetes/obesity
wild type
unmodified isolate of a species, well-characterized in the lab
mutant
differs to wild-type in that changes to DNA sequence have occurred
mutation
specific change/changes to a DNA sequence different to the wild-type
phenotype
observable trait
genotype
nucleotide sequence of a region of DNA
why are bacteria used as genetic models?
much is known about them
easy to manipulate
short generation time
vertical gene transfer
changes in DNA passed on to progeny
horizontal gene transfer
changes to DNA passed on from other bacteria/viruses
genetic transformation scientists
Fred Griffith
- conversion of non-pathogenic strept. pneum into a pathogenic
conjugation scientists
Lederburg and Tatum
- 2 e coli strains mixed and progeny isolated with both characteristics
transduction scientists
Zinder and Lederburg
- displayed bacterial viruses carrying DNA from one bacterium to another
Bacterial transformation
ability of bacterial cell to uptake DNA from other cells in the same environment
Competence
correct physiological state for recipient cell in order to uptake DNA
naturally/ artificially induced
purpose of transformation
increase functional/ metabolic capabilities to compete in environment
bacterial conjugation
gene transfer via cell-to-cell contact mating
conjugation mediation
conjugative plasmid can induce pilus/ transfer DNA
high frequency recombination strains
can transfer part of host chromosome to recipient
mob genes
allow for genetic transformation of a non-conjugative bacterial cell
stages of bacterial conjugation
donor cell attaches to recipient cell via pilus
pilus contracts
1 plasmid DNA strand attaches
recip and donor cell synthesize complementary strands to form an F+ cell
bacterial transduction
gene transfer via bacterial virus mediation
transducing particle
phage filled wiyh host and chromosomal DNA
stages of transduction
- bacteriophage injects DNA
- phage enzymes degrade host DNA
- cell produces more phages incorporating host/ phage DNA
- transducing particles transform other cells as donor DNA incorporated into recipient chromosome via recombination
transcription
decoding genetic info enmcoded by DNA into RNA
1 step process
decoding genetic info to proteins
2 step process (transcription/translation
bacterial genetic decoding
transcription and translation coupled
genes of related function often clustered on chromosome
transcription mediator
RNA polymerase
transcription 3 stages
initiation, elongation, termination
types of rNTPs
rATP, rUTP, rCTP, rGTP
Inititiation
RNA polymerase attaches to promoter sequence in DNA
elongation
RNA polymerase moves along template strand, decoding to RNA
termination
RNA polymerase recognizes terminating sequence and finishes transcription prior to strand separation
1 residue
1 amino acid= 3 ribonucleotides= 1 codon
number of possible codons per amino acid
64
number of proteins potential depending on reading frame
3
number of reading frames
2 strands with 3 proteins each
how is choice of reading frame determined
an ATG/AUG start codon
- ribosome binding site in front is complementary to 3’ end of 16S rRNA
2 stages of bacterial DNA replication
binary fission
DNA synthesis
DNA polymerase action in bacterial DNA replication
creates phosphodiester linkage between 5’OH end of DNA and 3’PO4 end on dNTP
DNA synthesis precursors
deoxynucleoside triphospates
dATP dGTP dCTP dTTP
bacterial replication
begins at oriC before proceeding bidirectionally (creating 2 replication forks) and finishing at terminus C (terC)
6 types of mutation
base-pair changes
deletions
inversions
insertions
frame-shifts
duplications
2 types of base-pair change mutations
transition/ transversion
base pair transition
pyrimidine> pyrimidine/ purine> purine
base pair transversion
pyrimidine<>purine
3 potential consequences of base-pair mutations
silent mutation (same sequence)
missense mutation (different sequence)
nonsense mutation (stop codon)
mutation frequency formula
m/N
no. mutants/ no. bacteria
mutant selection
general selection
1. isolate randomly distributed mutants
2. screen
specific selection
every isolated mutant of interest screened
negative selection
selects against muatnt growth
enrichment
negative selection use to promote growth of mutants and inhibiting wild-type/ competition growth w antibiotic
positive selection
selective conditions to promote mutant growth
(usually in resistance to a phage)
genomics
acquisition, storage, retrieval and analysis of DNA sequence
genome size range
(most common?)
0.13-14MBp
most common =3 MBp
E.Coli model strain
characteristics?
K12 strain
rapid growth, simple nutritional requirements
K12 relatives
meningitis
UTI
gastroenteritis
typhoid
plague
E.Coli chromosome size
4.6MBp
streptomyces coelicolor chromosome
linear chr
parts of streptomyces coelicolor chromosome
core (maintenance genes)
arms (variable extras)
ARTEMIS
visualisation tool displaying annotation files graphically
metagenomics
study of genetic material recovered directly from environmental samples
recombinant DNA industries
food, pharma, agriculture, medical research
food recombinant DNA examples
chymosin (cheese manufacture)
golden rice (enzymes promoting B carotene synthesis)
pharma recombinant DNA examples
human insulin (pig replacement)
HGH (cadaver replacement)
blood clotting factor VIII (haemophilia)
Hep B vaccine (yeast cell production)
medical research recombinant DNA examples
HIV antibody test
agriculture recombinant DNA examples
proteins (herbicide resistance)
glyphosphate (herbicide round-up)
resources for recombinant DNA tech
enzymes, DNA/ RNA, vectors, cells
types of enzyme used in recombinant DNA tech
restriction
taq polymerase
ligase
reverse transcriptase
restriction enzymes
cleave DNA at specific sequences
often recognise palindromic 4-8 Bp
DNA ligase
anneals
taq polymerase
amplification of DNA fragments
reverse transcriptase
converts RNA back to DNA
2 types of cleavage pattern
symmetrical (blunt end production)
asymmetrical (sticky end production)
2 types of DNA in recombinant DNA tech
insert/ vector
vector DNA
unique restriction sites
efficient oriC
gene expression regulatory sequences
plasmid size
2-200 kbp
vectors for larger DNA fragments
bacteriophages
cosmids/phagemids
cosmids/ phagemids
genetically engineered hybrids replicating as plasmid and packaged as bacteriophage
clone production steps
- prepare insert/vector
- ligate both
- transform recomb DNA into host
- select hosts containing DNA
isolation of specific inserts
cleaved via restriction nuclease
amplification of non-specific insert
cDNA copies total mRNA and reverse transcriptase used
amplification of specific DNA
PCR
Steps in DNA insertion
- isolate insert from RNA/DNA
- ligate insert into vector
- transform into host cell
- select recomb DNA hosts
ligation of insert into vector
cleaving of plasmid and ligase annealing of insert
transformation into host cell
- plasmid mixed in
- heat shock and CaCl induces competence of cells
- taken up by plasmid and selectively cultured
- recomb purified and expressed
- expression in host
selection of recomb DNA hosts
selective medium growth
insertional inactivation
DNA fragment in polylinker disrupts lacZ gene > inactive b galactosidase > no blue pigment detected by X-gal
how to check for recombinant DNA
Hybridization of ssDNA to probe
screening for protein expression
PCR
E.coli requirement for expression in host
expression vector
expression vector mechanism
contains promoter sequence recognised by host RNA polymerase and therefore expressed gene ligated at 3’ end
direct expression of recombinant protein
purification
investigation of protein function
expression of modified version of protein
cloned gene engineered to : change protein properties/ investigate fine details of a protein
non-coding DNA
introns
exons
intron
nucleotide sequence within gene
removed via splicing during RNA maturation
exon
gene region produced after intron removal
when would inactive form be expressed
if genomic DNA ligated into expression vector
bacterial expression systems advantages
simple
cheap
short generation time
large yield
bacterial expression systems disadvantages
can fail to fold crrectly, losing bio activity
toxic proteins to bacterial cell
no post-trans modifications
yeast expression systems advantages
simple
cheap
resembles mammalian cells
quick
pos trans modifications
yeast expression systems disadvantages
protease containing (degrades recomb proteins)
differing post-trans modifications
insect cell expression systems advantages
cheaper than mammalian
high-level expression
correct folding
post-trans modifications
insect cell expression systems disadvantages
post-trans modifications differ from mammalian cells
mammalian expression systems advantages
best for mammalian
correct folding
post trans modifications
mammalian expression systems disadvantages
expensive
complex cells
grow to lesser densities
example of expression for commercial use
diabetes
example of protein expression for further research
HIV
where’s insulin produced
beta cells of islets of langherhan in pancreas
bovine/porcine insulin considerations
side-effects
ethics
purification issues
contamination issues
insulin structure
2 polypeptide chains linked by disulfide bonds
synthesis of insulin procedure
folding stabilized by disulfide bonds, connecting peptide removed
recombinant insulin production
proinsulin gene (2introns) coding for mRNA which is reverse transcribed to cDNA, inserted into a recombinant plasmid and transforms bacterium
genetic model organisms wanted characteristics
short lifespan
readily available
amenable to genetic transformation
large offspring yield
small;
rapid development rate
e.coli cell
gram-negative, rod-shaped bacterium
generation time of E.coli
20-30 mins
e coli advantages
simple
safe
short generation times
e coli disadvantage as model organism
prokaryptic so differ to eukaryotic
homologue
gene related to another gene via descent from a common ancestoral DNA sequence
orthologue
genes from different species evolved from a common ancestral gene
paralogue
genes generated by a duplication event
gene knockout
gene sequences completely/ partially removed, inhibiting gene expression
gene knockdown
techniques interfering/ reducing gene expression
yeast budding/fission advantages
share characteristics of cell div cycle, gene expression, signalling pathways w humans
short life cycle
small
simple growth/ storage
easily transformable w plasmids
trivial construction of gene knockouts/ knockdowns
drosophilia melanogaster advantages
small
easy to collect
2 week life cycle
14000 gene encoding
easy to cross
phenotypic markers
transformable
research useful
p transposon
jumping gene used to transform drosophilia
drosophilia research areas
cell signalling
development
neurological disease
disease model
mutant model organism mimicking human phenotypes of disease
spinal muscular atrophy
neurodenegerative disease decreasing motor function
caused by mutation in smn gene
flys hold orthologous smn gene
alzheimers
accumulation of amaloid beta peptide
progressive neuron loss
C. elegans
small, non-parasitic nematode
transparent
easy to manipulate
both sexes
300 offspring pp
2-3 week lifespan
c. elegans dsRNA interference procedure
- dsRNA complementary to region of interest introduced
- process to short interfering siRNA’s
- prevtnion of expression of region of interest
zebra fish
transparent embryos
200 eggs weekly
difficult to sequence etc
knockdown possible w morphilinos
micro-injectible
zebra fish research areas
vertebrate development
neurobiology
toxicology
disease/ drug discovery
mus musculus
closely related to humans
identical gene composition offspring (lab inbreeding)
embryonic manipulation
knockouts available
mouse knockout process
- embryonic stem cells w one copy of target cell deleted
- injected into early embryo
- hybrid embryo introduced to mouse
- hybrid offspring
- further cycles > homozygous KO
C value paradox
gene number doesn’t increase linearly with genome size
percentage of human genome-wide repeats
44%
G-banding
mild proteolyis then GIEMSA
AT rich
R banding
heat denaturation then GIEMSA
GC rich
Q banding
quinacrine stain
AT rich
C-banding
BaOH then GIEMSA
Constitutive heterochromatin
alpha satellite
large arrays of repeated sequences
assembly site for kinetochore
centromere
positioning of centromere
metacentric
submetacentric
arcocentric
telocentric
short chr arms
p
long chr arms
q
telomere
specialised region at chr ends
function of telomere
allows distinguishment between real chromosome end/ unnatural so that unnatural is destroyed
end replication problem
end replication problem
3’ terminal isn’t copied > ssDNA overhang degraded
telomeres therefore prolong replicative senescence
telomerase
recognizes uncopied region
adds multiple copies of ‘TTAGGG’ repeat
replication machinery synthesizes other strand
histones
form octamer which DNA wraps to form nucleosome
euchromatin
relatively uncondensed
associated w active genes
heterochromatin
condensed
associated w silenced gene-poor regions
B-chromosomes
additional chromosomes possessed by some in population
holocentric
no singular cetromere, multiple kinetochores throughout
kinetochore
protein structure located at centromere, serving as attachment point for mitotic spindles
centriole
9 microtubule groups, generating mitotic spindle fibres
G2 of interphase
chromosomes extended
chromatin duplicated
chiasma
physical connection between 2 non-sister chromatids
leads to crossing over between pairs
meiosis prophase 1
leptopene
zygotene
pachytene
diplotene
diakenesis
synaptonemal complex
nucleoprotein zipper between paired homologous chromosomes
role in crossing over/ chiasma formation
disjunction
1/2 of tetrad migrates to each pole
oocytosis
begins in embryonic ovary and arrests in prophase 1
nondisjunction
aneuploid gametes
trisomy
3 copies of specific chromosome
mitosis vs meiosis
meiosis is reductional division
cant occur in haploids
2 successive divisions
mitosis 1 division
occurs in haploids/ diploids
sexual reproduction disadvantages
advantages
time and energy to find mate
potential breaking apart of favourable gene
better equipped for environmental changes
2 hypotheses of sex determination
protenos mode
lygaeus mode
heterogametic
producing unlikely gametes
homogametic
producing uniform gametes
klinefelter syndrome
tall with feminised physique
poor beard growth
low IQ
breast development
female pubic hair
testicular atrophy
osteoperosis
44 autosomes + XXY
Turner syndrome
short
constricted aorta
elbow deformity
no menstruation
brown spots
44 autosomes + 1X
human Y chromosome regions
PAR
SRY
MSY
PAR
MSY regions
euchromatin
centromere
euchromatin
heterochromatin
PAR
pseudoautosomal regions
sharing homology with X chromosome
synapse and recombine with x chromosome in meiosis
MSY
Male Specific Region of Y
doesnt synapse w x chromosome
SRY
sex determining region of Y
produces testes determining factor,triggers undifferentiated gonadal tissue of embryo to form testes
dosage compensation
females likely produce twice as much gene product for all X-linked genes
> mechanism required to equate X-linked gene product doses
Barr body
condensed X-chromatin
inactivated X chromosome
barr body formation
dosage concentration supported by X chromosome inactivation
lyonization
inactivation random at early point in development
once inactivated, all progeny cells have same X-chromosome inactivated
anhidrotic ectodermal dysplasia
x-linked mutation causing sweat gland absence
males have no sweat glands
females have mosaic of D/d sections over body
X chromosome inactivation mechanisms
initiated from XIC (X inactivation centre)
produces 2 non-coding RNA transcripts (Xist/ Tsix)
leads to packaging of 1 X chr into v dense, compacted form of chromatin
polycistronic
mRNA corresponding to multiple genes whose expression is also controlled by single promoter/terminator
prokaryotic promoter
sigma factor recognises sequence upstream of gene, positioning RNA polymerase
holoenzyme
sigma factor + RNA polymerase
no. of RNA polymerases in prokaryotes
1
no. of RNA polymerases in eukaryotes
3
RNA polym 1, 2, 3
RNA polym 1
ribosomal RNA
RNA polym 2
all protein-coding genes
RNA polym 3
non-coding RNA’s
RNA polym 2 promoters in eukaryotes
TATA box bound by transcription factor IID complex, recruiting RNA polym 2 and txn factors
5’ cap added while RNA transcribed
transcription termination
transcripts end 10-35nt downstream of signal AAUAAA
RNA cut via endonuclease, releasing DNA
poly(A) tail added
prokaryotic gene expression alteration ways
alternative sigma factors (different -35/-10 sequences)
how many sigma factors do E coli have
7
mutation of sigma factor
affects expression of set of gene regulating
eukaryotic various transcription factors
allows stimuli to affect each promoter
microarray
DNA corresponding to each gene in sequence dotted onto slide
mutation in intron
doesn’t affect coding sequence
could affect splicing
mutation in exon
alters coding sequence
intron/ exon boundaries function
define limits
recruit machinery removing introns from RNA (“spliceosome”)
capping
1st modification made to RNA polym 2- transcribed RNA
polyadenylation
addition of poly(A) tail to RNA transcript
small ribosomal sub-unit
reading of mRNA
large ribosomal sub-unit
houses synthetic petidyl transferase centre
ribosome function
finds ORF start and interprets codons so as to pair w tRNA
how does start codon recognition differ in pro/eu?
eukaryotic mRNA has a cap
Shine-dalgarno sequence
large sub-unit binds to small sub-unit, translates/ initiates on downstream AUG
continuous variation
e.g. height/ foot size
polygenic
Mendel’s first law
2 copies of each gene segregate in meiosis
Mendel’s second law
copies of each gene segregate independent of other genes in Meiosis
when do Mendel’s laws not apply
when both alleles are found on same chromosome
unlinked genes
meiosis independent assortment
linked genes
can’t assort independently
Hardy-weinberg formulae
p^2 + 2pq +q^2= 1
Hardy Weinberg assumptions
infinitely large population
random mating
no evolutionary forces acting
when does the Hardy-weinberg principle not work?
gene flow
genetic drift
non-random mating
natural selection
examples of autosomal recessive conditions
albinism
cystic fibrosis
phenylketoneuria
sickle cell
haemochromatosis
tay-sachs
phenylketoneuria
inability to metabolize phenylalanine
Tay-sachs
neurodegeneration due to lack of N-acetyl-hexosaminidase
cystic fibrosis
thick mucus build-up due to CFTR absence
haemochromatosis
excess iron accumulation due to mutation in HFE gene of which there are 2 mutant alleles
HFE gene
regulates hepcidin production
hepcidin
iron regulatory hormone determining iron absorption from food/store release
examples of autosomal dominant conditions
huntington’s
FASP
polycystic kidney disease
polydacyly
achondroplastic dwarfism
hypercholesterolaemia
achondroplastic dwarfism
FGFR3 mutation
failure to convert cartilage to bone
autosomal recessive pedigree
trait appears in progeny
horizontal pedigree pattern
autosomal dominant pedigree
trait appears in every generation
vertical pediree pattern
X-linked recessive disorders
haemophilia
muscular dystrophy
haemophilia
absence of factor VIII
blood doesnt clot
X-linked dominant disorders
hypophosphataemia
hypophosphatemia
vit-D resistant rickets
Leri-weill dyschondrosteosis
bone growth disorder
lack of SHOX gene copy
co-dominance
traits show up equally in F1
3 blood group alleles
I^a/I^b/i
incomplete dominance
F1 resembles neither parent
pleiotrophy
genes/alleles affecting more than one unrelated characteristic
e.g. manx phenotype
epistasis
gene modifying/masking phenotype of another gene
hypostatic
gene/allele being masked/modified
penetrance
number of members in population of specific genotype displaying the expected phenotype
expressivity
range of signs/symptoms that can occur in different people w genetic condition
polydactyly
incomplete penetrance as variable digits of progeny
complementation group
set of mutations mapping to same chromosomal locus, failing to complement when crossed
how is complementation skewed?
intragenic complementation
intragenic complementation
proteins w multiple functions forming multimers
linkage
genes on same chromosome, co-segregating in crosses
test-cross for 2 traits/genes
- construct double heterozygote
- cross w tester homozygous recessive for both traits
recombination frequency
(no. recombinants/ total progeny) *100
distance between 2 loci
1 centimorgan = 1 *10^6 Bp
syntenic genes
grouped in same way on chromosomes of 2+ species
synteny blocks
regions containing homologous genes
translocation
where 2 non-homologous chromosomes break and exchange fragments
acute myelogenous leukaemia
coding region for C-terminus of AML transcription replacement
required for haematopoiesis, coding region unrelated to other allele
centric fusion
2 telocentric chr fuse / generate new chromosome
robertsonian translocation
2 acrocentric p lost and 2q remaining fuse
result of robertsonian translocation
down syndrome/ patau syndrome
inversion
segment of chromosome becomes inverted with original position
2 types of inversion
pericentric
paracetric
paracentric
1 arm
pericentric
2 arms
result of chromosome inversions
small gene duplications/ large gene duplications prior to deletion of superfluous genes/ divergence of retained homologues
unequal crossover
strandbreak resulting in unequal crossover/ different no. repeat units
sister chromatid exchange
strandbreak on sister chromatids produces different repeat numbers
transcription factors in eukarya
bind motifs in promoter, promoting/inhibiting RNA pol II ability to initiate transcription
intron/ exon boundaries function
defines limits
recruit machinery removing introns from RNA
purpose of alternative splicing
can yield multiple products from each gene
bacterial translation
shine-dalgarno sequence recognised by small ribosomal subunit RNA via base-pairing
large binds to small sub-unit and translates downstream of AUG
small sub-unit binds CAP and large binds to first AUG
shine dalgarno function
allows independent translation of each ORF in polycistronic RNA
shine-dalgarno function
allows independent translation of each ORF in polycistronic mRNA
mutation in shine dalgarno
reduces specific ORF translation but not others
difference between pro/eu translation
pro is coupled / trans affects txn
eu is uncoupled/ trans doesn’t affect txn
haplotype
association of number of polymorphic markers
variable number tandem repeats cause
unequal crossover/ replication errors
microsatellites
GC-rich, variant repeats of ~5 nucleotides
e.g. SSR/ STR
Paternity testing
PCR testing of minisatellites
DNA-17 profiling
DNA-17 profiling
detects STR’S/ STR loci
SNP/ single nucleotide polymorphism
single base differences
produce new sites for restriction endonucleases
RFLP
Restriction fragment length polymorphism
CpG islands
associated w promoters
enriched w CpG sites
identified when sequence unknown due to restriction enzyme clustering
associated with 5’
SNP map functions
locating genes associated w phenotypes
diagnosis of problems/ phenotypes
international HAPMAP project
SNP maps of human genome
haplotypes used as tool for genotyping and examination
analysed via bead/gene chips
bead chips
PCR amplification of whole genome
fragments DNA into smaller pieces and hybridizes primers bound to beads on chip
genome wide association study pros
informs patient care/screening/ human history
insurance
gene identification influencing phenotypes
multigene family
group of genes descending from common ancestor and therefore having similar functions / genotypes
how do multigene families arise
duplication events followed by mutation generating alterations in function/expression
function of hox genes
encode transcription factors influencing expression patterns of genes during development
homeobox
180 nucleotide segment wncoding DNA binding ‘homeodomain’
primitive globin
monomeric
single O2 binding
thalassemia
alpha/beta chain production in unequal amounts leading to abnormal Hb
cause of thalassemia
mutation in a/b globin genes
severity depending on no. affected genes and mutation severity
B thalassemia
2b genes
1 mutated=minor
2 mutated= major
de novo generation
neutral/advantageous transcription of part of genome, evolving over time as new gene
In which cell-type is Baculovirus used as a cloning and expression vector for recombinant genes?
insect
Which enzyme is used to make a cDNA copy of mRNA?
reverse transcriptase
multiple cloning site
polylinker
