1
Q
why is genetic variation/instability important?
A
- fodder for natural selection-costly to individual but beneficial to population
- leads to the propagation of drug resistant microorganisms
- implications for human health: uncovering recessive genetic diseases, deregulation of normal genes, susceptibility/resistance to disease, response to treatment
2
Q
genetic variation in bacteria
A
- models same processes in our cells-recombination, new mutations, viruses, transposable elements
- mechanisms lead to induction and propagation of antibiotic resistance
- plasmids are a critical tool for recombinant DNA technology
3
Q
new mutations
A
- mistakes during DNA replication and DNA repair-greatest source of small lesions, 1/10^6 per nucleotide per replication in humans (9 in bacteria, 7 in yeast)
- chromosomal rearrangements caused by inappropriate recombination events and/or insertion of mobile elements-large lesions
4
Q
spontaneous mutations and natural selection in e coli
A
- haploid
- genome on dsDNA circular chromosome of 4-5,000,000 bp
- doubles every 20 min
- mutation rate is 1/10^9 per nucleotide per replication. in 10^9 cell there are likely to be many mutations represented in the pop
- can propagate resistance that way (gene was already there)
5
Q
bacterial gene transfer via mating/conjugation
A
- F+ bacteria can mate with F- bacteria
- F+ bacteria can form a sex pilus
- small epigenetic elements such as plasmids can be transferred via the pilus
- F+ status conferred by plasmids
- F plasmid carries sequences that allow it to be replicated and transferred to new host
6
Q
plasmids
A
- small, circular dsDNA molecules that are distinct from bacterial chromosome
- carry sequence elements that allow for replication and other goodies
- F plasmid carry genes required to make the sex pilus and transfer DNA to the recipient by rolling circle replication
- plasmids are used to manipulate, amplify, and purify exogenous DNA sequences
7
Q
rolling circle replication
A
- DNA strand cleaved
- one strand transfers to recipient cell, DNA synthesis in donor cell
- DNA synthesis also in recipient cell
- completion of synthesis and cell separation
8
Q
plasmids and drug resistance
A
- common way to spread resistance
- resistant cell carries F plasmid
- F plasmid integrates into bacterial chromosome
- mates to a non-resistant cell (sensitive) and transfers resistant gene
9
Q
bacterial transformation
A
- some bacteria (bacillus subtilis) can pick up DNA from their environment
- DNA may come from the lysis of other bacteria
- the exogenous DNA can be incorporated into the bacterial chromosome (recombination)
10
Q
homologous recombination in DNA
A
- reciprocal exchange of genetic information
- two homologous sequences align so they are in register
- both strands of each double helix are broken and rejoined to the homologue
- exchange can occur anywhere in the region of homology
- fidelity is high, the sequence at the site of exchange usually remains unaltered
11
Q
mechanism of homologous recombination
A
- strand nicking and exchange
- nicked strand invades other DNA and anneals to its bases and the displaced DNA binds with original DNA
- ligate nicked strands-DNA molecules joined by cross strand exchange
- proteins pull green up and purple behind and cut it (see slide)
- called Holliday junction
12
Q
integration of DNA by recombination
A
- can do two crossovers to integrate (replaces other DNA)
- F plasmid can crossover and integrate without replacing
13
Q
gene transfer by bacterial viruses aka bacteriphages
A
- viruses are parasites that cannot replicate themselves without a host
- genomes express coat proteins to package, protect and help deliver genome to new host, other activities for replication, express or integrate virus genome into host, and maybe genes picked up form previous host
- bacteriophage lambda-dsDNA virus that has been used to manipulate exogenous DNA sequences in E. coli
14
Q
latent vs lytic virus
A
-integration results in a latent stage referred to as the prophage
-integration occurs my site-specific recombination catalyzed by a virus encoded integrase
-attachment, injection–>integration into DNA and divides with DNA–>induction event turns virus potent
or
-attachment, injection, synthesis of viral proteins needed for new viruses and packaging–>lysis and release of new viruses
15
Q
movement of genes by transduction
A
- when bacteriophages are induced to excise themselves from the bacterial chromosome (in latent pathway), they can pick up flanking DNA
- this flanking DNA will be packaged into viral particles that can infect new hosts
- transfer of bacterial genes in this manner is called transduction
16
Q
transposable elements (transposons)
A
- integrate into the bacterial chromosome, frequently in multiple copies
- range in length from several hundred to several thousands of bp
- 10-20 transposons per bacteria
- codes for at least a transposase that catalyzes transposition
- may also carry antibiotic resistance genes that can be transferred to other cells by hopping into plasmids or bacteriophages
- insertion can disrupt a gene
17
Q
replicative vs non replicative transposition
A
-donor and target DNA, one part (red) goes into new DNA and remains in old DNA
vs.
-red part going to new DNA and no red part in old DNA
18
Q
transposons and genetic diversity
A
- may carry antibiotic resistant genes-multiple events can lead to amp of resistant gene, transposons can become infective by hopping into plasmid or bacteriophage
- insertion can disrupt a gene
- since transposon carries promotors, it can effect the expression of neighboring genes
- repeat sequences can confuse the homologous recombination apparatus leading to rearrangement of bacterial chromosome
19
Q
the truth about sex
A
- parents have each inherited 2 sets of chromosomes- diploid
- meiosis is a reduction division that produces haploid gametes
- gametes fuse to form the zygotes
- zygote develops into a diploid organism with a copy of each chromosome from each parent
- humans are extremely polymorphic so the product at the bottom now has a unique combination of genes
20
Q
independent assortment
A
- parental and maternal homologues are not identical, they carry different alleles
- maternal and paternal homologues separate independently from each other in meiosis
- therefore haploid gametes contain a mix of maternal and paternal chromosomes
- there are 2^n possibilities when n=# of chromosomes. 8.4 x 10^6 for me, 7.1 x 10^13 between two
21
Q
eukaryotic homologous recombination
A
- initiates with ds break
- Rad 50 complex resects 5’ ends leacing 3’ overhangs
- Rad51 facilitates strand invasion/exchange
- ligase and resolvase connect the ends
- many enzymes are shared for repair and recombination
22
Q
recombination during meiosis
A
- recombination enzymes are induced several fold during meiosis (actually on all the time)
- in humans there are 3 recombination events per chromosome per meiosis 3.5x10^41 possibilities for 2 people
23
Q
meiosis mutations
A
- all inherit 250-300 loss of function alleles: in frame small deletions, premature stops, splice site disruptions, reading frame shifts
- 50-100 variants known to cause inherited disorders
- rate of de novo mutations is `10^8
24
Q
transposable elements in humans
A
- move via RNA intermediate
- LINE-1,
- SINEs
- Alu sequence
- induced during meiosis
25
LINE-1
-6-8 kb long, 21% of genome, 850,000 copies encode its own reverse transcriptase, competent to transpose
26
SINEs
-100-300 bp in length, 13% of genome, 1.5 million copies use reverse transcriptase from LINEs
27
alu sequence
- 300 ncs lon, 5% of genome, 500,000 copies per haploid genes
- very few are competent to transpose
28
human transposons and genetic instability
- disrupt gene function by inserting in the coding region of an expressed gene (hemophilia)
- effect the expression of neighboring genes, their presence within genes tends to decrease expression
- provide sites for illegitimate recombination- unequal crossing over- gene amplification, exon amplification/deletion
- Alu sequences in particular define human DNA and have been used to clone human genes in other organisms
29
creation of gene families by unequal crossing over
- transposons create sites for miss-alignment during recombination and thereby an unequal crossover
- once there are multiple gene copies, the paralogs can become specialized be genetic drift
- globin gene in humans- double the globin exon and it differentiated
30
exon duplication/deletion by unequal crossing-over
-mis-alignment of homologs via transposons in introns, during recombination, causes both exon deletions and duplications
31
dystrophin gene
- exon duplication/ amplification lef to the creation of the dystrophin gene
- exon deletion causes some forms of muscular dystrophy.
- makes gene too long or too short
32
exon shuffling by transposable elements
-new genes with unique combinations of functions can be created by bringing together exons that code for functional protein motifs
33
retroviruses
- infective insertion elements
- require hosts to replicate
- genomes (ssRNA) packaged into a protein coat plus a few copies of the enzymes required to initiate virus replication
- coat protects genome and facilitates infection
- resemble retrotransposons in that the DNA that integrates is made form the RNA genome
- genomes typically code for the reverse transcriptase, coat proteins, and the integrase required to insert into the host genome
- see picture for life cycle
34
retrovirus lifecycle
- attaches
- releases capsid
- RNA with RT makes a DNA strand
- second strand synthesized
- integrated into host DNA
- synthesis and packaging of more viruses
35
retroviruses cause disease
- cell death-AIDS
- integration can disrupt important gene
- virus promoters are very active and can inappropriately activate expression of neighboring genes- carcinogenesis
- virus can pick up important genes from previous hosts, oncogenes and growth control genes
36
oncogenes were first discovered as genes picked up by retroviruses
- rous sarcoma virus: CT tumors
- chicken retrovirus
- picked up a constitutively active allele of the scr kinase growth control gene
- abl, erb-B, fes, fms, fos, jun, myc, raf, H-ras, rel, sis
- less commonly cause cancer in humans
MCP Unit 2
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