Unit II- Dynamic Genomes and the Creation of Genetic Diversity

Question 1

Why is genetic variation/instability important

Answer 1

-fodder for natural selection (can be costly to the individual but beneficial to the population)
-leads to the propagation of drug resistant micro-organisms
-implications for human health
(-uncovering of recessive genetic diseases
-deregulation of normal genes (cancer)
-susceptibility/resistance to disease
-response to treatment)

Question 2

Why you need to understand genetic variation in bacteria

Answer 2

• models the same processes in our cells (recombination, new mutations, viruses, transposable elements)
• the mechanisms lead to induction and propagation of antibiotic resistance
• to introduce plasmids, a critical tool for recombinant DNA technology

Question 3

New mutations- genetic variation

Answer 3

• mistakes during DNA replication and DNA repair (greatest source of small lesions: 1/10^9 per nucleotide per replication in bacteria, 1/10^7 in yeast, 1/10^6 in humans
• chromosomal rearrangements caused by inappropriate recombination events and/or the insertion of mobile elements (greatest source of large lesions)

Question 4

Spontaneous mutations and natural selection in E. coli

Answer 4

• haploid
• genome is encoded on a dsDNA circular chromosome of 4-5,000,000 base pairs
• doubles every 20 minutes
• mutation rate is 1/10^9 per nucleotide per replication. In 10^9 per nucleotide per replication. IN 10^9 cells there are likely to be many mutations represented in the population

Question 5

Gene transfer in bacteria via mating

Answer 5

• 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 is conferred by F plasmids

Question 6

Plasmids

Answer 6

• small circular, dsDNA molecules that are distinct from the bacterial chromosome
• carry sequence elements that allow for replication and other goodies (F Plasmids 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
• common way bacteria spread drug resistance
• 1/3 of Neisseria gonorhoeae isolates are pencillin resistant

Question 7

Bacterial Transformation

Answer 7

• some strains of 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 bacteria chromosome (recombination)

Question 8

Homologous Recombination in Bacteria

Answer 8

• this is the reciprocal exchange of genetic information (DNA)
• 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

Question 9

gene transfer by bacteria viruses (bacteriophases)

Answer 9

-viruses are parasites that cannot replicate themselves without a host

• genomes express:
• coat proteins that serve to package, protect and help deliver the genome to a new host
• other activities required to replicate, express or integrate the virus genome into the host chromosome
• possibly genes picked up from previous hosts

-bacteriaphage lambda: dsDNA virus that has been used to manipulate exogenous DNA sequences in E. coli

Question 10

Mechanism of bacterial recombination

Answer 10

• the process initiates with the alignment in register of two homologues, double stranded DNA sequences
• a nick is made in one strand allowing it to invade and anneal to the complementary strand of the homologue (called strand exchange)
• then the displaced strand is nicked and it anneals to the other homologue at which point the ends are ligated to complete formation of cross-strand exchange (Holiday junction)
• the DNA strands have to get the outside strands to cross each other
• then the crossing strand are cut and ligated to each other

Question 11

Integration of DNA by recombination

Answer 11

• double crossovers by homologous recombination can lead to integration of an exogenous DNA fragment into the bacterial chromosome
• this would lead to a stable transformation event
• in addition plasmids can integrate into the bacterial chromosomes via short regions of homology
• both mechanisms are used to transfer antibiotic resistance genes

Question 12

Latent versus Lytic virus

Answer 12

• integration results in a latent stage referred to as the prophage
• integration occurs by site-specific recombination catalyzed by a virus encoded integrase
• in lysis the virus just makes the cell make the proteins needed to make new virus and after they are packaged the cell explodes

Question 13

Movement of genes by transduction

Answer 13

• when the bacteriophages are induced to exise themselves from the bacterial chromosome, 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

Question 14

Transposons

Answer 14

• integrate into the bacterial chromosome, frequently in multiple copies
• range in length from several hundred to several thousands of base pairs
• 10-20 transposons per bacterium
• codes for at least a transposase that catalyzes transposition
• may also carry antibiotic resistance genes that can be transfered to other cells by hopping into plasmids or bacteriophages
• insertion can disrupt a gene

Question 15

Bacterial transposons

Answer 15

IS3, Tn3, and Tn10

• they can cut from the original site and insert into a new site: non-replicative transposition (cut-and-paste)
• or they can be copied by DNA replication and then insert into new site, amplification: replicative transposition

Question 16

Transposons contribute to genetic diversity

Answer 16

• may also carry antibiotic resistance genes (multiple transposition can lead to amplification of the antibiotic resistance gene, also transposons can become infective by hopping into a plasmid or into a bacteriophage)
• insertion can disrupt a gene
• since the transposons carries promoters, it can effect the expression of neighboring genes
• repeat sequences can confuse the homologous recombination apparatus leading to rearrangement of the bacterial chromosome

Question 17

Sex- mixing your grandparents alleles

Answer 17

• parents have each inherited 2 sets of chromosomes (ie. they are diploid)
• meiosis is a reduction division that produces two haploid gametes
• gametes fuse to form the zygote
• 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 alleles

Question 18

independent assortment

Answer 18

• the maternal and paternal homologous segregate randomly/independently from each other
• the haploid gametes contain a mix of maternal and paternal chromosomes

Question 19

Eukaryotic homologous recombination

Answer 19

• initiates with a double strand break
• Rad50 complex resects the 5’ ends leaving 3’ overhands
• Rad51 facilitates strand invasion/exchange
• Ligase and resolvase connects the end
• many enzymes are shared for repair and recombination

Question 20

Recombination and diversity

Answer 20

• recombination enzymes are induced several fold during meiosis
• in humans there are on average 3 recombination events per chromosome events per chromosome per meiosis
• we all inheriti 250-300 loss of function alleles
• the rate of de novo germline base substitution is ~10^8

Question 21

Transposable elements in humans

Answer 21

• three major known retrotransposons
• LINE1 ~6-8 kb in length, 21% of the genome, encodes its own reverse transciptase, competent to transpose
• SINEs- 100-300 bps in length, 13% of the genome (1.5 million copies), use the RT from LINEs to move
• Alu sequence: 300 ncs long, 5% of genome, 500,000 copies per haploid genome. Very few are competent to transpose
• transposition is induced during meiosis

Question 22

Transposons contribute to genetic instability

Answer 22

• they can disrupt gene function by inserting in the coding region of an expressed gene. One form of hemophilia is caused by an L1 insertion into the Factor VIII gene
• they can effect the expression of neighboring genes, there presence within genes tends to decrease expression
• they provide sites for illegitimate recombination aka unequal crossing over (gene amplificiation, exon amplification/deletion)
• Alu sequence in particular, uniquely define human DNA and have been used to clone human genes in other organisms

Question 23

Creation of genes families by unequal cross over

Answer 23

• transposons create sites for miss-alignment during recombination and thereby an unequal crossover
• once you have multiple gene copies, these paralogs can become specialized through genetic drift

Question 24

Exon duplication/deletion by unequal crossing-over

Answer 24

-mis-alignment of homologs via transposons in introns, during recombination, causes both exon deletions and duplications

Question 25

The Dystrophin gene

Answer 25

• exon duplication/amplification led to the creation of the dystrophin gene
• exon deletion causes some forms of muscular dystrophy

Question 26

Exon shuffling by transposable elements

Answer 26

-new genes with unique combinations of functions can be created by bringing together exons that code for functional protein motifs

Question 27

Infective insertion elements: the retroviruses

Answer 27

• viruses are parasites that require hosts to replicate
• their genomes (single stranded RNA) are packaged in a protein coat plus a few copies of the enzymes required to initiate virus replication
• the coat protects the genome and facilitates infection
• resemble retrotransposons in that the DNA that integrates is made from an RNA genome
• genomes typically code for the reverse transcriptase, coat proteins and the integrase required to insert in to the host genome

Question 28

How can retoviruses cause disease

Answer 28

• cell death e.g. AIDS
• integration can disrupt an important gene
• virus promoters are very active and can inappropriately activate the expression of neighboring genes: carcinogenesis (HTLVs)
• virus can pick up important genes from previous hosts, for example oncogenes/ growth control genes
• oncogenes were first discovered as genes picked up by retroviruses