21. Recombination I

Question 1

what is the action of the Trp operon?

Answer 1

• feedback inhibition (repression)
• operator: recognised by trp repressor
• if repressor binds to operator, it physically blocks RNApol from transcribing the gene

Question 2

how does the Trp operon operate feedback inhibition?

Answer 2

• repressor only binds and blocks transcription in presence of tryptophan
• tryptophan binds to repressor, changes shape to become active
  – is co-repressor; as it switches repressor to active state

Question 3

what levels of tryptophan are needed?

Answer 3

• low levels means no bind
• no attachment to DNA and blocking of transcription
• allows transcription of trp operon by RNApol

Question 4

what are plasmids?

Answer 4

• small DNA molecules found in bacterial cells
• physically separated from bacterial chromosomal DNA
• most commonly found in bacteria
  – small circular dbl-stranded DNA molecules
  – can also be present in achaea and eukaryotic organisms

Question 5

what are the characteristics of plasmids?

Answer 5

• replicate indipendantly of chromosome
• relatively few genes
• their genetic informations is not essential to host
  – bacteria lack them function normally

Question 6

what is the characterisation of plasmids?

Answer 6

• single vs. multi-copy plasmids
  – 1 plasmid/cell vs. >40 per cell
  – has consequence of expression of genes present on these plasmids
  – if 40 copies of plasmid encoding antibiotic resistance, likely that cell produces a lot of proteins providing resistance
• conjugative plasmids have genes for pili
  – and can transfer copies of themselves to other bacteria during conjugation

Question 7

what are fertility factor plasmids?

Answer 7

• 94.5 kb, carries genes for cell attachment and plasmid transfer during conjugation
• most of transfer genes carried in tra operon (21 genes)
  – many direct formation of sex pili that attach F+ cell (donor carrying F plasmid) and F-cell
• other gene products aid DNA transfer

Question 8

how for bacteria exchange genetic material?

Answer 8

• horizaontal gene transfer

Question 9

what is horizontal gene transfer?

Answer 9

• movement of gene(s) between matyre independent organisms
  – one-way transfer from donor to recipient

Question 10

what are the three main ways of DNA movement in bacteria?

Answer 10

• transformation (direct uptake)
  – bacteria take up DNA from environment and incorperate it into their genome
• transduction (phages play a role)
  – movement of DNA between bacteria by viruses
• conjugation (pili)
  – direct transfer of DNA by bacteria usually via plasmids
  – from one cell to another by the means of a physical bridge

Question 11

what is transformation?

Answer 11

• uptake of naked DNA molecule/fragment by cell
  – and incorperation of molecule into recipient chromosome in heritable form
  – when bacteria lyse, they release considerable amounts of DNA into surrounding environment
  – if fragment touches competent cell, it can be bound to cell and take up
• transformation in random
  – any portion of genome may be transferred between bacteria
• this is exploited in molecular biology
  – means of introducing DNA, plasmids into bacteria host system

Question 12

what is the process of transformation?

Answer 12

• DNA fragments taken up by recipient cell
• recombination occurs between donor DNA and recipient DNA
• double-stranded DNA binds to plasma membrane
• enzymes cut DNA
• other enzymes break down one DNA strand
• a protein binds the DNA
• DNA replaces portion of bacterium’s DNA

Question 13

what is the process of artificial transformation?

Answer 13

• antibiotic-sensitive bacterial cell
• treated with CaCL2
  – to permeabilise cell walls
• adding plasmid DNA
• transformed bacterial cell is formed
• selection on bacterial growth medium containing appropriate antibiotic

Question 14

what is genetic transformation and what examples are used?

Answer 14

• genetic alternation of cell(s)
• direct uptake and incorporation of DNA
  – bacteria
  – yeast and filamentous fungi
  – insect cells
  – mammalian cell or tissue culture
  – plant cell or tissue culture

Question 15

how are plants transformed?

Answer 15

• microparticle bombardment as means of transforming
• gene gun
• makes use of micron-sized particles for DNA delivery (gold/tungsten particles)
  – widely used technique as overcomes problems of transforming higher plants due to cell walls
  – employed to transform macroalgae and plants

Question 16

what is agrobacterium mediated transformation?

Answer 16

• agrobacterium tumefaciens
  – soil borne, Gram negative bacterium
• causes Crown Gall Disease in dicotyledonous plants
  – unique mechanism of DNA transfer and integration of transferred DNA (T-DNA) into host-plants genome
• Ti-plasmid encodes
  – T-DNA (cancer causing genes)
  – vir region (virulence region for plant attachment)
  – plasmid replication features
• ‘stripped down’
  – cancer-forming genes ‘dis-armed’
  – vector still able to mobilise Ti-plasmid and transfer foreign gene(s) into host plant(s)