General problem
· Bacteria don't
have sex. Normal mechanism of cell division is clonal (one cell produces
many identical offspring).
What possibilities exist for transfer of DNA from one cell to
another? How common are they
in nature?
· Originally
thought that bacteria lacked any significant gene transfer. But discovery
of
R-plasmid transfer was shocking revelation
of rapid gene transfer, not just among
individuals of same species but among quite different
genera of bacteria.
HISTORY:
1. British biochemist Frederick Griffith - 1920's: worked with Strep.
pneumoniae:
· Two strains with different
appearances:
· Strep. Pneumoniae (S) was heat-killed and combined with R bacteria and injected together into mice =killed. Therefore, a "Transforming factor" must be present.
3. Lederberg and Tatum - 1946:
• Able to isolate two E. coli mutants (auxotrophs)
• bio- Met- X Thr-
Leu-
• Mix and plate in minimal medium
lacking all 4 components
• bio+ Met+ Thre+ Leu+
• CONCLUSION: Definitely there is genetic
exchange.
4. Lederberg and Zinder: Used the U-TUBE EXPERIMENT (1950's)
• GOAL: to demonstrate if cell to cell contact
is necessary
• CONCLUSION: Yes, cell to cell contact is
necessary, therefore CONJUGATION and
not TRANSFORMATION
5. Zinder and Lederberg:
• GOAL: demonstrate that conjugation occurs
in other organisms, Salmonella typhimurium
• Back to the U-tube experiment
• Cell to cell contact not required
• DNAse treatment didn't affect the process ----------
TRANSFORMATION
rule out!
• Absorbed out by "sensitive cells" or antibodies
--------- TRANSDUCTION!
Genetic Engineering relies critically on ability to move DNA from one
cell to another. Any modem biologist should be familiar with basic mechanisms,
possibility of extending these with laboratory "tricks".
1. RECOMBINATION
·Combining
genetic information from 2 individuals to form a new one which is different
from either parent.
·Distinguish
two stages of gene transfer: (1) getting DNA from Donor cell to recipient
cell;
(2) getting DNA integrated into
recipient (or into a different type of stable form, typically
a plasmid). Even if (1) and (2)
happens, no detectable result unless recombination also happens.
Why would such a system ever evolve? Probably as a repair
mechanism for situations
when cell faces double stranded damage to chromosome
(can't be fixed by normal repair
mechanisms, since don't have a remaining "good strand"
to copy from). But if cell had
duplicated chromosome but not yet divided, could
copy good DNA from other chromosome,
use to replace damaged section.
1. THREE MECHANISMS FOR DNA TRANSFER
Transformation, transduction, conjugation. Not clear
how important these are in nature. In laboratory they are very important.
A. DNA TRANSFORMATION
· Uptake of DNA fragments from medium
surrounding cell.
· Requires specific proteins
in cell membrane, and energy.
· Only found naturally in certain
cells; e.g. Pneumococcus, Hemophilus, etc. Not found in E.
coli. (This is changing and now some people
think it does happens in E. coli!!!)
· Why did
this evolve? Maybe as way to scrounge DNA from dead cells, save trouble
of
having to make nucleotides from scratch.
· Cells that
can take up DNA are said to be competent. Requires induction of
several genes
("competence factors") depending on the bacterium
*-DNA-binding proteins
*-envelope-associated exonuclease
*-competence-specific proteins
(prevent nuclease attack?)
· Typically occurs
during exponential or late exponential growth, shuts off in stationary
phase.
· Easier to
do competence with PLASMIDS: Why? 4 circular VS linear DNA!!!.
· E. coli
can be made competent by treatment with calcium chloride ->4
renders membrane
permeable
B. TRANSDUCTION
· A bacteriophage serves as the vehicle
for the transfer of bacterial DNA.
Two types:
a) Generalized transduction
·Phages
normally replicate in bacteria, producing many copies of phage DNA
· This is known as a
lytic
cycle. Many copies of phage DNA are packaged heads and
released upon bacteria lysis.
· Host
DNA is often degraded, and occasionally, piecesof partially degraded
bacterial
DNA of the correct size are packaged
inside phage coat proteins, phage erroneously packs
up a "mistake" = transducing phage.
· This
"mistake" phage can't cause infection; but it can be transferred
to a different
bacterium, get DNA into cell without
risk of being degraded in environment
· Even if this occurs,
chances are slim that successful expression of DNA will occur --- still
needs to undergo recombination. If most
cells are killed by phage, not much use.
b) Specialized transduction:
· Involves
"temperate" phages ® integrate in
the host chromosome
· Integration
always
at a particular site ® prophage
· A bacterium
carrying a prophage is lysogenic.
· Upon
induction two possibilities: a) Normal phage genes ®
lytic
b) Portion of the phage genes (95%) +bacterium genes
(5%)
C) CONJUGATION
· Conjugation
= plasmid-directed transfer of DNA from one cell to another.
· Cell to cell contact is necessary
· Episome ®
a plasmid that can exist extrachromosomally or as part of the chromosome.
PROPERTIES OF PLASMIDS
· Circular DNA elements, always double-stranded
DNA
· Can occur in as few as 1 copy per
cell (single copy plasmids) to as many as several dozen
(multicopy plasmids)
· Variable sizes; small plasmids
about 0.1% size of host chromosome, large plasmids can be
as much as 10% the size of host chromosome. Smaller
plasmids have few genes (30 or less)
· Ubiquitous; almost all cells isolated
in nature carry plasmids, often more than one kind.
· Have a replicon (origin for DNA
replication), number of copies per cell regulated. Large
plasmids typically only 1-5 copies/cell (stringent
control); small plasmids ~ 10 - 50
copies/cell (relaxed control)
· Many plasmids are incompatible;
if one is present, cell cannot support another plasmid of
same compatibility group.
· Not essential to cell under all
circumstances; can be "cured" by agents that impair DNA
replication ---- > cured cell lacking plasmid. Can
be spontaneously lost over time unless
some selection makes plasmid valuable to cell.
· Extend range of environments in
which a cell can live (e.g., by degrading antibiotics, or
providing enzymes for digestion of novel catabolites).
Plasmids can be classified in two classes:
a) Conjugative ® possess tra
genes (attachment, sex pilus)
b) Nonconjugative ® lack tra
genes
EXAMPLES OF PLASMIDS
·R
PLASMIDS: carry antibiotic resistance genes (enzymes that modify or
degrade
antibiotics) -- plasmids
with these genes are called R factors
• F PLASMIDS: Fertility plasmids--> code
for the Sex pilus
• COL PLASMIDS: contains genes for bacteriocins
• VIRULENCE PLASMIDS: Genes for toxins ---->
harmful to other hosts
• METABOLIC PLASMIDS: genes for enzymes to
degrade different substrates: heavy
metals, hydrocarbons, etc.
CONJUGATIVE PLASMID TRANSFER
· Some plasmids
have specific transfer genes, can copy DNA, transfer one copy to a plasmid-
cell
· Sex pilus:
rigid fiber, sticks out from cell wall. Acts as recognition molecule to
locate
sensitive cell (plasmid minus). When + and - cell
are attached by pilus, cells pulled
together. (Grappling hook analogy).
· Plasmid DNA
replicates (remember "rolling circle" replication) during transfer; one
copy
remains in donor cell, identical copy transferred
to recipient.
· Once recipient
receives plasmid, it grows pili, becomes a donor. One plasmid could
potentially infect a whole population, convert all
cells to plasmid-containing cells.
1. F+ x F- = BOTH F+
· F+ contain the F plasmid
and the sex pilus (determined by genes in the tra region)
· Transfer is
always unidirectional; always moves from F+ cell (donor)
to F- cell (recipient).
2. MATING OF Hfr X F- = RECIPIENT F-
· Transfer occurs with fairly high
frequency of recombination (Hfr = abbreviation).
• Very rarely, F plasmid becomes integrated into host DNA = Hfr.
Very stable, doesn't "pop"
back out
• Still retains ability to transfer to another cell, but in doing so,
drags
host DNA with it
• Transfer always originates at same point (due to location of F factor
in chromosome at one
site)
·In
E coli
, when Hfr mates with F-, transfer of entire chromosome takes
~100 minutes, but
very rare . Only part of the F factor is transferred,
so recipient remains F-. Location of
different genes can be mapped by time of transfer.
3. MATING OF F' x F-
• Since plasmid is an episome, it can leave the bacterial chromosome
and take bacterial
chromosomal genes.
• The recipient becomes F'
• Hfr ® F' x F- = Both
F'