Unit 2 notes

bewilderedvoyageBiotechnology

Dec 12, 2012 (4 years and 8 months ago)

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Unit 2 notes

Transfer of DNA from one cell to another

Importance

Second mechanism for acquisition of NEW genes

Mutation was the first mechanism discussed

Has created medical problems



Genes for antibiotic resist
ance

often passed on



Genes with ability to
cause disease

Transformation



Definition



transfer of DNA through open environment



Source of DNA



dead, lysed cells



Lysis produces random fragments of DNA that transfer between cells, but recipient cells must
satisfy certain requirements



Any part of the g
enome can be transferred



Recipient cells



Must be “competent”

to absorb genetic material


can’t absorb huge pieces of DNA

o

Even a single gene
-
sized piece of DNA is huge



Natural competency

(vs. artificial)

o

Only observed in small group of genera



EX:



Streptoco
ccus



Bacillus



Pseudomonas

o

Depends on:



growth stage



particular DNA receptors on surface of cell



need certain genes in call to create DNA receptors on surface of cell in the first
place

o

Uptake system



Binds DNA outside of cell



Usually linear fragments (as
opposed to?)



Double stranded DNA is absorbed


one strand degraded, the other enters the
cell



Once inside cytoplasm, one strand aligns with
homologous regions

(complementary bases) in chromosomes



Then integrated into chromosomes



Outcomes



If doesn’t integra
te


degraded in cytoplasm



If does integrate


gain of genetic info

o

Cross
-
species transfer



Pathogen can gain drug
-
resistant gene from a non
-
pathogen (our cells maybe?)



Depends on degree of relatedness



If donor and recipient cells are more closely related,
higher chance of
finding homologies



Genes can traverse species lines

o

Net result of transfer



Potential gain of:




drug resistance



Ability to cause disease

o

Example:
Streptococcus
pneumonia



Griffith experiments (1920s)



2 strains of Streptococcus pneumonae

(causes pneumonia)



Capsulated


anti phagocytic (beats white blood cells)



Non
-
encapsulated


avirulent (could not cause disease)



Griffith was trying to create vaccines



Combined 2 strains


encapsulated cells were dead and non
-
encapsulated were
alive



He
expected mice injected with this mixture to be healthy, but mice died



Something had transferred the ability to make capsules to the avirulent cells



Avery Oswald (1940s) showed that the transferred factor
was DNA



Determines that DNA contains genetic materia
l (not proteins, as previously
supposed)



Artificial competency

o

Cells are treated to make cell membrane more permeable for DNA

o

Methods to makes cells competent



Calcium chloride (CaCl2)



Electroporation



Brief pulse of high voltage



(what do these do?)

o

Use in
genetic engineering

o

Significance of plasmid DNA

(circular)



Usually used in genetic engineering



Less likely degrade



Doesn’t have to integrate into chromosome (?)



Self
-
duplicates


builds up lots of copies



Easy to remove



Easy to paste genes in to plasmids
(?)



Used to put
any

gene into bacteria



Get lots of product (protein)

Transduction



Definition



DNA transfer from recipient to donor via bacteriophage (virus)

o

“phage” for short



Potential for cross
-
species transfer

o

Range of virus

o

Some viruses infect few spec
ies, some have broad host species range



Use in genetic engineering

o

Package genes into phage

o

All done in a test tube

o

Use virus to deliver genes



Types of transduction

o

Generalized transduction



During VIRUS replication in host cells

(donor)



Possibility that f
ragments of double
-
strand BACTERIAL DNA

can be a
ccidentally
packaged in new virus particle



Random process



Packages fragments from all parts of chromosome



In other words


bacterial genes come from anywhere in chromosome



Progeny (new virus)

-

r
elease
d

from lysed host

(donor cell dies at end of
replication cycle)



New phage b
ind
s

to next host cell



Injects bacterial DNA into host cell



Injected DNA



Remains double
-
stranded



If particle carries bacterial DNA, cannot replicate, cannot kill host, host
(potentia
lly gains DNA)



Possible fates of DNA in recipient



1
-

may be degraded


no transfer of genes



2
-

may be incorporated into chromosome

o

Double stranded DNA is integrated if contains gene of 2 (?)

o

Contains new info for recipient

o

Specialized transduction



Begins
with



Lysogenic or temperate phages



Lysogenic phase


will integrate DNA into host chromosome



Usually occurs at specific locations



Now referred to as prophage (as opposed to phage?)

o

Viral DNA is copied with chromosome

o

During cell division



Outcomes

o

1
-

copyin
g (of viral DNA) can continue for generations with no
production of new particles

o

2
-

viral DNA



May be excised from chromosome



Viral DNA directs production of new particles



Flip to lytic cycle


particles released



Specialized t
ransduction occurs



When
excision of viral DNA is faulty



Excised prophage DNA

a
ccidentally carries
adjacent
bacterial DNA



Now has viral DNA


b
acterial DNA recombinant



Will be packaged into new virus particles



When injected into next host cell

o

“DNA may integrate into recipient gen
es

o


“defective” DNA can’t direct another lytic cycle

o

Instead recombinant DNA incorporated into recipient
chromosome

o

Recipient gains bacterial cells

o

Mechanism only transfers c
ertain specific bacterial genes

o

Prophage DNA

w
as originally inserted in specific l
ocation

o

Excision


only get immediately adjacent, or flanking, bacterial
gene sequences

Conjugation



“Bacterial mating”



DNA is transferred

d
uring direct cont
act

between cells



Done u
sing pilus called

s
ex pilus or F (fertility) pilus

o

Hollow protein tube

o

DNA moves through pilus



Need several genes

t
o build F pilus structure

o

Including tra (transfer = t) genes



Transfers often involve

o

Transfer of “transposons”



Description of transposons

o

Transpositional elements

o

Segments of DNA that move around genomes

o

Chromosome goes to another spot in chromosome (?? Fix)

o

May be copied independently of the rest of the DNA

o

“insertion sequences” insert copies of DNA into new location

o

In order to copy transposon


must be already incorporated into chromosome or
plasmid DNA



Genes in transposons m
ay be limited to

minimum needed to act as transposon

o

Only those genes needed for moving and inserting



But may also include:

o

1
)

Genes needed for conjugation



Tra genes to build pilus



If transposon is part of plasmid


creates conjugate
d plasmid or
F
-
factor
plasmid

o

2) Genes involved in “resistance”



If in plasmid, creates resistance or
R plasmid



Genes for antibiotic resistance

o

3) Could be both



Genes for conjugation AND genes for drug resistance



Get…
conjugative

R Plasmid




Types of
conjugation

o

All types MAY include transfer of inserted transposons

o

F+ X F
-

mating



F+ “donor” strain



Carries “F plasmid” or “F Factor”



F plasmid at minimum


carries genes for conjugation (tra genes)

o

Genes to build F pilus

o

Process transfers

copy
of F plasmi
d to recipient cell



Donor DOESN’T LOSE, donor is aliv



At minimum recipient gains F plasmid and ability to do conjugation



Recipient goes from F
-

to F+

o

BUT plasmid

m
ay carry more information than just “tra”



Plasmid can/do spread R genes



Drug resistance gene
s



Drug and heavy metal resistance



Between cells in mixed species population

-

cross species lines



MAJOR mechanism in acquiring drug resistance

o

Restriction:



Usually no F+ X F+ conjugation



F plasmid has genes for surface proteins that INHIBIT this exchange

o

Hfr mating



Hfr = High frequency recombinant

o

During Hfr conjugation



Conjugation genes from 2 sources



Cells copy and transfer
chromosomal
genes



Incorporated into CHROMOSOME

(not plasmid)



Usually coming from integrated transposon (may have once been a
plasmid)



Cells copy (so donor doesn’t lose!) chromosomal DNA

o

Replication of DNA begins in middle of conjugated genes (F Factor
genes)

o

1
st



some F factor genes

o

2
nd



all the rest of the chromosome

o

3
rd



last of F factor genes

o

No. of chromosomal genes tran
sferred



Depends on time pilus is maintained



Takes about 90 min to transfe all DNA



Usually don’t get 90 min


only some genes transferred in this case



Usually not all F factor genes get transferred so recipient remains F
-