BIOL 3301 - Genetics Ch19 - Regulation of Gene Expression in Prokaryotes
BIOL 3301 - Genetics Ch9B - Transduction St Nb
Transcript of BIOL 3301 - Genetics Ch9B - Transduction St Nb
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Transduction
Use of phages to carry bacterial
genes into new host cells
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Figure 6-15 Copyright © 2006 Pearson Prentice Hall, Inc
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Transduction
! "peciali#ed transduction $ % or a si&ilar
phage '() carriers the bacterial genes
!*enerali#ed transduction $ P+ or otherphage heads carry the bacterial genes
instead of phage genes
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a&bda -%. phage
! Te&perate phage that infects E.coli cells
! /irulent phages -T2, T, etc. $ 1ill their hosts
! Te&perate phages $do not necessarily 1ill their
hosts Phage '() incorporates itself into thehost chro&oso&e lysogenic infection
! Phage '() replicates along with bacterial '() prophage
! Phage '() can initiate a lytic infection in whichphage '() re&ains independent of the hostchro&oso&e
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a&bda -%. phage
! 'uring lysogenic infection, % '() inserts
into a site called att3 between gal and bio
genes in 4 coli
! 5utagens can induce lytic cycle
! % '() usually ecise perfectly
! "o&eti&es carry out ecess '() $ hostgenes
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a&bda -%. phage
! "ince the roo& a7ailable inside a phage head is li&ited,the a&ount of etra '() is only about +08 of the si#e ofphage '()
! 9 '() is about : 1b
! The &ore host '() is included, the &ore phage '() is&issing
! The result is a transducing phage, a particle thatcontains a reco&binant '()
! The phage '() part that is &issing in case bio gene isincluded is a regulatory region re;uired for lysogeny butnot for lytic cycle
! Transducing phage can replicate lytically to for& pla;ues
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9 Phage
! If the % '() pic1s up gal locus on one
end, it loses genes on the other end that
are essential for the lytic cycle
!
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Transduction
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"peciali#ed Transduction
! Can be used instead of con=ugation to
fro& &erodiploids for co&ple&entation
tests
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P+ Phage 5ediation of *enerali#ed
Transduction
! Instead of phage '(), the phage heads are
in7ol7ed
! The phage head for&s around a piece of host
'(), producing a transducing particle that candeli7er this '() to a new host
! 'onated host '() can reco&bine with the
recipient>s chro&oso&e
!
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P+ Phage
! Contains ?+: 1b '()
! P+ head can contain up to ?+: 1b ofdonor '()
! This is 28 of the whole E. coli geno&e
! )bout @: a7erage si#e genes
! The chance of finding a gi7en host locus ina gi7en phage particle is at &ost002-28of '(). 000A-0A8ofphages are transducing. B @2 +0 2:
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5apping 5utations
! ne of applications is to perfor& a
co&ple&entarity test
! Transduce a '() fro& leu- cell into leu-
cell
! )re the &utations at the identical
nucleotide sitesD
! If not, how far are theyD
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Transduction
! Process of bacterial reco&bination
&ediated by bacteriophages
! +?:2, Einder and ederberg, "al&onella
typhi&uriu&
! "train )22 $ phe- trp –met+ his+
! "train ) $2 $ phe+ trp + met- his-
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Transduction
! Utube with filter that pre7ented cell
contact
! Found prototrophs only in )22
! )2 produced filterable agent only when
growing together with other strain
! F) could not pass through the filter withs&aller pores
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Transduction
! Conclusions
$ *enetic reco&bination was &ediated by
bacteriophage
$ 3acteriophage P22 is a prophage
$
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Te7en phages
! +?+: $ F G Twort
$ 3acteriophage $ a bacterial 7irus
!+?A $ 'elbruc1, phages T+ $ T@ -t fortypeJ.
! The &ost ob7ious feature is a polyhedral
head which contains the phage geno&e
-+@: 1b, double stranded.
! Tail fibers and a baseplate
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Te7en phages
! 5i bacteria and phage, add &olten agar
and pour on top of solid &edia
! 3acteria grows, for& a lawn
! 3acteriophage infects and lyse bacteria $
pla;ues are for&ed
! Count pla;ues $ deter&ine the nu&ber ofinfectious phages -pfu.
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Crossing Phages
! 4peri&entK
$ Ttos cells and Ttor cells
$ T2h rL and T2hL r phages
$ 5i together
$ (onparental types producedK
! T2 hL rL
! T2 h r
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5apping
! )ll four possible co&binations of pla;ues
can be obser7ed on the &iture of
bacterial cells
! (onparental pla;ues bred true $
reco&bination occurred
! Isolated se7eral r &utations $ r+, r2, etc
! Crossed with h &utant
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5apping
! r@ and h $ +2A8 reco&bination
! r+A and h $ +@8 reco&binant
! r+A and r@ D $less than +2A8! r@ and and r+A are different genes
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Fine "tructure 5apping
! T r &utants $ Hershey &apped to region
rII
! rII &utants grew on E.coli strain 3 but did
not grow on strain M -%. containing phage
in its chro&oso&e
! 3en#er used this conditional lethality to
study rII locus
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Fine "tructure 5apping
! If all &utants are in one gene, they are
7ery close together
! Used conditional lethality to find &utants $
analy#ed thousands
! 'etected a reco&bination fre;uency of
one in a &illion
! The finding that reco&bination could occur
within a gene was 7ery i&portant one
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Fine *ene 5apping 3en#er
! 3en#er through his study of fine gene
&apping was able to de7elop two basic
techni;ues of genetic analysis, allowing
hi& to etend and rapidly construct hisfine structure &aps
$ 'eletion analysis
$ Co&ple&entation analysis
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'eletion 5apping
Figure 6-23 Copyright © 2006 Pearson Prentice Hall, Inc
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Figure 6-24 Copyright © 2006 Pearson Prentice Hall, Inc
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Bacteriophage T4 can be used to do "Deletion apping" e!perients
#here recobination is used to deterine #here a particular delection
has occurred$
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%opleentation
This techni;ue was designed by 3en#er todefine the gene in ter&s of function
! Ghen doing fine gene structure analysis, suchas in phages, co&ple&entation allows todeter&ine whether two &utants are in thesa&e gene
! 5ore specifically, 3en#er wanted to find outwhether the entire rII region acted as a singlefunctional unit or whether is &ade up ofsubunits that function independently
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Cistrans test
! Two strains of E.coli , both lac-
! Con=ugate cells to for& a &erodiploid
! If two &utations are on different genes,then the &erodiploid will be Lac+
-co&ple&entation.
!If &utations are in the sa&e gene,&erodiploid will be lac-
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Cistrans test
! "o&eti&es can get co&ple&entation of the
&utations of the sa&e gene $ intragenic or
intracistronic co&ple&entation
! The &utations can be in trans position $)bNa3! r in cis position $ abN)3 on the sa&e
chro&oso&e
! "ey&our 3en#er called it cistrans test andsuggested the ter& cistron
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Cis Test
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Trans Test
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Cistrans test
! 3en#er did cistrans test on bacteriophage
$ infected bacteria with two &utant
phages
! Co&ple&entation groups $ two for rII
locus
! (a&ed the groups cistrons
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%opleentation
! If there are two different &utations in therII se;uences of two different phages, bothwill produce the &utant phenotype
! Howe7er, after perfor&ing double infectioneperi&ents in the non per&issi7e strainM, depending on the location of the&utation, they can or cannot co&ple&enteach other restoring the lost function
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%opleentation
! If they are in the sa&e functional subunit, they
will be still &utant, unable to lyse strain M
! Thus, function is not restored
! If they are in different functional subunits,function will be restored resulting in the wild type
phenotype -lysis of strain M. This is because
each will contribute a wor1ing subunit, thus
resulting in the lysis of strain M
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%opleentation
! Two groups of &utants were defined bythese tests, ) and 3
! 5utants in group ) co&ple&ented with
those of 3, whereas no co&ple&entationwas obser7ed within &utants of eachgroup
! 4ach group &apped in separate ad=acentregions in the rII locus which was thensplit in two sub units, ) and 3
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Cistrons
! 3ased on this test, the units of function
were na&ed cistrons, ter& which deri7es
fro& cistran test
(ow we 1now that one cistron codes forone polypeptide chain
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&ecobination 's
%opleentation
!
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Figure 6-2( Copyright © 2006 Pearson Prentice Hall, Inc
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Figure 6-21a Copyright © 2006 Pearson Prentice Hall, Inc
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Figure 6-21b Copyright © 2006 Pearson Prentice Hall, Inc
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Figure 6-22 Copyright © 2006 Pearson Prentice Hall, Inc
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Fine "tructure 5apping
! 3en#er identified @ different regions of rII locus
! Crossed a new point &utation with each of
the se7en deletion &utants!
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