BIOL 3301 - Genetics Ch9B - Transduction St Nb

8/18/2019 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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! 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



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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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! 3en#er identified @ different regions of rII locus

! Crossed a new point &utation with each of

the se7en deletion &utants!


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BIOL 3301 - Genetics Ch9B - Transduction St Nb

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