MOLECULAR AND CELLULAR BIOLOGY, Oc . 2006, p. 7451–7465 Vol. 26, No. 20
0270-7306/06/$08.00⫹0 doi:10.1128/MCB.00684-06
Copy igh © 2006, Ame ican Socie y o Mic obiology. All Righ s Rese ed.
An hp 1 Poin Mu a ion Tha Impai s T ansc ip ion and mRNP
Biogenesis wi hou Inc easing Recombina ion
䌤
Pablo Hue as, Ma ı´a L. Ga cı´a-Rubio, Ral E. Wellinge , Rosa Luna, and And e´s Aguile a*
Depa amen o de Gene´ ica, Facul ad de Biologı´a, Uni e sidad de Se illa, A d. Reina Me cedes 6, 41012 Se illa, Spain
Recei ed 20 Ap il 2006/Re u ned o modi ica ion 13 June 2006/Accep ed 4 Augus 2006
THO/TREX, a conse ed euka yo ic p o ein complex, is a key playe a he in e ace be ween ansc ip ion
and mRNP me abolism. The lack o a unc ional THO complex impai s ansc ip ion, leads o ansc ip ion-
dependen hype ecombina ion, causes mRNA expo de ec s and as mRNA decay, and e a ds eplica ion
o k p og ession in a ansc ip ion-dependen manne . To ge mo e insigh in o he in e connec ion be ween
mRNP biogenesis and genomic ins abili y, we sea ched o HPR1 mu a ions ha di e en ially a ec gene
exp ession and ecombina ion. We isola ed mu an s ha we e ba ely a ec ed in gene exp ession bu exhibi ed
a hype ecombina ion pheno ype. In addi ion, we isola ed a mu an , hp 1-101, wi h a s ong de ec in an-
sc ip ion, as obse ed o lacZ, and a gene al de ec in mRNA expo ha did no display a ele an hype e-
combina ion pheno ype. In THO single-null mu an s, bu no in he hp 1 poin mu an s s udied, THO and i s
subuni s we e uns able. In e es ingly, in con as o hype ecombinan null mu an s, hp 1-101 did no cause
e a da ion o eplica ion o k p og ession. T ansc ip ion and mRNP biogenesis can he e o e be impai ed by
THO/TREX dys unc ion wi hou inc easing ecombina ion, sugges ing ha i is possible o sepa a e he
mechanism(s) esponsible o mRNA biogenesis de ec s om he u he s ep o igge ing ansc ip ion-
dependen ecombina ion.
Gene exp ession equi es he co ec coupling o ansc ip-
ion o mRNA p ocessing and expo (7, 17, 30, 31). The inal
ou come o ansc ip ion is a ibonucleop o ein pa icle
(mRNP) consis ing o an mRNA molecule bound o p o eins,
which la e unc ion in expo , mRNA su eillance, and ans-
la ion. T ansc ip ion and assembly o he mRNP a e igh ly
coo dina ed wi h he expo o mRNA (42). A numbe o
p o eins ha e been iden i ied ha a e impo an o he co ec
assembly o expo -compe en mRNP pa icles, including he
THO complex, composed o Tho2, Hp 1, M 1, and Thp2, as
iden i ied in Saccha omyces ce e isiae (9). THO has also been
pu i ied om humans and D osophila, in which Hp 1 and Tho2
homologues a e p esen (32, 40). In yeas and humans, i has
been shown ha THO is pa o he TREX complex, which
includes he Sub2/UAP56 and Y a1/Aly mRNA expo ac o s
(40). Func ional connec ions be ween THO and he Sub2 and
Y a1 expo ac o s ha e been shown by syn he ic le hal phe-
no ypes o double mu an s and by mul icopy supp ession (11,
19, 40).
Null mu an s o he THO complex a e impai ed in ansc ip-
ion elonga ion (8, 23, 36). Addi ionally, hese mu an s exhibi
a ple ho a o pheno ypes, including ansc ip ion-dependen
hype ecombina ion (2), nuclea mRNA e en ion (40), and
as mRNA deg ada ion (22, 46), as well as syn he ic le hali y
in combina ion wi h mu a ions a ec ing ansc ip ion elonga-
ion such as sp 4 (35) and wi h RNA expo mu a ions such as
mex67-5and y a1-1(19, 40). Al oge he , hese da a sugges
ha THO plays a c ucial ole in mRNP me abolism a he
in e ace be ween ansc ip ion and mRNP expo . Mu an s
wi h changes a ec ing he Sub2-Y a1 componen o TREX,
he Mex67-M 2 mRNA expo ac o , he Nab2 hnRNP, o
he Thp1-Sac3-Sus1 RNA expo complex exhibi ansc ip ion
and gene ic ins abili y pheno ypes simila o hose o THO
mu an s (12, 14, 19).
The THO complex is exclusi ely ec ui ed o ansc ibed
ch oma in (20, 40, 46). Rec ui men o he Sub2 and Y a1
componen s o he THO/TREX complex o ansc ibed DNA
is pa ially dependen on THO (46). In con as o THO, bind-
ing o Sub2 o ansc ibed ch oma in is RNA dependen (1).
The ele ance o THO in he ea ly s eps o mRNP biogenesis
is suppo ed by he obse a ion ha ansc ip ion impai men
in hp 1⌬mu an s is supp essed i he nascen mRNA is sel -
clea ed by an a i icially enginee ed hamme head ibozyme. In
hese mu an s, he nascen mRNA can o m RNA-DNA hy-
b ids (R loops) linked o bo h impai men o ansc ip ion and
ansc ip ion-associa ed ecombina ion (18). The obse a ion
ha deple ion o he e eb a e ASF/SF2 splicing ac o in
chicken DT40 cells and human HeLa cells also leads o
genomic ins abili y linked o R loop o ma ion (21) indica es
ha a numbe o mRNA p ocessing enzymes may ha e a ole
in p e en ing RNA-dependen s uc u es ha igge genome
ins abili y. Ou cu en iew is ha he THO complex pa ici-
pa es in co ansc ip ional assembly o expo -compe en
mRNP du ing ansc ip ion elonga ion and con ibu es o he
p e en ion o he RNA om o ming s uc u es ha hampe
ansc ip ion and could comp omise genome s abili y (3).
Despi e he link be ween impai ed ansc ip ion and hype -
ecombina ion in THO mu an s, i is no clea whe he im-
pai ed gene exp ession by i sel is su icien o induce hype -
ecombina ion o whe he hype ecombina ion is possible in
he absence o impai ed gene exp ession. Thus, we sea ched
o THO mu an s comp omised in gene exp ession ha did no
a ec gene ic in eg i y and ice e sa. Hype ecombina ion
* Co esponding au ho . Mailing add ess: CABIMER, A . Ame ico
Vespucio s/n, 41092 Se ille, Spain. Phone: 34 954 468372. Fax: 34 954
461664. E-mail: [email p o ec ed].
䌤
Published ahead o p in on 14 Augus 2006.
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mu a ions ha did no ha e a signi ican e ec on ansc ip ion
we e no ound. Ins ead, we isola ed a new allele, hp 1-101
(L586P), which causes a s ong dec ease in ansc ip accumu-
la ion bu does no show a ele an hype ecombina ion phe-
no ype. The in i o s abili y o THO and i s subuni s was
s ongly educed in THO-null mu an s such as hp 1⌬bu no in
hp 1 poin mu an s. The absence o associa ed hype ecombi-
na ion seems o be due o he ac ha , in con as o hp 1⌬,
ansc ip ion impai men in hp 1-101 does no hampe epli-
ca ion o k p og ession. Ou esul s indica e ha THO dys-
unc ion can cause ansc ip ion de ec s ha a e no linked o
impai men o eplica ion o k p og ession, opening he pos-
sibili y ha THO links op imal mRNP biogenesis wi h an-
sc ip ion elonga ion, apa om i s e ec on genomic in eg i y.
MATERIALS AND METHODS
S ains and plasmids. The s ains used in his s udy a e lis ed in Table 1. All
o he s ains a e isogenic o W303-1A, excep LAU3-10A and LAU3-10D, which
a e congenic seg egan s om a SChY58A ⫻AYW3-1B c oss. S ains
WH101-1A and WH103-1A, isogenic o W303-1A bu ha bo ing he hp 1-101
and hp 1-103 alleles in he HPR1 ch omosomal locus, we e ob ained by gene
eplacemen in SChY58a (hp 1⌬::KAN). Plasmids pRS313, pRS316 (38), YCp70,
YCpA13 (4), pRS314GLlacZ (15), YEp351-SUB2, pCM184-LAUR (19),
pRS316-GAL1lacZ, pSCh202, pSCh204, pSCh206 (8), pPHO5-Rib
⫹
-lacZ
(Rib
⫹
), pPHO5- ib
m
-lacZ ( ib
m
), pGL-Rib
⫹
, pGL- ib
m
(18), and pRWY005
(43) ha e been desc ibed p e iously. Plasmid pRS313-GZ was cons uc ed by
cloning he GAL1p ::lacZ usion om pRS316-GAL1lacZ in o pRS313 in i o as
p e iously desc ibed (29). pRS316-HPR1 was ob ained by cloning he 6.55-kb
HindIII HPR1 agmen om YCpA13 in o he HindIII si e o pRS316. Plasmids
YCphp 1-101 o YCphp 1-104 we e ob ained by in i o cloning o linea mu-
agenized HPR1 PCR agmen s in o YCp70 in yeas s ain LAU3-10A as de-
sc ibed below.
New hp 1 alleles ob ained by andom mu agenesis. In i o mu agenesis o
HPR1 was pe o med acco ding o e e ence 27, wi h some modi ica ions. PCR
ampli ica ion o HPR1 was done unde subop imal condi ions wi h 3.25 mM
MgCl
2
, 0.25 mM MnCl
2
, 0.2 mM dTTP, 0.2 mM dGTP, 0.2 mM dCTP, 0.04 mM
dATP, and 1.75 U o Expand High Fideli y Taq polyme ase (Roche). YCpA13 (1
␮g) was used as he empla e, and he oligonucleo ides 5⬘GCCACGTTTGTT
TACTCG 3⬘and 5⬘CTATCATCCAACGCTTCC 3⬘we e used as p ime s. The
LAU3-10A s ain (hp 1⌬), p e iously ans o med wi h pRS313-GZ, was co-
ans o med wi h he pu i ied, PCR-mu agenized HPR1 mix u e and he linea
BamHI-XbaI agmen o YCpA13, which co esponds o YCp70 ca ying 200 bp
o HPR1 lanking sequences on each side. In i o ecombina ion be ween he
PCR-induced hp 1 alleles and he linea ec o led o o ma ion o he YCphp 1
plasmids in he cells. T ans o man s hus ob ained we e pla ed on syn he ic
comple e (SC) medium wi hou his idine and leucine con aining limi ed amoun s
o adenine (1.5 mg/li e ) o isualiza ion o ed sec o s esul ing om ecom-
bina ion o he ch omosomal leu2-k::ADE2-URA3::leu2-k epea sys em. Colo-
nies we e hen eplica ed on o SC wi hou his idine and leucine con aining 2%
galac ose o allow exp ession o he GAL1p ::lacZ usion. On he ollowing day,
a solu ion con aining 0.25 M phospha e bu e (pH 7.5), 0.1% sodium dodecyl
sul a e (SDS), 400 ␮g/ml 5-b omo-4-chlo o-3-indolyl-␤-D-galac opy anoside (X-
Gal), and 0.5% aga ose was added and ␤-galac osidase ac i i y was sco ed by blue
colo o ma ion a e 2ha 30°C. Colonies ans o med wi h ei he YCpA13 o
YCp70 we e used as posi i e (wild ype) o nega i e (hp 1⌬) con ols, espec i ely.
Pu i ica ion o he THO complex and analysis o i s subuni s. The THO
complex was pu i ied by using he C- e minally andem a ini y pu i ica ion
(TAP) epi ope- agged Tho2 p o ein and ollowing s anda d p ocedu es (33) and
using speci ica ions p e iously epo ed (19). M 1 was de ec ed by Wes e n
analysis o pu i ied THO complexes p e iously sepa a ed by 10% SDS-poly-
ac ylamide gel elec opho esis (PAGE) and ans e ed o nylon memb anes.
A e ea men wi h phospha e-bu e ed saline con aining 0.1% Tween 20 and
5% milk, p o eins we e de ec ed wi h an i-M 1 an ibody and pe oxidase-conju-
ga ed goa an i- abbi immunoglobulin G (IgG). Blo s we e washed wi h phos-
pha e-bu e ed saline–0.1% Tween 20 and de eloped by enhanced chemilumi-
nescence eac ions (Ame sham). The Tho2-TAP epi ope usion was de ec ed in
whole cell ex ac s wi h pe oxidase an ipe oxidase (PAP; Sigma) an ibody by
ollowing he manu ac u e ’s ins uc ions as desc ibed p e iously (19).
2D gel analysis o eplica ion in e media es. Cells we e g own o e nigh o an
op ical densi y a 600 nm o 0.2 o 0.3 in SC medium con aining 1% glyce ol-
lac a e and 1% a inose as a ca bon sou ce. A e b inging he cul u e o 2%
galac ose, cells we e g own o ano he 3.5 o 4 h p io o DNA isola ion. DNA
o 100 ml o cells was isola ed wi h ce yl ime hylammonium b omide (44),
dissol ed in a inal olume o 100 ␮l o T is-EDTA, and s o ed a 4°C. P io o
analysis, 50 ␮l o DNA was double diges ed wi h SmaI-SacI. A e diges ion,
DNA was p ecipi a ed and subjec ed o wo-dimensional (2D) gel elec opho e-
sis as desc ibed p e iously (6), wi h he ollowing modi ica ions. DNA was sep-
a a ed o 20 h in he i s dimension on a 0.5% aga ose gel a 1 V/cm and o
14 h in he second dimension on a 1.2% aga ose gel a 4 V/cm and 4°C in he
p esence o 0.3 ␮g/ml e hidium b omide. A e alkaline ans e o he DNA on o
a Hybond-N
⫹
memb ane, he memb ane was hyb idized agains a
32
P-labeled
1.2-kb agmen ob ained by PCR co e ing ARS1 and pa o he 3⬘-lacZ gene.
Memb anes we e exposed on a Fuji 3600 Phospho imaging sys em and analyzed
wi h he imaging so wa e supplied.
ChIP. Rec ui men o Sub2, Tho2, and RNAPII o ch oma in was de e mined
by ch oma in immunop ecipi a ion (ChIP) analyses on he endogenous PMA1
gene in yeas s ains con aining a 5⬘-end TAP epi ope- agged e sion o ei he
he SUB2 o he THO2 gene inse ed a i s ch omosomal locus, as p e iously
desc ibed (16). B ie ly, immunop ecipi a ions we e pe o med wi h Ig-Sepha ose
o TAP epi ope- agged p o eins o wi h an i-Rpb1-CTD monoclonal an ibody
TABLE 1. Yeas s ains used in his s udy
S ain Geno ype Sou ce o e e ence
W303-1A MATaade2-1 can1-100 his3-11,15 leu2-3,112 p1-1 u a3-1R. Ro hs ein
AYW3-1B MAT␣ade2 can1-100 his3 p1 u a3 leu2-k::ADE2-URA3::leu2-k13
U768-4C W303-1A hp 1⌬::HIS3 R. Ro hs ein
SChY58a W303-1A hp 1⌬::KAN S. Cha´ ez
WH101-1A W303-1A hp 1-101 This s udy
WH103-1A W303-1A hp 1-103 This s udy
BSU-S2T-6D MATaade2-1 leu2 hp 1⌬::HIS3 SUB2-TAP 19a
LAU3-10A MATaade2-1 his3-11,15 p1-1 u a3-1This s udy
leu2-k::ADE2-URA3::leu2-k hp 1⌬::KAN
LAU3-10D MAT␣ade2-1 his3-11,15 p1-1 u a3-1 leu2-k::ADE2-URA3::leu2-kThis s udy
WMC1-1A W303-1A mex67-519
WMH1 hp 1⌬::KAN mex67-5isogenic o W303-1A and ans o med wi h pRS316-HPR1 This s udy
WWT2T W303-1A THO2-TAP 19a
WTT3-4A W303-1A THO2-TAP hp 1-101 This s udy
WTT3-4C W303-1A THO2-TAP hp 1⌬::KAN This s udy
STT2 W303-1A THO2-TAP sub2⌬::HIS3 19
MTT1 W303-1A THO2-TAP m 1⌬::KAN S. Jimeno
WTT4-5C W303-1A THO2-TAP hp 1-103 This s udy
WHYL.2A W301-1A hp 1⌬::HIS3 GAL1p ::YRL454 19a
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8WG16 (Be keley An ibody Company) and p o ein A-Sepha ose o RNA poly-
me ase II (RNAPII). The GFX pu i ica ion sys em (Ame sham) was used o he
las DNA pu i ica ion s ep. We used 20- o 27-bp oligonucleo ides o PCR
ampli ica ion o PMA1 sequences. Fo Tho2 ec ui men , we ampli ied wo
agmen s, localized a posi ions 292 o 321 and 2273 o 2242, whe eas o Sub2
and RNAPII ec ui men , we only ampli ied he 2273- o-2242 agmen . In bo h
cases, we used he PCR o he in e genic egion a posi ions 9716 o 9863 o
ch omosome V as a nega i e con ol. The PCR p oduc s ob ained we e elec o-
pho esed in a 15% ac ylamide gel, s ained wi h e hidium b omide, and quan i ied
in a Fuji FLA-3000. The ela i e abundance o each DNA agmen was calcu-
la ed as he a io o each DNA agmen o he in e genic- egion quan i ica ion
esul s o he p ecipi a ed ac ions no malized wi h espec o he co esponding
a ios o he inpu ac ions.
Fo analysis o RNAPII p ocessi i y, a s ain ha bo ing ei he YLR454 o
lacZ unde he con ol o he GAL1 p omo e was used (23). RNAPII abun-
dances a di e en posi ions o he genes we e assayed as indica ed, by using
ChIP wi h an i-Rpb1-CTD monoclonal an ibody 8WG16 and p o ein A-
Sepha ose as p e iously desc ibed (23). We used 20- o 30-bp oligonucleo-
ides o PCR ampli ica ion o wo agmen s o YLR454, posi ions 3 o 43
and 7621 o 7674; o wo agmen s o lacZ, posi ions 715 o 1025 and 2247
o 2467; and o he 9716- o-9863 in e genic egion o ch omosome V ha was
used as a con ol. Real- ime quan i a i e PCR was pe o med wi h SYBR
g een dye in he 7500 Real Time PCR sys em o Applied Biosys ems by
ollowing he manu ac u e ’s ins uc ions.
Miscellanea. Recombina ion equencies we e calcula ed as p e iously de-
sc ibed (18). Fo each geno ype, he ecombina ion equencies a e gi en as he
a e age and s anda d de ia ion o he median ecombina ion alue ob ained
om wo o h ee di e en ans o man s wi h 6 o 12 independen colonies pe
ans o man . ␤-Galac osidase and phospha ase assays and No he n analyses
we e pe o med acco ding o p e iously published p ocedu es (18). mRNA ex-
po assays we e pe o med by in si u poly(A)
⫹
RNA localiza ion wi h a syn he ic
digoxigenin-labeled oligo(dT)
20
as p e iously desc ibed (5, 14).
RESULTS
Isola ion o hp 1 mu a ions wi h di e en ial e ec s on gene
exp ession and gene ic ins abili y. Mu agenic PCR-gene a ed
hp 1 alleles we e cloned in o he YCp70 plasmid by in i o
homologous ecombina ion (see Ma e ials and Me hods). hp 1
mu a ions ha di e en ially a ec ed gene exp ession and e-
combina ion we e sc eened by a colo assay based on he
ch omosomal leu2-k::ADE2-URA3::leu2-kdi ec - epea con-
s uc (4). Wi h his cons uc , wild- ype cells o m whi e col-
onies (Ade
⫹
) whe eas hp 1 cells o m ed-sec o ing colonies
(Ade
⫹
Ade
⫺
). De ec s in gene exp ession we e de e mined
wi h a GAL1p ::lacZ usion cons uc which esul s in a da k
blue colo in exp ession-compe en wild- ype colonies and a
ligh blue colo in exp ession-de icien hp 1 mu an colonies.
Sc eening o 5,346 ans o man colonies pe mi ed he iden-
i ica ion o ou hp 1 mu an s (hp 1-101 o hp 1-104) which,
upon subsequen analyses, we e e i ied as being p e e en ially
hype ecombinan o de ec i e in lacZ exp ession. The hp 1
alleles we e sequenced, and he mu a ions esponsible o he
amino acid changes o each allele we e de e mined (Fig. 1A).
hp 1-101 ha bo s wo poin mu a ions, bu only one (L586P)
leads o amino acid subs i u ion. In e es ingly, al hough he
p ima y sequence o he Hp 1 p o ein is no pa icula ly con-
se ed, his mu a ion lies in a highly conse ed egion (Fig.
1B). hp 1-102 has a ⫺1 ameshi ha leads o a p ema u e
s op codon and a ca boxy- e minally unca ed Hp 1 p o ein.
hp 1-103 and hp 1-104 ha bo mul iple poin mu a ions leading
o 12 and 6 amino acid subs i u ions, espec i ely.
Nex , ecombina ion equencies and ␤-galac osidase ac i -
i ies we e de e mined in a LAU3-10A hp 1⌬s ain ca ying he
leu2-k::ADE2-URA3::leu2-kand GAL1p ::lacZ sys ems, e-
spec i ely, and ans o med wi h he YCp70::hp 1 se ies o
plasmids (hp 1-101 o hp 1-104), YCpA13 (HPR1), o he
emp y ec o YCp70 (hp 1) (Fig. 1C). hp 1⌬cells display a
s ong educ ion in ␤-galac osidase ac i i y (a 25- old de-
c ease) ha co ela es wi h a s ong inc ease in ecombina ion
(773- old abo e he wild- ype le el). Like hp 1⌬cells, hp 1-101
and hp 1-102 cells exhibi a signi ican educ ion in ␤-galac o-
sidase ac i i y (4.3- and 6.7- old dec eases, espec i ely) which,
in con as o hp 1⌬, was accompanied by a weak inc ease in
ecombina ion (5- and 50- old, espec i ely). Ins ead, ␤-galac-
osidase ac i i y in hp 1-103 and hp 1-104 cells was poo ly
a ec ed (1- o 2- old dec eases), while ecombina ion alues
(239- and 567- old abo e he wild- ype le el) we e close o he
alues ob ained in hp 1⌬. We he e o e decided o u he
analyze hp 1-101 and hp 1-103 in o de o unde s and he basis
o he di e en pheno ypes hey p oduce.
hp 1-101 causes a gene exp ession de ec no associa ed wi h
hype ecombina ion, whe eas hp 1-103 a ec s bo h gene ex-
p ession and ecombina ion. The s eng h o he gene exp es-
sion de ec and hype ecombina ion pheno ypes o hp 1⌬has
been shown o be dependen on he DNA sequence (10). Thus,
ansc ip ion h ough he lacZ bac e ial gene is se e ely com-
p omised in hp 1⌬and leads o s ong hype ecombina ion,
bu he ansc ip ion de ec and hype ecombina ion pheno-
ypes a e weak o ba ely de ec able in o he DNA sequences,
such as ha o PHO5. To unde s and he connec ion be ween
he gene exp ession and ecombina ion pheno ypes obse ed
in he hp 1 poin mu an s, we u he analyzed he mos ep-
esen a i e alleles, i.e., hp 1-101 (s ong gene exp ession de ec
and weak hype ecombina ion) and hp 1-103 (weak gene ex-
p ession de ec and s ong hype ecombina ion). Thus, we con-
s uc ed isogenic s ains ha bo ing single ch omosomal copies
o hp 1-101 (WH101-1A) and hp 1-103 (WH103-1A) a he
HPR1 locus by gene eplacemen .
Recombina ion analysis in he L-lacZ sys em, con aining
lacZ lanked by leu2 di ec epea s, e ealed ha , compa ed o
hp 1⌬( ecombina ion 87- old abo e he wild- ype le el), hy-
pe ecombina ion was weak in hp 1-101 (8- old) and mode a e
in hp 1-103 (20- old) (Fig. 2A). Nex , we analyzed ecombina-
ion in he L-PHO5 and GL-lacZ sys ems, he la e o which is
he same as L-lacZ excep ha ansc ip ion is d i en by he
GAL1 p omo e . Wi h ei he ac i ely ansc ibed L-PHO5 o
GL-lacZ unde ep essi e condi ions (2% glucose), we ob-
ained wild- ype ecombina ion le els in he L-PHO5 and GL-
lacZ sys ems in bo h hp 1-101 and hp 1-103, espec i ely.
These esul s indica e ha hype ecombina ion was ansc ip-
ion and sequence dependen in bo h mu an s. In o de o
de e mine whe he ansc ip ion impai men and hype ecom-
bina ion in hese mu an s a e associa ed, we analyzed an-
sc ip le els in he same sys em used o measu e ecombina-
ion. No he n analysis showed simila L-PHO5 ansc ip
le els o all o he s ains (Fig. 2B). Impo an ly, in he hp 1-
103 mu an he ansc ip le els o lacZ mRNA exp essed om
he L-lacZ sys em only eached 75 o 80% o he wild- ype
le els, which clea ly di e s om he ␤-galac osidase esul s
ob ained in he GAL1p ::lacZ sys em (Fig. 1C and da a no
shown). Thus, when ansc ip ion was analyzed in he same
sys em in which hype ecombina ion was de ec ed, he hp 1-
103 mu an showed he same pheno ypes as hp 1⌬, al hough o
a lesse ex en . As he L-lacZ and GAL1p ::lacZ sys ems di e
in he leng h o he ansc ibed DNA sequence, i seems ha
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he hp 1-103 e ec on ansc ip ion was de ec able in long
DNA sequences. This is consis en wi h p e ious obse a ions
indica ing ha hype ecombina ion is only obse ed in associ-
a ion wi h a de ec able ansc ip ion de ec (2).
RNAPII p ocessi i y is impai ed in hp 1-101 mu an s. Since
hp 1-101 did no display a signi ican hype ecombina ion phe-
no ype compa ed wi h hp 1⌬, we wonde ed whe he his mu-
a ion leads o a eal impai men in RNAPII ansc ip ion, as
is he case o hp 1⌬s ains. Fi s we de e mined gene exp es-
sion om GAL1p ::lacZ and GAL1p ::PHO5 in hp 1-101 mu-
an cells compa ed o wild- ype and hp 1⌬cells. As shown in
Fig. 3A, whe eas ␤-galac osidase ac i i y was clea ly dimin-
ished in hp 1-101, acid phospha ase was no a ec ed. This
pa e n is simila , al hough o a lesse ex en , o he esul s
ob ained o he null hp 1⌬s ain.
Nex , we de e mined whe he he educ ion o mRNA le els
in hp 1-101,asinhp 1⌬, was di e en , depending on he gene
es ed. We analyzed he mRNA le els in a ious open eading
ames (ORFs) d i en by he s ong GAL1 p omo e . These
ORFs included lacZ om Esche ichia coli (3 kb and 53% GC
con en ); PHO5 (1.5 kb and 40% GC), YAT1 (2 kb and 51%
GC), and YRL454 (8 kb and 41% GC) om S.ce e isiae; and
LAC4 (3 kb and 41% GC) om Kluy e omyces lac is. As shown
in Fig. 3B, hp 1-101 causes a educ ion as g ea as ha p o-
duced by hp 1⌬in he mRNA le els o lacZ and YRL454 bu
has li le e ec on PHO5, consis en wi h he enzyma ic anal-
ysis (Fig. 1). Howe e , i has only a mode a e e ec on YAT1
and LAC4 mRNA le els. These esul s sugges ha he
s eng h o he de iciency in mRNA accumula ion in hp 1-101
cells is also ORF dependen .
In hp 1⌬s ains, RNAPII p ocessi i y, de ined as he abili y
o RNAPII o a el he en i e leng h o a gene, is impai ed
(23). Al hough mRNA le els we e a ec ed in hp 1-101,i was
concei able ha RNAPII p ocessi i y was no impai ed in his
s ain, he e o e sepa a ing RNAPII p ocessi i y om hype -
ecombina ion. To es his hypo hesis, we de e mined he p o-
cessi i y o RNAPII in hp 1-101 along he lacZ and YRL454
genes by ChIP. This was pe o med wi h an an i-Rpb1 an i-
FIG. 1. Gene ic cha ac e iza ion o hp 1 poin mu a ions ha di e en ially a ec ansc ip ion and ecombina ion. (A) Schema ic ep esen-
a ion o he amino acid subs i u ions o he hp 1 alleles s udied. Mu a ions ha change he amino acid sequence a e ep esen ed as black ba s.
(B) P o ein alignmen o he egion su ounding L586 in se e al Hp 1 o hologs. The Hp 1 p o ein sequences used belong o he ollowings
o ganisms: S.ce e isiae wild ype and hp 1-101 mu an (Sc WT and Sc 101, espec i ely), Saccha omyces bayanus (SBay), Homo sapiens (Hsap),
Mus musculus (Mmus), Xenopus lae is (Xlae), B achydanio e io (B e ), D osophila melanogas e (Dmel), Saccha omyces kluy e i (Sklu), Caeno-
habdi is elegans (Cele), A abidopsis haliana (A ha), Dic yos elium discoideum (Ddis), Neu ospo a c assa (Nc a), and Schizosaccha omyces pombe
(Spom). (C) ␤-Galac osidase ac i i y and ecombina ion equencies o he LAU3-10A (hp 1⌬) s ain con aining he leu2-k::ADE2-URA3::leu2-k
ecombina ion sys em and ans o med wi h pRS313-GZ (con aining he GAL1p :::lacZ usion) and wi h ei he YCpA13 (HPR1), YCp-hp 1-101
o YCp-hp 1-104 (hp 1-101 o hp 1-104), o he emp y ec o YCp70. The pe cen age o ␤-galac osidase ac i i y wi h espec o he wild- ype le el
( aken as 100%) is shown. Each alue ep esen s he a e age o wo o h ee independen expe imen s. The ecombina ion equencies (10
⫺4
) a e
a e ages o wo o ou median equencies ob ained om he same numbe o luc ua ion expe imen s.
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body and quan i a i e s anda d and eal- ime PCRs a he 5⬘
end and 3⬘end o each coding sequence. As shown in Fig. 4,
RNAPII is signi ican ly educed owa d he 3⬘end o lacZ in
hp 1⌬, as well as hp 1-101 (Fig. 4A). In YRL454, RNAPII was
signi ican ly educed a he 3⬘end in he hp 1⌬s ain and o a
lesse ex en in he hp 1-101 s ain (Fig. 4B). These esul s a e
consis en wi h he mRNA accumula ion esul s obse ed o
each gene and indica e ha RNAPII p ocessi i y is diminished
in hp 1-101. Thus, we conclude ha impai ed RNAPII p oces-
si i y by i sel is no su icien o lead o hype ecombina ion.
Ins ead, in hp 1-103, RNAPII p ocessi i y along lacZ was no
a ec ed (Fig. 4B), in ag eemen wi h he exp ession da a (Fig.
1C), bu i was a ec ed o he long (8-kb) YLR454 ORF o he
same ex en as in hp 1-101 (Fig. 4B). This is consis en wi h he
conclusion ha hp 1-103 a ec s ansc ip ion o a mino de-
g ee bu su icien ly o cause ansc ip ion-dependen hype e-
combina ion.
Nuclea accumula ion o poly(A)
ⴙ
RNA in hp 1-101 and
hp 1-103 mu an s. The ansc ip ional de ec o THO-null mu-
an s such as hp 1⌬has been shown o be associa ed wi h
nuclea accumula ion o poly(A)
⫹
RNA caused by de ec s in
mRNA expo (19, 40). The unc ional ela ionship o THO
wi h mRNA expo is p o ided by he syn he ic le hali y o
RNA expo ac o mu an s in he absence o a unc ional
THO complex, as demons a ed by he hp 1 mex67-5 sdouble
mu an s. To explo e whe he he hp 1-101 and -103 poin mu-
an s we e a ec ed in RNA expo , we i s de e mined he
capaci y o he new mu a ions o escue he syn he ic le hali y
o he hp 1⌬mex67-5mu an . Fo his, we cons uc ed hp 1⌬
mex67-5s ains ca ying a URA3-based plasmid con aining
wild- ype HPR1. These s ains we e ans o med wi h a plas-
mid con aining ei he mu an allele hp 1-101 o hp 1-103, and
he capaci y o each allele o escue hp 1⌬mex67-5le hali y
was de e mined by g ow h in SC medium plus 5- luo oace ic
acid (FOA). As shown in Fig. 5A, he abili y o each hp 1 allele
o escue hp 1 mex67-5le hali y is linked o i s ansc ip ional
de ec . hp 1-103, which had li le e ec on gene exp ession,
almos comple ely escued he syn he ic le hali y, whe eas
FIG. 2. Recombina ion in hp 1-101 and hp 1-103 mu an s. (A) The isogenic s ains W303-1A (WT), WH101-1A (hp 1-101), WH103-1A (hp 1-103),
and SChY58a (hp 1⌬), ca ying he mu an alleles in eg a ed in he ch omosomal HPR1 locus, we e ans o med wi h plasmid pSCh204 (L-lacZ
ecombina ion assay) o pSCh206 (L-PHO5 ecombina ion assay). G ay boxes ep esen LEU2 epea s ha lank ei he he PHO5 o he lacZ ORF, as
indica ed. The whi e box ep esen s he p omo e , and he whi e solid a ow ep esen s he CYC ansc ip ion e mina ion si e. Shown a e ecombina ion
equencies unde ep essed condi ions (glucose) in cells ans o med wi h plasmid pRS314GLlacZ, con aining he GL-lacZ ecombina ion assay, in
which ansc ip ion is unde he con ol o he GAL1 p omo e . The ecombina ion equencies shown a e a e ages o wo o ou di e en expe imen s.
(B) No he n analysis o he L-lacZ and L-PHO5 ecombina ion sys em in isogenic s ains W303-1A (WT [wild ype]), WH101-1A (hp 1-101), and
SChY58a (hp 1⌬) ans o med wi h ei he pSCh204 o pSCh206, espec i ely. Fil e s we e hyb idized wi h ei he a lacZ o aPHO5 p obe and wi h he
25S RNA p obe. All da a we e no malized wi h espec o he RNA signal. The a e ages o wo expe imen s a e plo ed. A.U., a bi a y uni s.
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hp 1-101, which con e s s ong ansc ip ion de ec s, did no ,
sugges ing a di ec co ela ion be ween ansc ip ion and im-
pai ed mRNA expo . Analysis o o he hp 1 alleles isola ed in
his s udy, such as hp 1-102 and hp 1-104, con i med his co -
ela ion be ween ansc ip ion de ec and syn he ic le hali y
wi h mex67-5(da a no shown).
We nex analyzed he nuclea expo o poly(A)
⫹
mRNA by
in si u hyb idiza ion wi h a 20-me oligo(dT) luo escen p obe
in he wild- ype, hp 1-101,hp 1-103, and hp 1⌬s ains (Fig.
5B). Incuba ion o he cells a 37°C led o nuclea poly(A)
⫹
mRNA accumula ion in hp 1⌬and hp 1-101 cells. Howe e , in
hp 1-103 cells, mRNA accumula ion was only obse ed a e
6 h o incuba ion a 37°C. These esul s indica e ha he gene
exp ession de ec in hp 1⌬and hp 1-101 cells di ec ly co e-
la es wi h hei mRNA expo de ec .
T ansc ip ion impai men in hp 1-101 and hype ecombi-
na ion in hp 1-103 a e dependen on he nascen mRNA mol-
ecule. The ansc ip ion impai men and hype ecombina ion
pheno ypes o hp 1⌬cells ha e been shown o be dependen on
s uc u es media ed by he nascen mRNA (18). To assay he
FIG. 3. T ansc ip ion analysis o hp 1-101 e sus he wild ype and hp 1⌬as a unc ion o GC con en and he leng h o he ansc ibed DNA
sequence. (A) Isogenic s ains W303-1A (WT [wild ype]), WH101-1A (hp 1-101), and SChY58a (hp 1⌬), ca ying he mu an alleles in eg a ed
in o he ch omosomal HPR1 locus, we e ans o med wi h pRS316-GAL1lacZ (GAL1p ::lacZ usion) o pSCh202 (GAL1p::PHO5 usion).
␤-Galac osidase o phospha ase ac i i y was de ined as he pe cen age ( o lacZ o PHO5, espec i ely) o he wild- ype ac i i y, which was aken
as 100%. The mean and s anda d de ia ion o h ee independen expe imen s a e plo ed. (B) No he n analysis o mRNA le els in di e en
plasmid cons uc s con aining lacZ (pRS416GAL1lacZ), PHO5 (pSCH202), LAC4 (pSCH255), and YAT1 (pSCH247) unde he con ol o he
GAL1 p omo e in s ains W303-1A (wild ype), WH101-1A (hp 1-101), and U768-4C (hp 1⌬). Fo he analysis o ansc ip ion o he YLR454
gene, s ain WHYL.2A (hp 1⌬GAL1p::YLR454) was ans o med wi h he emp y ec o YCpA70 (hp 1⌬) o wi h plasmid YCpA13 (HPR1)o
YCp:hp 1-101 (hp 1-101). O he s de ails a e he same as hose in Fig. 2B.
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e ec o he nascen mRNA on ansc ip ion, we used he
p e iously de ined Rib
⫹
and ib
m
cons uc s (Fig. 6A), in
which a PHO5-Rib-lacZ ansc ip ional usion con aining ei-
he a wild- ype (Rib
⫹
) o a mu a ed ( ib
m
) Hamme head
ibozyme sequence was placed unde he con ol o he GAL1
p omo e (18). In bo h cons uc s, a 2.2-kb long mRNA is
ansc ibed, bu in he Rib
⫹
cons uc he ac i e hamme head
ibozyme clea es he ansc ip , libe a ing a 1.8-kb mRNA.
Meanwhile, RNAPII a els a he , p oducing a 0.6-kb
mRNA. No he n analysis o he ib
m
cons uc e ealed ha
less ansc ip was accumula ed in hp 1⌬and hp 1-101 cells
han in wild- ype and hp 1-103 cells. Howe e , ibozyme clea -
age in he Rib
⫹
cons uc led o an inc ease in he esul ing
0.6-kb ansc ip so ha simila mRNA le els we e ound in all
s ains.
Appa en ly, ibozyme clea age was capable o inc easing he
amoun o mRNA ansc ip s in hp 1⌬and hp 1-101 cells. In a
p e ious wo k, we showed ha ibozyme clea age was capable
o pa ially supp essing he hype ecombina ion pheno ype ob-
se ed in hp 1⌬cells. Thus, we asked whe he ibozyme clea -
age migh also be su icien o educe he hype ecombina ion
pheno ype obse ed in hp 1-103 cells. Hype ecombina ion
was assayed wi h he GL-Rib
⫹
and GL- ib
m
epea sys ems
con aining PHO5, ollowed by ac i e Rib
⫹
o inac i e ib
m
sequences be ween 0.6-kb-long leu2 di ec epea s, espec i ely
(Fig. 6B). In he GL- ib
m
sys em, ecombina ion was s ongly
inc eased in hp 1⌬(285- old), mode a ely inc eased in hp 1-
103 (17- old), and poo ly a ec ed in hp 1-101 (3- old). In all
h ee s ains, ecombina ion le els we e educed wo- o h ee-
old by he ibozyme-media ed clea age o he nascen mRNA.
The e o e, bo h he ansc ip ion de ec o hp 1-101 and he
hype ecombina ion o hp 1-103 a e simila ly dependen on
he nascen mRNA, as shown o hp 1⌬mu an s.
Di e en ial s abili y and ec ui men o he THO complex in
hp 1-101 and hp 1-103 e sus THO-null mu an s. The THO
complex is a highly s able complex which can be pu i ied wi h
sal concen a ions as high as 1.2 M and emains s able in i o
in mu an s o he TREX complex such as sub2⌬(9, 19). How-
e e , he in eg i y o he THO complex has no been s udied in
mu an s o he THO subuni s. To de e mine whe he he di -
e en pheno ypes o hp 1-101,hp 1-103, and he THO-null
mu an s we e ela ed o he s abili y o THO, he complex was
pu i ied om he di e en mu an s ains. Wi h a TAP
epi ope- agged Tho2 p o ein, he complex was pu i ied om
whole cell ex ac s de i ed om wild- ype, hp 1-101,hp 1-103,
hp 1⌬,m 1⌬, and sub2⌬s ains by wo-s ep a ini y pu i ica-
ion (19, 33). Wes e n blo analysis wi h a PAP an ibody
showed ha he amoun o TAP-Tho2 was simila in whole cell
ex ac s o hp 1-101 and wild- ype cells (Fig. 7A). Al hough
TAP-Tho2 seems sligh ly educed in hp 1-103 and sub2⌬and
he amoun o Tho2 a ies be ween expe imen s (Fig. 7A), i
was simila in all cases o ha in wild- ype cells. In con as , in
FIG. 4. RNA p ocessi i y is a ec ed by hp 1-101 in a manne s ongly dependen on he GC con en and mode a ely dependen on he leng h
o he ansc ibed DNA sequence. (A) ChIP assays o RNAPII a he lacZ gene. Expe imen s we e pe o med wi h s ains W303 (WT [wild ype]),
WH101-1A (hp 1-101), and U768-4C (hp 1⌬) ans o med wi h plasmid pRS416GAL1lacZ con aining lacZ unde he con ol o he GAL1
p omo e . ChIPs wi h egions 1 and 2 and an in e genic egion as a con ol we e pe o med in wo di e en ans o man s, and PCRs we e epea ed
ou imes o each ans o man . The a io o DNA in egions 1 and 2 was calcula ed om he DNA amoun s in egions 1 and 2 ela i e o he
DNA amoun ob ained om he in e genic egion. Values we e no malized o he amoun o DNA in egion 1 in each s ain, which was se o 100%
( o de ails, see Ma e ials and Me hods). (B) ChIP assays o RNAPII a he YLR454 gene unde con ol o he GAL1 p omo e . The expe imen s
we e pe o med wi h s ain WHYL.2A (hp 1⌬GAL1p::YLR454) con aining ei he plasmid YCpA13 (HPR1) o YCphp 1-101 (hp 1-101)o he
emp y ec o YCpA70 (hp 1⌬). ChIPs we e pe o med wi h wo di e en ans o man s each, and quan i a i e PCRs we e epea ed wice o each
ans o man . Values we e no malized o he amoun o DNA in egion 1 o each ans o man , which was se o 100%.
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hp 1⌬and m 1⌬, loading o simila amoun s o cell ex ac
on o he gel did no pe mi de ec ion o a signal co esponding
o he ull TAP-Tho2 p o ein (Fig. 7A). Only when 10- old
mo e whole cell ex ac was loaded was a weak signal co e-
sponding o he TAP-Tho2 p o ein de ec able (Fig. 7A). As
THO2 ansc ip le els ha e been ound o be simila in wild-
ype and hp 1⌬cells (13), he dec ease in Tho2 mus esul
om a pos ansc ip ional e ec , sugges ing ha THO subuni s
may only be s able in he o m o a complex.
Analysis o he TAP-Tho2 pu i ied THO complex by g adi-
en SDS-PAGE e ealed ha in he wild ype, hp 1-101,hp 1-
103, and sub2⌬, he ou THO subuni s (Tho2, Hp 1, M 1,
and Thp2) we e p esen (Fig. 7B). Howe e , in hp 1⌬and
m 1⌬, only he TAP-Tho2 p o ein was en iched by he TAP
sys em. Thus, i one o he co e p o eins o THO is missing, he
in eg i y o he THO complex is g ea ly educed. In o de o
ind ou whe he he emaining p o eins o m a leas some
kind o complex, we used an an i-M 1 an ibody o de ec
FIG. 5. mRNA expo de ec s o hp 1 mu an s. (A) Supp ession o hp 1⌬mex67-5syn he ic le hali y. S ain WMH1 (hp 1⌬mex67-5)
ans o med wi h URA3-based plasmid pRS316-HPR1 was addi ionally ans o med wi h LEU2-based plasmids YCpA13 (HPR1), YCp-hp 1-101
(hp 1-101), and YCp-hp 1-103 (hp 1-103) o he emp y ec o YCp70 (hp 1⌬). T ans o man s we e spo ed in 10- old se ial dilu ions on selec i e
medium wi h o wi hou FOA. Pla es we e scanned a e 3 days o incuba ion a 30°C. No e ha only ans o man s ha bo ing alleles supp essing
he syn he ic le hali y a e able o g ow on SC medium-FOA pla es. (B) Analysis o nuclea poly(A)
⫹
RNA localiza ion in W303-1A (wild ype),
WH101-1A (hp 1-101), WH103-1A (hp 1-103), and U768-4C (hp 1⌬) log-phase cul u es cul i a ed o 3 o 6ha 37°C, as indica ed. Samples we e
immobilized in Te lon-coa ed slides and hyb idized i s wi h a digoxigenin-labeled 20-me oligo(dT) and second wi h a luo escein-conjuga ed
an i-digoxigenin an ibody o in si u de ec ion o mRNA. Nuclei we e s ained wi h 4⬘,6⬘-diamidino-2-phenylindole (DAPI). U, u acil; L, leucine.
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emaining aces o M 1 in he TAP-Tho2 pu i ied samples.
Simila amoun s o M 1 we e de ec ed in wild- ype, hp 1-101,
hp 1-103, and sub2⌬cells, whe eas no signal was de ec ed in
hp 1⌬and m 1⌬cells when simila amoun s o TAP-pu i ied
Tho2 we e loaded (Fig. 7C). Howe e , when 10- old mo e
Tho2 p o ein was loaded on o he gel, M 1 could be de ec ed
in hp 1⌬TAP-Tho2 pu i ied ac ions (Fig. 7C). These esul s
indica e ha he THO complex is uns able i one o i s com-
ponen s is absen , such as Hp 1 o M 1, bu is s able in bo h
he hp 1-101 and hp 1-103 mu an s, as well as in mu an s in
which a TREX componen , such as Sub2, is absen .
Since, con a y o hp 1⌬, THO is s able in hp 1-101 and
hp 1-103 cells, we wonde ed i THO was also ec ui ed in o
ac i e ch oma in in hese mu an s. Wi h he same TAP-Tho2
s ains as be o e, we ound ha hp 1-101 and hp 1-103 e-
c ui ed he TAP-Tho2 subuni o THO o a ansc ip ionally
ac i e PMA1 gene (Fig. 8) a le els clea ly abo e ha o hp 1⌬
mu an s. The e o e, he wo poin mu a ions analyzed a e no
null alleles and o m THO complexes ha a e compe en o
ch oma in ec ui men .
Sub2 is poo ly ec ui ed o ac i e ch oma in in bo h hp 1-
101 and hp 1-103 mu an s. The ac s ha he p esence o THO
a he si e o ansc ip ion elonga ion helps ec ui Sub2 o
ansc ibed ch oma in (1, 46) and ha mul icopy SUB2 is able
o supp ess hp 1⌬(11, 19) a e consis en wi h he hypo hesis
ha Sub2 ec ui men o he nascen mRNA plays a key ole in
gene exp ession. As THO is as s able in bo h hp 1-101 and
hp 1-103 mu an s as in wild- ype cells, we wonde ed whe he
hei espec i e pheno ypes could be caused by di e en le els
o Sub2 ec ui men o ansc ibed ch oma in. While es ing
his possibili y, we i s no iced ha wi h a lacZ-URA3 ans-
la ional usion unde he con ol o he e p omo e (LAUR
sys em) (19), SUB2 o e exp ession supp essed he ansc ip-
ional de ec s o bo h he hp 1-101 and hp 1-103 mu an s (Fig.
9A). This was consis en wi h he possibili y ha Sub2 was no
e icien ly ec ui ed o ansc ibed ch oma in in bo h mu an s.
Again, hp 1-103 showed a ansc ip ion de ec in ex a-long
DNA sequences (Fig. 9A) ha ein o ces he conclusion ha
hp 1-103 is a leaky a he han a sepa a ion-o - unc ion mu a-
ion.
Sub2 ec ui men o ac i e ch oma in was di ec ly assayed
by ChIP in s ains ca ying a SUB2-TAP epi ope usion a he
SUB2 ch omosomal locus wi h Sepha ose IgG and an i-
RNAPII an ibodies. As shown in Fig. 9B, he e was no signi -
ican di e ence in he ec ui men o RNAPII o he cons i u-
i ely ansc ibed PMA1 gene be ween he wild- ype and hp 1
mu an s ains es ed. Ne e heless, ec ui men o Sub2-TAP
was as ine icien in hp 1-101 and hp 1-103 cells as in hp 1⌬
cells (55 o 60% o he wild- ype le el). The e o e, we can
conclude ha he speci ic gene exp ession pheno ype o hp 1-
FIG. 6. Nascen -mRNA dependency o he ansc ip ion and hy-
pe ecombina ion de ec s o hp 1 mu an s. (A) Kine ic analyses o
ansc ip ion ac i a ion o he ib
m
and Rib
⫹
usions in s ains
W303-1A (WT [wild ype]), WH101-1A (hp 1-101), WH103-1A (hp 1-
103), and U678-4C (hp 1⌬). The PHO5- ib
m
-lacZ ( ib
m
) and PHO5-
Rib
⫹
-lacZ (Rib
⫹
) ansc ip ional usions we e unde con ol o he
GAL1 p omo e (GAL1
p
). They con ain an ac i e o inac i e ( espec-
i ely), syn he ically made, 52-bp ibozyme (Rib), ollowed by a 266-bp
agmen o he U3 gene o p e en he clea ed mRNA om deg a-
da ion and he 369-bp P uII 3⬘-end lacZ agmen a he un ansla ed
egion o PHO5 (posi ion ⫹1405). Samples we e collec ed 2 h a e
galac ose addi ion o ac i a e ansc ip ion. Elec opho esis was pe -
o med wi h o maldehyde-aga ose gels ( ib
m
) o u ea-ac ylamide gels
(Rib
⫹
), and hyb idiza ion was wi h a U3 p obe, co esponding o he
ib
m
and Rib
⫹
egion in he scheme. All da a we e no malized wi h
espec o he endogenous U3 signal. The a e age o h ee di e en
expe imen s is plo ed. A.U., a bi a y uni s. (B) Recombina ion e-
quencies o wild- ype, hp 1,hp 1-101, and hp 1-103 cells con aining he
ecombina ion sys ems GL-Rib
⫹
(black ba s) and GL- ib
m
(g ay ba s)
a e shown. Each ecombina ion equency is he median alue o six
independen colonies. The a e age median alue o wo o ou expe -
imen s and he s anda d de ia ion a e plo ed.
VOL. 26, 2006 hp 1 SEPARATION-OF-FUNCTION MUTATION 7459
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