• No results found

Mapping of functional regions of murine retrovirus long terminal repeat enhancers: enhancer domains interact and are not independent in their contributions to enhancer activity.

N/A
N/A
Protected

Academic year: 2019

Share "Mapping of functional regions of murine retrovirus long terminal repeat enhancers: enhancer domains interact and are not independent in their contributions to enhancer activity."

Copied!
9
0
0

Loading.... (view fulltext now)

Full text

(1)

Copyright © 1989,American SocietyforMicrobiology

Mapping of Functional Regions of Murine Retrovirus

Long

Terminal

Repeat Enhancers:

Enhancer Domains Interact and Are

Not

Independent in Their Contributions

to

Enhancer

Activity

TOMHOLLON't AND FAYTH K. YOSHIMURA2*

Department of Microbiology' andDepartment of Biological Structure,2 University of Washington, Seattle, Washington 98195

Received 22November 1988/Accepted 8 April 1989

We have used deletion andrecombinant longterminalrepeat(LTR)mutantstoexamineenhanceractivity differences between LTRs of the nonpathogenic Akv and the thymus lymphomagenic MCF13 murine

retroviruses.Deletionmutantanalysisrevealed that major control regions for MCF13 and Akv LTR enhancer

activity were similar but not identical. For both LTRs, major control regionswere distinctly different ina murine T-cellandafibroblastcell line.Recombinantenhancer analysis showed that LTRs could be divided into three regionscapableofaltering the levelofenhanceractivitythroughcooperativeorantagonisticinteraction.

The contribution ofeach regiontoenhancer activity was dependenton itscontext withrespect to the other

regions. LTRenhancer function indifferentcelltypesappearstobe the resultofthe interaction of enhancer modular elements.

Among the murine retroviruses which do not contain an oncogene within theirgenomes are viruses witha range of pathogenic potential, including those which replicate

with-out harming their hosts. Those murine leukemia viruses (MLVs) which are pathogenic are able to generate diseases which generally involve specific tissue types. To understand which viral genome sequences control disease specificity, recombinant retroviruses have been made by combining different regions of the genomes of retroviruses with dif-ferent diseaseproperties. Bycomparingdiseases inducedby the recombinants and their parental viruses, the major determinant of histological disease specificity has been mapped to DNA sequences within the U3regions of retro-viral LTRs (6-8, 13-15, 22, 23). Within these U3regions are tandemdirect repeat sequences which have been shown to betranscriptional enhancers (19, 20). Thisfinding has sug-gested thatenhancers are important determinants of

retro-viral disease specificity.

The implied linkage between enhancers and disease has been strengthened bythe observation that the exchange of tandem repeat sequences with a small adjacent DNA

se-quence in Moloney and Friend MLVs will change the diseasesthevirusescause(23).Moloneyviruscausesthymic lymphomas, and Friend virus causes erythroleukemias. A recombinant virus with Friend tandem repeat sequences and Moloney structural genes caused erythroleukemias. The

converse viral construction containing Moloney tandem repeat sequencesand Friend structural genes causedthymic

lymphomas.

Li and colleagues (23) also observed that mu-tant MLVs with one instead of two tandem repeats take muchlonger to induce disease than wild-type viruses. This observation has been supported by work from several groupswhich have shown with gene expression assaysthat long terminal repeats (LTRs) that have deleted onetandem repeatlose about half of their enhanceractivity(11,19, 32). Furthermore, stable and transient gene expression assays

* Correspondingauthor.

tPresentaddress:InstitutPasteur,75724Paris Cedex15,France.

haveshown thatstimulationof geneactivityby enhancersof leukemogenic murine retroviruses is strongest in the cell type in which they induce disease (2, 3, 38).

Previous work in our laboratory has shown by means of stable gene expression assays that enhancer activity of the thymus lymphomagenic MCF13 MLV LTR is greatest in murine T-cell lines (38). That work also showed that the LTR of the nonpathogenic Akv virus does not have this T-cell-specific expression property, despite its 81% DNA

se-quencehomology withthe MCF13 LTR.

Studies of enhancersofDNAand RNA tumor viruses and the immunoglobulin heavy chain gene have shown that enhancersarecomposedof modules 15 to 20 basepairs(bp) in length which can further be subdivided into sequence motifs that are binding sites for trans-acting transcription-regulatoryproteins(16, 21,28, 33, 35). Avarietyof sequence motifs forbinding trans-acting proteins have beenfound in the Moloney MLV LTR(35). MCF13 and Akv LTRs have sequences similartotheMoloney motifswithin their tandem repeats, including those for nuclearfactor 1-CCAAT

tran-scription factor binding (26), the glucocorticoid-responsive element (5), the simian virus 40 (SV40) core consensus

sequence (17), and several other DNA-binding proteins which, to date, have only been described for murine

retro-viral LTRs. Akv and MCF13 enhancer mechanisms, then,

arealso likelyto depend oninteractions with transcription-regulatory proteins. Whichproteins may be involved in the T-cell preferential expressionof MCF13 is unknown. Nor is there any knowledge of modular enhancer elements within these LTRs.

Inthis report, we have made enhancer deletion mutants

and recombinant enhancers composed of both MCF13 and Akv LTR sequences in order to begin mapping sequences responsible for MCF13 and Akv enhancer differences. We

tested these constructions with transient gene expression assays in a murine T-cell and a fibroblast cell line. The results of theseexperiments suggest that enhancerfunction

of murine leukemia viruses in different cell types is theresult

of the interaction of enhancer modular elements. 3353

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

MATERIALS ANDMETHODS

Cell lines. NIH3T3 murinefibroblastsweregrownin10% fetal bovine serum (FBS) (Hyclone

Laboratories,

Logan, Utah)-minimal essential medium (GIBCO

Laboratories,

GrandIsland,N.Y.). L691 cellsare aC57/Leaden radiation-induced thymic lymphoma murine cell line (24); they were

grownin5% FBS-RPMI 1640(GIBCO).

Transfection and analysis of gene expression. L691 cells were washed and suspended in RPMI without serum at

107

cells in 0.4 ml pertransfection

experiment. Then,

10

jig

each ofchloramphenicol acetyltransferase

(CAT)

construct

plas-mid and

3-galactosidase

plasmid containing the SV40

early

promoter (pCH110) (12) was mixed with cells to be

trans-fected. Transfection procedures were performed by means ofelectroporation, essentially as described by Potter et al. (29), except that a cylindrical electroporation chamber de-sign was used which improved transfection

efficiency

about fivefold over cuvette chamberdesigns (T. Hollon and F. K.

Yoshimura, submitted for publication).

Trypsinized

NIH 3T3 cells were washed and

suspended

in RPMI without serum at3 x

106

cells in 0.4 ml pertransfection

experiment.

Then4 ,ug eachof CATconstructplasmid andpCH110was

mixed withNIH3T3

cells.

tobe transfected.

Electroporation

conditions were the same as for L691 cells. All

plasmids

transfected were in supercoiled form. Electroporated L691 cellswere placed in RPMI plus

penicillin-streptomycin plus

5% FBS for 2 days before cell

protein

extracts were har-vested. Electroporated NIH 3T3 cells were placed in mini-mal essential mediumplus 10% FBS plus

penicillin-strepto-mycin and were alsoharvested after 2 days.

Protein extract harvests and

nonchromatographic

CAT assays were done essentially as described

by Sleigh

(34), except that protein extracts for CAT assays were heat treatedat65°Cfor 10 min in the presence of 5 mM EDTA

(4)

in order to denature cellular enzymes interfering with the CATassay. The averagecountsperminute of

14C-acetylated

chloramphenicolextractedfor MCF13 LTR

(clone M2)

CAT assayswas29,800cpmin L691 cellsand

13,700

cpmin NIH 3T3 cells.

3-galactosidase

activity measurements wereused

to correctCAT activitymeasurementsfor variationsdueto

transfection efficiencies. Each

3-galactosidase

assay was

done by using 100 jig of

protein

extractin 0.25 M Tris

(pH

7.8)plus 10

V1

50 mMCPRG

(chlorophenol

red

P-D-galacto-pyranoside; Boehringer Mannheim

Biochemicals,

Indianap-olis, Ind.)-70

VI

Z buffer

(25)-deionized

water to a total volume of 100

VI.

Zbuffer is 0.06 M Na2HPO4

7H20-0.04

M

NaH2PO4.

H20-0.01 M KCl-1 mM

P-mercaptoethanol

(pH 7.0). The assaywas done at 37°C for 150 min, and the i-esulting

P-galactosidase

activitywas read at 574nm. Plas-mids were transfected an average of 12times each in L691 cells and 9 times each in NIH 3T3 cells.

Plasmid constructs. The LTR+CAT constructions were made in several steps according to standard

procedures.

Constructions were verified by analysis with appropriate restriction endonuclease digestion procedures. The

SphI-BamHI DNA fragment from pSV2CAT (9) containing the SV40 promoter without enhancer and the CAT gene was

ligated into the SphI and HindIll sites ofpUC18after first making BamHI and HindIII ends blunt with Klenow

frag-ment treatment. This plasmid was called pUCCAT. PstI-DraIfragments ofMCF13 and Akv LTRs were ligated into theXbaI site ofthepoly-cloning site of pUCCATto create

M2PrS andA2PrS;PstI andXbaI sites wererendered blunt in orderto achieve ligation. To make clones A2 and M2, a

PstI-HindIII fragment was first removed from pUCCATin

orderto removethe SV40promoter.

Next,

theHindlll site at the 5' end of the CAT gene was made blunt ended

by

Klenow

fragment

treatment.PstI-SmaI LTR

fragments

were

then

ligated

intotheCATvector to

replace

thedeleted

SV40

promoter; thiscreated A2and M2.

Deletion mutants. A2 and M2 were eachcut with EcoRV and

religated

in the absence of the EcoRV-EcoRV

fragment

withinthe tandem repeats.This

generated

AAAand

MMM,

which each haveonetandem repeatdeleted. BamHI-EcoRV

fragments

weredeletedfromAAAand MMMtocreateOAA and

OMM;

BamHI and

ApaI

fragments

were deleted to createOOAandOOM

(BamHI

wasin the

pUC18

poly-cloning

site).

AOA and MOMweremade

by

deleting

the

EcoRV-ApaI

fragments

from AAA andMMM.PrMwasmade

by

deleting

theBamHI-DraI

fragment

from M2.

Recombinant enhancers. PstI-EcoRV sequences were

ex-changed

between MMM and AAA to create AMM and MAA.

EcoRV-ApaI

sequences were

exchanged

between

MMMand AAAtomake MAM and AMA. AAM and MMA resulted from

exchanging

ApaI

toSmaIsequences between AAA and MMM.

RESULTS

Experimental

strategyand constructionofmutants.

Figure

1 shows a

comparison

of the Akv and MCF13 LTR

se-quences involved in the DNA constructions used in this

study.

Sequences

present as tandem direct repeats in the LTRs are enclosed in brackets. MCF13 and Akv tandem repeats are 69 and 99

bp,

respectively.

Our

previous

work

using

stable gene

expression

assays

suggested

that sequence differencesbetween these LTRswere

responsible

fora5-to

10-foldincrease of MCF13 LTR enhancer

activity

overAkv in murine T-cell lines

(38).

Although

tandemrepeats in the U3

region

of other MLVs have been shown to function as

enhancer elements

(20),

Laimins et al.

(19)

have demon-strated that tandemrepeats maynotbe the sole locations of enhancer

activity.

Therefore,

our

operational

definition of LTR enhancers has allowed for the

possibility

that not all enhancer

activity

is

provided by

theMCF13 andAkvtandem repeats.

Figure

2 shows ourenhancer constructions. For ease of

manipulations

involving

cloning,

all deletion and

recombi-nant enhancer mutants were

generated

from constructs

containing only

oneofthetandem repeats from eachLTR.

To

begin

to map

regions

responsible

for LTR enhancer

activity,

we made use ofrestriction enzyme sites found at similar

positions

in thetwoLTRs. EcoRVand

ApaI

restric-tion sites

(Fig.

1)

within each tandemrepeatare23

bp

apart

and straddle

SV40-like

enhancercore consensus sequences

(17).

We therefore refer to this DNA segment as the core

region.

EliminationofDNA segments

terminating

inone or theotherof theserestriction sitescreatesdeletionmutantsin which

comparable

regions

have been lost from Akv and MCF13 LTRs. We reasoned that

by

comparing

enhancer activities of deletion mutants with the

original LTRs,

we

would be able to map LTR

regions controlling

enhancer

activity

in different cell types andto

identify regions

respon-sible for the differencesbetween thetwoenhancers.

To create recombinant

enhancers,

we

again

took advan-tageof the EcoRV and

ApaI

sites,

which

effectively

allowed

us todivide eachLTR into threeparts: sequences 5' tothe core

region

(PstI-EcoRV),

core

region (EcoRV-ApaI),

and sequences 3' to the core

region, including

the promoters

(ApaI-SmaI).

For convenience we call these the

5',

core, and 3' LTR

regions.

MCF13 and Akv DNA segments cut

on November 10, 2019 by guest

http://jvi.asm.org/

(3)

PstI

CTGCAGTAACGCCATTTTGCAAGGCATGGAAAAGTACCAGAGCTGA.GTTCTCAAAAGTC...ACAAG

G A T G AAACAAGA

EcoR V Core

GAAGTTTAGTTAAAGAATAAGGCTGAA*AAAACTGGGACAGGGGCCAAACAGGATATC4iTGGTCGAIGCA

A AG ... G A GT C TA A |

ApaC

CCTGGGCCCCGGCTCAGGGCCAAGAgAGATGGTACTCAGATAAAGCGAAACTAGCAGCAGTTTCTGGAA

TA C C C A]T T AC A A

DraI

MCF13 AGTCCCACCTCAGTTTCAAGTTCCCCAAAAGACCGGGAAA.

AACCCCAAGCCTTATTTAAACTAA~~

Akv CC AG AACTGTC CA G G T G TC C

MCF13 CAGCTCGCTTCTCGCTTCTGTAACCGCGCTTTTTGCTCCCCAGCCC ATAAGGTAAAAACCCCACA

Akv C A G T G

SmaI

CTCGGCGCGCCAGTCATCCGATAGACTGAGTCGCCCGGG C

B. MCF13 or Akv LTR

TATA CCAAT Cap Site

I

I

11

_ _

I~II I~I 'I"t tr

I

Il~~~cl

Pr1

p

I I

E A

I I

E A

U3

I I

D S

U5 R

FIG. 1. Comparison of Akv and MCF13 LTR nucleotide sequences and structure of LTRs. (A) Akv and MCF13 LTR nucleotide

sequencesbetweenthe PstI andSmaI sitesarealigned for comparison. Sequence differencesareshown for the AkvLTR,withblankspaces foridentical bases and dots for deletions. The sequencesarefrom thepublished data of Van Beverenetal.(37) and Yoshimuraetal.(38). Nucleotidespresentastandemdirectrepeatsin the LTRsareshown withinbrackets, but onlyasinglerepeatfor each LTR isshownhere. Boxes surround theSV40-likecoresequencesandCCAAT and TATA motifs in thepromoter.(B) Generalizedstructureof the MCF13 and AkvLTRs.Restrictionendonuclease sitescommontothe Akvand MCF13 LTRsarePstI(P), EcoRV (E), ApaI (A), DraI (D), and SmaI(S).

LTRs usedinconstructionstodirectCATgenetranscriptionwerePstI-SmaI LTR fragments. The PstI sitestarts atbase36atthe 5' end of eachLTR; the SmaI sitestarts atabout33bases 3'toeachcapsite. Thehorizontalarrowsshow thetandem directrepeats;boxes marked Carethe23-bpcoreregion between the EcoRV and ApaI sites. The DraI site is5bp5'toeachCCAAT box. The DraI-SmaIregion includes

thepromoter(Pr). Arrows marking R and US LTR regionsaretoscale with the U3 region.

withoneortheother restrictionenzyme wererecombinedto create LTRs containing enhancers that contained both

MCF13 and Akv sequences. Comparing recombinant en-hancers would let us ask what effect each region had on enhancerfunctionwhenlinkedtodifferentsequencesofAkv

or MCF13 origin. We also made constructions which ex-changedLTR enhancers and promoters, enablingusto ask whether these enhancers work differently with different

promoters. Enhancers and promoters were exchanged at a DraIsite 5bp5'tothe CCAATboxof each promoter.

WeusednonchromatographicCAT transientgene expres-sion assays (34) to test enhancer activity. Results of CAT

assaysofwild-typeLTRenhancersin themurineL691 T-cell

andNIH3T3fibroblastcell linesareshown inFig. 3. In the T-cell line, CAT activity of the MCF13 LTR with two tandem repeats (M2) is 30 times greater than that of the construct withonlytheMCF13 promoter(PrM).The

differ-encebetweenthesesameclones in NIH 3T3 cellsis50-fold. The Akv enhancer attached to the MCF13 promoter (A2PrM)is20 timesasactiveasPrM in the T-cell line and 75

times as active in fibroblasts. This confirms our previous

report (38) that both LTRs contain sequences which are

capable ofenhancing transcriptional activityfrom a murine leukemia viruspromoter.That thesesequencesare function-ingasenhancers issupported by previouswork in whichwe haveshown that thisactivityisorientationindependent (38), ahallmark of enhancers (17).

When we compared the relative strengths of the two LTRs, we found that M2produced2.4timesasmuch CAT

activity as theAkv LTR (A2) in L691 cells. In fibroblasts,

their strengths were reversed; Akv was 2.7 times more active than MCF13. Between celllines,the LTRs differedin relative strength by about sixfold, as can be seen by com-paringM2 and A2 activities in NIH 3T3 and L691 cells(Fig. 3).

LTR enhancers show promoter preference. We created A2PrM and M2PrA (the MCF13 enhancer and the Akv promoter),which exchangeenhancer and promoter regions (Fig. 2), totest whetherthe enhancers functioned indepen-dently of their promoters. Berg et al. (1) reported that MCF13

A.

Akv

MCF13 Akv

MCF13

Akv AA G

MCF13 Akv

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.612.101.507.76.430.2]
(4)

M2

I

IH

iHI[

IPi

I

A2

IU2PrA I IIIII

:

AmpC

LTI CAT

A2PrM

M2PrS

I

III

M

A2PrS

PrM

DG

DELETfON MUTANTS

OAA

AOA

OOA

P EAO D S

ff'_C4-' -b

~cowe

AAA-MAM AMA

MAA 11

AMM ~ :

MMA

I I I

P EA D S

[image:4.612.70.306.66.398.2]

44-PC4-3' -*

FIG. 2. MCF13 and Akv LTR constructions used for transient expression CAT assays. All LTRs were cloned into a

pUC18-derivedplasmid containing the CATgene.Theplasmid designatthe

upperright illustrates the general form of these constructions. At the

upperleftareLTRs consisting of PstI-Smal fragments. Open boxes represent MCF13 regions; Akv regionsare stippled. Boxes below

thosemarked C and PrepresenttheEcoRV-ApaI coreregion and

thepromoter,respectively. M2 and A2arethewild-type LTRs; the 2denotes thepresenceof both tandemrepeats,shown witharrows.

PrM, PrA, and PrSarethe MCF13 and Akv LTRpromotersand the SV40earlypromoter(hatched), respectively. M2PrA is the MCF13 enhancer withAkvpromoter;asimilar notation describes the other

enhancer-promoter recombinations. MMM and AAA correspondto

LTRs withonetandemrepeatremovedfrom M2 and A2. Each letter M or A stands fora 5' (PstI-EcoRV), core (EcoRV-Apal), or 3'

(ApaI-SmaI) LTR region. For the deletion mutants, a0is usedto

denote a deleted region. Restriction enzyme sites important to

deletion mutant and recombinant enhancer construction are PstI

(P), EcoRV (E), ApaI (A), DraI (D), and SmaI (S). Arrows below therestriction sites indicate the 5',core,and 3'regions.

enhancers can sometimesfunction differently with different promoters. Akv and MCF13 promoters differ by 5 bp be-tween the CCAAT box andthe transcription initiation site (38), although their CCAAT and TATAbox sequences are identical.

Thedifference between M2 and M2PrAwaslessthan17% in both cell lines (Fig. 3), which we judged not to be

significant. The A2versusA2PrMcomparison showed larger differences. A2PrM had 40%moreactivitythanA2in Tcells and 45% less activity than A2 in fibroblasts. These differ-ences also correlate with ourdata showingthat the MCF13 LTR has greater activity than Akv in T cells but less in

fibroblasts. For these reasons, it appears that the effect of the MCF13 promoter on the enhancers may be of

signifi-'~- 200

|

100

0.

M2 A2 M2PrA A2PrM M2PrS A2PrS PrM FIG. 3. CAT activities of wild type LTRs and enhancer-pro-moterrecombinants. L691 T- and NIH 3T3fibroblast murine cell linesweretransiently cotransfected with LTR+CAT plasmids and 3-galactosidase (pCH110) plasmids.Nonchromatographic CAT ac-tivity measurements (34) are counts per minute of 14C-acetylated chloramphenicol extracted from CAT assays with ethyl acetate. CATactivities measured for these transfections werecorrected for transfectionefficiency variation with ,3-galactosidaseactivity mea-surements. Theresults ofmultipleindependent cotransfections are presented as averages. Error bars show standard errors of the means.The levelofMCF13 wild-type LTR (M2)activity has been set at100for each cellline,and all other CATactivityaverages are expressed relativetothe M2 levels. ForFig.3, 4,and 5, theaverage countsperminute of14C-acetylatedchloramphenicol extractedfor M2 CATassays was 29,800 cpm inL691 cells and 13,700cpm in NIH 3T3 cells.

cance.Whenwerecombined enhancers with theSV40 early

promoter (M2PrS and A2PrS), we found that in both cell

lines MCF13 and Akv had at least 10 times more activity

with their own promoters than with theSV40promoter.

DifferentLTRregionscontrol enhanceractivity in aT-cell

and a fibroblast cell line. To map regions important for enhanceractivity in each cell line, we made deletion mutants whichprogressivelydeleted similarregionsfrom the MCF13 and Akv LTRs. We first deleted one of the two tandem repeatsequences from each LTRto createMMM and AAA (Fig. 2). Inournomenclature, each letter MorA stands for

a5', core, or3'region. In thedeletion mutants, 0 is usedto

denotea deleted region. The 5' region from PstI to EcoRV wasdeleted to create OMM and OAA. Deleting5' and core

regionsfrom PstIthroughtheApaI sites resulted in OOM and OOA. The core region included between EcoRV andApaI was deleted in MOM and AOA. By comparing M2 with

MMM,OMM,MOM, andOOM in both cell lines andsimilarly for Akv mutants, contributions of the deleted regions to

enhanceractivitywere determined.

CAT activities of M2 and its deletion mutants are

com-paredinFig.4. InL691 T cells(Fig.4A),neither theloss of

oneof the tandem repeats(MMM)northefurther loss of the 5'region (OMM) causedmorethana15%decline in enhancer activity from the M2 level. Loss ofthe coreregion(MOM), however, caused a70% declineinactivity fromthe levelof the M2clone.Surprisingly, deleting both5' andcoreregions inOOMdidnotlower enhanceractivitytothe MOMlevel,but rather, the activity of OOM was twice the activity of MOM. This suggested that the MCF13 5' region is suppressing enhancer activity in MOM. On the other hand, this result

couldbe duetoantagonistic interactionsof 5' and 3'regions

no longer separated by the core region. Comparison of deletion mutants with MCF13 promoterPrM showed that the major control regions ofM2 enhanceractivity are the

core region and sequences between it and the promoter. A

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.612.325.565.81.216.2]
(5)

L691

L~~~-MMM AAA OMM OAA MOM AOA OOM OOA PrM

M2 A2 MMM MA OMM OM MOM AOA OOM OOA PrM

FIG. 4. CAT activities of deletion mutants in L691 T cells and NIH 3T3fibroblasts. Barsshowaverage CAT activities of MCF13 and AkvdeletionmutantsinL691 cells(A)andNIH3T3cells(B).

MCF13-derived mutantsare

represented by

stippled

barsand mu-tantsof Akv

origin

areshown

by

open bars.

second enhancer tandem

repeat

seemedtocontribute littleto

MCF13 enhancer

activity

in L691cells.

In NIH3T3

fibroblasts,

MCF13 deletionmutantsshowed adifferent

pattern

(Fig.

4B).

The lossofonetandem

repeat

inMMMlowered enhancer

activity by

onethirdfromthe M2

level. OMM had

only

halfthe

activity

ofM2. MOM had

only

20% of the

activity

ofM2. Whereas OOM had 60% ofM2

activity

in L691

cells,

it had

only

10% ofthat in NIH 3T3

cells. In the

fibroblast

cell

line,

the

major

control

regions

of MCF13 enhancer

activity

reside ina second tandem

repeat

and in the 5' and core

regions.

A

comparison

ofA2andits deletionmutantsin L691cells

(Fig.

4A)

showed that the deletion of one tandem

repeat

(AAA)

or5'

(OAA)

or core

region

(AOA)

is without

signifi-cant effect.

OOA,

in which 5' and core

regions

have been

deleted,

had

nearly

70% more enhancer

activity

than

A2,

whichmayindicatethataregionrepressingenhancer

activ-ity

has been removed fromOOA. In L691

cells, then,

mostof theenhancer

activity

ofA2is controlled

by

sequencesinthe

3'

region.

Itis

interesting

that inL691

cells,

OOA has

greater

activity

than AOA andOOM has

greater

activity

than MOM.

These

parallel

results with different LTRs

support

our

sug-gestion

that sequences in the 5'

region

may be

inhibiting

enhancer

activity.

In NIH3T3

cells,

AAAhad

only

60% ofA2

activity

dueto the deletion of one tandem

repeat

(Fig.

4B).

Additional deletions ofthe 5'

region

(OAA),

core

region

(AOA),

and both

5' and core

regions

(OOA)

showed

large

declines in

activity

fromthe AAA and A2 levels. In this cell

line,

the

controlling

-A

regions

ofenhancer

activity

are a second copy ofa

repeat

sequence and 5' and core

regions.

Regions responsible

for differences in MCF13 and Akv enhancer activities. We have also derivedinformationabout the

regions

ofthe LTRwhichaccountforthedifferences in enhancer activities between MCF13 and Akv in the L691 T-celllineand in NIH 3T3fibroblasts. In L691

cells,

M2had 2.4 times the CAT

activity

of A2

(Fig.

4A).

When one tandem

repeat

was deleted from each

LTR,

MMM

activity

was

greater

than the

activity

of

AAA,

but thedifferencewas about halfofthedifferencebetween M2 and A2.

Comparing

5'

region

deletionmutants, OMMwasmoreactivethan

OAA,

and thedifferencewas about75% ofthedifferencebetween

M2and A2. But when thecore

region

was

deleted,

compar-ison ofMOM and AOA and

comparison

of OOM and OOA showed that the

greater

activity

associated with MCF13

sequences had

disappeared.

Thus,

regions

most

important

for the MCF13 and Akv LTR difference seemed to be a second copy ofthe tandem

repeat

and the core

region.

InNIH 3T3

cells,

A2 CAT

activity

was2.7times as

great

as M2

activity

(Fig.

413).

When one tandem

repeat

was deleted from each

LTR,

the

activity

of AAA exceeded

MMM

activity,

butthis differencewasabout halfthe

differ-encebetween A2 and M2. The OMMversusOAA

comparison

showed that the

original

difference between A2 and M2

activity

waseliminatedwhen 5'

regions

weredeleted.

Com-parison

ofthecore

region

deletionmutantsshowed that AOA

had more

activity

than

MOM,

which

suggested

the core

region

did notdeterminethe differencein Akv and MCF13 enhancer activities. OOA had

greater

activity

than

OOM,

which

suggests

that the 3'

region

also contributed to the difference between Akv and MCF13 in NIH 3T3 cells.

However,

the presence of a second tandem

repeat

and sequences within the 5'

region

seemed to be the

major

determinant ofthe difference inLTR enhanceractivities in this cell line.

Comparing

recombinant enhancers. With enhancer con-structs

MAM, AMA, MMA,

AAM, MAA,

and AMM cre-atedfrom

parts

ofMMMand

AAA,

wewishedtoassessthe relative contribution of each

region

to enhancer

activity

when associated with sequences from another LTR.

Com-parisons

ofcore

region

deletion mutants MOM and AOA in

Fig.

4A indicated thatcore

regions

accountforsome ofthe differences between MMM and AAA in L691 cells. We wishedtoknowwhether eachrecombinantenhancer withan MCF13core

region

would have

greater

activity

in L691cells than the

corresponding

Akv core

region

recombinant. Four

comparisons

ofcore

region exchanges

are

possible:

MMM

versus

MAM,

AMA versus

AAA,

MMA versus

MAA,

and

AMM versus AAM. Evidence that an MCF13 core

region

recombinant

always

has

greater

activity

than its Akv core

region

counterpart

would

suggest

that thecontribution ofthe

core

region

to enhancer

activity

is

independent

of the

sequences

surrounding

it. On the other

hand,

if some en-hancers with Akv core

regions

are more thanor

equally

as active as their

counterparts

with MCF13 core

regions,

the

core

region

contributionmaybe

dependent

on

adjacent

5'or

3'

region

sequences.

Figure

5A shows the CAT results from four core

region

exchanges

inL691 cells. MMMhas 47%more

activity

than

MAM,

which is

comparable

to the difference between the activities ofMMM and AAA. The AMA versus AAAand

MMA versus MAA

comparisons,

however,

showed a smalleror no

change

when Mand Acore

regions exchanged

places.

In

addition,

the AMM

comparison

with AAM showed that the Akv core

region

could

greatly

stimulate

8

qp-I

9

R

!9

CC

4c1)

0!i

8

iou

I

v-N

R 100

w

2:

1. cc

so

6

0-1

M2 A2

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.612.57.299.75.376.2]
(6)

A

onn

_

L691

I.-.

100

X-NIH3T3

'P 200

I-_ I 1o1 l l | | I

MMM MAM AMA AM MMA MM AMM MM PrM FIG. 5. CATactivities of recombinant LTR enhancers. Recom-binant enhancers arearranged toshow CATactivitychangesfrom

exchanging core regions in L691 cells (A) and NIH 3T3 cells (B).

Stippled bars represent recombinant enhancers with MCF13 core

regions;recombinant enhancers with Akvcore r-egionsareshownby

openbars. Fourcomparisons ofcoreregion exchangesare shown-,

lefttoright: MMMversus MAM, AMAversusAAA, MMAversus

MAA,and AMMversusAAM.Thepromoter PrM is shown insolid bars. The recombinant enhancers can also be compared for the effects of 5' or 3' region exchanges. The 5' region exchanges are

MMM versus AMM, MAA versus AAA. MAMversus AAM. and

MMA versus AMA. The 3' region exchanges are MMM versus

MMA. AAM versus AAA. MAM versus MAA. and AMM versus

AMA.

enhancer activity. Conversely, this comparison indicated that the MCF13 core regionwas depressingenhancer

activ-ity. This wasa surprisingresult notpredicted by analysis of the deletion mutants of Fig. 4A. Enhancers with MCF13

coreregions apparently did not always havegreater activity

in L691 cells than those with Akv core regions. These data suggest that whether an Akv or MCF13 core region

de-creases, increases, ormakes no change in enhancer activity

dependson the context of enhancer sequences with respect

toother regions.

Data from the AOA versus MOM comparison in Fig. 4B

indicated that the core region was not responsible forAAA being more active than MMM in fibroblasts. This informa-tion would predict that core region exchanges in NIH 3T3 cells would havelittle effectonenhanceractivity. Of the four

core region exchangesshown in Fig. SB,three showed little

change. But the AMM and AAM exchange showed that a core region canhave alarge effect on enhancer function in

NIH 3T3 cells.Aswith L691cells,the dataindicate thatcore

region exchanges did not always have the same effect on

enhanceractivity.Inboth celllines, thecoreregionseems to

be influenced by sequences in otherregions.

The recombinant enhancer measurements also allowed comparison ofthe effects ofexchanging5' regions. In L691 cells, comparisons of5'regionexchanges (Fig. 5A) showed resultssimilartothose inNIH 3T3cells (Fig. SB). InbothT

cells and fibroblasts, the MMM versus AMM comparison suggested that the 5' MCF13 region increases enhancer activity. In both cell lines, theMAM versus AAM compar-isons indicated a decline in activity due to the 5' MCF13 region. In both cell lines, two comparisons, MMA versus AMA and MAA versusAAA, did not show much change. The data suggest that in the right sequence context the

almost silent 5' enhancer domains in L691 cells can be activated. In eithercell line,a 5' region can cooperate with other regions to stimulate activity, interact antagonistically to suppress enhancer activity, or make no change. As with the core region, the data indicate that the 5' region

contri-bution to enhancer activity is not independent ofthe other

sequences.

When wecompared 3' regionexchangesin L691cells (Fig. SA), we found one large change in activity, i.e., the AAM

and AAA exchange. The other exchanges (MMM versus

MMA, MAM versus MAA, and AMM versus AMA) indi-catedonlysmallchangesinactivity.For NIH 3T3 cells(Fig. SB) exchanges, MMM versus MMA, MAM versus MAA, and AMM versus AMA indicated the 3' Akv region

stimu-lated enhancer activity. The fourth exchange, AAM versus

AAA, did not show an increase in activity by the 3' Akv

region.When3' enhancerregionsareexchanged, promoters

are exchangedaswell. Itispossiblethat the promoters have influenced these results. Experiments described earlier (Fig. 3) showed Akv enhancer sequences could be influenced by MCF13 promoters. In any case, our data show that a 3'

enhancer region plus promoter exchange does not always give the same result. It seems that the activity of the 3' region (perhaps with some promoter influence) can be

af-fectedby the 5' and core regions.

DISCUSSION

We have examined differences in enhancer activities

be-tween LTRs of the nonpathogenic Akv and the thymus lymphomagenic MCF13 retroviruses. These experimentsare afirst step toward pinpointing LTR sequences that control

enhancer activity and tissue-specific expression and that

may possibly contribute to viral pathogenicity. We have found thatwithin acellline, similar,butnotalwaysidentical,

LTR regions are the major control regions of enhancer activity. Despite the fact that this is not a fine mapping of

LTRenhancers, the regulatoryregions inthe twocell lines

were clearly different. This finding suggests that there are

modular functional divisions within each LTR. Regions controlling differences between Akv and MCF13 enhancer activities inthetwocell lineswerealsonotidentical,further

suggestingseparable functional elements in LTRenhancers. More refined mappingwill be neededto preciselylocate the modular elements.

Functional attributes of LTR regions. We observed dif-ferent contributions that a second copy of the enhancer tandem repeatcanmaketoenhanceractivityin different cell lines. Infibroblasts, deletion ofone tandem repeat in either the AkvorMCF13 LTR resultedinlossof activity similarto the50%activitylosspreviously reportedfor other retroviral

LTRs (11, 19, 32). In contrast, in the T-cell line loss ofone

tandem repeatdid not significantly reduce either MCF13 or

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.560.38.280.72.369.2]
(7)

Akv enhancer function. Li et al. (23) demonstrated that deletion of an LTR tandem repeat substantially delays the onset of Moloney virus-induced thymic lymphoma and of Friend MLV-induced erythroleukemia. While the disease caused by Moloney viruses with one or two tandem repeats did not change, 20% of the Friend viruses with a deleted repeat caused myelogenous or megakaryocytic leukemia. The induction of these new leukemias by the mutant Friend virus suggests that in the Friend LTR the deletion of one tandem repeat may have affected the tissue-specific expres-sion of this enhancer. This result may have some bearing on ourfinding that in both cell lines a second copy of the repeat sequence contributed to the overall activity differences

between Akv and MCF13 LTRs.

Our findingthat the differences among LTRs in the T-cell line was partly attributable to the core region (Fig. 4A) was particularly interesting. The SV40 core consensus sequences of Akv (GTGGTCAA) and MCF13 (GTGGTCGA) differ only at the seventh base. Sequence changes in the core region of the SV40 enhancers have been shown to greatly change enhancer activity (39). Experiments by Schirm et al. (31) have suggested that core sequences differing by only one base could be used to assemble enhancers with very different patterns of tissue-specific expression. Tissue-preferential expression by Akv and MCF13 LTRs may be more compli-cated than differences in core motifs, however. The 23-bp

EcoRV-ApaI

segment we call the core region for conve-nience contains the site for at least one other DNA-binding

tr-ans-acting

protein. This DNA-binding protein has been identified by Speck and Baltimore (35) for the Moloney MLV LTR. They have shown that this protein, named leukemia virus factor C (LVc), binds to the sequence CCTGC. The MCF13 sequence CCTGG (a four of five match) and Akv sequence CTAGG (a two of five match) are at LTR positions corresponding to the location of the Moloney LVc motif. The LVc motifs in the core region near theApaI site contain the only sequence mismatches in this region besides the

SV40 core motifs. The poor two of five match for Akv suggests that LVc does not bind to this Akv sequence. Thiesen and colleagues (36) have shown that nucleotide differences in the Moloney and Friend MLV LTR LVc regions contribute to tissue-specific differences in transcrip-tional activity of Moloney and Friend LTRs. Moloney and Friend core motif sequences are identical. It is

possible,

then, that sequence differences in either the core or LVc motifs contribute to the difference between MCF13 and Akv LTR function or, alternately, that both motifs do so to-gether.

Our deletion mutant analysis indicated possible repressor activity in L691 T cells. The00A deletion mutant was more active than the intact Akv LTR, A2. Furthermore, 00A

activity was twice as great as AOA activity and, likewise, 00Mactivity was twice as great as the activity of MOM (Fig. 4A). These results suggest that each LTR has a repressor active in the 5' region in L691 cells. In view of our recom-binant enhancer results which showed that 5' regions in L691 cells could increase, decrease, or make no change in en-hancer activity, this suppression of activity may depend on interactions with other enhancer regions. Whether the re-pression we have observed has relevance for viral replica-tion or pathogenicity we do not yet know.

In comparing this work with our previously published work showing T-cell-specific activity of the MCF13

en-hancer, we observed several important differences. Our previous report used stable gene expression assaysbased on the G418 resistance

gene,

Neo'r, to show that the MCF13

enhancer was 5- to 10-fold more active in T cells than the

Akvenhancer;infibroblasts, thetwo LTRshad nearlyequal activity. In this report, using transient expression CAT

assays,wehavefound asixfold difference between Akvand MCF13 enhancer strength, but this difference is distributed between the two cell lines we tested. In the T-cell line, MCF13 is2.4 timesas active as Akv, and in NIH 3T3 cells, Akv is approximately 2.7 times more activethan MCF13. It is possible that the difference between our stable and tran-sient assay resultsis duetodifferentdesignsofourNeo' and

CAT plasmid constructions. However, several laboratories have noted that stable and transient gene expression assays are not always comparable in results and that widely used reporter genes, such as tk, CAT, and Neor, apparently can interact with promoter sequences so that assays with dif-ferent reporter genes sometimes give different results (10, 18, 27). Nevertheless, our past and present data have dem-onstrated that the MCF13 LTR is more active than Akv in T-cell lines.

Cooperation of LTR regions. The deletion mutant data (Fig. 4) showed that the 5'LTRregions in L691 cells and the 3' regions in NIH 3T3 cells made no substantial

contribu-tions to enhancer activity. From this data, it could be predicted that exchanging5' regions in recombinant

enhanc-ers would not influence enhancer activity in L691 cells. Similarly, 3' region exchanges should have little effect in

NIH 3T3 cells. Unexpectedly, we found instead that each

Akv or MCF13 region was capable of stimulating or

sup-pressingenhancer activity orof making no significant

differ-ence upon exchange. Regions which the deletion mutant data indicated were functionally almost silent could in some recombinations substantially affect enhancer activity. We suggest that in the wild-type LTRs each region is relatively activeorinactivein partbecauseofthe influence oftheother regions. The three regions seem to act in concert to control enhanceractivity, asenhancer modules within them interact cooperatively or antagonistically to raise or lower enhancer

activity. This would mean that the magnitude of LTR enhanceractivity and possibly the tissue-specific expression

attributable to any one module would not necessarily

indi-cate its functions in combination with other modules from

either its cognate or other enhancers.

Our conclusions are supported by the experiments of Rochford et al. (30), demonstrating that recombining en-hancer sequences can have unpredictable effects on viral function. By deleting the B domain of the polyomavirus enhancer from the polyomavirus genome and recombining

the remaining A domain with the lymphotrophic Moloney

virus enhancer, they obtained polyomavirus expression in

the pancreas, an organ in which neither parental virus is expressed. An example such as this together with our own data suggests that interaction among LTR enhancer

mod-ules, rather than solely a sequence change within a binding

motif, may be responsible for the MCF13 LTR contribution to lymphomagenesis. The unexpected activities of the

recombinant enhancers in CATassays suggest that some of the enhancers might have interestingpathological properties in retroviruses.

In summary, our experiments are the beginning of a

definition ofthefunctionalproperties ofsequence motifs and

modules withina pathogenicand anonpathogenic LTR. Our deletion mutant analysis hasprovided evidenceoffunctional

divisions within LTR enhancers. The unexpected

interac-tions within the recombinant enhancers suggestthat further studies of how enhancer modules work together will be

on November 10, 2019 by guest

http://jvi.asm.org/

(8)

important for betterunderstanding of how enhancers control transcription.

ACKNOWLEDGMENTS

We thank Kurt Diem and Lisa Garbrick for expert technical

assistance. pCH110 was a kind gift from Frank Lee and Gordon

Ringold. We thank Richard Palmiter and Ron Reeder for critical reading of the manuscript.

T.H. waspartially supported by a Public Health Service

Molec-ular and Cellular Biology Predoctoral Training Grant from the National Institutes of Health. This work was supported by Public

Health ServicegrantCA44166from the National Institutesof Health

to F.K.Y. and by a Department of Energy grant (DE-FG 06086 ER60409). Duringasubstantialpartoftheperiod that this workwas

performed. F.K.Y. was a Scholar of the Leukemia Society of

America.

LITERATURE CITED

1. Berg, P. E., Z. Popovic, and W. F. Anderson. 1984. Promoter dependence of enhancer activity. Mol. Cell. Biol. 4:1664-1668. 2. Bosze, Z., H.-J. Thiesen, and P. Charnay. 1986. A transcrip-tional enhancer with specificity for erythroid cells is located in the long terminal repeat ofthe Friend murine leukemia virus.

EMBO J. 5:1615-1623.

3. Celander, D., and W. A. Haseltine. 1984. Tissue-specific

tran-scription preference as a determinant of cell tropism and

leu-kaemogenic potential ofmurine retroviruses. Nature (London)

312:159-162.

4. Crabb, D.W., and J. E. Dixon. 1987. A method forincreasing

the sensitivity ofchloramphenicol acetyltransferase assays in

extracts of transfected cultured cells. Anal. Biochem. 163: 88-92.

5. DeFranco, D., and K. Yamamoto. 1986. Twodifferent factorsact separately ortogetherto specifyfunctionally distinct activities

at a single transcriptional enhancer. Mol. Cell. Biol. 6:993-1001.

6. DesGroseillers, L., and P. Jolicoeur. 1984. The tandem direct

repeats within the long terminal repeat of murine leukemia viruses are the primary determinant of their leukemogenic

potential. J. Virol. 52:945-952.

7. DesGroseillers, L., and P. Jolicoeur. 1984. Mapping the viral

sequencesconferring leukemogenicityand disease specificity in Moloney and amphotropic murine leukemia viruses. J. Virol. 52:448-456.

8. DesGroseillers, L., E. Rassart,and P. jolicoeur. 1983. Thymo-tropism of murine leukemia virus is conferred by its long

terminal repeat. Proc. Natl. Acad. Sci. USA 80:4203-4207. 9. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982.

Recombinant genomes which express chloramphenicol

acetyl-transferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051. 10. Graves, B. J., S. P. Eisenberg, D. M. Coen, and S. L. McKnight.

1985.Alternate utilizationoftworegulatory domains withinthe

Moloneymurine sarcomavirus long terminal repeat. Mol. Cell. Biol.5:1959-1968.

11. Graves, B. J., R. N. Eisenman, and S. L. McKnight. 1985.

Delineation of transcriptional control signals within the

Molo-neymurinesarcoma virus long terminalrepeat. Mol.Cell. Biol.

5:1948-1958.

12. Hall, C. V., P. E. Jacob, G. M. Ringold, and F. Lee. 1983.

Expressionand regulation of Escherichiacoli1acZgenefusions

in mammaliancells. J. Mol. Appl. Genet.2:101-109.

13. Holland, C. A., J.Wozney, P. A. Chatis,N. Hopkins, and J. W.

Hartley. 1985.Construction ofrecombinants between molecular

clonesof murineretrovirus MCF247 and AKV: determinant of

an in vitro host range property that maps in the long terminal

repeat. J.Virol. 53:152-157.

14. Ishimoto, A., A. Adachi, K. Sakai, and M. Matsuyama. 1985. Long terminal repeat of Friend-MCF virus contains the se-quenceresponsiblefor erythroid leukemia. Virology141:30-42.

15. Ishimoto, A., M. Takimoto,A. Adachi, M. Kakuyama,S. Kato,

K. Kakimi, K. Fukuoka, T. Ogiu, and M. Matsuyama. 1987. Sequences responsible for erythroid and lymphoid leukemia in the long terminal repeats of Friend-mink cell focus-forming and Moloney murine leukemia viruses. J. Virol. 61:1861-1866.

16. Jones, N. C., P. W.J. Rigby,andE.B.Ziff. 1988. Transacting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev. 2:267-281.

17. Khoury, G., and P. Gruss. 1983. Enhancer elements. Cell 33:313-314.

18. Koltunow, A. M., K. Gregg, andG. E. Rogers. 1987. Promoter efficiency depends upon intragenic sequences. Nucleic Acids Res. 15:7795-7807.

19. Laimins, L. A., P. Gruss, R. Pozzatti, and G. Khoury. 1984. Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus. J. Virol. 49:183-189.

2)0. Laimins, L. A., G. Khoury, C. Gorman, B. Howard, and P. Gruss.1982. Host-specific activation of transcription by tandem repeatsfor simian virus 40 and Moloney murine sarcoma virus. Proc. Natl. Acad. Sci. USA 79:6453-6457.

21. Lenardo, M., J. W. Pierce, and D. Baltimore. 1987. Protein-binding sites in Ig gene enhancers determine transcriptional activity and inducibility. Science 236:1573-1577.

22. Lenz, J., D. Celander, R. L. Crowther, P. Patarca, D. W. Perkins, and W. A. Haseltine. 1984. Determination of the leukaemogenicity of a murine retrovirus by sequences within the longterminal repeat. Nature(London) 308:467-470. 23. Li,Y.,E.Golemis, J. W. Hartley, and N. Hopkins. 1987.Disease

specificity ofnondefective Friend and Moloney murine leuke-mia viruses is controlled by a small number ofnucleotides. J. Virol. 61:693-700.

24. McGrath, M. S.,E. Pillemer, D. Kooistra, and I. L. Wiessman. 1980. The role of MuLV receptors on T-lymphoma cells in lymphoma cell proliferation. Contemp. Top. Immunobiol. 11: 157-184.

25. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring HarborLaboratory, ColdSpringHarbor. N.Y. 26. Nagata, K. R., R. A. Guggenheimer, T. Inomoto, J. H. Lichy,

and J.Hurwitz. 1982. Adenovirus replication in vitro: identifi-cationofahost factor thatstimulates synthesisof the pretermi-nal protein-dCMP complex. Proc. Natl. Acad. Sci. USA 79: 6438-6442.

27. Novak,T.J., andE.V.Rothenberg. 1986. Differential transient and long-term expression of DNA sequences introduced into T-lymphocyte lines. DNA 5:439-451.

28. Ondek, B., L. Gloss, and W. Herr. 1988. The SV40 enhancer contains two distinct levels of organization. Nature (London) 333:40-45.

29. Potter, H., L. Weir, and P. Leder. 1984. Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes byelectroporation. Proc. Natl. Acad. Sci. USA 81:7161-7165.

30. Rochford, R., B. A. Campbell, and L. P. Villarreal. 1987. A pancreas specificity results from the combination of polyoma-virus and Moloney murineleukemia virus enhancer. Proc.Natl. Acad. Sci. USA 84:449-453.

31. Schirm, S., J. Jiricny, and W. Schaffner. 1987. The SV40 enhancercan bedissected into multiple segments, each with a different cell type specificity. Genes Dev. 1:65-74.

32. Schulze, F., E. Boehnlein, and P. Gruss. 1985. Mutational analysesof the Moloney murine sarcoma virus enhancer. DNA 4:193-202.

33. Sen, R., and D. Baltimore. 1986. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46:705-716.

34. Sleigh,M.J. 1986.Anonchromatographic assay for expression of the chloramphenicol acetyltransferase gene in eucaryotic cells. Anal. Biochem. 156:251-256.

35. Speck, N.,and D. Baltimore. 1987. Six distinct nuclear factors interact with the 75-base-pair repeat of the Moloney murine leukemiavirus enhancer. Mol. Cell. Biol. 7:1101-1110.

on November 10, 2019 by guest

http://jvi.asm.org/

(9)

36. Thiesen, H.-J., Z. Bosze, L. Henry, and P. Charnay. 1988. A DNA element responsible for the different tissue specificities of Friend and Moloney retroviral enhancers. J. Virol. 62: 614-618.

37. Van Beveren, C., E. Rands, S. K. Chattopadhyay, D. R. Lowy, and I. M. Verma. 1982. Long terminal repeatof murine

retro-viral DNAs: sequence analysis. host-proviral junctions, and

preintegration site. J. Virol. 41:542-556.

38. Yoshimura, F. K., B. Davison, and K. Chaffin. 1985. Murine leukemia viruslongterminalrepeatsequences canenhancegene activityinacell-type-specific manner. Mol. Cell. Biol.

5:2832-2835.

39. Zenke, M., T. Grundstrom, H. Matthes, M. Winthzenth, C. Schatz,A.Wildeman,and P.Chambon. 1986.Multiplesequence

motifs are involved in SV40 enhancer function. EMBO J. 5:387-397.

on November 10, 2019 by guest

http://jvi.asm.org/

Figure

FIG.1.forNucleotidesAkvsequenceseachCBoxestheLTRs are Comparison of Akv and MCF13 LTR nucleotide sequences and structure of LTRs
FIG. 2.deletionthethedenoteenhancer(P),PrM,enhancer-promoter(ApaI-SmaI)expressionderivedrepresent2thoseupperSV40MupperLTRs denotes or MCF13 and Akv LTR constructions used for transient CAT assays
FIG. 4.andtantsNIHMCF13-derived CAT activities of deletion mutants in L691 T cells and 3T3 fibroblasts
FIG.5.effectsbars.openMAA,leftbinantexchangingregions;MMA.MMMMMAStippledCAT activities of recombinant LTR enhancers

References

Related documents

This mutant retains only 15 to 20% of its transactivation activity when compared with wild-type E2F-1 (12; data not shown) and is still able to bind DP-1 (Fig. Since this mutant

Nucleic acid sequencing has distinguished between five human immunodeficiency virus type 1 (HIV-1) and two human immunodeficiency virus type 2 (HIV-2) subtypes (13); the

The two cows from which the isolates were derived were seropositive for BIV on Western blot assay and sero- positive for bovine leukemia virus (BLV) and seronegative for

Tat-Rev fusion protein to activate HIV-1 LTR-dependent gene expression via the RRE SLIIB RNA target, while the AN/Rev mutant displayed a barely detectable level of Tat function

the amount in the original inoculum; (ii) also detected in the liver was the viral antigenomic RNA, which is complementary to the genomic RNA found in virions, and is diagnostic

The cell line tion of the VP1 residues involved in B-cell antigenic sites 1 generated in response to PV2 was poliovirus specific and and 3, the amino acid variability of this

Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the

To determine whether mutant forms of gB were recognized by HSV-specific, H-2b-restricted CTL, 51Cr release assays were performed with lymphocytes from HSV-infected C57BL/6 mice