re1at1ve p051t10n and 5tren9th5 0f p01y(a) 51te5 a5 we11...

Re1at1ve p051t10n and 5tren9th5 0f p01y(A) 51te5 a5 we11 a5 tran5cr1pt10n term1nat10n are cr1t1ca1 t0 mem6rane ver5u5 5ecreted 1x-cha1n expre5510n dur1n9 8-ce11 deve10pment 6a6r1e11a 6a111,1 Jeffrey W. 6u15e, 2 M1chae1 A. McDev1tt , 1 Ph111p W. 7ucker , ~ and J05eph R. Nev1n51

1H0ward Hu9he5 Med1ca1 1n5t1tute, 7he R0ckefe11er Un1ver51ty, New Y0rk, New Y0rk 10021 U5A; 2Departrnent 0f M1cr0610109Y, 50uthwe5tern Med1ca1 5ch001, Un1ver51ty 0f 7exa5 Hea1th 5c1ence Center, Da11a5, 7exa5 75235 U5A

Dur1n9 8-ce11 d1fferent1at10n, there 15 a dramat1c 5w1tch 1n the RNA pr0duct5 0f the 1mmun09106u11n ~ heavy cha1n tran5cr1pt10n un1t. 1n the mature 8 ce11 there 15 r0u9h1y e4ua1 pr0duct10n 0f the ~5 and the ~m RNA, wherea5 1n the ant160dy-5ecret1n9 p1a5ma ce11 there 15 near1y exc1u51ve pr0duct10n 0f the tx5 RNA. A p1a5m1d c0nta1n1n9 the ent1re ~ tran5cr1pt10n un1t wa5 pr0per1y re9u1ated when a55ayed 6y tran51ent tran5fect10n 1n a 8 1ymph0ma and a p1a5macyt0ma. 1n c0ntra5t, n0 5uch re9u1at10n wa5 065erved w1th 5eparate p1a5m1d5 that c0u1d pr0duce 0n1y 0ne 0r the 0ther RNA. 1n5tead, the ~m p01y(A) 51te wa5 ut1112ed m0re eff1c1ent1y than the tx5 p01y(A) 51te, 1rre5pect1ve 0f the ce11 type. We a150 f0und that tran5cr1pt10n term1nat10n pr10r t0 the ~m p01y(A) 51te 1n p1a5macyt0ma5 c0ntr16ute5 t0 preferent1a1 pr0duct10n 0f ~L5 RNA 1n the5e ce115. F1na11y, reduc1n9 the d15tance 6etween the tw0 p01y(A) 51te5 1mpr0ved the u5e 0f the ~m 51te at the expen5e 0f the u5e 0f the t~ 5 1n 8 1ymph0ma ce115, 5u99e5t1n9 a c0mpet1t10n f0r a 11m1t1n9 fact0r. 5uch c0mpet1t10n wa5 n0t apparent 1n p1a5macyt0ma5. We c0nc1ude that re1at1ve p01y(A) 51te 5tren9th and the p051t10n 0f the p01y(A) 51te5 w1th1n the tran5cr1pt10n un1t, c0up1ed w1th a chan91n9 c0ncentrat10n 0f a 11m1t1n9 fact0r, a5 we11 a5 tran5cr1pt10n term1nat10n pr10r t0 the ~m p01y(A) 51te, a11 p1ay a r01e 1n determ1n1n9 the expre5510n 0f the ~ 10cu5 dur1n9 8-ce11 deve10pment.

[Key W0rd5: 19M; p01y(A) 51te ch01ce; tran5cr1pt10n term1nat10n]

Rece1ved March 2, 1987; rev15ed ver510n accepted, May 8, 1987.

Due t0 the c0mp1ex1ty 0f the pr0ce55 0f mRNA 610- 9ene515 1n an an1ma1 ce11, the re9u1at10n 0f the 0utput 0f a 9ene can take many f0rm5 (Nev1n5 1983; 81rn5t1e1 et a1. 1985; Pad9ett et a1. 1986). 7h15 15 part1cu1ar1y true f0r c0mp1ex tran5cr1pt10n un1t5 that 9enerate mu1t1p1e mRNA pr0duct5.0ne 5uch examp1e 15 the 1mmun09106- u11n heavy-cha1n 10cu5. F0110w1n9 a55em61y 0f the var1- a61e re910n 9ene c0mp0nent5, the ~ heavy cha1n 0f 19M appear5 1n the cyt0p1a5m 0f pre-8 ce115. After 119ht-cha1n 9ene rearran9ement, 119ht cha1n5 and ~ heavy cha1n5 are a55em61ed 1nt0 19M, wh1ch 15 1ater c0-expre55ed 0n the ce11 mem6rane (m) w1th the c105e1y 11nked d0wn5tream 9ene pr0duct, 19D. 7he pre-8, 1mmature 8 (m19M +), and mature 8 (m19M + m19D +) ce115 have the capac1ty t0 pr0- duce tw0 f0rm5 0f the p. pr0te1n; a heavy cha1n that can 6e 5ecreted a5 an ant160dy m01ecu1e and a re1ated heavy cha1n that can 6e 1n5erted 1n the mem6rane a5 a recept0r f0r ant19en. When the mature 8 ce11 enc0unter5 ant19en, there 15 an 1nduct10n 0f pr011ferat10n and maturat10n that u1t1mate1y re5u1t5 1n term1na1 d1fferent1at10n and a 5w1tch t0 e55ent1a11y exc1u51ve pr0duct10n 0f the 5e-

creted f0rm 0f the ~ heavy cha1n. A5 15 the ca5e f0r a11 0f the heavy-cha1n 9ene5, the mRNA5 enc0d1n9 the mem- 6rane and 5ecreted f0rm5 0f the 19M heavy cha1n (~m and ~5 RNA) der1ve fr0m the 5ame tran5cr1pt10n un1t. 7hey d1ffer 6y 5e4uence5 at the 3• term1nu5 that are enc0ded 1n d15t1nct ex0n5 and 5pec1f1ed 6y d15t1nct p01y(A) add1- t10n 51te5 (A1t et a1. 1980; Ear1y et a1. 1980; R09er5 et a1. 1980). 7hu5, RNA pr0ce551n9 at a1ternate p01y(A) 51te5 and 5u65e4uent 5p11c1n9 event5 90vern the pr0duct10n 0f the5e tw0 d15t1nct mRNA5.

0u r a1m ha5 6een t0 1dent1fy the event5 1n mRNA 610- 9ene515 that are re9u1ated dur1n9 8-ce11 maturat10n and that re5u1t 1n the 5e1ect1ve pr0duct10n 0f the ~m and p-5 mRNA5. L1ke1y p05516111t1e5 1nc1ude fact0r5 that 5pec1f1- ca11y rec09n12e the p01y(A) 51te5 0r fact0r5 that c0ntr01 the extent 0f tran5cr1pt10n (1.e., term1nat10n fact0r5) dur1n9 the maturat10n 0f a 8 ce11. We have emp10yed p1a5m1d5 c0nta1n1n9 the 1ntact ~ 10cu5 and der1vat1ve5 w1th 1501ated p01y(A) 51te5 t0 pr06e f0r the pre5ence 0f 5pec1f1c fact0r5. 1n fact, we f1nd n0 ev1dence f0r re9u1a- t10n v1a p01y(A) 51te-5pec1f1c fact0r5, 6ut rather we c0n-

6ENE5 • DEVEL0PMEN7 1:471-481 • 1987 6y C01d 5pr1n9 Har60r La60rat0ry 155N 0890-9369/87 $1.00 471

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6a111 et a1.

c1ude that the re1at1ve aff1n1ty 0f the p01y(A) 51te5 f0r a c0mm0n fact0r and the1r p051t10n 1n the tran5cr1pt10n un1t are the key e1ement5 1n determ1n1n9 expre5510n 1n mature 8 ce115. When ce115 term1na11y d1fferent1ate 1nt0 p1a5ma ce115, tran5cr1pt10n term1nat10n 15 1ar9e1y re5p0n- 5161e f0r exc1u51ve pr0duct10n 0f the 5ecreted mRNA.

Re5u1t5

A55ay5 f0r ~ expre5510n and re9u1at10n

7 h e p1a5m1d p~ (6r055ched1 and 8a1t1m0re 1985) c0n- ta1n5 a funct10na11y rearran9ed ~ 9ene 1n5erted 1nt0 an expre5510n vect0r c0nta1n1n9 the p01y0ma ear1y re910n t0 a110w rep11cat10n a5 a mean5 0f amp11fy1n9 accumu1at10n 0f the mRNA5 and a m0u5e h15t0ne H4 9ene t0 5erve a5 an 1nterna1 c0ntr01 (F19. 1). We have a55ayed expre5510n 0f p~ 6y tran5fect10n 1nt0 e1ther a repre5entat1ve 0f a mature 8 ce11 (the 8-1ymph0ma ce11 11ne M12.4) (La5k0v et a1. 1981) 0r a term1na11y d1fferent1ated p1a5ma ce11 (the p1a5macyt0ma J558L) {01 et a1. 1983). 51nce 60th 11ne5 have 105t expre5510n 0f the1r end09en0u5 heavy cha1n5 (~ and 0L, re5pect1ve1y) we can a55ay p~ 1n the a65ence 0f 6ack9r0und. RNA wa5 prepared 48 hr after tran5fect10n and a55ayed f0r 12 RNA 5e4uence5 6y 51 ana1y515.

0 u r 1n1t1a1 exper1ment t0 a55e55 whether the tran5fec- t10n 0f the p~ p1a5m1d 1nt0 the M12 and J558L ce11 11ne5

pr0duced the expected re9u1at10n 15 5h0wn 1n F19ure 1C. A 51n91e 51 pr06e, a5 dep1cted at the 60tt0m 0f the f19ure, wa5 capa61e 0f mea5ur1n9 60th ~ and 9-m. 7he ~5 RNA pr0tected the 3•-end-1a6e1ed pr06e up t0 the p01y(A) 51te y1e1d1n9 an 51 fra9ment 0f 410 nuc1e0t1de5. 7he ~m RNA pr0tected the pr06e t0 the p01nt 0f the d0n0r 5p11ce 51te w1th1n the C~4 ex0n y1e1d1n9 an 51 fra9ment 0f 225 nuc1e0t1de5. We have a150 u5ed a 5epa- rate pr06e t0 detect the ~m RNA 3• end and f1nd the 5ame 4uant1tat1ve re5u1t 6y th15 ana1y515 a5 w1th the pr06e that detect5 the ~-m 5p11ce (data n0t 5h0wn). 7ran5- fect10n 0f the p~ p1a5m1d 1nt0 the M12 8-1ymph0ma ce11 11ne y1e1ded appr0x1mate1y e4ua1 am0unt5 0f the 125 and 9-m RNA. 1n c0ntra5t, pr0duct10n 0f the ~5 RNA d0m1- nated when the ~ p1a5m1d wa5 tran5fected 1nt0 the p1a5- macy t0ma J558L. 7he chan9e 1n the rat10 0f the RNA5 var1ed 6etween 1 : 5 and 1 : 8 0ver the c0ur5e 0f a num6er 0f a55ay5. We theref0re c0nc1ude that the tran5- fect10n a55ay 0f the ~ 9ene c105e1y appr0x1mate5 the re9- u1at10n 0f ~m and ~, RNA pr0duct10n.

7ran5fect10n a55ay5 f0r 51te-5pec1f1c pr0ce551n9 fact0r5

We have te5ted p055161e mechan15m5 f0r p01y(A) 51te ch01ce 1n the 19 10cu5 thr0u9h the a55ay 0f der1vat1ve5 0f the p~ p1a5m1d. 7he p1a5m1d P~5 c0nta1n5 the ~, p01y(A) 51te 6ut n0 0-m 51te (F19. 2A). C0nver5e1y, the ~ p01y(A)

A

H4

P01y0m0 7

~5PA I

L V0J C~1C~2C~3 C~4~ --1 • m :::~:1

Pr06e w

~5 3• end w

/~m 5p11ce x

Fm PA

m1 m2 ~ K1A V1A

~5 PA

1 C~.4

610n1

4 1 0 n t

223nt

8

~5- m

P-m- 11J

•••••" - 5 2 7

e - 4 0 4

- 309

- 2 4 2 "=• - 2 3 8

--,. - 2 1 7

- 2 0 1

em - 1 9 0

. . . . 1 8 0

0

/~5 m R N A m 1 ~ ~ " " ~ H ~ ~ A A A 0

,•. ,.-" ........ "••" . . . . ", H 4 - N F m m R N A 1 ~ "~1~1~6~'~1 . . . . . . . . . . . . . . . . "12:2~ 1222.-AAA

F19ure 1. 5tructure 0f the P1~ p1a5m1d and expre5510n dur1n9 tran51ent tran5fect10n a55ay5. (A) 7he p~ p1a5m1d (6r055ched1 and 8a1t1m0re 1985) c0nta1n5 a rearran9ed mu 9ene (9.1 k6), a m0u5e h15t0ne H4 9ene (0.6 k6), and the ear1y re910n 0f p01y0ma (3.6 k6) 1n a p8R322 vect0r (2.0 k6). Arr0w5 1nd1cate d1rect10n 0f tran5cr1pt10n. 5h0wn 6e10w 15 the ~ tran5cr1pt10n un1t and the ex0n 5tructure5 0f the 1t5 and ~m mRNA5. (8) 51 mapp1n9 0f RNA5 pr0duced fr0m M12 and J558L ce115 tran5fected w1th the p~ p1a5m1d. A60ut 20 ~9 0f t0ta1 RNA wa5 hy6r1d12ed t0 a 3•-end-1a6e1ed pr06e dep1cted at the 1eft. Hy6r1d12at10n t0 the ~5 mRNA pr0tect5 a fra9ment 0f 410 nuc1e0t1de5 [t0 the ~, p01y(A) 51te]. Hy6r1d12at10n t0 the ~m mRNA pr0tect5 a fra9ment 0f 223 nuc1e0t1de5 (t0 the ~-m 5p11ce d0n0r). H4 RNA pr0duced fr0m p~ 1n each tran5fect10n wa5 4uant1tated 6y 5• 51 ana1y515 (6r055ched1 and 8a1t1m0re 1985).

472 6 E N E 5 • D E V E L 0 P M E N 7




Deve10pmenta1 c0ntr01 0f 19M expre5510n

A Y-5 PA Y-m PA

L VDJ Cy.1 Cy2Cy.3 Cy.4 J• m1 m21 PF ~ • 1 1 ::.:•:4 [1/A V1A

P~5

P~m ~ ~/A 9//A 1

~5-

~m-

PP•m -1-

PY" m PY•5 PY•5 p/.L / / ~ - - - - - 7 / ~ - - 7 f /

• ~ - 3 1 0 • ~ 0

~F:.-:-~): " 1 - 2•42 ~ ~ 238

1 1 - 20=

1 2 3 4 5 6 7 8

F19ure 2. 5tructure and expre5510n 0f p9. p1a5m1d5 w1th 51n91e p01y(A) 51te5. (A) 7he p~t 5 p1a5m1d wa5 c0n5tructed 6y de1et1n9 a 8c11-Ec0RV fra9ment 1eav1n9 1190 nu- c1e0t1de5 d0wn5tream 0f the ~t5 p01y(A) 51te. 7he p~t m p1a5m1d wa5 c0n5tructed 6y de1et1n9 an Ec0RV-Xh01 fra9ment 1eav1n9 1ntact the 5p11ce d0n0r 5e4uence f0r ~m 1n the C~4 ex0n. {8) 7ran5fect10n5 and 51 a55ay5 were perf0rmed a5 de5cr16ed 1n F19. 2. F0r the a55ay5 5h0wn 1n 1ane5 5 and 6, e4ua1 am0unt5 0f pp.= and p~5 were c0tran5- fected.

51te ha5 6een de1eted fr0m the p1~m p1a5m1d. 1n each ca5e, the RNA that can 6e pr0duced 15 0f n0rma1 5tructure. When the5e p1a5m1d5 were tran5fected 1nt0 M12 ce115 and J558L ce115, the re5u1t5 5h0wn 1n F19ure 28 were 06- ta1ned. F1r5t, there wa5 n0 ce11 preference f0r RNA pr0- duct10n. F0r 1n5tance, the 1eve1 0f p.~ RNA fr0m p1~5 wa5 the 5ame 1n tran5fect10n 0f M12 and J558L; 1~m RNA fr0m p1~m wa5 e4ua1 1n 60th ce11 type5. An 1n5pect10n 0f H4 RNA pr0duct10n fr0m the var10u5 p1a5m1d51nd1cated that there were n0 effect5 fr0m tran5fect10n eff1c1ency 0r p1a5m1d c0py num6er that c0ntr16uted t0 the 1eve15 0f 1mmun09106u11n RNA5. 51nce there 15 n0 ce11-5pec1f1c expre5510n fr0m P1% 0r p12~ 1t w0u1d appear un11ke1y that the p-JWm 5w1tch wa5 due t0 5pec1f1c fact0r5 act1n9 at the p01y(A) 51te5.1n add1t10n, the 1~ pr0duct10n d0m1nated 0ver 1% 1n each ca5e, 1nc1ud1n9 when the tw0 p1a5m1d5 were c0tran5fected. Part1cu1ar1y 5tr1k1n9 wa5 the rat10 0f 1~m/p.5 1n J558L ce115. When der1ved fr0m 5eparate p1a5m1d5, the 1~m RNA wa5 51xf01d m0re a6undant than the 1% RNA. H0wever, when the 1~m RNA wa5 pr0duced fr0m the p~ p1a5m1d {1~m p051t10ned d0wn5tream 0f 1~), there wa5 f1ve t1me5 m0re 1% RNA than P-m. C1ear1y, the p051t10n w1th1n the tran5cr1pt10n un1t appear5 cr1t1ca1.

7he a60ve re5u1t5 5u99e5ted that the p-m p01y(A) 51te may 6e 5tr0n9er than the ~% p01y(A) 51te and 0ffered an

exp1anat10n f0r the near-e4ua1 u5e 0f the tw0 p01y[A) 51te5 1n the M12 1ymph0ma ce115. Name1y, the 1neff1- c1ency 0f the 1~5 51te c0u1d a110w tran5cr1pt5 t0 90 un- c1eaved and thu5 ava11a61e f0r c1eava9e at the ~,, 51te. 1f the 1~m 51te wa5 5tr0n9er, the5e ••ava11a61e tran5cr1pt5•• c0u1d 6e pr0ce55ed at h19her eff1c1ency, ff true, then p1ac1n9 the 1~m 51te f1r5t 1n the tran5cr1pt10n un1t 5h0u1d e11m1nate u5e 0f the 1~5 51te. 5uch an exper1ment wa5 perf0rmed u51n9 the p1~m/5 p1a5m1d dep1cted 1n F19ure 3A. 7he tran5fect10n eff1c1ency 0f the p1~5 p1a5m1d wa5 10w c0mpared w1th the p12 p1a5m1d a5 jud9ed 6y the reduced H4 519na1. 7h15 wa5 a repr0duc161e f1nd1n9 w1th th15 p1a5m1d, the rea50n f0r wh1ch 15 n0t c1ear. H0wever, the eff1c1ency wa5 the 5ame 1n tw0 ce11 f1ne5 and the cr1t1ca1 a5pect 0f the exper1ment 15 the rat10 0f 1~ t0 1~,,. 7here wa5 n0 apparent u5e 0f the d0wn5tream 1~ p01y(A) 51te fr0m the p1~m/~ p1a5m1d when 3• end5 were a55ayed a5 5h0wn 1n F19ure 38. A1th0u9h 1t 15 p055161e that a mRNA u51n9 the 1~ p01y{A) 51te m19ht 6e un5ta61e due t0 an a1tered ex0n 5tructure, we c0u1d f1nd n0 ev1dence what50ever f0r u5e 0f the 1~ p01y(A) 51te, even 1n a 10n9 exp05ure 0f the aut0rad109ram {1ane5 3• and 4•).

Fr0m the5e re5u1t5 we draw 5evera1 c0nc1u510n5. F1r5t, there 15 n0 ev1dence f0r tran5-act1n9 fact0r5 that are p01y(A) 51te 5pec1f1c. 5ec0nd, the 1•m p01y(A) 51te appear5

6ENE5 • DEVEL0PMEN7 473




6a111 et a1.

A

PP. PP-m1. /• / f 1

PA ~ . ~ ,

V/A 9/A 1

/~mPA~ /~51A :••.:: V / A ....... 1

970nt 1242n1 P-m Pr06e x x p.= Pr06e

270nt 159nt P-m PA 1~ ~ p.= PA

3 0 9 -

2 4 2 ~ 1 m 1 , e - ,u.mPA

238 - 2 1 7 -

2 0 1 - - - - 1 9 0 - 180 - 4,=

1 6 0 - e =

1 4 7 - ~ ~ - p.5PA

1 2 3 4

- H 4

tyL •

3• 4•

F19ure 3. 5tructure and expre5510n 0f the P~m/5 p1a5m1d. (A) 1n the p~.~ p1a5m1d the 8c11 fra9ment c0nta1n1n9 the ~ p01y(A) 51te wa5 p1aced 710 nuc1e0t1de5 d0wn5tream 0f the ~m p01y1A) 51te. 7he 51 pr06e5 u5ed f0r the a55ay 0f 9~ and ~m 3• end5 are dep1cted at the 60tt0m 0f the f19ure. (8) 51 mapp1n9 0f RNA5 pr0duced fr0m M12 and J558L ce115 tran5fected w1th the p ~ p1a5m1d. Hy6r1d12at10n t0 ~-5 and 9.m RNA pr0tect5 fra9ment5 0f 159 nuc1e0t1de5 [t0 the ~5 p01y1A) 51te] and 270 nuc1e0t1de5 [t0 the p.= p01y1A1 51te]. Lane5 3• and 4• are 3 x 10n9er exp05ure5 0f 1ane5 3 and 4.

t0 6e 1ntr1n51ca11y 5tr0n9er (m0re eff1c1ent} than the ~5 p01y(A) 51te. When the p01y(A) 51te5 were 0n 5eparate p1a5m1d5, the ~m RNA wa5 the d0m1nant pr0duct and 1f the 1am 51te were p1aced 1n fr0nt 0f the 1~5 51te w1th1n the 5ame tran5cr1pt10n un1t, we c0u1d f1nd n0 ev1dence f0r u5e 0f the ~5 p01y(A) 51te. 7h15 re5u1t thu5 pr0v1de5 a 6a515 f0r c0mpet1t10n 6etween tw0 11nked p01y(A) 51te5.

D1fferent1a1 tran5cr1pt10n term1nat10n w1th1n the p tran5cr1pt10n un1t dur1n9 8-ce11 maturat10n

1f p1a5ma ce115 reta1n the a6111ty t0 eff1c1ent1y u5e the ~1, m p01y(A) 51te, then what 15 re5p0n5161e f0r the 065erved

dec11ne 1n 1t5 u5e when 11nked t0 ~5~ 7ran5cr1pt10n ter- m1nat10n c0u1d 6e a mechan15m, 6ut prev10u5 rep0rt5 have 0ften ar9ued a9a1n5t a tran5cr1pt10n term1nat10n mechan15m f0r the 9-m t0 ~5 5w1tch. Danner and Leder 11985} rep0rted expre5510n 0f mem6rane c0nta1n1n9 RNA 1n p1a5macyt0ma5 6ut the5e exper1ment5 d1d n0t mea5ure tran5cr1pt10n rate5; they 0n1y dem0n5trated the pre5ence 0f the5e 5e4uence5. Ruether et a1. 11986) d1d mea5ure tran5cr1pt10n and 5u99e5ted that expre5510n 0f an aden0v1ru5-60me ~ 10cu5 1n p1a5macyt0ma ce115 d1d n0t term1nate pr10r t0 the ~m ex0n5. L1kew15e, Yuan and 7ucker [19841 d1d n0t f1nd ev1dence f0r tran5cr1pt10n ter-

474 6ENE5 • DEVEL0PMEN7





m1nat10n 1n the ~ 10cu5 1n 11p0p01y5acchar1de (LP5)- 5t1mu1ated 1ymph0cyte5 wh1ch pr0duce 1ar9e am0unt5 0f p,~ RNA. 1n c0n51der1n9 p055161e rea50n5 why the5e tran5cr1pt10n mea5urement5 may n0t have detected ter- m1nat10n, 1t 6ecame c1ear that 5evera1 fact0r5 c0u1d p1ay a r01e. 7 h e ch01ce 0f hy6r1d12at10n pr06e5 c0u1d 6e an 1mp0rtant c0n51derat10n. 1f tran5cr1pt10n pr0ceeded un- a6ated thr0u9h the ~ ex0n5 6ut 5t0pped ju5t pr10r t0 the ~m p01y(A) 51te, f0rmat10n 0f the ~m RNA w0u1d 6e pre- vented 6ut tran5cr1pt10n term1nat10n w0u1d n0t 6e 5c0red un1e55 the pr06e a55ayed 5e4uence5 1mmed1ate1y adjacent t0 the p01y(A) 51te. 5uch pr06e5 were n0t u5ed 1n the prev10u5 5tud1e5 c1ted a60ve. 1n add1t10n, the pr0- duct10n 0f 5ter11e ~ tran5cr1pt5 0ften 1n1t1ated 1n the J1-1-C~ 1ntr0n (A1t et a1. 1982; Ne150n et a1. 1983; Lenn0n and Perry 1985) c0u1d 065cure 5pec1f1c term1nat10n event5 1n ce11 11ne5. 7ha t 15, there 15 n0 way t0 d15t1n- 9u15h tran5cr1pt10n fr0m a pr0duct1ve a11e1e fr0m a6er- rant tran5cr1pt10n that der1ve5 fr0m a n0npr0duct1ve a1- 1e1e. A1th0u9h 1n 50me 1n5tance5 1t appear5 that 5ter11e tran5cr1pt5 may 06ey the n0rma1 term1nat10n 519na15, th15 may n0t a1way5 6e the ca5e. W1th th15 1n m1nd, we exam1ned the extent 0f tran5cr1pt10n acr055 the ~. 10cu5, u51n9 tran51ent tran5fect10n5 0f M12 and J558L ce115 w1th the p~ p1a5m1d where end09en0u5 ~. tran5cr1pt10n d0e5 n0t c0ntr16ute t0 the tran5cr1pt10n mea5urement, and u51n9 a5 0ne 0f the pr06e5 f0r hy6r1d12at10n a 5ma11 DNA fra9ment 0f 200 nuc1e0t1de5 that 5pan5 the ~m p01y(A) 51te (F19. 4).

M12 ce115 and J558L ce115 were tran5fected w1th the p1a5m1d and nuc1e1 were 1501ated 40 hr 1ater. Na5cent cha1n5 were 1a6e1ed 6y 1n v1tr0 1ncu6at10n, and the nu- c1ear RNA wa5 1501ated and then hy6r1d12ed t0 f11ter5 6ear1n9 M13 pr06e5 repre5ent1n9 var10u5 re910n5 0f the tran5cr1pt10n un1t. 0 n e 5uch examp1e 15 5h0wn 1n F19ure 4 and a 4uant1tat10n 0f the re5u1t5 0f 5evera1 1ndependent exper1ment5 15 91ven 1n 7a61e 1. Ana1y515 0f tran5cr1p- t10n 0f the p~ p1a5m1d 1n M12 ce115 revea1ed e4u1m01ar rate5 acr055 the ent1re tran5cr1pt10n un1t; thu5, n0 ev1- dence f0r term1nat10n. 1n c0ntra5t, tran5cr1pt10n 0f p~ 1n J558L ce115 dr0pped 5harp1y near the V~m p01y(A) 51te thu5 1nd1cat1n9 ce11-5pec1f1c term1nat10n pr10r t0 the ~m p01y(A) 51te. 0 f c0ur5e, th15 1nterpretat10n depend5 0n there 6e1n9 n0 5pec1f1c 1055 0f the5e 5e4uence5 (de9rada-

7a61e 1. Re1at1ve tran5cr1pt10n rate5 acr055 the ~. 10cu5 1n M12 and J558L tran5fect10n5

Re1at1ve tran5cr1pt10n rate5

DNA pr06e

Ce11 11ne a 6 c d e

M12 1.0 1.2 0.9 0.6 1.1 J558L 1.0 0.7 0.5 0.1 0.1

Hy6r1d12at10n 0f 1a6e1ed nuc1ear RNA t0 510t 610t5 0f 5en5e- 5trand M13 DNA5, a5 5h0wn 1n F19. 4, were 5canned w1th a den51t0meter. 7he den51t0meter va1ue5 were c0rrected f0r the 512e 0f the hy6r1d121n9 DNA and n0rma112ed t0 the va1ue f0r the ••a•• pr06e. 7he data 5h0wn are the avera9e 0f three 1ndependent exper1ment5. 8ack9r0und hy6r1d12at10n a5 mea5ured w1th M13 DNA wa5 undetecta61e 1n the exp05ure5 emp10yed.

t10n) 1n the 1501ated nuc1e1, a p05516111ty jud9ed t0 6e very un11ke1y 91ven pa5t re5u1t5 w1th 1501ated nuc1e1. 7here wa5 50me ev1dence 0f term1nat10n 1n the re910n 6etween the ~5 and ~m p01y(A) 51te5 6ecau5e hy6r1d12a- t10n t0 pr06e c wa5 reduced 50%. H0wever, w1th the pr06e that 5panned the ~m p01y(A) 51te (pr06e d), 1t wa5 c1ear that very few p01ymera5e5 pr0ceeded t0 th15 p01nt. Furtherm0re, the d1fference 1n tran5cr1pt10n rate5 acr055 the ~m p01y(A) 51te 1n M12 c0mpared w1th J558L wa5 51x- t0 tenf01d, thu5 fu11y acc0unt1n9 f0r the chan9e 1n m R N A 1eve15. 7heref0re, we c0nc1ude fr0m th15 data that term1nat10n 0f tran5cr1pt10n pr10r t0 the ~m p01y(A) 51te can 6e a 519n1f1cant fact0r 1n the 5w1tch 1n pr0duc- t10n 0f ~-m and ~5 mRNA5 when 8 ce115 under90 term1na1 d1fferent1at10n t0 p1a5ma ce115. 7h15 c0nc1u510n 15, 1n fact, c0n515tent w1th a recent ana1y515 0f the extent 0f tran5cr1pt10n 1n a num6er 0f hy6r1d0ma5 and p1a5macy- t0ma5 wh1ch 1nd1cated that 1n many ca5e5 tran5cr1pt10n term1nat10n c0u1d 6e a maj0r fact0r 1n the dec11ne 0f ~m RNA pr0duct10n 1n 19M-5ecret1n9 ce115 (Ke11ey and Perry 1986). 1n th05e ca5e5 where there wa5 11tt1e term1nat10n, tran5cr1pt10n may we11 have der1ved fr0m n0npr0duct1ve a11e1e5. A1ternat1ve1y, there may 51mp1y 6e 50me ce11u1ar var1at10n 1n the extent t0 wh1ch term1nat10n c0ntr16ute5 t0 the p1a5ma ce11 phen0type. 0 u r exper1ment5 1nd1cate that tran5cr1pt10n term1nat10n certa1n1y can 6e a fact0r.

• •

~5 PA

0 6

M12

J558L

VAVA 4 • -

c d e

/ / / 11 1 1 •

1

F19ure 4. 7ran5cr1pt10n rate ana1y515 0f pp. expre5510n 1n M12 and J558L ce115. 5h0wn at the t0p 0f the f19ure 15 a map 0f the ~ tran5cr1pt10n un1t and the M13 pr06e5 u5ed f0r hy6r1d12at10n. 7he exact der1vat10n 0f the pr06e5 15 de5cr16ed 1n Mater1a15 and meth0d5. 7he M13 DNA pr06e5 were 1mm061112ed 0n n1tr0ce11u- 105e f11ter5 and u5ed f0r hy6r1d12at10n 0f RNA 1a6e1ed 1n v1tr0 fr0m nuc1e1 0f M12 and J558L ce115 tran5fected w1th p~. 70 the r19ht 0f each 5pec1f1c pr06e {5en5e 5trand1 15 the hy6r1d12at10n t0 an M13 pr06e 0f the 0pp051te 5trand. F1na11y, hy- 6r1d12at10n t0 M13 DNA w1th n0 1n5ert y1e1ded n0 detecta61e hy6r1d12at10n.





6a111 et a1.

8 J558L

A F5PA Ec0R~ FmPA I !

L V0J CF1C#2 C#3 C/j. 4~ m1 m2 $ --1 • 1 1 ~:~-1 1~/,/~ V1A 6---- P~

k~5-

. . . . . . . • V/A V/A } ~ p/.LA300

:•:•:••:•: . . . . 1V./2 1f//.•.•2 ]---- py.A750

- - [ ~ . . . . . . . . . . . . 1P•/A v /~ t - - - - p~A1000

F19ure 5. 5tructure and expre5510n 0f p~x 1ntr0n de1et10n mutant5 1n J558L tran5fect10n5. (A) 5chemat1c d1a9ram 0f the 5tructure 0f the 1ntr0n de1et10n5.5h0wn 15 the 5tructure 0f the w11d- type p9 and the p051t10n 0f an Ec0RV 51te w1th1n the C1x4/M1 1ntr0n that wa5 u5ed f0r 8a131 de1et10n. 8e10w 15 dep1cted the 5tructure 0f three de1et10n mutant5 1n wh1ch 300 nuc1e0t1de5 (p~xA300), 750 nuc1e0t1de5 (p~xA750), and 1000 nuc1e0t1de5 (p~xA1000) have 6een de1eted. 7he 5tructure 0f the5e de1et10n5 ha5 6een determ1ned 6y re5tr1ct10n ana1y515 and thu5 the exact end p01nt5 0f the de1et10n are n0t kn0wn. (8) 51 mapp1n9 0f RNA5 pr0duced 1n J558L ce115 tran5- fected w1th p9 and the 1ntr0n de1et10n5. RNA5 were a55ayed a5 de5cr16ed 1n F19. 1.

F r n - ~°~ 1 ~ 1 1

H 4 - ~

De1et10n 0f a p0rt10n 0f the 11~-1a m 1ntr0n a1ter5 tran5cr1pt10n term1nat10n 1n J558L ce115 and 1ncrea5e5 1~m RNA pr0duct10n

Fr0m the preced1n9 re5u1t5, we c0nc1ude that the 1n- crea5ed rat10 0f 1x~ t0 1xm RNA 1n J558L ce115 15 1n 1ar9e part due t0 term1nat10n 0f tran5cr1pt10n pr10r t0 the 1xm p01y(A) 51te. 1f 5uch term1nat10n were med1ated 6y a ce11-5pec1f1c fact0r rec09n121n9 a c/5-act1n9 1x 5e4uence, then e11m1nat10n 0f the tar9et 5e4uence 5h0u1d have the 5ame effect a5 the a65ence 0f the fact0r (a5 1n M12 ce115) and thu5 revert expre5510n t0 that 5een 1n the M12 ce115. We have te5ted th15 p05516111ty 6y 9enerat1n9 de1et10n5 1n the 1ntr0n 6etween the 1x~ p01y(A) 51te and the 1xm ex0n5, a5 5h0wn 1n F19ure 5A. 7he5e p1a5m1d5, a10n9 w1th the w11d-type p1x p1a5m1d, were tran5fected 1nt0 J558L ce115 and 1x RNA expre5510n wa5 a55ayed 60th 6y 51 ana1y515 and 6y tran5cr1pt10n rate mea5urement. A5 5een 6ef0re, 1x~ RNA wa5 the pred0m1nant pr0duct 0f the p1x p1a5m1d. H0wever, a5 5e4uence wa5 de1eted fr0m the 1nterven1n9 re910n, there wa5 a chan9e 1n the rat10 1n fav0r 0f the 1xm RNA 5uch that the p1x-1000 p1a5m1d pr0- duced an e4ua1 am0unt 0f pxm and 1x~ RNA (F19. 58). Fur- therm0re, we n0te that the chan9e 1n 1xd1xm rat10 wa5 n0t at the expen5e 0f the 1x~ RNA; pr0duct10n 0f the 1x~ RNA rema1ned c0n5tant. Rather, a5 the 512e 0f the de1e- t10n 1ncrea5ed, there wa5 a para11e1 1ncrea5e 1n the 1xm RNA pr0duct10n re5u1t1n9 1n a r15e 1n the 1eve1 0f t0ta1 px RNA. A5 5h0wn 1n F19ure 6 and 5ummar12ed 1n 7a61e 2, th15 1ncrea5e 1n 1xm RNA pr0duct10n 1n the J558L ce115 wa5 acc0mpan1ed 6y an 1ncrea5ed fre4uency 0f tran- 5cr1pt10n 0f the 5e4uence5 1mmed1ate1y d0wn5tream 0f the 1x m p01y(A) 51te. 7he rate 0f tran5cr1pt10n 0f the

d0wn5tream 5e4uence5 1n the p1x p1a5m1d wa5 0n1y 10% that 0f the C~ ex0n 5e4uence5. H0wever, tran5cr1pt10n 0f the ~x m d0wn5tream 5e4uence 1ncrea5ed f1vef01d fr0m the p1x-1000 p1a5m1d. We thu5 c0nc1ude that the de1et10n 0f 1nterven1n9 5e4uence 6etween the 1x~ p01y(A) 51te and the 1xm ex0n5 a1ter5 the extent 0f tran5cr1pt10n 0f the 10cu5 1n J558L ce115 and a5 a re5u1t a110w5 eff1c1ent pr0duct10n 0f 1xm m R N A 1n the p1a5macyt0ma. 7h15 re5u1t mu5t a150 mean that pr0ce551n9 0f tran5cr1pt5 fr0m p1x t0 pr0- duce 1x5 RNA 15 n0t 100% eff1c1ent. 0therw15e, the 1n- crea5e 1n 1xm pr0duct10n w0u1d have re5u1ted 1n a de- crea5e 1n 1x5.

Ana1y515 0f 11~11a m RNA pr0duct10n 1n M12 ce115 d1rected 6y 1ntr0n-de1et10n mutant5 pr0v1de5 ev1dence f0r p01y(A) 51te c0mpet1t10n

F1na11y, we have a150 a55ayed f0r the pr0duct10n 0f 1x5 and 1xm RNA fr0m the var10u5 de1et10n mutant5 1n the M12 1ymph0ma ce11 11ne. 1n the5e ce115, there wa5 11tt1e 0r n0 term1nat10n 0f 1x tran5cr1pt10n pr10r t0 the 1xm p01y(A) 51te (F19.4). 7hu5, the 519na1 f0r term1nat10n that 15 rec09n12ed 1n J558L ce115 15 apparent1y n0t ut1112ed 1n M12 ce115, 1nd1cat1n9 that the de1et10n5 5h0u1d have n0 effect 0n tran5cr1pt10n 0f the 1xm p01y(A) 51te 5e4uence5 1n the M12 ce115. 7h15 wa5 1n fact true (data n0t 5h0wn). L1kew15e, we ant1c1pated that m R N A pr0duct10n 1n M12 ce115 w0u1d 6e unaffected 6y the de1et10n5. H0wever, 5uch wa5 n0t the ca5e a5 5h0wn 1n F19ure 7 and 5umma- r12ed 1n 7a61e 3.1n fact, there wa5 a dramat1c chan9e 1n the rat10 0f 1x5/1xm RNA a5 a funct10n 0f de1et10n 0f the 1ntr0n 5e4uence5. H0wever, the nature 0f the chan9e 1n rat10 wa5 4u1te d1fferent fr0m that 5een 1n the J558L ce115.1n the M12 ce115, the 1eve1 0f 1xm RNA 1ncrea5ed at

476 6 E N E 5 • D E V E L 0 P M E N 7




D e v e 1 0 p m e n t a 1 c0ntr01 0{ 1 9 M expre5510n

A

m

~.5PA

m m m ~:.~1

1,-,~--,~-~; 0 6

8 p# p/~. A 3 0 0 PF A 750 pp. A 1000

- + - + - + - +

..... ~ ~ , , ~ *m~

~ m - 6

~ - P y

~m PA

~12A

~,~-11 1 e Pv

F19ure 6. 7ran5cr1pt10n rate ana1y515 0f p~ 1n- tr0n de1et10n mutant5 1n J558L tran5fect10n5. (A) pp. 10cu5 and the der1vat10n 0f the M13 pr06e5 emp10yed f0r hy6r1d12at10n. (8) Pr06e5 a, 6, and e were the 5ame a5 th05e u5ed 1n F19. 4. 7he Py pr06e 15 a p01y0ma-5pec1f1c 5e4uence that 11e5 1mmed1ate1y d0wn5tream 0f the ~ 5e4uence 1n pr06e e. Mea5urement5 0f tran5cr1pt10n rate5 1n 1501ated nuc1e1 were a5 de5cr16ed 1n F19. 4 and Mater1a15 and meth0d5. Lane5 1a6e1ed + and - refer t0 hy6r1d12at10n t0 5en5e and ant15en5e M13 DNA c10ne5, re5pect1ve1y.

the expen5e 0f the p.~ RNA; the t0ta1 p. RNA rema1ned c0n5tant. 7h15 w0u1d 6e expected 1f there wa5 n0 chan9e, a5 a funct10n 0f the de1et10n5, 1n the 0vera11 rate 0r extent 0f tran5cr1pt10n, wh1ch 1ndeed wa5 the ca5e. 7he 1ncrea5ed ~m RNA pr0duct10n c0up1ed w1th a de- crea5ed pr0duct10n 0f ~5 RNA 5u99e5t5 a c0mpet1t10n f0r a c0mm0n 11m1t1n9 fact0r re5p0n5161e f0r the pr0duct10n 0f the tw0 RNA5 that 15 affected 6y the am0unt 0f 1ntr0n 5e4uence 1n the tran5cr1pt10n un1t. Furtherm0re, the c0mpet1t10n 15 ce11 5pec1f1c 51nce 1t 15 0n1y ev1dent 1n the M12 ce11 w1th n0 ev1dence f0r 5uch 1n the J558L ce11, 1nd1cat1n9 that the c0mm0n fact0r 15 n0 10n9er 11m1t1n9 1n the5e ce115.

D15cu5510n

0 u r data 5u99e5t that, t09ether w1th tran5cr1pt10n term1- nat10n, re1at1ve p01y(A) 51te 5tren9th c0up1ed w1th p051- t10n 0f the p01y(A) 51te w1th1n the tran5cr1pt10n un1t are cr1t1ca1 fact0r5 1n determ1n1n9 the expre5510n fr0m the 10cu5. Fr0m the ana1y515 0f ~ p1a5m1d5 c0nta1n1n9 51n91e p01y(A) 51te5 1t appear5 un11ke1y that there are ce11-5pe- c1f1c fact0r5 f0r each p01y(A) 51te. 7hat 15, we f0und n0 ev1dence f0r ce11-5pec1f1c re9u1at10n when the p01y(A) 51te5 were n0t t09ether 1n the 5ame tran5cr1pt10n un1t. 7he m05t 5tra19htf0rward 1nterpretat10n 0f the data 15 that there 15 a 51n91e fact0r that 15 ut1112ed 6y 60th p01y(A) 51te5, and the 1neff1c1ency 0f the P-5 51te en5ure5 the ava11a6111ty 0f tran5cr1pt5 t0 6e pr0ce55ed at the P-m 51te. Prev10u5 exper1ment5 ut11121n9 the 1ate aden0v1ra1 tran5cr1pt10n un1t had 1nd1cated that p01y(A] 51te f0rma- t10n 0ccurred dur1n9 tran5cr1pt10n; that 15, the na5cent RNA cha1n 15 the 5u65trate f0r c1eava9e (Nev1n5 and Darne11 1978). 7he 0rder 0f the p01y(A) 51te5 1n a tran- 5cr1pt10n un1t thu5 6ec0me5 a cr1t1ca1 fact0r 51nce there 15 p01ar1ty t0 the pr0ce55 [the pr0x1ma1 p01y(A) 51te 15 5een 6ef0re the d15ta1 51te]. Fr0m the data pre5ented here, we w0u1d c0nc1ude that the ~5 p01y(A) 51te 15 rather weak and theref0re a110w5 a num6er 0f tran5cr1pt5 t0 90

unc1eaved at th15 51te. 1n c0ntra5t, the ~m p01y(A) 51te appear5 t0 6e 4u1te eff1c1ent 5uch that tran5cr1pt5 that 90 unpr0ce55ed at the 1x5 51te m19ht we11 6e pr0ce55ed at the ~-m 51te. 1n the mature 8 ce11 th15 re5u1t5 1n near-e4ua1 u5e 0f the tw0 p01y(A) 51te5.1n the p1a5ma ce11, the u5e 0f the d0m1nant 1xm 51te 15 reduced 6y tran5cr1pt10n term1- nat10n, thu5 y1e1d1n9 pred0m1nant1y P~5 RNA.

7he ana1y515 0f 1x RNA expre5510n fr0m the 1ntr0n de- 1et10n5 pr0v1de5 further 1n519ht 1nt0 th15 pr0ce55.8y re- m0v1n9 5e4uence5 1n the 1ar9e 1ntr0n 6etween the tw0 p01y(A) 51te5, there wa5 an 1ncrea5ed pr0duct10n 0f fxm RNA and a 51mu1tane0u5 decrea5e 1n 1x5 RNA 1n the M 12 ce11 11ne. 7h15 0ccurred 1n the a65ence 0f any chan9e 1n tran5cr1pt10n 0f the 10cu5. We w0u1d 0ffer the f0110w1n9 a5 an exp1anat10n f0r th15 065ervat10n. A1th0u9h p01y(A) 51te 5e1ect10n 11ke1y take5 p1ace 0n the na5cent tran5cr1pt (Nev1n5 and Darne11 1978), 1t may n0t 0ccur 1n5tant1y. 7hu5, the re1at1ve ••nearne55•• 0f tw0 p01y(A) 51te5 and the1r re1at1ve ••5tren9th•• may d1ctate the1r fre4uency 0f u5e. 1n th15 1n5tance, the 5tr0n9er ~m p01y(A) 51te 15 6r0u9ht c105er t0 the weak ~t, 51te 6y v1rtue 0f the de1e- t10n5. We 5u99e5t that th15 chan9e 1n 5pac1n9 15 5uff1- c1ent t0 a110w the ~m p01y(A) 51te t0 6e rec09n12ed 6ef0re the P-5 51te 15 pr0ce55ed. Why, then, d0e5 th15 n0t 0ccur 1n the J558L ce115• C1ear1y, c0mpet1t10n can 0n1y take p1ace 1f a c0mm0n fact0r 15 11m1t1n9. A1th0u9h we f0und n0 ev1dence f0r a r15e 1n a fact0r 1n the exper1ment5 em- p10y1n9 51n91e p01y(A) 51te5, ana1y515 0f the 1ntr0n de1e- t10n5 1n the M12 ce11 11ne 1ndeed 5u99e5t that c0mpet1- t10n 0ccur5 when tw0 p01y(A) 51te5 are 11nked 1n the 5ame tran5cr1pt10n un1t. Furtherm0re, the data 5u99e5t that the fact0r 15 n0 10n9er 11m1t1n9 1n the J558L ce115.

7w0 0ther rep0rt5 have recent1y de5cr16ed chan9e5 1n ~ / ~ rat105 a5 a funct10n 0f 1ntr0n de1et10n5. 1n 0ne 5tudy, the maj0r1ty 0f de1et10n5 had n0 effect 0n ~ pr0- duct10n 1n p1a5macyt0ma5 (Danner and Leder 1985). 0n1y when the p.~ p01y(A) 51te wa5 1mpa1red wa5 there an 1ncrea5e 1n ~ RNA. H0wever, the 1ar9e5t de1et10n wa5 n0 m0re than 350 nuc1e0t1de5, a 512e that pr0duced 11tt1e





6a111 et a1.

7a61e 2. Re1at1ve tran5cr1pt10n rate5 acr055 the ~t f0cu5 1n J558L tran5fect10n 0f 1ntr0n de1et10n5

Pr06e

P1a5m1d a 6 e Py

P1* 1.0 0.7 0.10 0.06 pp.A300 1.0 0.6 0.15 0.11 p~A750 1.0 0.6 0.40 0.34 p~A1000 1.0 0.8 0.53 0.45

Hy6r1d12at10n 0f 1a6e1ed nuc1ear RNA t0 510t 610t5 0f 5en5e- 5trand M13 DNA, a5 dep1cted 1n F19. 6, were 5canned w1th a den51t0meter. 7he va1ue5 were c0rrected f0r the 512e 0f the hy- 6r1d121n9 5e4uence and n0rma112ed t0 the va1ue f0r the ••a•• pr06e. 7he data pre5ented are the avera9e 0f three 1ndependent exper1ment5.

7a61e 3. p~ RNA pr0duct10n fr0m 1ntr0n de1et10n5 1n M12 and J558L

M12 J558L

P1a5m1d ~5 1•m ~m1[~5 ~5 ~m P-m/6t5

p~t 1.0 0.6 0.6 1.0 0.10 0.1 p~A300 0.92 0.8 0.9 1.0 0.22 0.2 p1*A750 0.37 1.3 3.5 0.8 0.64 0.8 p~tA1000 0.25 1.4 5.6 0.9 0.98 1.1

Pre5ented are re1at1ve 1eve15 0f ~t 5 and ~t m RNA5 determ1ned 6y den51t0metr1c 5cann1n9 0f aut0rad109ram5 0f 51 ana1y5e5 a5 de- p1cted 1n F195.5 and 7.7he data are n0rma112ed t0 the va1ue 0f the 1*5 RNA expre55ed fr0m the p~ p1a5m1d 1n each tran5fect10n.

effect 1n 0ur exper1ment5. M0re recent1y, Peter50n and Perry (1986) have rep0rted exper1ment5 re9ard1n9 the r01e 0f 1ntr0n 5e4uence5 1n the re9u1at10n 0f the ~t 9ene. 5pec1f1ca11y, 1ar9er de1et10n5 0f 1ntr0n 5e4uence5 a1tered the 1*5/1*m rat10 1n fav0r 0f J*m, thu5 c0n515tent w1th the re5u1t5 we have de5cr16ed here. H0wever, the5e auth0r5 d1d n0t mea5ure tran5cr1pt10n rate5 n0r the a6501ute 1eve15 0f the tw0 RNA5, rather ju5t rat105.8a5ed 0n the1r re5u1t5, Peter50n and Perry (1986) 5u99e5ted that there wa5 a c0mpet1t10n 6etween the f0rmat10n 0f the C~t4 t0 M1 5p11ce and the rec09n1t10n 0f the 1*5 p01y(A) c1eava9e 51te. 7hey ar9ued that de1et10n 0f 1ntr0n 5e4uence, 6y reduc1n9 the d15tance 6etween the 5p11ce d0n0r and ac- cept0r, m19ht 1mpr0ve the eff1c1ency 0f 5p11c1n9. 7h15 c0nc1u510n 15 1n c0ntra5t t0 0ur 5u99e5t10n that the c0n- tr01 may 1nv01ve p01y(A) 51te c0mpet1t10n. At the pre5ent t1me there 15 n0 c1ear ev1dence t0 a110w a d15- cr1m1nat10n 6etween the5e tw0 p055161e mechan15m5, a1th0u9h we w0u1d p01nt 0ut that there 15 n0 ev1dence f0r an effect 0f 1ntr0n 512e 0n 5p11c1n9 eff1c1ency. A150, we d0 f1nd that when the tw0 p01y(A) 51te5 are 5eparate, and thu5 ~m 5p11c1n9 cann0t c0mpete a9a1n5t ~5 p01y(A) 51te f0rmat10n, the pr0duct10n 0f 1*m RNA 9reat1y ex- ceed5 that 0f 1*~ (F19. 2). 7he 51mp1e5t 1nterpretat10n 15 that the ~m p01y(A) 51te 15 5tr0n9er than the f,~ and th15, c0m61ned w1th the1r re1at1ve p051t10n w1th1n the tran- 5cr1pt10n un1t, 15 re5p0n5161e f0r the near-e4ua1 expre5- 510n 1n a mature 8 ce11.

7he mechan15m f0r tran5cr1pt10n term1nat10n 1n th15

10cu5, and 1n part1cu1ar the re9u1ated term1nat10n, re- ma1n5 065cure a5 1t d0e5 f0r the 0ther examp1e5 0f p01y- mera5e 11 term1nat10n. H0wever, an ana1y515 0f the effect 0f the 1ntr0n de1et10n5 0n term1nat10n d0e5 pr0v1de 50me 1n519ht 1nt0 the pr0ce55. F1r5t, the de9ree 0f re11ef 0f term1nat10n wa5 9radua1 and wa5 a funct10n 0f the 512e 0f the de1et10n. 7h15 065ervat10n c0u1d 6e rec0n- c11ed 1n at 1ea5t tw0 w a y 5 . 7 h e r e c0u1d 6e mu1t1p1e ter- m1nat10n 519na15, the rem0va1 0f a 51n91e 0ne 0f wh1ch d0e5 n0t e11m1nate term1nat10n. H0wever, the1r effect mu5t 6e add1t1ve 51nce term1nat10n wa5 9radua11y reduced a5 the de1et10n5 1ncrea5ed. We fav0r an a1ternat1ve exp1anat10n. 7he de1et10n5 may n0t have rem0ved a ter- m1nat10n 519na1 6ut rather ju5t a 5pacer 5e4uence 6e- tween the 519na1 and the 51te 0f term1nat10n. F0r 1n- 5tance, 1f a 5e4uence e1ement d1rected term1nat10n at a d15tance r0u9h1y 1 k6 d0wn5tream, 1rre5pect1ve 0f the 5e4uence at that p051t10n, then de1et10n 0f 500 nuc1e0- t1de5 6etween the 519na1 and the term1nat10n 51te w0u1d 5t111 re5u1t 1n term1nat10n 1 k6 d0wn5tream 0f the 519na1, 6ut n0w at a 5e4uence 500 nuc1e0t1de5 further d0wn- 5tream than the 0r191na1 51te. 7hu5, 5e4uence5 n0t n0r- ma11y tran5cr16ed w0u1d n0w 6e tran5cr16ed a5 a re5u1t 0f the de1et10n; 1n th15 ca5e, the ~1, m p01y(A) 51te. What m19ht th15 519na1 6e• A 11ke1y cand1date 15 the p01y(A) 51te, 1n th15 examp1e the ~t5 p01y(A) 51te. Ev1dence 5up-

M12

.4-11U1J0

F19ure 7. Expre5510n 0f the p1x 1ntr0n de1et10n mutant5 1n M12 ce11 tran5fect10n5.51 mapp1n9 0f RNA5 pr0duced 1n M12 ce115 tran5fected w1th the 1ntr0n de1et10n5. RNA5 were a55ayed a5 de5cr16ed 1n F19. 4.





p0rt1n9 th15 c0me5 fr0m ana1y5e5 0f 60th the ~3-91061n tran5cr1pt10n un1t (Fa1ck-Peder50n et a1. 1985) and the a-91061n tran5cr1pt10n un1t (Wh1te1aw and Pr0udf00t 1986) where 1t wa5 c1ear that term1nat10n depended 0n a funct10na1 p01y(A) 51te. 7h15 1nterpretat10n 15 a150 c0n- 515tent w1th the expre5510n 0f p1a5m1d5 1n wh1ch the 1x5 p01y(A) 51te ha5 6een de1eted. 80th 0ur exper1ment w1th the P1xm p1a5m1d (F19. 2) and the 51m11ar exper1ment 0f Danner and Leder (1985) 1nd1cated that there 15 eff1c1ent pr0duct10n 0f 1xm RNA 1n p1a5macyt0ma5 when the 1x~ p01y(A) 51te 15 de1eted.

A1th0u9h there 15 ev1dence f0r tran5cr1pt10n term1na- t10n fr0m mea5urement5 0f the 1x 10cu5 1n p1a5macy- t0ma5 and hy6r1d0ma5 (Ke11ey and Perry 1986), a 51m11ar 5tudy 0f the 8 10cu5 1nd1cated that term1nat10n d1d n0t p1ay a r01e 1n the 5w1tch fr0m mem6rane t0 5ecreted RNA expre5510n (M11carek and Ha11 1985). F1rm c0nc1u- 510n5 c0ncern1n9 the ~ 10cu5 may 6e premature 51nce 0n1y 0ne p1a5macyt0ma wa5 5c0red and the a55ay5 have n0t 6een d0ne 1n the a65ence 0f any p0tent1a1 6ack- 9r0und tran5cr1pt10n. H0wever, 1t 15 a150 p055161e that the c0ntr01 0f ~m expre5510n 6y tran5cr1pt10n term1na- t10n 15 due t0 the un14ue arran9ement 0f the 1x-~ 10cu5. 7 h e p. and 8 10c1 are tran5cr1pt10na11y 11nked and the ce5- 5at10n 0f 8 RNA pr0duct10n dur1n9 8-ce11 maturat10n 15 the re5u1t 0f tran5cr1pt10n term1nat10n pr10r t0 the ex0n5 (Yuan and 7ucker 1984; Mather et a1. 1984). P05- 5161y th15 un14ue tran5cr1pt10n un1t c0mp1ex1ty d1ctate5 a c0ntr01 0f term1nat10n n0t f0und f0r the 0ther heavy- cha1n 10c1.

C0ntr01 0f the 11 tran5cr1pt10n un1t dur1n9 8-ce11 de ve10pm en t

Fr0m a11 0f the data pre5ented here, we pr0p05e the f01- 10w1n9 a5 a 11ke1y 5cenar10 f0r the event5 that d1ctate expre5510n 0f the 1x tran5cr1pt10n un1t. 1n the 8 ce11 that 15 expre551n9 r0u9h1y e4ua1 am0unt5 0f 5ecreted and mem6rane-60und 1x cha1n5, tran5cr1pt10n pr0ceed5 un- a6ated thr0u9h 1xm, 9enera11y term1nat1n9 ju5t up5tream 0f the 8 ex0n5, a1th0u9h 50me tran5cr1pt5 c1ear1y pr0ceed thr0u9h the 8 ex0n5 (Mather et a1. 1984; Yuan and 7ucker 1984). 7he rat10 0f 1x5 t0 1xm RNA pr0duct10n 15 d1ctated 6y the re1at1ve 5tren9th5 0f the p01y(A) 51te5 and the fact that the 1xm 15 51tuated c0n51dera61y d0wn5tream 0f the 1x~ 51te. 1n a pre-8 ce11 where the 1eve1 0f 1xm 15 c0n51dera61y 9reater than 1x~ we w0u1d pred1ct that there 15 a very 10w am0unt 0f a p01y(A1 51te fact0r 5uch that the 1neff1c1ent 1x5 p01y(A) 51te 15 rare1y u5ed. When the mature 8 ce11 enc0unter5 ant19en and d1fferent1ate5 1nt0 a p1a5ma ce11, we 5u99e5t that tw0 event5 take p1ace. F1r5t, there 15 a further 1ncrea5e 1n a p01y(A) 51te fact0r (6ey0nd that wh1ch ha5 0ccurred 1n the chan9e fr0m a pre-8 ce11 t0 mature 8 ce11) t0 en5ure even 9reater u5e 0f the 1x~ p01y(A) 51te. And, t0 e11m1nate any c0mpet1t10n 6y the 1xm 51te and thu5 further 1ncrea5e the u5e 0f the 1x5 51te, tran5cr1pt10n term1nate5 pr10r t0 the 1xm p01y(A) 51te, thu5 e11m1nat1n9 1t fr0m the pr1mary tran5cr1pt. 1n- tere5t1n91y, 1t 15 p055161e t0 rec0nc11e the5e tw0 phe- n0mena a5 a 51n91e event 1f 1ndeed term1nat10n 15 tr19- 9ered 6y the u5e 0f the p01y(A) 51te a5 5u99e5ted a60ve.


7ha t 15, a5 the 1x5 p01y(A) 51te 15 u5ed w1th 9reater fre- 4uency due t0 an 1ncrea5ed 1eve1 0f fact0r, term1nat10n en5ue5. A5 term1nat10n decrea5e5 the 5ynthe515 0f the 1xm p01y(A) 51te, there 15 1e55 c0mpet1t10n w1th the 1x5 and thu5 9reater u5e 0f the 1x5 p01y(A) 51te and theref0re m0re term1nat10n. 5uch a mechan15m c0u1d c1ear1y dr1ve the expre5510n t0 exc1u51ve pr0duct10n 0f 1x5.

F1na11y, 1t 15 a150 kn0wn that 1n add1t10n t0 the 5e1ec- t1ve u5e 0f the 1x5 p01y(A) 51te 1n p1a5ma ce115, there 15 a 1ar9e 1ncrea5e 1n 1x m R N A when 8 ce115 mature t0 p1a5ma ce115 w1th0ut a c0mmen5urate 1ncrea5e 1n tran- 5cr1pt10n (Yuan and 7ucker 1984; Ke11ey and Perry 1986; 6er5ter et a1. 1986). We have n0t 065erved 1ar9e 1n- crea5e5 1n t0ta1 1x m R N A 1n the tran5fected M12 and J558L ce115, a re5u1t c0n515tent w1th prev10u5 065erva- t10n5 (6r055ched1 and 8a1t1m0re 1985). A5 5u99e5ted 6y the5e auth0r5, the m05t 11ke1y exp1anat10n 15 the nature 0f the a55ay, a tran51ent tran5fect10n w1th a rep11cat1n9 p1a5m1d. 1f the 1ar9e 1ncrea5e 1n p. RNA n0rma11y 5een when 1ymph0cyte5 d1fferent1ate 15 due t0 1ncrea5ed RNA 5ta6111ty, then a 5h0rt-term a55ay m19ht n0t a110w 5uff1- c1ent t1me f0r the effect t0 6e e5ta6115hed. 7h15 1nterpre- tat10n 15 further 5upp0rted 6y 0ur 065ervat10n that 5ta61y tran5fected J558L ce115 pr0duce at 1ea5t 10-f01d m0re 1x RNA than 5ta61e M12 tran5fectant5 (J. 6u15e et a1., 1n prep.). 7hu5, 1t 15 4u1te p055161e that three 5eparate a5pect5 0f m R N A 6109ene515 c0ntr16ute t0 the f1na1 1x5 RNA-d0m1nant phen0type 0f the p1a5ma ce11:chan91n9 c0ncentrat10n 0f a p01y(A) 51te fact0r, tran5cr1pt10n ter- m1nat10n t0 e11m1nate u5e 0f the 1xm p01y(A) 51te, and 1ncrea5ed 5ta6111ty 0f the 1x RNA.

Mater1a15 and m e t h 0 d 5

Ce11 cu1ture and tran5fect10n5

7he J558L ce11 11ne wa5 9r0wn 1n DME 5upp1emented w1th 10% feta1 ca1f 5erum. 7he M12 ce11 11ne wa5 9r0wn 1n RPM1 c0n- ta1n1n9 10% feta1 ca1f 5erum. 7ran5fect10n5 were perf0rmed a5 de5cr16ed 6y 6r055ched1 and 8a1t1m0re (1985). 7he ce115 were harve5ted after 40 hr and t0ta1 RNA wa5 1501ated and pur1f1ed a5 de5cr16ed 6y Ch1r9w1n et a1. (1979).

C0n5truct10n 0f p1a5m1d5

7he DNA c0n5truct referred t0 1n th15 paper a5 p~ 15 the p~A3 p1a5m1d de5cr16ed 6y 6r055ched1 and 8a1t1m0re (1985). 70 c0n- 5truct the p~x 5 p1a5m1d, the 1.8-k6 Ec0RV-Xh01 fra9ment wa5 de1eted. 70 c0n5truct the p~tm p1a5m1d, the 1.2-k6 8c11-Ec0RV fra9ment wa5 de1eted. 70 c0n5truct the P~w p1a5m1d, the 1.4- k6 8c11 fra9ment c0nta1n1n9 the ~x~ p01y{A) 51te wa5 1n5erted 1nt0 the Xh01 51te 0f the PP-m p1a5m1d. 7he 1ntr0n de1et10n5 were created 6y 8a131 d19e5t10n 5tart1n9 at the Ec0RV 51te. 7he extent 0f the de1et10n5 wa5 determ1ned 6y re5tr1ct10n en2yme mapp1n9.

51 nuc1ea5e a55ay5

Appr0x1mate1y 10-30 ~9 0f t0ta1 RNA wa5 hy6r1d12ed w1th a2p-end-1a6e1ed pr06e5. 7he hy6r1d12at10n temperature wa5 46°C f0r the W-5pec1f1c pr06e5 and 50°C f0r the H4-5pec1f1c pr06e. 7he hy6r1d5 were d19e5ted w1th 50 un1t5 0f 51 nuc1ea5e (PL 810chem1ca15) at 37°C f0r 1 hr. 7he pr0tected DNA fra9- ment5 were ana1y2ed 6y e1ectr0ph0re515 1n 8% acry1am1de-urea 9e15.





6a111 et a1.

7ran5cr1pt10n ana1y515 1n 1501ated nuc1e1

F0r each tran5cr1pt10n rate mea5urement, 2 -4 x 105 ce115 were tran5fected and harve5ted 40 hr 1ater. 7he pr0cedure5 f0r 1501a- t10n 0f nuc1e1, 1n v1tr0 1ncu6at10n, and 1501at10n and hy6r1d12a- t10n 0f 1a6e1ed nuc1ear RNA have 6een de5cr16ed prev10u51y (We6er et a1. 1977; H0fer and Dame11 1981). F0r each a55ay, appr0x1mate1y 2 x 108 nuc1e1 were 1ncu6ated w1th 500 ~C1 0f [a2P1U7P f0r 15 m1n at 30°C. Nuc1ear RNA wa5 1501ated 6y h0t phen01 extract10n and 6ef0re hy6r1d12at10n wa5 part1a11y 6r0ken 6y 1ncu6at10n 1n 0.2 N N a 0 H f0r 15 m1n 0n 1ce. N1tr0ce11u105e f11ter5 6ear1n9 the var10u5 M13 51n91e-5tranded (55) DNA pr06e5 were prepared a5 de5cr16ed 6y Kafat05 et a1. (1979). 7he M13 pr06e5 u5ed were: pr06e a der1ve5 fr0m a cDNA 5pann1n9 part 0f the C~2 ex0n {fr0m a Pvu11 51te), the C~u3 ex0n, and part 0f the C~4 ex0n (t0 the P5t1 51te) and ha5 a t0ta1 1en9th 0f 761 nuc1e0t1de5; pr06e 6 15 a 610-nuc1e0t1de P5t1-H1nd111 fra9ment c0nta1n1n9 the ~ p01y{A) 51te; pr06e c 15 a 916-nuc1e0t1de Kpn1-H1nc11 fra9ment; pr06e d 15 a 200-nuc1e0t1de R5a1 fra9- ment; pr06e e 15 a 400-nuc1e0t1de 5ph1-Xh01 fra9ment; pr06e Py 15 a 1400-nuc1e0t1de Xh01-Ec0R1 fra9ment 0f p01y0mav1ru5 DNA. A11 pr06e5 were c10ned 1nt0 the p01y11nker 0f M13 mp18 and M13 mp19.

A c k n 0 w 1 e d 9 m e n t 5

We thank Dr. Rud01ph 6r055ched1 f0r the 91ft 0f the pw p1a5m1d a5 we11 a5 f0r vah1a61e adv1ce. We a150 thank Car011ne 610ver and Chhaya Da5 f0r techn1ca1 a5515tance. 6 .6 . wa5 5upp0rted 6y fe110w5h1p5 fr0m EM80 and the 5w155 Nat10na1 5c1ence F0undat10n. M.M. 15 an M57P fe110w. 7he w0rk wa5 5upp0rted 6y Nat10na11n5t1tute5 0f Hea1th 9rant5 A1 18016 and 6 M 31689 t0 P.7. and 6 M 35894 t0 J.N.

Re1erence5

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A1t, F.W., N. R05en6er9, V. Enea, E. 51den, and D. 8a1t1m0re. 1982. Mu1t1p1e 1mmun09106u11n heavy cha1n 9ene tran- 5cr1pt5 1n A6e150n mur1ne 1eukem1a v1ru5 tran5f0rmed 1ym- ph01d ce11 11ne5. M01. Ce11. 8101. 2: 386-400.

81rn5t1e1, M.L., M. 8u5511n9er, and K. 5tru6. 1985.7ran5cr1pt10n term1nat10n and 3•-pr0ce551n9: 7he end 15 1n 51te. Ce11 41: 349-359.

81attner, F.R. and P.W. 7ucker. 1984. 7he m01ecu1ar 610109Y 0f 1mmun09106u11n D. Nature 307: 417-422.

Ch1r9w1n, 7.M., A.E. Pr2y6y1a, R.J. MacD0na1d, and W.J. Rutter. 1979. 1501at10n 0f 6101091ca11y act1ve r160nuc1e1c ac1d fr0m 50urce5 enr1ched 1n r160nuc1ea5e. 810chem15try 18: 5294-5299.

Danner, D. and P. Leder. 1985. R01e 0f an RNA c1eava9e/p01y(A) add1t10n 51te 1n the pr0duct10n 0f mem6rane-60und and 5e- creted 19M mRNA5. Pr0c. Nat1. Acad. 5c1. 82: 8658-8662.

Ear1y, P., J. R09er5, M. Dav15, K. Ca1ame, M. 80nd, R. Wa11, and L. H00d. 1979. 7w0 mRNA5 can 6e pr0duced fr0m a 51n91e 1mmun09106u11n ~ 9ene 6y a1ternat1ve RNA pr0ce551n9 pathway5. Ce11 20: 313-319.

Fa1ck-Peder5en, E., J. L09an, 7. 5henk, and J.E. Dame11, Jr. 1985. 7ran5cr1pt10n term1nat10n w1th1n the E1A 9ene 0f aden0- v1ru5 1nduced 6y 1n5ert10n 0f the m0u5e 8-maj0r 91061n ter- m1nat0r e1ement. Ce11 40: 897-905.

6er5ter, 7., D. P1card, and W. 5chaffner. 1986. Dur1n9 8-ce11

d1fferent1at10n enhancer act1v1ty and tran5cr1pt10n rate 0f 1mmun09106u11n heavy cha1n 9ene5 are h19h 6ef0re mRNA accumu1at10n. Ce11 45: 45-52.

6r055ched1, R. and D. 8a1t1m0re. 1985. Ce11-type 5pec1f1c1ty 0f 1mmun09106u11n 9ene expre5510n 15 re9u1ated 6y at 1ea5t three DNA 5e4uence e1ement5. Ce11 41: 885-897.

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Kafat05, F.C.C., W. J0ne5, and A. Ef5trad1ad15. 1979. Determ1na- t10n 0f nuc1e1c ac1d 5e4uence h0m01091e5 and re1at1ve c0n- centrat10n5 6y d0t hy6r1d12at10n pr0cedure. Nuc1e1c Ac1d5 Re5. 7: 1541-1551.

Ke11ey, D.E. and R.P. Perry. 1986. 7ran5cr1pt10na1 and p05t- tran5cr1pt10na1 c0ntr01 0f 1mmun09106u11n mRNA pr0duc- t10n dur1n9 8 1ymph0cyte deve10pment. Nuc1e1c Ac1d5 Re5. 14: 5431-5447.

La5k0v, R., J.K. K1m, V.L. W00d5, P.E. McKeever, and R. A50f5ky. 1981. Mem6rane 1mmun09106u11n5 0f 5p0ntane0u5 8-1ymph0ma5 0f a9ed 8AL8/c m1ce. Eur. J. 1mmun01. 11: 462-468.

Lenn0n, 6 .6 . and R.P. Perry. 1985. C~-c0nta1n1n9 tran5cr1pt5 1n1t1ate heter09ene0u51y w1th1n the 19H enhancer re910n and c0nta1n a n0ve1 5•-n0ntran51ata61e ex0n. Nature 318: 475- 478.

Mather, E.L., K.J. Ne150n, J. Ha1m0v1ch, and R.P. Perry. 1984. M0de 0f re9u1at10n 0f 1mmun09106u11n mu and de1ta cha1n expre5510n var1e5 dur1n9 8 1ymph0cyte maturat10n. Ce11 36: 329-338.

M11carek, C. and 8. Ha11. 1985. CeU-5pec1f1c expre5510n 0f 5e- creted ver5u5 mem6rane f0rm5 0f 1mmun09106u11n 9amma 26 mRNA 1nv01ve5 5e1ect1ve u5e 0f a1temate p01yadeny1a- t10n 51te5. M01. Ce11. 8101. 5: 2514-2520.

Ne150n, K.J., J. Ha1m0v1ch, and R.P. Perry. 1983. Character12a- t10n 0f pr0duct1ve and 5ter11e tran5cr1pt5 fr0m the 1mmun0- 9106u11n heavy-cha1n 10cu5:Pr0ce551n9 0f ~m and ~5 mRNA. M01. Ce11. 8101. 3: 1317-1332.

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G Galli, J W Guise, M A McDevitt, et al. secreted mu-chain expression during B-cell development.transcription termination are critical to membrane versus Relative position and strengths of poly(A) sites as well as

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