Lengths of transcriptional units coding for the heat shock proteins in Drosophila melanogaster

J. ,VoZ. Biol.

(1979) 132, 141-161

Lengths of Transcriptional Units Coding for the Heat Shock Proteins in Drosophila melanogaster JONATHAN ~.CARLSONAND

DAVID E. PETTIJOHN

Department of Biophysics and Genetics University of Colorado Medical Center 4200 East Ninth Avenue B-121 Denver, Col. 80262, U.S.A.

The lengths of the transcription units coding for tllr Drosophila ,melanogaster lleat shock proteins were determined using tlrc ultraviolet promoter mapping techunit lengths of nique. The figures were in good agreement with the transcription three of the heat shock cytopla.smic RNA species as determined by the sanie technique. The rates of gene ina,ctivation were first-order with respect to ultraviolet irradiation dose, suggesting that there are single genes of a given species in each transcriptional unit. The length of the transcription urlit coding for the majol heat shock protein, the 70,000 M, protein. was found to be 6.8 x IO3 bases. The length of the transcription unit of the 2.6 x 103-base messenger RNA coding for tile 70,000 M, protein was fourld to be 5.5 x lo3 to 6.5 x lo3 bases by two independent methods and is in good agreement wit11 the determitlation for the prot,ein. In general, the transcription units contained 3.5 to 10 times as much DNA as is needed to code for the amino acid sequence of t,llo respective proteins. Tile cytoplasmic R,NA branscription units were 1.5 to 6.5 times the molecldar lengths of the IYspectivc: RNA species. The results suggest that tllcre is appreciable post-tratlscriptional cuttingoftlleprimary transcriptioti product oftl~sc~grncs. Tllr lengtlls of these transcription units are abollt, one-third to orlr-qrlarter the lttngtll of DNA in an avera,ge-sized chromomere.

1. Introduction In Drosophila ,melanogu.ster, as well as in ot’her eukaryotes, the size of the transcription unit’s t,hat code for messenger RNB ha,s been a, matter of intensive investigation for several years now. The polyt’ene chromosomes present in some tissues of Drosophila contain about 5000 to 6000 bands or chromomeres. and estimates of the number of genes by genetic techniques suggest tha,t there is about one gene per band (Judd & Young, 1973; Hochman, 1973; LeFevre, 1973,1974). This is consistent with the number of mRNA sequences detectable in Drosophila cells (Levy & McCarthy, 1975) and, therefore, it has often been suggested that each band represents a transcription unit. Each band contains, on the average, 20 to 25 kbi- of DNB per DN,4 copy (Laird, 1973): and hence a transcript’ of an entire band would be 20 to 25 kb long. Direct’ visualization of active non-ribosomal transcription units by electron microscopy has shown transcription units of a wide range of sizes w&h t,he larger ones easily containing

142

J. 0.

C!dRLSOS

.\;‘\‘I_, I).

Ii:. I’ETTl
enough DNA to span an average-sized band (i\;IcKnight, B Xller. 197~: IArti CY: Chooi, 1976). It is not, clear, however. which of these transcript’ion unit#s codt~ fol mRNA precursors, since 6n the average only about 200& of t’hr: mass and dO(!,, of tilt\ sequence diversity of the het,erogeneous nuclea,r RSA is convrrtt~d to mRNA (L~rlgyt~l & Penman. 1975; Levy et al., 1976). The average size of nuclear RNA isolated from /1roso@Zu is about8 4 to 6 kl). whicll is significantly smaller tha,n would be expected for a continuous transcript, of thta DNA in an average-sized chromomere. The average size of thcl messenger RNA from Drosophila cells is two- to t)hreefold smaller than t,he average nuclear RNA (Lcngyel & Penman, 1975; Lamb S: Laird, 1976). and would only requirr 5’);, t’o locks, of thr DNA in an average band to code for its sequence. It, is therefore not clear wht:thw mRNA precursors are transcribed frorn relatively large transcript~ional units and t,hcrr processed by post,-transcription nucleolytic cleavage to rnRSA siw. or \vhtthw thr, mRNA precursors are transcribed from small, mR&\‘A-sized t’ranscript,iorlal units. In order to determine t,he lengths of individual transcription unit,s. ux ha,vr: w~~tl the technique of nhraviolrt, promot,er mapping. 1:ltravioIet ra,diat)ion is know11 t#o introduce t,ranscription-terminating lesions ra,ndomIy in DKA (Sauerbier. 1975.1976). The probability that a given gene will be expressed after ultraviolet irradiatiou is determined by the ultraviolet dose and the distanw of t’hc gent& from t,hr prornotc1 (Brautigam & Sauerbier, 1974 ; Sauerhier. 1976). Thereftwe. by monitoring tht> decrease in synthesis of either t’he RNA or t,he protein product of the gene w&h the ultraviolet dose, one rnay estirnate the distance of t,he gene from it-s promoter. This method has been used successfully in studying tra,rlscril)t,ioll~Ll organization in bacteria (Hackett c1: Sauerhirr. 1974). I~acteri~~plingc (Brautigant & Saucrbier. 1973,1974; Hercules $ Sauerbier, 1973,1974: Pollock et ~1.. 197X). n~amn~aliat~ ~11s (Hackett & Sauerbier, 1975: Giorno & Bauerbicr. 1976,197s : (:oldherg ut (I,!.. 1977~ : Gilmore-Hebert et al.. 1978) and animal viruses (Ball $ Whit,e. 1976: Ahr&atn & Banerjee, 1976; Goldberg et al.. 1977b: Brrk & Sharp, 1977). Since the purpow ot this work is t)o det)ermine t’he length of the primary branscript ncocssary for the synthesis of a given protein, it is dwirable to rnonit,or the final prottain products. I~‘trr this t,o be feasible, t,he mRNA for the protein must not’ be made prior t,o or during tht: nltraviolet irradiation, since polysomcs containing the messagtb made bcfow riltraviolet irradiation would continue t,o make protjein af%cr irradiation and thfa li fctirnw for specific messages arc generally not known. Thus. it, is nrwsswry for the synthwis of the protein product,s to be easily inducible a,ftcr irradiatJion. larvae t,o high temperat8urw aad a variety of other t,twttExposure of Drosophila ments arc known to induce a discrete set of puffs on t,hc polytene chromosomes (Ritossa, 1962,1964; bshhurner, 1970: Lecnders & Berendes. 1972: van Breugal. 1966). The induction of t’hese puffs is accompanied by a redist,ribution of RNh ptAymerase molecules to these puffed regions (Jamrioh pf (16.. 1977) and by t~he appeararw~ of a new set, of RNA and protein molecules (TissiBres of al., 1974: Lewis of al.. 1975: Biessmann et al., 1978a.b). The new RNA species hybridize ir/, situ to the same Ix&~ that puff (Spradling et al.; 1975,1977: McKenzie rt ab.. 1975), and diwct the synthesis of heat shock proteins in iw vitro transIat,ion y&ems (McKenzie dz Mrselson. 1977: Moran et (11.. 1978: Mirault et al.. 1978). The induction of thr twq’ R?\‘A and pt’ot.clin species is not tissue-spccitic (TissiPrw et nl.. I974 : Botmcr B, Pardw. 1976) a.nd is also seen in Drosoph.~ib cell cult~urex (Sprxtlling et uZ.. 197.5 : McKrnzic it al.. 1976). Furtlwrmore, this induction is at the transcriptional IwI~I. sinw both t’Iit> nuclear ;uitl

TRANSCRIPTION

UNIT

LENGTHS

IN

DROSOPIEILd

14.3

cytoplasmic RNA from heat-shocked cells show similar in situ hybridization patterns, which are quite different from the normal pattern (Lengyel & Pardue, 1975). The inducibility of the heat shock proteins at the transcriptional level makes it’ possible to use the ultraviolet dose mapping technique to determine the length of the primary t’ranscripts used in their synthesis. In this paper we report the determination of t#he length of the primary transcripts for seven of the heat shock proteins.

2. Materials and Methods (a)

Cell culture

growth

conjditions

melanogaster cells used in these experiments were Schneider’s line 2 The Drosophila (Schneider, 1972), which were a gift from Alan Blumenthal. They were grown in plastic tissue culture flasks (Falcon) on Schneider’s medium (Pacific Biologicals) supplemented with 15% dialysed fetal calf serum (Gibco), which had been heated to 55 to 60°C for 40 min. Cultures were maintained between lo6 and 2 x lo7 cells/ml at 23 to 25“C and had a cell doubling time of about 40 h. (b)

Ultraviolet

irradiation

conditions

Log phase cells (5 x lo6 to 107/ml) were suspended in their growth medium with a rnbbel policeman and 1 to 2.5-ml samples of cell suspensiotl were irradiated at a dose of about 13 erg/mm2 per s for the appropriate time on a IO-cm diameter watch glass with a G.E. G,T,/l low pressure mercury lamp. The cells were stirred constantly during irradiation with a magnetic stirrer to assure uniform exposure to the ultraviolet light. After irradiation, the cells were kept in the dark until the end of the labeling period to prevent photoacti\-ated repair of ttlr lesions (Trosco 85 Wilder, 1973). (c) Heat

shock labeling

conditions

For experiments involving protein synthesis after heat sllock, cellular DNA was prelabeled for 15 to 20 II with 0.1 &i of [3H]thymidine/ml (50 Ci/mmol; Schwarz Mann) to provide an internal standard for the number of cellular equivalents of heat shock protein in the samples. The cells were irradiated as described above, collected by centrifugation and resuspended in 0.1 to 0.15 vol. Schneider’smediurnwithout methionine, arginine, tryptopIIan or serum. The cells were placed in a 37°C incubator for 40 to 60 min to allow decay of pre-existing polysomes and accumulation of new polysomes. After heat shock, [35S]methionine (100 to 500 &i/mmol; Amersham Searle) was added to a concentration of 100 to 300 pCi/ml and incubation was continued at 37°C for 1 to 2.5 11. Labeling was stopped bl the addition of 3 ml of ice-cold 0.075 M-NaCl, 0.024 M-EDTA. For experiments involving heat shock-induced RNA sythesis, cells were prelabeled witlr 0.05 to 0.1 &i of [14C]uridine/mI (50 mCi/mmol: Schwarz Mann) to label ribosomal RN4. which provided an internal standard for tile number of cellular equivalents of heat shock cells were placed at 37°C in a shaker bath and, after RNA in the samples. After irradiation, 5 tnin, [3H]uridine (20 to 40 Ci/mmol; Schwarz Mann) was added to a concentration of 50 &i/ml. Incubation was continued for 2 11 at 37°C and terminated by plunging the tube irrt,o ice. For determination of acid-precipitable counts, portions of the samples were pipetted int,o ice-cold 0.075 M-NaCl, 0.024 XI-EDTA. Cells were collected by centrifugation and resllsperrded in either 0.075 ax-NaCl, 0,024 >I-EDTA or 0.1 x-NaCl, 0.01 M-MgCl,, 0.03 M-Tris (pH 7.5) and lysed by the addition of either sodium dodecyl sulfate or NP40 (Shell); 50, tric~ttloroact~tic acid. 0.01 M-sodium pyrophosphate was added and, after 20 to 30 min on ice, tile precipitate was collect,ed on Whatman GFjC filters, washed with 5% trichloroacetic acid and etllanol a,nd counted in a toluenr based scintillation cocktail. (d) Preparation

of samples

for

gel electrophoresis

Protein samples were prepared for electrophoresis essentially by the method of Tiss&res et al. ( 1974). After labeling, the cells were collected by centrifugation at 5000 rovs/min for 5 Init). The pellet was resuspended in 4 to A drops of 0.075 At-NaCl, 0.024 M-EDT-~ and

144

.I-. 0.

G1RLSON

:\Nl>

I).

l!:. I’ETTIJOHN

2.5 ml of LO’& tricllloro;l,cf:tic: acid was atltletl. Aft+lr 20 Inill OII ic:cb. tllcs procipitat,t. \vas collected by cnrrt8rif~q@iori at, IO.000 rclvs/min for IO Inill. ‘1’111~ppollet, wa.s rrausperrded ill 2 ml of ice-cold absohltc! ~tlllarlol and, >lft,r>r IO to 20 Illitl 01) iccx. collected k)p centrifugatiot~ ‘I’l~c precipitate was resuspended in 2 ml of ice-cold chlor~ for 10 min at 10,000 revs/mill. form/methanol (1: 1, v/v) and, after 10 to 20 min on icp, was a,gain collected by centrifilgation for 10 min a,t, 10,000 revs/min. T11e prrcipitatc IVAS dried in a va~num desiccator an(t 100 ;, glvct‘rol. . 5o/0 p-lnr~r~!apt~octlr;tnc,l. dissolved in 50 ~1 of 27; sodilun dodecyl sulfate. 0.0625 u-Tris (pH 6.8), 0~001 ?;, brompllenol blurb. Total cyt,oplasmic RXA was prepared for electroplrorc:sis c>ssentially l)y tllc> rnet#llodx of Lengyel et al. (1975) and Spradling et al. ( 1977). After labeling the ~11s were collectrd 1,) centrifugat,ion for 5 Inin at 1500 revs/mirr and resuspended irl IO 1111elf ior-c-ok1 0.1 31-Nn(‘t. 0.03 mI-Tris.HCl (pH 8.3), 0.01 M-M&I, and collt~ctcti t)y ct,rtt,l,ifilglltic,rl again at> 1500 revs/minfor 5 min. The tolls were resuspended in 1 ml of 0. I M-Na(:l, 0.03 M-Tris. HCl (pH8.3). 0.01 M-M@,, 25 pg polyvinyl sulfate/ml, 10-:’ &I-spormioo. O.L.5 ) 9;) tliet~lylpyrocarborl~t~~,. The samples were transferred to a 1 ml Teflon glass llornogenixer, thr detergent NP40 \vas added to 0.5% a,nd cells were broken with 5 to 10 strokes of the pest,le. Nuclei were centrifuged out at 2500 revs/min for 5 min : tjhelr 0.12 ml of S?‘:) sodium dodecyl sulfat,e, 0.02 WI-EDTA was added to tllcx supernatant, and, aft,rr IO ruin, tIllis was cxtractad with phellol,! chloroform ( I : I. \-iv) containing I y/i, Isosmyl alcotrol. ‘I%(~ aqur’ous phase was rt-rxtractc~tl se\-era1 t,imes until there was little or no visible interfacial nlnterial. RNA IV-BYprc&pitatacl from the aqueolls phase by the addition of 2 to 3 volum~~s of absolute c+tlanol alid incubatic,,) at - 20°C o\-rrnight. The precipitate \vas collected by carnt,rifugation at 10,000 ra\-s/min for I II. The precipitate was dried in a, VA~CIIIIII~ drsiccator and dissolved in a solutioo corltuillillg (pH 8.3). (1.004 >z-EDTA and 0.1 u/o sodiutn do7 nr-urea,, 10% stlcrose, 0.09 M-Tris-hora,tt~ clecyl sulfate. (c) (Zel electrophoresis,

ccutoradioyraphy

and j~~~orogra&q

t 0 (‘1LL I’roteill samples \vcr(L a~~lyzcad 011polpac~I,ylatnidt ~qlsla.bs (0.8 t,r) 1 1tn11 ,* I3 cm or 0.8 to 1 mm Y 13 cln \i 25 cm) using the discontiImous sodium dodecyl sulfatc-corttaining buffer system described by Laemmli (I 970). The running gels were either 10% or 12.5’:;, acrylamide and the stacking gels were 50/;, acrylamide. Samples were heated for 2 to 3 min in a boiling waterbath before loading the gel. The gels were run at 12.5 mA until the tracking dye was about 5 mm from the bottom of the gel. After electrophoresis, the gels ~e;‘l’t> stained with Coomassie blue and dried onto filter paper. Radioactivel,y labeled proteins wcw detected by autoradiography on X-ray film. RNA samples were analyeed on polyacrylamide gel slabs of ttlca sarn~~ dimolsions as IISCYI for the protein gels. The gels and buffers were prepared as described by Spradling et a.1. (1977). Acrylamide concentrations were 3%. 4% and 2.5 to !io,i, gradients. Samples w(‘re heated to 60°C for 5 min before loading on the gel. Eloctrophoresis was performed at 100 \ for 10 to 20 h, depending OII the experiment. After electrophoresis, the gels mere processed for fluorography by the method of Homier & Laskey (1974) and fluorographed a,t - 70°C using Kodak X-Omat XR5 film pm-exposed to assure linear film response (Laskey & Mills, 1975). Autoradiographs and fluorographs were scanned with an Ortec densitometer. Relative areas under the peaks were determinod by tracing t,he peaks onto tracing paper, cutting the tracings out and weighing tllem. The peak areas xvcre Ilormalized to the number of [“H]DNA counts in the gel sample irl tllu cast of tl~c~ protnill gels, or thr arcs nrrdrr tl~tb ribosomal RNA peaks irl the case of the RNA prls, to allow caompe,risotl of tile amouut (of each species in different salnples. (f)

Growth

and isolatiovn

oj plasmid

l)AVA

Escheri&ia ooZi K12 strains HE%101 (ckDml03C) and HBlOI (rkDmlO3D) were a gift Moselson. 011. from David Hognrss. E. coli strain HRlOl (229.1) was a gift from Matthew tures were grown in D buffcxr (55 mM-potassiuni phosphate (pH 7.2), 7.5 msz-(NH),SO,. 0.4 mivr-MgSO,, 2 mM-sodium citrate) with 2 “/o (w/v) glucose, 0.5g0 (w/v) Difco Casamino acids and 10 pg thiamin . HC!l/ml. Chloramphenicol was added to 170 pg/ml at a cell density of about 6 x lO*/ml, and cells were harvested 16 to 20 h later. Cleared lysates were prepared by the method of Clewell & Helinski (1969). After extraction with phenol and precipitation

TRANSCRIPTION with

EDTA,

TJNIT

LENGTHS

TN DR080I’HILA

otharrol, the DNA was rrsl~spclldcd in 50 rnkl-Na(‘1, brought t.o 51.5’y0 (w/\-) (%(‘I atld 300 ILK t%tlidililn

115

IO rnnl-‘l’ris. HCl (pH 7.5), 1 rn%It)rolriitl6*/tnl. Hird cetlt,rifitgr~d at,

30,000 rPvs/niin ill at1 SWAO. I rotor for 60 t#o70 II. ‘I’ll<%to\\,(>r k)irlld co~lta,inirig tlio plnsmicl DNA was collected DNA was dialyzed EDTA. Work wittl

and ethidium was rrmovctl t,y sc:\,cral ext,ract,ions with rr-butanol. Tllrl against and stored in 50 mmNaC1, 10 mM-Tris.HCl (pH 7.5), I In>lrecombinant DNA plasmids was condnctrd under P2, EKl contn.i,unr~tlt

conditions. (g) A’ucleic

acid hybridization

Nucleic acid hybridization was performed essentially by tllc procedures of Vogelstein CY Gillespie (1977) or Casey & Davidson (1977). Plasmid DNA in 50 m&~-NaCl, 10 mM-Tris (pH 7.5), 1 mM-EDTA was made 10 rnx[ irk MgSO, and reacted with EcoRI (Boehringer Mannheim) for plasmid 229.1 or HirbdIII (Boehringcar Mannhcirn) for plasmids ckDml03( and ckDml03D. The DNA was extracted witti ptlcnol, precipitated wit11 ethanol and dissolved in either 80% (v/v) formamide, 0.4 nr-NaCl, 0.04 M-PIPES (pH 6.4), 0.001 M-EDTA or 70% (v/v) formamide, 0.36 x-potassium phosphate (pH 6.8). RNA labeled with C3H]uridine dissolved in the same solvent was mixed with the DNA and the samples were lleated iti a boiling waterbath for 5 min and immediately placed in a waterbath at the hybridization temperature (50 to 52°C). Hybridization reactions (20 to 50 ~1) ~verc done either in sealed glass capillaries or in 1.5 ml polypropylene Eppendorf centrifuge tubes. Hybridization was continued for 3 to 20 h with no difference in the level of llybridization. DNL4 was judged to be in excess, since halving or doubling the DN’A concentration made no difference irl the level of hybridization. The hybridization reaction \va,s terminated by the addition of 200 to 300 ~1 of cold 0.45 *I-NaCl, 0.045 M-sodium citrate altd RNase A (Worthingtou) to 250 pg/ml. The RNase reaction was done at 37% for 90 min. The entire reaction mixture was passed through an agarose A 15 m (Bio-Rad) column (1 cm x 30 cm) equilibrated with 0.15 &I-NaCl, 0.015 M-sodium citrate. Fractions (1 ml) were collected and counted with 3a70 complete counting cocktail (RPI). Radioactivity that passed through the column with the void volume was scored as hybridized RNA, while radioactivity included in the columrl was scored as unhybridized RNA. Hybridization background was determined by assaying identical mixtures minus DNA. This background was subtractrd from the measured radioactivity in the uncorrected hybrids.

3. Results (a) RNA

and protein

synthesis after heat shock

The time-course of RNA synthesis after heat shock in cultured Drosophila cells was oxamined by determining the amount of [3H luridine incorporated into acid-precipitable material at various times after hea.t shock. Tnitially, label is incorporat,ed rapidly but’ the rate levels off at lat,er times (Fig. 1(a)). This leveling off is due, at least, in part, to a rapid decrease in the rate of Rl!GA synthesis. As shown in the bar graph (Fig. l(a)), the amount of [3H]uridine incorporat’ed in Is-minute pulses administered during successive intervals after heat shock rapidly drops to lO”/b to 15% of that’ synthesized during the first 15 minutes. This is consistent wit,h the data presented 1)~ Spradling et al. (1977). The kinetics of incorporatjion of label into nuclear and cytoplasmic RNA is shown in Figure l(b). Nuclear RKA is initially labeled more rapidly and then the labeling begins to level off, while the cytoplasmic RKA steadily increases for a longer time. This is similar to the nuclear and cytoplasmic la,beling behavior in non-heat-shocked Drosophda cells (Lengyel 8: Prnman. 1977). All the experiments to be described here involving RNA labeling were performed during the first two hours after heat shock, when the amount of labeled RNA in the cytoplasm is still increasing. The kinetics of protein synthesis after heat shock were determined by following the amount of [35S]methionine incorporated into acid-prrcipitable material (Fig. l(c)).

J. 0.

CARLSON

AND

D. E.

Time hn)

Time (min)

(a)

(b)

PETTIJOHN

Time

(mln)

Cc)

FIG. 1. Kinetics of RN.4 and protein synthesis after heat shock. Cells were suspended in growth medium at 3 x 106 to 107/ml; 12.5 t,o 25 pCi of [3H]uritlinc/ml was added and cells were immediately placed in a 37,C wat,erbath. At the appropriate times. 1 ml samples were removed and trichloroacetic acid-precipitablc radioactivity was detwminccl as described in Materials and Blethods. The units are arbitrary u&s derived by normalizing t,hri number of acid-precipitable counts at each point. t,o the number of counts incorporated during the first 16 min of labeling. Each point is a mean of 2 t’o 3 determinations at t,hat t,ime. To determine the rate of synthesis at different times after heat shock, 1 ml samples of cells wow ln~lw labeled for 15 min with 12.5 &i of [3H]uridine at appropriate times after t,he temperature was raised to 37°C. The bar graph indicat’es the number of counts incorporated in that 15 min interval. t,o provide an int,ernal (b) Cells were pre-labeled for 16 h with 0.01 &i of [14C]thymidine/ml standard for the number of cells in the samples, heat shocked, and labeled at 37’C with [3H]ur.idine. At the appropriate times, samples were removed and labeling was terminated. The cells were lysed with 0.5% NP40 and nuclei and cytoplasm were separated as described in Materials and Methods. Nuclei and cytoplasm were preclpitat,ed separat,ely with trxiohloroacetic acid and countr~l on GFjC filters. The 3H radioactivity in each sample was normalizrtl to the IV! radioactivity at t,hat time point. (a) Nuclear RNA; (:;) cyt,oplasmic RNA. (c) Cells were suspended, collected by centrifugation and resuspandctl at a wmcrntratio~r of about 107/ml in Schneider’s medium lacking met,hionine, arginine, tryptophan and swum. Thta temperature was raised to 37°C and, after 40 min, 65 PCi of [35S]methionine/ml was added. ;\I approprmt,e times after the label was added, samples WOPCremoved and prepared for &ct rophoresis as described in Materials and Methods. A portion of each sample was counted in a Tritoll XlOO/toluene scintillation cocktail t,o obtain the above dat,a. (a)

In these experiments, label was added 40 to 60 minutes after raising the temperature to allow the decay of polysomes present before heat shock and a build-up of nen polysomes. The data therefore show the time-course of protein synthesis af’t,ctl addition of label rather than after the increase in temperature. [35S]nlethionine wa:: incorporated into acid-precipitable material linearly for up to eight hours. All cxprriments involving protein labeling described in t,his paper were performed within thr first four hours after heat shock. (b) qffect of ultraviolet

irradiation

on synthesis

qf’ heat shock

RNA

and protein

When cells are irradiated with ultraviolet light, transcription-terminating lesions are introduced into the DNA and the rate of RNA synthesis decreases. Figure 2(a) shows the effect of ultraviolet irradiation on the nuclear and cytoplasmic RNA fractions shocked

induced and pulse

by heat labeled

shock.

In these

experiments

for two hours with

the cells were

irradiated,

[“HI uri d ine. The synthesis

heat

of nuclear

TRANSCRIPTION

UNIT

LENGTHS

IN

147

DROSOPHILA

RNA was much more sensitive to ultraviolet irradiation than was the synthesis of cytoplasmic RNA. This might be expected, since the majority of cytoplasmic RNA synthesized during heat shock sediments at 4 to 5 S (Spradling et al., 1975). These RNAs are presumably synthesized from small transcript’ion units and, therefore, are relat’ively resistant to ultraviolet irradiation. The synthesis of heat shock proteins was decreased sha,rpl,y by ultraviolet irradiation of cells prior to heat shock (Fig. 2(b)). This is to be expected, since the proteins synthesized after heat shock are primarily coded for by RNA induced a,fter irradiation.

(c) EJfect of ultraviolet

irradiation protein

on synthesis and RNA

of ins&&id

heat shock

.species

Preparations of total cellular proteins from heat shocked and [35S]methioninelabeled cells were analyzed on sodium dodecyl sulfate/polyacrglamide gels and autoradiographed. As previously noted, only a small number of protein bands were labeled during heat shock (McKenzie et al., 1975). Seven protein species reproducibly incorporated enough label to allow quantitation from the autoradiographs. These proteins were numbered in order of increasing mobility on sodium dodecyl sulfate/ polyacrylamide gels (Fig. 3). The species and their apparent molecular weights are: Pl, 82,000; P2, 70,000; P3, 67,000: P4 through P7. 28,000 to 22,000. These molecular weights are in good agreement with those determined by others (McKenzie & Meselson. 1977; Moran et al., 1978, Mirault et al., 1978). As shown in Figure 3, the synthesis of each of these proteins was sensitive to ultraviolet irradiation. The amount of each protein at each dose was quantitated by densitometry. normalized to the number of cellular equivalents in the sample as described in Materials and Methods and then normalized t’o the amount synthesized in t,he unirradiated sample. When the logarithms of these values were plotted z’ersw the dose, st’raight lines were obtained (Fig. 4). This indicated that the rate of synthesis decreased exponentially with dose. as expected for the synthesis of any individual species if there was one copy of the gene per promoter. The slopes and corellation coefficients for the dose response lines for each of the proteins determined in several independent experiments are shown in Table 1. The slopes of the least-squares lines for proteins PI, P2 and P3 were quite reproducible in four separate experiments. Since the labeling time for these four experiments was varied between 1 and 2.5 hours, t’he reproducibility of the inactivation slopes makes it unlikely that any of the ultraviolet repair mechanisms known in Drosophila (Trosco & Wilder, 1973) interfered with the inactivation of protein synthesis. The amount of label incorporated and the resolution on gels of proteins P4 bhrough P7 was such that accurate quantitat,ion was possible in only two of the four experiments. Upon raising the temperature of Drosophila cells to 36 to 37°C a number of discrete species of cytoplasmic RNA are synthesized. Several of these hybridize in situ to the bands t’hat puff during heat shock and are known to function as mRNA in the symhesis of the heat shock proteins (Spradling et al.? 1975,1977; McKenzie et al., 1975; McKenzie & Meselson, 1977; Moran et al.. 1978: Mirault et al., 1978: Schedl Pf al., 1978). Tn order to reinforce the determinat’ions of the lengths of the transcription unit’s for the prot,eins, the ultraviolet, sensitivity of the synthesis of heat shock cytoplasmic RNA species was also investigated. Cells were exposed to ultraviolet irradiat’ion, heat shocked and labeled with L3H]uridine. Cytoplasmic RNA was isolated from

148

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11,

0

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1).

I

40

I,

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L’ETTIJOHS

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60 80 Dose ( s 1

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I

too

I20

FIG. 2. Effect of ultraviolet irradiation on heat shoclc RN.4 and protein synthesis. (a) Cells were labeled for 15 to 18 h with 0.06 to 0.1 &i of ~14C]uridine/ml and then suspended in growth medium and irradiated with different. doses ELS described in Materials and Methods. The cells were then placed in a 35°C waterbath and, 5 min lat,er, 50 $,Y of [3H]uridine/ml was added. ilfter labeling for 2 h at 37”C, the cells were collrctec( by centrifugation and fractionated into nuclei and cytoplasm. Portions of the nuclear and cytoplasmic fraction were precipitated with trichloroacet,ic acid and radinactivit,y was drtcrmined. The number of “H counts in the samplr was normalized to the total number of ‘V: conntd for that time point, ~tncl then normalized to thcx zero dose value to obt,ain the percentage ww doso incorporation. Each point on the graph is au average of 5 t,o 7 determinations at that dose. (0) Nuclear RNA: ( 1) cytoplasmic RNA4. (b) Cells were labeled for 15 to 18 h with [3H]thpmidinr~ at 1 pL(li/ml, suspended in growth medium and irradiated as described for (a). After irritdizLtion, t,htL cells were collected by &ntri. fugation and resuspended in 0.1 vol. Schneider’s medium without methionine, arginine. trypt)ophan or serum. After 40 min to 1 h at, 37’C, [W]methionine was added a,t 25 to 75 &i/ml an(l labeling was allowed to proceed for :I const,ant time pcriotl wt 37°C (I to 2.5 h, depending on t,hc% experiment). The cells were then collected, lyserl and prepared for electrophoresis as described in Materials and Methods. Each point is an average of I to 4 tktnrminations.

these samples and analyzed on 5 r\I-urea/p?olyacryl~~,r~lide gels (Fig. 5). The nlajor bands are those of the 14C-labeled ribosomal RNA st,andards. The RNA species. designated Rl, R2 and R3. were analyzed by densitometry, since they had the same electrophoretic mobilities relative to the Drosophila 18 S rRNA as some of the poly(A)-containing heat shock mRNAs analyzed by Spradling et al. (1977). To provide accurate quantitation of each RNA species at each ulbraviolet, dose, it, was necessary to normalize the amount of RKA to an internal standard. This standard was provided by labeling rRNA before irradiation with [14C]uridine. The amount of mRNA in the sample was normalized to the amount’ of prta-labeled rRNA in that sample. This method necessitated the use of t~ot,al qvtoplasrnic RNA rather t,h;tn poly(,~)-containirlg RNA on t,he gels. Although this simplitird the pinificatiol~ proctdure, it complicaLted t,he gel pattern. The electrophoretic mobilities of the rRNA species were fairly close

TRANSCRIPTlOX

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PI -

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P4 P5P6P7-

FIG. 3. Polyacrylamitlo gel elrctrophorwis of heat ~1~~~~~1; protcsins synthesized after ultraviolet irradiation. irratliatctl wit,h ultraviolet light, heat Drosophila cells were pro-labeled with 13H]thymidinr, shocked, labeled with [%]methionine, and the protjcins were prepared for electrophoresis as described in Materials and Methods. Electrophoresis was performed on a 12.50/O polyacrylamide slab gel and the gel was dried and autoradiographed. The Figure shows an autoradiograph of 7 protein preparations that have been irradiated with different doses of ultraviolet light. Approximately equal amounts of cellular equivalents of protjrin were applied to each slot. The dose of irradiation (in seconds) is given above each slot. The protein bands xc designated with the nomenclature used in the text.

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Dose ( s 1 FIG. 4. Effect of ultraviolet irradiation on Drosophila hrat shock protein species. Autoradiographs of polyacrylamide gels such as t,hat shown in Fig. 3 were scanned with a densitometer. The relative areas under each prot,ein peak were determined and normalized to the number of cellular equivalents of protein applied to the gel as described in Materials and Methods. These values were then normalized to the amount, of protein in tho unirradiated sample and the logarithm of this value was plotted against the dose. The Figure shows these plots for the heat shock proteins PI through P7. Each point on the graph represents the mean value of 1 to 4 determinations at that dose. The error bars represent the range of values of all the data. The lines drawn have slopes and y-intercepts equal to the averages of t,hr slopes and y-intercepts of the least-squares lines for each of the experiments.

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DrosophiZn cells wvl~r’c’ prc-labeled with ~‘“L’]uritlin~, ~rrarliatcd xvith ultraviol& light, heat shocked and labeled with [3H]uridinc. (‘ytoplasmic RNA was prepared for electrophoresis at 100 V for 10 h on 8%2.5 % to 5 4; gradient gel as described in Materials and Methods. The Figure shou.5 a fluorograph of the gel. The first 6 slots show total ryt~oplasrnia RNA from lwr-labeled cells that were given different doses of ult,ravioM lightJ before hmt shock. The doses (in seconds) are shown above the slots. The slot labeled D shows the total cytoplanmic RNA labeled for 20 h with [3H)uridine at 25°C. The slot, labeled M shows toktl mouse cytoplasmic RNA used fur molecular weight standards. The positions of the RSAs that’wwr quankltakd ZII-P sh~uw. 1

TRANSCRIPTION

UNIT

LENGTHS

IN

DROSOPHILA

153

to the mobilities of species RI and R2, which resulted in some ambiguity in quantitation due to uncertainty in background corrections. Densitometer tracings of the fluorograms allowed identification of the RNA species : however, errors of up to 300/i;, due t’o background correction could have been present in the quantitation of any given measurement for R2. Errors in quantitation of Rl could have been even great’er. due to it’s low concentration. Although these background corrections introduce greater uncertainty than was present in the determinations of heat shock proteins (Fig. 4), the measurements of R2 were reproducible within the standard deviations given in Table 1. Also, these background corrections were nomina,l for R3. R3 probably contained more than one component, since increasing ultraviolet’ dose skewed t’he peak toward the higher molecular weight’ portion. although t,wo peaks in this region are not, clearly resolved on t)he gels. The dose response curves for Rl, R2 and R3 arc shown in Figure 6. The decrease in synthesis for t’he three species is exponential with ult8rdviolet dose, indicating a single gent’ per promoter. The slopes and correlation coefficients of the least-squares lines for several experimentIs a,re shown in Table 1. In some of the ultraviolet inactjivation curves, most) notably in the data for R2 (Fig. 6), a, shoulder was evident in the low dose range. The slopes tabulated in tjhtL data of Table 1 are t’he final slopes, ignoring the early data points which suggest a shoulder. A possible explanstion for this shoulder is developed in t’he Discussion. The ult’raviolet sensit’ivity of the synthesis of one of the heat’ shock RNA species was also quantitated by RNA-DNA h>rbridization. For these experiments RKA isolated from cells irradiated for different times was h,vbridized to an excess of DNA from a plasmid containing a portion of a specific heat shock gene. The hybridizat~ions \\‘(‘re done in solution at high tttnpcraturc: in the presc’nctb of formamidn using

101

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FIG. 6. Effect of ultraviolet irradiation on Drosophila heat shock RNA species. Fluorographs of polyacrylamide gels such as that shown in Fig. 5 were scanned with a densitometer. The relative areas under RNA peaks Rl, RZ and R3 WWP determined for each sample and these values were normalized to the area under the rRNA peaks and then to the amount of the appropriate species in the m&radiated sample. The logarithm of this value was plotted against the dose. The Figure shows these plots for the heat shock RNAs Rl, Rd and R3. Each point on the graph represents the mean value of 1 to 3 determinations at that dose. The ewor bars represent the range of values obtained in the different repeat experiment,s. The lines drawn have slopes and y-intercepts equal t,o the averages of t,he slopes and y-intercepts of the least-squares lines for each of the experiments.

154

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conditions where the DNA-DNA duplex is unstable but where the RNA-DNA duplex can form (Casey & Davidson, 1977; Vogelstein & Gillespie, 1977). To test this technique using RNA transcribed from a defined transcription unit,. the effects of ultraviolet1 light on the transcription of Drosophilic rRNA was investigated. Total cellular RNA was isolated from samples of cells t,hat had bec~r irradiated for different doses and labeled with [3H-juridine at 25°C. The amountj ot labeled rRNA in each sample was quantit’ated by hybridization to plasmids ckDml03C and ckDmlO3D. which contain portions of t’he I). n~eZa~so~&rt 28 S a,nd 18 S rRNA genes, respectively (Glover 6.~Hognexx, 1977). When thr amount of RNA that hybridized to each of the plasmids was det’ermined and plotted against the dostb, as in Figures 4 and 6, the curves were linear (slopes given in Table I ). The ina,&vation slopes averaged over four determinations were -0*0056 and --0.0134 s ~ 1 for the I8 S and 28 S species, respectively. This agrees well \vith thcl values of --OW.59 aud -0.0135 s-l determined previously by electrophoretio analysis (Carlson et nl.. 1977). This t’echnique was next applied t,o a heat shock cytoplanmic RNA. RNA samples were hybridized to pla.smid 229.1. which contains a portion of the gent> coding for the 3’ end of the 2.6 kb heat shock induced mRKA (Livak et cnl., 1978). This RNA is known to hybridize to ba.nds 878 and 87C irk situ (Spradlinp ef al.. 1977: Henikoff $ Meselson, 1977). and to code for the 70,000 molecular weight, 1’2. Cptopla.smic. RNA was prepared from samples of cells that had been irradiated with differentj doses. htaat The amount, of RNA Ilybridizing to 229.1 shocked and labeled with [ 3H]uridine. DNA at ea,ch ultraviolet dose is plotted on a logarithmic scaltl in Figure 7. ‘I’htb Irastsquares slopes for six separate hybridization experiments arc tabulated and averaged

E’IG, 7. Effect of ultraviolet irradiation on the synt,hesis of HNA hybridizat,ion to plasmid 229.1 DNA. RNA isolated from cells that had been pm-labeled with ~14C]urrtline, irradiated with ditierenl doses of ultraviolet light, heat shocked and labeled with [3H] uridine were hybridized to an excess of plasmid 229.1 DNA as described in Materials and Methods. The amount that hybridized in each sample (an average of 1.73% to 0.72% of the input counts depending on the ultraviolet dose) was normalized to the l*C pre-label in the sample and corrected for background determined in a mock hybridization reaction in which the DNA had been omitted (average of 0.33% of the input counts). The corrected value was normalized to the value in the unirradiated sample and the logarithm of this was plotted against the doso. Each point on the Figure represents a mean of 6 determinations at that dose and the error bars represent the range of values obtained in repeat experiments. The line drawn has a slope and y-intercept equal t,o the averages of t,he slopes and y-intercepts of the least-squares lines for 6 experiments.

TRANSCRIPTIOK

CJNIT

LENGTHS

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155

DROSOPHILA

in Table 1. It may be seen Ohat mea,n values differ by about SO”,; from those obtained for R2 by electrophoretic analysis. Reasons for this difference mill be discussed below. The slope of the ultraviolet inactivation line directly reflects the distance between the promoter and the gene that codes for the specific protein or RNA species. If the inactivation slope is known for a transcription unit of known size, it may be used as a standard to estimate the lengths of the transcription units for other gene products. The ribosomal RNA precursor of Drosophila is about 8 kb in length and has an inactivation slope under the same irradiation conditions of -0.0135 s-l (Carlson et al.. above). The average inactivation slope, 1977) or -0.0134 s-l (data mentioned standard deviation and estimated transcription unit length relative to the rRNA transcription unit are shown in Table 2 for all of the targets investigated. Also shown are minimum expected transcription unit lengths based on the apparent molecular weight#s of the gene products on gels. It’ can readily be seen that the lengths of the transcription units for the proteins and RNAs are much larger than would be required to simply code for the specific amino acid or nucleotide sequence. TABLE 2 Estimated

lengths

m (s-1) Pl P:! P3 P4 P5 PG P7 Rl R2 R3 229.1 hybrid

- 0.0149 -0.0115 --0.0147 -0~0109 - 0.0103 -0.0113 -0~0113 - 0.0075 -0.0109 -0.0108 .- 0.0084

of heat shock transcription

S.1). (s-1) 0~0005 0~0015 0.0015

0.0017 0.0026 0.0017

1 W) 8.8 6.8 8.7 0.5 6.1 6.7 6.7 4.5 6.5 6.4 5.0

units a (kb) 2.2

1.9 1.8 0.8 0.i 0.6 0.6 2.9 2.6 0.9

m, Mean inactivation slope from Table 1; the standard deviation about the mean is from Table 1: I, the length of the t,ranscription unit based on the rRNA precursor having an inactivation slope of -0.0135 s-1 and a length of 8 kb; s, minimum expected transcription unit length calculated from the apparent moleculnr weight of the protein or RNA t&mated from its electrophoretic mobility.

The best evidence correlating a given heat) shock protein with a specific heat shock mRNA is for the most abundant heat shock protein. the 70,000 molecular weight protein designated P2 in this work. This protein is known to be coded by an mRNA of about’ 2.6 kb from translation studies in vitro (McKenzie & Meselson, 1977; Moran et al., 1978; Mirault et aZ., 1978). This RNA, which is presumably the RNA designated R2 in this work, is known t,o hybridize in situ to bands 87A and 87C on polytene chromosomes (Henikoff & Meselson, 1977; McKenzie & Meselson, 1977; Spradling Pt al., 1977). These bands are known to contain the genes coding for protein P2, since flies that carry deletions of both these bands are unable to synthesize P2 (Ish-Horowitz et IzZ., 1977). Furthermore, plasmids that contain DNA from these bands specifically interf’ere wit’h the synthesis of P2 in vitro in hybrid arrested translation assays

J. 0. CBRLSON

1.56

ANI‘,

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I’tr:TTt.JOHN

(Schedl ct trl., 1978: Livak p/ rrl., 1978). l’la~smid 229. I ~rsocl fi)r the hyl,ridizat,ioll studies in this work is complcrnmtar~ to t’hr: 3’ Ix&on of’ t,his RNA (Livak of r/l.. 1978). lt is interost~ing. th&?forr. to c:ompnrt: t’hc t)arget sizes of t,h(x prot,ein 1’2. thtk . RNA R2 and the RNA I-1.vtxl ‘d’lzmg t’o plasmid 229.1. The avtbrage target size of protein P2 is 6.8 kb. while the avemge target size for the RNA R% determined I)? electrophoretic analysis is 6.5 kb. The average target size for RNA hybridizing to plasmid 229.1 is 5 lib. It should he noted. however. &at while the polya~crplamidc gel analyses measure the t,arget size only for complet,ed products. the hybridization analysis could also detect the synt,hesis of incompIet,e RNA produotjs that, are trrminat’ed within the transcriptiona,I unit, that codes for R2 and 1’2. Plasmid 229.1 contains about 1 kb of DNA complementary t,o the 3’ end of t,he 2% kb RNA R2. The hybridization anal,vsis would detect) a,11transcripbs that included a portion of this 1 kb sequence. and the target size would represent an averagr: target size for all transcripts terminating within that) region. To csompare the target’ size of RNA hybridizing to 229.1 to the other two targets lye should. therefore. add 0.5 kb bo the former target. This gives an estimate for the length of the transcription unit’ of 5.5 kh. A comparison of the Iengt’hs of t,he transcription unit d&rrmiuc~d by the t hrcc methods is shown in Figure 8. The thrfle determinations agrer rc~asonab1~ ~~(~11.

R2 Promoter

P2

J! 0

I

2

3

I 4

5

6

7

I 8

Length (kb) FIG. 8. Target. sizes of P2, R2 and 229.1 hybridizing DNA. The transcription unit, coding for protein P%, RNA R2 and RNA hybridizing to planmid 229.1 is shown diagrammatically. The target sizes determined bp polyacrylamide gel analysis of heat shock protein P2, by polyacrylamide gel analysis of heat shock RNA R2, and hybridization of heat shock RNA to plasmid 229.1 extends from the promoter on the left. to the center of t,hcL appropriate bracket on t’he right. The uncertainty in the tlet,erminution (1 standard deviation on either side of the mean) is indicated by the widt,h of the brackets and by thr oxt,ent of vertical, rightward and leftaward crosshatching for I‘2, R2 and 229.1. wqwtjivcly.

Data linking other heat shock RKAs to specific heat, shock proteins is less firm Messenger RNA that behaves like Rl on polyacr,vIamide gel electrophoresis a.nd sucrose gradient centrifugation directs t,he synthesis of the 82,000 molecular weight protein Pl in protein synthesis reactions ill. vitm (McKenzie $ Meselson, 1977 : Moran et al., 1978: Mirault et al., 1978). The target sizes for protein PI and RNA Rl art’ 8.8 kb and 4.5 kb, respectively. These target sizes are significantly different : however. the target size for Rl is based on a single determination a,nd, because of the background corrections discussed above, could include signiticant error. The low molecular weight heat shock proteins, P4 through P7, are coded bv an unresolved set of mRNAs designated 12 S RNA by several authors (McKenzie & Meselson: 1977; Moran et al., 1978; Mirault et al., 1978) or A4-A5 by Spradling et al. (1977). These are probably the RNAs designated R3 in this work. The target, sizes of P4 through P7 range from 6.1 to 6.7 kb and the target size for R3 is 6.4 kb. Here the target sizes for the proteins and RNA are in good agreement.

TRANSCRIPTION

UNIT

LEKGTHA

IN

DROSOPHILd

167

4. Discussion The ease with which the he& shock genes ma,y he induced makes them attractive for the study of gene expression in Drosophila,. As a step toward understanding the processes involved, we undertook a study of the lengths of the transcriptional units involved in the synthesis of the heat shock prot,eins using t,he techniques of ultraviolet promoter mapping. Since the heat shock response is induced at the transcriptional level, it was feasible to examine both the ultraviolet sensitivities of the production of the final protein products as well as the mRNA. In order to convert the ultraviolet inactivation rates to a length of DNA measured in kb, t)he D. melanognster ribosomal RNA transcription unit was used as a standard. This transcription unit has been well-characterized in terms of morphology (Laird et al., 1976; Laird & Chooi, 1976), DNA structure (Glover I% Hogness, 1977; White & Hogness, 1977; Wellauer $ David, 1977; Pellegrini et al., 1977) and processing of the primary transcript (Jordan et al., 1976; Levis $ Penman, 1978). The ultraviolet sensitivity of the synthesis of the large and small rRNA species as determined by polyacrylamide gel electrophoretic analysis (Carlson et al., 1977) and by hybridization analysis (this work) agree well. It might be argued that since rRNA and mRNA are transcribed in the cell by different RNA polymerases, the sensitivity of the two polymerases to ultraviolet-induced lesions might be different. However, data in mammalian systems suggest that ultraviolet irradiation induced lesions terminate transcription by the two polymerases at about the same efficiency (Giorno & Sauerbier, 1976; Goldberg et al., 1977a; Gilmore-Hebert et aZ., 1978). Indeed, virtually all polymerases that have been examined seem to be affected at about the same level (Sauerbier, 1976). We do not believe, therefore, that this introduces a serious error in our results. In some of the ultraviolet inactivation curves, notably in the data for R2 and the hybridization to plasmid 229.1, there was a shoulder in the low dosage region. A shoulder may be interpreted as follows (Hackett & Sauerbier, 1974). Suppose there were more than one copy of a given transcription unit and that these transcription units were not normally transcribed at maximal rates. If damage to one of the units could be compensated by more frequent transcription of the undamaged copies of the branscription units, a shoulder would be expected in the dose response curve until t’he compensation is no longer possible. The final slope in the portion of the curve beyond the shoulder would reflect the true target size of the transcription units. This interpretation is plausible, since it is known that there are several copies of the gene coding for protein P2 and RNA R2 located at two different sites in the Drosophila genome (Henikoff & Meselson, 1977 ; Livak et al., 1978). Thus, for the data sets that exhibited a shoulder, the low dosage points were omitted from the least-squares analysis. In principle, a shoulder could also result’ from repair of the transcription terminating lesions. Although the studies described above indicated that DNA repair processes did not influence these results, the experiments do not exclude this possibility. In this case. one would have expected shoulders on all of the inactivation curves; however, this was not observed. In any case, the final slopes in the curves would be appropriate for the measurements described here. The target sizes of the heat shock protein products correspond to transcription unit lengths of between 6 and 9 kb when compared to t’he target size and transcription unit length of the rRNA precursor. Tn the cases where a target size for a protein and the corresponding mRNA species could be measured, the agreement was reasonably good.

1.58

.J. 0.

CARLSOS

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1). E. I’ETTI,JO.HS

This gives us confidence that we are actually measuring t’he leng& of t,he t,ra,nscriytion units for the p&ems. As determined in these experimerns. the transcription unit. lengths for the prot~eins include between 3.5 and 10 times as much DNA as is needed to code for the amino acid sequences of the respective proteins. The transcriptiou units of the RNAs are between 1.5 and 6.5 t,imes the apparent moleculw lengths of the corresponding RKAs. These relationships het)ween tJranncription unit length autl mRNA length are similar to those determined for hulk mRKA in HeLa cells (Goldberg cutal.. 1977~) and for several discrebe mRNA species in mouse myeloma cells (Giorno $ Sauerbier. 1978: Gilmore-Hebert Pt uI., 1978). Furthermow. the t,arget sizes do not show a direct relationship to the relative sizes of t.ht: proteins or RNAs themselves. Por example. protein P2. which has a molecular \veight of 70,000. comes from a transcription unit of about) the same size as the transcription unit, for the 22,0t)0 molecular weight’ protein 1’2. The lengths of the heat shock t,ranscription units would imply7 that’ the mRNAs for the proteins arc derived from larger primary transcript8s, as is the cast for a number of eukaryotic RNAs (Hastes & Aviv. 1977: Curtis it clb.: 1977: Tilghman et nl., 197X: Gilmore-Hebert, $ Wall. 1978; Gilmore-Hct,er.tS PL ccl.. 1978). An alternative interpretation is that, some sort, of activator gene product or products, s:uch as have been proposed by Britten and Davidson and co-workers (sw Davidson rt ($1.. 1975), must. be synthesized in order to facilitatt t’he expression of t,he genes. .1n this case. the target size couid reflect, ttw longt,hs of both the activator gene and the heat, shock protein gene as wet I. The 70.000 molt~cular weight heat, shock protein 1’2 is known t’o no coded by the 2.6 kh mRKA R2, which is homologous to DNA seyuences present at bands X7A and 87C on polytem chromosomes. Each of the two loci probably contains more than one copv of the gene for t8hcl 2.6 kh mR?CA (Henikoff & Mcsclson. 1977). The linearity of t,he inact)ivation curve for the p2 and R2 gene productas shown here argues against the possibility t!hat more than one actively t,ranscribrd It2 gene can be cotranscrihcd on a single transcription unit. Locus 87C also contains a numtw of copies of a tau clemly repeated I .,i to 1.6 kh sequence, called c@. which is homologous to a hetwopenrous size class of RNA that has no known function (Lis et al., 1978). The relationship bet\zwn thcw two classes of RNA is not known: however, Livak ef tel. (I!t%) have isolat’ed a plasmid tl1n.t contains a set of at, least, t’wo and possibly three tandemI> rrpea,ted copies of the x/3 sequence situated about 0.8 kb from the 5’ end of one of tilt, copies of the R2 mRNA gene. The target sizes of P2. R2 and the 229.1 hybridizing RNA are all consist,ent with transcription startring within the afi tandem repeats and ending at the 3’ end of t,he nucleotide sequence coding for P2. Thus. at tca,st ow of thr @ sequcncw and one R2 gene could he on the same transcription unit. The ;LIM)v~~ model assumes that the Rb coding gene that is linked with the E/I sequenws is actualI> transcribed. I tI is not kno\vn whether all of t
TRANSCRIPTION

UNIT

LXNGTHS

IN

DI~O,)‘Of’IilL.-l

I*>9

transcript must be synthesized in order to co-ordinate expression of the two loci and perhaps the other heat shock genes as well. The t’ranscription unit lengths for the heat shock proteins and RNBs are definitel>. smaller t’han the amount of DNA per DNA copy in an average sized polytene chromosome band (20 to 25 kb). It should be mentioned that the ultraviolet transcription mapping method cannot eliminate the possibility that the prot’eins and RKAs corms from even larger Oranscription units if the extra length is on the 3’ end of the transcript and is not essential for gene expression. The transcript,ion unit lengths a,rta. however. within the size distribution of heterogeneous nuclear RNA in r)). rr/eZn,/ogaster cells (Lengyel & Penman, 1975; Lamb & Laird, 1976) and are larger than th(c awrttge mRNA molecule. The transcription unit lengths are close to the size of tlrt “40 S” RNA isolated from vitamin B6 or heat-induced salivary gland puffs from Urosophila hydei (Bisseling et al., 1976; Lubsen et al., 1978). It is also interr!stinp. although perhaps coincidental, t’ha,t the t’arget sizes arc approximately the sizt estimated for the combined control and structural elements for xanthine dehydrogenase in 11. mehogaster (Chovnick et al., 1977).

This work was supported by a research grant (GM-l 8243) and a postdoctoral fello\~shlp (P32-HDO5098) from the National Institutes of Health. Tltc allthors thnljk Dr \Valt~(~r Sauerbier for allowing the use of his facilities for ultraviolet irradiation xrltl Drs Mcselsorr and D. Hopness for providing the plasmids used in this work.

REFERENCES Abraham, G. & Banerjee, A. K. (1976). Proc. Nat. Acad. Sci., C.$.A. 73, 1X)4-I%@. S&burner, M. (1970). Chromouoma (Berlin), 31, 356-376. Ball, L. A. & White, C. N. (1976). Proc. Nat. Acad. Sci., C.i)‘..4. 73, 442-446. Bastos, R. N. & Aviv, H. (1977). Cell, 11, 641.-650. Berk, A. J. & Sharp, P. A. (1977). Cell, 12, 45-55. Biessmann, H., Levy, W. B. & McCarthy, B. J. (I 97%). Proc. ~Vat. dcarl. Xcl:., U.S.=l.

75,

Biessmann. H., Wadsworth, S., Levy, 1%‘. B. and McCarthy, B. J. (197%). Cold Spri?q Harbor Symp. Qua&. Biol. 42, 829-833. Bisseling, T., Berendes, H. D. & Lubsen, N. H. (1976). Cell, 8, 299.-304. Bonner, W. M. & Laskey, R. A. (1974). Eur. J. Biochem. 46, 83-88. Banner, .J. J. & Pardue, M. L. (1976). Cell, 8, 43--X). Brautigam, A. R. & Sauerbier, W. (1973). J. c’irol. 12, 882--886. Brautigam, A. R. & Sauerbier, W. (1974). J. Viral. 13, 1110-l 117. Carlson, J., Ott, G. & Sauerbier, W. (1977). J. Mol. Biol. 112, 353-357. Casey, J. & Davidson, N. (1977). Nucl. Acids Res. 4, 1539-1552. Chovnick, A., Gelbart, W. & McCarron, M. (1977). Cell, 11, I- 10. Clewell, D. B. & Helinski, D. R. (1969). Proc. iVat. Acad. Xci., G.S.il. 62, 115!~~1166. Curtis, I’. tJ., Mantei, N., van den Berg, J. & Weissmann, C. (1977). rroc. IV&. Acarl. Sci.. U.S.A. 74, 3184-3188. Davidson, E. H., Klein, W. H. & Britten, R. .J. (I 977). Develop. Hiol. 55, 69- 84. Gilmore-Hebert, M. & Wall, R. (1978). Proc. ,!Vat. Acad. Sci., U.S.A. 75, 342 -345. Gilmore-Hebert, M., Hercules, K., Komarony, M. & Wall, R. (1978). J’roc. Xat. Acatl. Sci.. U.S.A. 75, 604k-6048. Giorno, R. & Sauerbier, W. (1976). Cell, 9, 775-783. Giorno, R. & Sauerbier, W. (1978). Proc. Nat. Acad. Sci., U.S.A. 75, 43744358. Glover, D. M. 6t Hogness, D. S. (1977). Cell, 10, 167-176. Goldberg, S., Schwartz, H. & Darnell, J. E.. .lr (1977a). Proc. Sat. .-lcarl. 9ci., Ci.S.A. 74. 4520-4523.

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AND

11. IX. PETTIJOHN

Goldberg, S., Weber, J. & Darnell, J. E., Jr (19776). Cell, 10, 617-621. Hackett, P. B. & Sauerbier, W. (1974). A7ature (London), 251, 639-641. Hackett, P. B. & Sauerbier, W. (1975). J. Mol. Biol. 91, 235-256. Henikoff, S. & Meselson, (1977). Cell, 12, 441-451. Hercules, K. & Sauerbier, W. (1973). ,7. V+oZ. 12, 872.-881. Hercules, K. & Sauerbier, W. (1974). .7. l’irol. 14, 341~-348. Hochman, B. (1973). Cold Spring Harbor Symp. f&ant. BioZ. 38. 581- 589. Ish-Horowitz, D., Holden, J. J-. & Gehring, W. J. (1977). Cell, 12, 6433652. Jamrich, M., Greenleaf, 9. L. & Bautz, E. K. F. (1977). Z’roc. Sat. Acad. Sci.. I’.S.il. 74, 2079-2083. Jordan, B. R., Jourdan, R. bt Jacy, B. (1976). J. ilrlol. Biol. 101. 85 105. Judd, B. H. & Young, M. W. (1973). CoZd Spring Harbor Symp. Qzhant. BioI. 38, 57% 579. Laemmli, U. K. (1970). Nature (Lon,don), 227, 680.-685. Rev. Genet. 7. 1777204. Laird, C. D. (1973). Anna. Laird, C. D. & Chooi. W. Y. (1976). Chromosoma (Berli?~), 58, 193218. Laird, C. D., Wilkinson, L. E.. Foe. 1’. E:. $ Chooi, W. Y. (1976). Chromosoma (Berlin), 58, 169-192. Lamb, M. M. & Laird, c’. D. (1976). Biochem. Genet. 14, 3.55~-371. Laskey, R. A. S: Mills, A. D. (1975). Eur. J. Biochem. 56, 335-341. Leenders, H. J. & Berendes, H. D. (1972). Chromosoma (Rerlin), 37, 433 444. LeFevre, G., Jr (1973). Cold Spring Harbor Symp. Quan,t. HioZ. 38, 591 -599. LeFevre, G., Jr (1974). Annu. Rev. Genetics, 8, 51l62. Lengyel, J. A. & Pardue, M. L. (1975). .J. Cell BioZ. 67, 24Oa. Lengyel, J. &. Penman, 8. (1975). Cell, 5, 281- 290. Lengyel, ,J. A. & Penman, S. (1977). 1)eveZop. BioZ. 57, 243 253. Lengyel, J ., Spradling, A. & Penman, S. (1975). In Methods in (‘e/l Hiology ( I+oscot,t,, I).. ed.), vol. 10, pp. 195 208, Academic Press, New 1-o&. Levis, R. 1,. & Penman, 8. (1!)78). .J. Mol. RioZ. 121, 21% 238. Levy, W. B. & McCarthy, B. J. (1975). Biochemistry, 14, 2440 2446. Levy, W. B., aohnson. C. B. & McCarthy, B. J. (1976). ,\TzrcZ. Acida Rex. 3, 177771789. Lewis, M., Helmsing, P. .I. & Ashburner, M. (1975). I’TOC. A’at. .4cad. Scl:.. 7r.S.A. 72, 36043608. Lis, J. T., Prestidge, L. & Hogness, D. S. ( 1978). Cell, 14, 90 I !)I!). Livak, K. J., Freund. R., Schweber, M., Wensink, 1’. (‘. & Mesrlson, M. (1978). 1 ‘rec. LVat. Acad. Sci., U.S.A. 75, 5613.-5617. Lubsen, N. H., Sondermeijer, P. J. A., Pages, M. & Alonso. C. ( 1978). Ch~romoswna (Berlin). 65, 199~-212. McKenzie, S. L. & Meselson, M. (1977). .J. Mol. Biol. 117, 279 283. McKenzie, S. L., Henikoff, 8. & Meselson, M. (1975). Proc. Nat. Acad. Sci., ci.ij’.A. 72, 1117-1124. McKnight, S. L. & Miller, 0. J., *Jr (1976). Cell, 8, 305 3 19. Mirault, M.-E., Goldschmidt-Clermont. M., Moran, I,., Arriyo. A. I’. k ‘I’issieres, A. (1978). Cold Spring Harbor Symp. Quant. BioZ. 42, 819 -827. Moran, L., Mirault, M.-E., Arrigo, 8. P., Goldschmidt-Clcrinont, M. & Tissierrs. A. ( 1978). Phil. Trans. Roy. Sot. ser. B, 283, 391-406. M., Manning, J. & Davidson, N. (1977). Cell, 10. 213 224. Pellegrini, Pollock, T. J., Tessman, I. & Tessman, E. S. (1978). .I. ,zloZ. Biol. 124. 147- 160. Ritossa, I?. (1962). Experimentia, 18, 571 573. Ritossa, F. (1964). Exp. Cell Res. 35, 601.-607. Sauerbier, W. (1975). R&at. Res., 651- 652. Sauerbier, W. (1976). Adwan. Radiat. BioZ. 6, 49SlO6. Schedl, P., drtavanis-Tsakonas, S., Steward, R., Gehring, W. J., Mirault, M.-E., GoldSchmidt-Clerrnont, M., Moran, L. bz Tissieres, L4. (1978). Cell, 14, 921-929. Schneider, I. (1972). J. Embryol. Exp. Morph. 27, 353 ~365. Spradling, A., Penman, S. & Pardue, M. L. (1975). Cell, 4, 39&404. Spradling, A., Pardur, M. L. & Pennlair. 8. (1977). ./. .z/oZ. HioZ. 109. 55%587.

TR4NSCRIPTION

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DROSOPHILB

161

Tilghman, S. M., Curtis, P. J., Tissikes, D. C., Leder, P. & Weissman, C. (1978). l’roc. Nat. Acad. Sci., U.S.A. 75, 1309-1313. Tissi&es, A., Mitchell, H. K. & Tracy, U. M. (1974). J. i%fol. Biol. 84, 389-398. Trosco, J. C. & Wilder, K. (1973). Genetics, 73, 297- 302. van Breugal, F. M. A. (1966). Genetics, 37, 17-28. Vogelstein, B. & Gillespie, D. (1977). Biochem. Biophys. Res. Commun. 75, 1127-I 132. Wellauer, P. K. & David, I. B. (1977). Cell, 10, 193~-212. I+‘hite, R. L. & Hogness, D. S. (1977). Cell, 10, 177-192.