A Drosophila heat shock gene from locus 67B is expressed during embryogenesis and pupation

,I. Mol. Hid. (19X7)198, 235-240

A Drosophila Heat Shock Gene from Locus 67B is Expressed During Embryogenesis and Pupation Daniel Pauli and Chia-Hwa Tonka Department of Molecular Biology, LTniz?ersity of Geneva SO Quai Ernest-Ansermet, 1211 Geneva 4. Switzerland (Received 22 June 1987) LVe present

a detailed

characterizat’ion

of the strurt,ure and expression of gene ,‘3 from the Northern blol, analysis reveals a major poly(A)+ transcript during two stages of development: mid-embryogenesis and beginning of pupation. Aft’er heat shock. the abundance of this mRNA is increased and small amounts of larger RXAs representing alternate terminations of the major transcript appear. In Schneider 3 tissue culture cells, beside the major transcript, we also observe small amounts of the larger RNAs after a heat shock. The sequencing of cDl?;A and genomic clones shows an intronless t,ranscription unit with one long open reading frame. The deduced polypeptide has 169 amino acids. It shares a strong homology with the four small heat shock proteins in the region also conserved in the mammalian a-crystalline B2 chain. In gene 3, this homology is rrstri&ed to the first 50 residues along the conserved 83 amino acid stretch. Two heat shock regulatory elements are localized upstream from the gene.

67R locus of Drosophila

melanogaster.

1. Introduction The Drosophila melanogaster chromosomal locus 67B contains seven heat-inducible genes clustered within 15 kb (Ayme & TissiBres, 1985). Four of them encode the small heat shock proteins hsp22, synthesized 23. 26 and 27, which are strongly following different stresses including elevated & temperatures (TissiBres et al., 1974; Ashburner Honner. 1979; Petersen et al., 1979; Craig & ef >lc(‘a,rthy. 1980: (‘orces et at.. 1980; Wadsworth al.. 1980; Voellmy et al., 1981). The protein products of the three other genes, called 1, 2 and 3 according to Sout’hgate et al. (1985), have not so far been identified. In contrast to their co-ordinate induction by heat shock. the seven small hsp genes are expressed at very different levels during several developmental stages. All. and especially hsp23 and gene I, are transcribed in early pupae (Sirotkin & Davidson, 1982: Mason et aE., 1984: Ayme & TissGres, 1985). hsp26 and 27 genes are expressed in the ovary nurse cells and their transcripts are stored in maturing oorytes where they remain during the first four hours of embryogenesis (Zimmerman et al.. 1983). A detailed analysis of the tissue distribution of hsp26 mRXA during development revealed a complex pattern of expression in spermatocytes. nurse cells, epithelium. imaginal discs, proventriculus and central nervous system (Glaser et al.. 1986). Furthermore, treatment, of Drosophila tissue culture cells or imaginal discs with t)he molting hormone B-ecdysone, induces the

transcription of the four small hsp genes but not that of genes 1. % and 3 (Ireland & Rerger, 1982; Ireland et al.. 1982). The sequencing of the four small hsp genes and of gene 2 has revealed that they are very similar: close to the C terminus of their predicted proteins, they share a stretch of 83 amino acids which is also conserved in the mammalian a-crystalline B2 chain (Ingolia & Craig, 1982; Southgate et al., 1983; Ayme & TissiBres, 1985). Ayme & TissiGres (1985) also reported a weak cross-hybridization between genes 2 and J and some of the small hsp genes, suggesting that the seven heat shock genes clustered at the 6712 locus derive from a common ancestral gene. Tn t,his paper, we analyze further gene 3. We find t,hat by contrast with the others. gene 3 is expressed during mid-embryogenesis. As reported (Sirotkin & Davidson, 1982; Ayme & TissiBres, 1985) we also observe a transcript,ion in early pupae and after a heat shock. DNA sequencing shows that it has no intron and could encode a polypeptide of 169 amino acids. We note a strong homology with the small hsp’s along the first 50 amino acids that are also conserved in the mammalian a-cr)-stalline.

2. Materials and Methods Schneider 25°C at a Schneider’s with 1O0h

line 3 tissue culture cells were maintained at concentration between IO6 and 107/ml in Lkmyhilu medium (GTBC0) supplemented (v/v) heat-inactivat,rd fetal calf serum

Embryos

Larvae

Purse

adult

67B/ I kb

hsP 27

------;--a* ---------------*

Figure 1. Organization of the locus 67B and map of the gene 3 transcription unit. The filled arrows represent the small hsp genes. Genes called I, 2 and 3 according to Southgate et al. (1985) are shown by open arrows. These 3 genes were previously named 1, 4 and 5. respectively (Sirotkin & Davidson. 1982). In the detailed map of gene 3, the thick arrow represents the major transcript and t,he broken arrows the 2 minor heat-inducible mRh’As. kb. lo3 bases or base-pairs; bp. base-pairs. ((:TKC0). Cells were recovered by centrifugation and resuspended in 0.1 cculture volume of 60 tnM-KU. 15 tnMPia(‘l. 15 tnM-Tris . HP1 (pH 7.5). 0.3 mM-sperminr. 1 trt~spermidine. 1 tnM-EDTil. 0.1 rnM-ICGTA. I IllMtlithiothreitol. After addition of an equal volume of 0.5 w Sa(‘1. 25 mM-ED’I’A and 1 Ib (w/v) SDS. cells were Iysed by vigorous vortcxing. Following :i rxtrac~tiotrs with lthettol/chloroform/isoatnglatcohol (50 : 50 : 1, by vol.) RX& were precipitated at -20°C’ overnight with 3 vol. rt.hanol. Total nucleic acids were rxt,racattd from tht> Oregort-R stock of I). mdanoynsfrr as descaribrd by Aytttta B Tissi&res (1985). Embryos. Ist, and 2nd inst,ar tarvarl NY~IYcollec%rd on agar plat,rs ~~~vrretl wit tt baker’s >.r;tst and grown to the appropriate stage at 24°C’. Early and late 3rd instar larvae were collected as described b? Mason et al. (1984). The early pupal satttples (8ontaitt white prepupae and pupae up to 1%h-old at 20°CI. Thra late pupal samples c~orrespond to pupae ttiorf’ that1 IOO-Itold at %O”(‘. Adults were I-wcxrk-ok. l’ol,v(:\)’ RX,\ was prepared according t,o Maniatis rt nl. (1982). Rl\;.As were elertrophoresed on 1.2°,0 (w/v) agarosr gels csontaining 2.2 M-formaldehyde. transferred onto ttitroc>rllulose filters and hybridized with sittglr-stt,atltlrcl ~xobes prepared front phapr >I13 clotrrs as tlrsc~t,il,~~d (Aymr & TissiPres. 1!)85). Probfl X contains sryuc~c~s from +45 t)o +468 and probe 1%frotn + I 139 t.o + 1503 (S;(YLFigs 1 and 4). ;\t the stringent c~ontlitions ust~t for hvbridization and washings. t.hey do not c~ross-tt~l)ritliz(, a:ith other genes of loc~un67R

(b) I’ritttc,r p.rtrrasiott frttd S, tttcxppiti!g l’ritner rxtension analyses using probr (’ (f45 t,o + 283) were performed as described by l’auli et nl. ( 1986). The restriction fragment, Hind111 (- 138)-HindII (+468). 5’ end-labrlled at, the Hind11 Bitt’, was annealed overnight at’ 50’(’ with 50 pg of total nucleic acids and digested at 37°C’ for 30 min with 100 units of S, nuclrase according to Maniatis of nl. (1982). Both methods gavrx a similar initiation site of transcription (co-ordinat,e + 1 in Fig. 4). (c) Sequencing

Sequencing was performed by the dideox.ynucleotide chain termination technique (Sanger et nl., 1977, 1980) using either Ml3 clones of selected restrict,ion fragments

Figure 2. 1)rvelopmental csxltression, Tot)at nuc+~c~ al,ids (10 &lane) were analyzed by Sorthertt hlottittp using probe Ai as described in Materials and Mrthocls. For the embryonic samples, the figures represent tirrtca in tr after egg laying at %‘(‘. E. rarly: 1,. tatr. or progressivr drlt~tions with Es0111 (tienikofl’. I!)&&) for the downstream HitldIIIEcoRI fragment. A full-length cDICdZ clottr was isolated from an (>arly pupal lihrarj (Poole rt rrl.. 1985).

3. Results Figure I depicts the general organization of’ locus and a detailed map of thtb 67B of Il. melanogaster gene 3 t’ranscription unit with some of t,hr singlestranded probes used. It should be noted t,hat genes 2 and 3. named according to Southgate rt al. (1985). were called -1 and 5, respecatively. by Sirotkirr K! Davidson ( 198%). Probe A which covers the 5’ rind of gene ;i’ (WY ttelow) was used t,o study it)s t*xprension during development (Fig. 2). A poly(A)’ mK,N=\ of about 1075 nuclrot~ides was detec%ed during midembryogenesis and early pupation. ‘I’t.~\ns~rif,tion itt the embryos starts about seven hours nft’er egglaying at 24°C’ and reaches a maximum at, 10 t,o 12 hours. The amount’ of mK?iA then decreases and is barely detectable during larval stages. l’rarrscription resumes in late t)hird instar attd is maximal in early pupae. The abundance of gene S rnRNA is then sitnilar to that of’ hsp26 or 27 transcripts. Like t’hr other small hsp RNAs (Masott rt al., 1984). it, diminishes during t,hc pupal period. X.0 t’ranscripts were found in adults. either freshI> eclosed or one week old. Tf a heat shock at WY is performed at arty stagtk of development 1 except (aarl?; embryogenesis which is refract’ory t,o heat shock (Zimmerman ut al.. 1983), a great,er amount of ~VIIP 3 rnRSA is detected. Figure 3(a) shows t,wo selected examples. Two larger transcripts of 2300 and 1600 nuc1eotidr.s are also found in heat -shocked samples. Thesth

Small

EMB 12-18 h \-

c

HS

(a)

Early pupae c

HS

Early pupae 7-z

(b)

Heat Shock Gene Expressrd

SL3 I C

HS HS I 2

\

(c)

Figure 3. Heat shock induction. (a) Poly(A)+ RSA (I ~g/latw) ot’ wtrtrol (C’) or heat shock (HS; 36°C fat 4.5 mitt) rtnbryos (ESIH. 12 to 18 h) and early pupw wtw analywtl by Northern blotting using probe A. (b) As (a). hut using probe I% which is loc~alized downstream from thr tttajor tranwriI)t (ser Fig. 1). (c) Xortliwri blot of total RNA (IO &lane) extracted from Schneider line 3 (Sh3) tissue c*ultur’e c*rlls and yobed with yobe A. Crlls \+tw eit,hrr kr[,t at, 25 ‘(‘ (C‘) or heat-shocked at, 36°C for 1 (HSl) ot 2 (HS,“) ti.

mRKAs are drtwted w-it h probes situated downstreatn from the major transcription unit (Fig. 3(b)). but not, with upstream probes. We c&mate their abundance to be about 1 to 2 ?;G of that of’ the matjor t rarwript Similar length heterogt~twit iw have Iwrt found for hsp23. 26 and 27 trtR,XA synt hrsizrd during heat shock in Drosophila tissue wlturr wils (Kerger rt al.. 1985). M’r found an optimal tcJmperatSurc of induction of 35”(‘. similar to that of the small hsp pcw+s (dat’a not showtlJ. Thr, t~xprwsio~i of ptwr ~7 was also invrstigatrd in Droso~hil~~ tissue cwlturr ~~41s (Schneider line 3). As sh0wr1 in Figrtrts 3((n). tlw basal level of t,ranscription in CWII~rol cells is inweawd by hcwt shock. ‘I%~ I'itrP Iargw m RNA found in stressed organisms ;tr~ also prrsrnt in heat -shock4 tissue wltuw wlls.

in, Embryos

237

EMB

‘C

Late 0uDae

Early Duoae

14-18 h HS’

‘C

HS’

‘C

HS’

(a ) hsp27 9 G -

‘C

Gene/

M-SIIP-LLHLARELDH M-*LST-**S*VD**QE MAN*PL-**S**DD*GMA*LPMFWRM*E*MAM-*L**FI*,,**E**H-

Gene3

M-PD**FV*N*-DSP*S

- 404

hsp27 hsp26 hsp23 hsp22 Gene/ CC-Cry

VGKDG-FQVCMDVSQFKPN * * * * * -*******A****S I************H***S *N***-YKLTL**KDY--S *NRN*-***S*N*K**AA* LE**R*S*NLN*KH*S*E

- 309

Gene 3

SN*Q*N*E*HL**GL*Q*G

hsp26 hsp23 hsp22

HS’

622- 622 527 (d3&n

527-

( b)

hsp27 hsp26 hsp23 ha22 (b)

(a)

Figure 5. lIetermination of the transcription initiat,ion site. (a) Primer extension and (b) S, mqping were f)erformed a,s described in Materials and Methods. Srr the legend to Fig. 3 for abbreviations. Fragment, sizes are indicxated in base-pairs.

(c) l’r&in

coding

region

The putative polypeptide encoded by gene d has 169 amino acids and a calculated molecular weight of 18,792. Tt’ is a basic polypeptide (19.5q;I hasi( and loo/;, acidic amino acids) rich in histidintx (11.296). A comparision with the four small hsp‘s and gene 1 reveals two homolo;;ods regions, both at’ the nucleotide and amino acid ievels, localized at similar positions tIo where homologies have ~MW described for the small hsp’s and gene 2 (Tngolia, & d nl.. 1983; Aymr Nr (%a&, 1982; Southgate TissiBres, 1985): amino ad residues (1) The first, 15 N-terminal constitute a small hydrophobic region which was noted to share a weak resemblance with some signal peptides and transmemhrane domains (131ohel k I)obherst,ein, 1975: Ovchinnikov it nl., 1979: Southgate et nl., 1983). The best homology (36O,,,) is found wit,h hsp27 (Fig. 6(a)): (2) A stretch of 83 amino acid residues near t’he (’ terminus is homologous bet’ween the small hsp’s and the mammalian a-csrystalline R2 chain. In thcx case of the gene
4. Discussion Resides localized

their heat, inducibility. at, the chromosomal

the seven genes IOCWS 67K of

Gene/. a-cry Gene3

hsp27 hsp26 hsp23 hsp22 Gene/ CC-Cry

EREDGHGMI -QRHFVRKYT **Q*D**H* -)f*****R*K k** *D* *Fb-T** * * *R*.\ QQ*AEQ*GYSS***LGR*V *K*****v*-s***I***I **Q*E**F* -S*E*H***R

Gene3

*+thhD**HV-S****PAVS

hsp27 hsp26 hsp23 hsp22 Gene/ CL-Cry

LPKGFDPN----EVVSTVS V*D*YKAE----Q***QL* **P*YEA,,----K*A**L* **D*YEAD----K*S*SL* h***yh'**----**H**L* I*ADV**L----AIT*SL*

Gene 3

AAQ*VRFGCHCFHF*GGW*

hsp27 hsp26 hsp23 hsp22 Gene/ a-cry

SDG-VLTLKAPPPPS * * * -***VSI*K*QA * * * -***I*V*K**A D**-***ISV*N**G * * * -I**V***Q*LP ***-***VNG*RKQA

Gene3

*QTHGS*ISF-QGGA

Figure 6. Homologies in the protein coding regions between t,hr 4 small hsp’s. genes 1, .? and thr bovine r-crystalline 132 chain. Xst,erisks represent residues identical t.o that of the hsp27 amino acid seyurncr which is taken as reference. Some gaps ( -) havr heen intjrodured for a better alignment. Data are from Southgat,c, rt al. (1983) for hsp27. 26, P3 and 22, Ayme OzTissigrrs (198.5) for gene I. and Van der Ouderaa P/ ul. (1973) for thr r-crystalline. (a) First 15 N;-t,rrminal residut~s. (1)) (:rnt’ral homology from amino aAd 85 to 167 iti hsp17. X4 t’o 166 in hspP6. 66 to 148 in hsp23, 59 to 111 in hspld. I22 to PO4 in gene I. 70 to 152 in z-crystallint, and 75 to I62 in gene 3. Boxes show 2 prominent hydrophobic and hydrophilic peaks. Il. mrhnognxtrr show a csomplex pattrrn of’ stage during normal and tissue-specific expression tlrvelopmrnt (Sirotkin & Davidson. 1982; Zimmerman et al., 1983; lMason et al., 1984; Ayme & TissiGres, 1985; Glaser et (II.. 1986). All a,re rxprexsetl at vrrv different levels during the heginning of

Small Neat Shock Gene Expressed

Table 1 Homology

between the small hsp’s. gu&es 1. 3 an,d a-crystalline

hsp% hsp27 hsplfi hsp”3

hspl2 (he (:enr

I .‘:

66 (7X)

hsp%Y hspl:! 70(78) 72 (78)

45(44) 51 (46) 57 (46)

Z-PrW Grnc I (kne 3 tallinr 67 (64) 53 (54) 54 (52) 40 (36)

43 46 41 31 37

(62) (62) (56) (4%) (52)

48(50) 53 (56) 48 (52) 39 (40) 46 (44) 35 (50)

The perwntages of homology at the amino acid level are givrn for thr entir, 83 amino acid stretch conserved with the bovine a-cryxtallinr and. in pwenthrses, for the first 50 of thesv rrsidurs. pupation. hspPR and gene 1 are also transcribed in young adults (Mason et al., 1984: Ayme & TisGres. 1985). hsp26 and 27 genes are expressed in the ovaries of mature females; their transcripts are then transported i&o the oocytes where they remain during the first four hours of embryogenesis (Zimmerman et nl.. 1983). Our results extend the transcriptional complexit’y of locus 67K. We find that gene 3 has two peaks of expression during development: in mid-embryogenesis with a maximum at 10 to 12 hours after egg laying and in the beginning of pupation. With the exception of the hsp83 gene. whose expression is almos;t const.ant during all of development (Mason et al.. 1984), it is the first example of the transcription of a heat shock qene during Drosophila embryogenesis. The rxpressmn of gene 3 in early pupae has been reported (Sirotkin $ Davidson. 1982: Ayme 8; Tissitres. 1985). The abundance of its R’XA at this stage is approximat+y similar to that of hsp26 or 27 trans(3ript.s. After a heat shock at 36°C‘. the amount of gene 3 transcript is increased at at1 developmental stages except early embryogenesis, which is refractory to heat shock (Zimmerman rt tel., 1983). In addition to the major transcript present during development. smsll amounts of two larger RKAs arc’ then d&rc+ed. Their analysis reveals that they represent alternate terminations of the major mRS.\. Similar< 3’ length heterogeneity has been described for hsp23, 26 and 27 genes (Kerger it nl.. 19%). We also investigated the expression of gene 3 in /)rosophiln tissue culture cells (Schneider line 3) and found that its basal t’ranscription level is increased by heat shocak. The result is in disagreement with a tjrrvious rel)ort of Ireland rt al. (1982) who did not detect itiiy gs(lne -3 exprtGon in Schneider line 3 caells. It is possible that our use of more sensitive sing&t randtad probes explains this discarepancy. The f)NX squt:n&lg dat,a reveals several similarities t)t~tween gene .S and the small hsp genes. \VV notca the present+’ of’ a single initiation site 01’ 1ranscaript ion. the itt)seric~c~ of introns and a long .I + l’-ricati 5’ untranslatrd lradrr sequence, Two ht rclt r.hes tromcllt,p~,u,s t,) heat shoc+k tqula,tor?-

in Embryos

239

element,s are present in t’he first 130 base-pairs upst,ream from the CAP site. A third one is found in t,he leader. The fact that they have only seven matches out of ten to the consensus CTnCrAAnnTTCnAG (Pelham, 1982, 1985; Hienz. 1985) could explain the poor heat inducibility of gene ,P. Homologies between the four small hsp’s, genes I and 3 were found in their putative coding regions. First. they share a weak homology along their 15 IVt,erminaf amino acids. These hydrophobic* domains have also a faint resemblance with the signal peptide of the human pro-insulin and the transmembrane region of bacteriorhodopsin (see Sout,hgate it al.. 1983). Similar hydrophobic N-t,erminal st rrtvhes have brrn reported in small hsp‘s from other organisms including soybean (Schiifl rt al.. 19X1), Caenorh,ahditis elegans (Russnak B Candido, 1985: Jones et a/.. 1986) and humans (Hickey et al.. 1986). Second. close to the C’ terminus. the four small hsp and gene 2 proteins share a region of 83 amino acids. \vhich is also conserved in bovine x-crystalline (Ayme $ TissiBres. 1985). \Vit,h the put,ative gene 3 polypeptide, an homology of 50 t,o 600,, is restricted t,o the first 50 residues. Srverthelesss. t,he two most conserved regions, a hydrophobic peak followed by a large hydrophilic stretch (boxes in Fig. 6) are present in the gene :I polvpeptide. 20 heat-inducible genes have been identified on the left nor on the right of the seven heat shock genes at the 67R locus (Ireland rf 01.. 1982; our unpublished results). Since gene % shows no homology with the small hsp’s (11. Pauli, (‘.-H. Tonka d A. *Lyme, unpublished results), our st)udy complet,es the structural characterization of heat shock genes located at the 6713 locus and having a homology with mammalian cc-crystalline. The protein product of gene 3 has not yet been identified. Its calculated molecular weight of 18.792 suggests a protein similar in size to hsp22 (19.705). One rea,son why this protein has not been observed so far could be that it’s rnRNA is not preferentially translated at ele\-atrd temperature. The sryuenc*rs responsible for rffic.ient translation have been lowlizrtl in the first 20 trwnsc.ribrd nuc~leotides (Hult mark it rrl., 19X6) w hrw hsp70 and the small hsp gc~t~es sh;lre homologies whic.h are lveakrr in gc’tlrk .3. The protluc~tioi~ of’ antibodies t’rvnt polyf)eptities t~xt)re’s& in bac%etia mav help to iclent ifv the genr 3 fwotvin. \V? thanli 21. Tissitrrs for his itltrrwt and t,nc~ouragemriit throughout this work: 0. .lcxnni and Y. Del&to for preparing t,hr Figures: P. Spirrrr. .I Vazquez anal Ii I)eLotto for t,heir o-itival reading of’ thr manusc-ript: T,. Kauvar and T. Korntwy for the c-1)S.A litwary. This work was supportrti 1)~.thr, Swiss Satiorral Sc,iv,,c.,l Foundaticbn.

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