The genes for silk fibroin in Bombyx mori

J. Mol. Biol. (1972) 70, 637-649

The Genes for Silk Fibroin in Bumbyx mori YOSHIAIU SCZUKI,~

L. PATRICK GACIE$ AXD DOXAT.,D D. BROWN

Department of Embryology, Carnegie Institution Baltimore, Md. 21210, U.S.A.

of Washington

(Received 6 March 1972) The genes for the protein silk fibroin were quantitatod by hybridizaton of purified fibroin messenger RNA with the DNA from scveral tissues of the silkworm BO~P&JX nun-i. Recauso of its high G-(-C content, fibroin DNA has a highcr buoyant density in CsCl than ribosomal DNA or main band DSA. Interaction of B. mm-i DNA wit,h Ag+ effected an even better separation of fibroin genes in Cs,SO, centrifugation; the fibroin gonos bind loss Ag+ than rDNA or bulk DNA and thorcforc have a lower density. The fibroin mRXA which hybridized to B. tori DNA of high C-1-C content hsd the same unusual bssc composition as the input mRNA and the same characteristic oligonuoleotide profile after RNasc T, digestion. The relative abundance of DNA nuclcotido sequonccs complementary to fibroin mRNA wn.s t.ho same in DNA from tho animal’s cam=, the middle silk gland or the posterior silk gland whcro fibroin is synthosizod in W&O. This f&ding rules out specific gene amplification as an explanation for the specialized synthesis of fibroin by posterior silk gland cells. Hybridization saturation experiments showed that O-OO22o/o of B. naori DX,4 is homologous to silk fibroin mRNA. From this value, the estimated molecular weight of fibroin mRNA (2 to 4 x lOa daltons) and the approximate size of the B. mori haploid genome (0.5 pg DNA) we calcu1at.c that thcro arc bctwoon ono and thrco fibroin gcnos per haploid complement of DNA. In a period of only 3 to 4 days each fibroin gene gives rise to 10’ molecules of n-&KA which in turn produoc lo9 molecules of silk fibroin. This “amplification” must be the result of transcriptional and translational controls including tho efficient ut.ilization of a stable messenger RNA.

1. Introduction One mechanism which has been proposed for the regulation of specific genes is their selective replicat,ion in those cells in which they arc expressed (Schultz, 1965; Brown & Dawid, 1968). Specific amplifiaat.ion of t,he rihosomnl Ri\‘A genes is known to occur in of DNA in Drosophilu oocytes (Brown & Dawid, 1968; Gall, 1968) and polytenization is restrict,cd to replica.tion of the euchromatic portion of the genome (Gall, Cohon $ Polan, 1971; Dickson, Boyd & Laird, 1971). The important question remains, however, whether genes coding for spccializcd proteins arc amplified specifically in the cells

which express these genes. The

1JoStCriOr

silk

gla.nd

of the silkworm

~OWZ&JX nlori

presents

itself

as an ideal

t Yroeont adciross: National Tnstitutc of Houlth of Japan, Kemioseki, Shinagawn-ku, Tokyo 141, Japan. $ Present address: Department of Cell Biology. Roohe Institute of Molecular Biology, Nutley. N.J. 07110, U.S.A. 637

638

Y.

SUZUKI,

L.

P. GAGE

AND

D. D. BROWN

tissue with which to answer this question. The gland which consists of a single cell type undergoes extensive DNA synthesis during its differentiation without concomitant cell division-a process termed polyploidization. During the first 3 to 4 days of the last larval instar there is about a four- to tenfold increase in DNA content so that each cell contains as much as 0.17 pg of DNA (Gillot & Daillie, 1968; Tashiro, Morimoto, Matsuura & Nagata, 1968), which we estimate to be between lo5 to lo6 times more DNA than a diploid cell. Soon after DNA synthesis has stopped the posterior gland cells change from synthesizing a variety of proteins to the synthesis of a single protein -silk fibroin (about 300 pg/cell). This sequence of cessation of DNA synthesis followed by synthesis of specific protein(s) is characteristic of many differentiated cells. One explanation for this sequence of events could be that the specific genes to be expressed are amplified just before their expression. The silk fibroin system, besides being an exaggerated example of cell specialization, has other features which make the analysis of the fibroin genes possible. The messenger RNA (mRNA) for fibroin can be labeled to high specific activity with 32P04 and it has been purified and characterized by partial sequence analysis (Suzuki & Brown, 1972). Fibroin consists primarily of alternating glycine and alanine residues (Lucas, Shaw & Smith, 1957,1962; Lucas & Rudall, 1968). The major codons for these ammo acids in the mRNA have been shown to be GGU and GGA for glycine and GCU for alanine (Suzuki & Brown, 1972). Therefore, the mRNA and the fibroin genes have an unusually high G + C content (60%). This fact has made it possible to separate the fibroin genes from the bulk of B. tori DNA by buoyant density oentrifugation. This partial purification of the fibroin genes enhances the specificity with which they can be detected by hybridization. We report here the identification and quantitation of the hybrid formed between fibroin mRNA and B. mori DNA. The abundance of DNA sequences complementary to fibroin mRNA has been measured in the DNA isolated from three tissues, one of which is the posterior silk gland which synthesizes fibroin in viva.

2. Materials and Methods (a) Pur$cation of jbroin 32P-labeled mRNA The methods of raising B. mori larvae, and in viva labeling, purification,

and identification of fibroin 32P-labeled mRNA have been published (Suzuki & Brown, 1972). Purification begins with 2 cycles of sucrose gradient fractionation of phenol-extracted whole cell RNA (Fig. l(a) and (b)). The mRNA sediments between 45 and 65 s, usually as one, but sometimes as two, broad peaks. The mRNA within these two peaks has the same base oligonucleotide profiles after composition (60% G+C with 40% G) and yields identical RNase T1 digestion. When these two RNA’s were denatured and centrifuged in a sucrose gradient containing 70% formamide they sedimented at the same rate, about 32 s (Fig. l(o)), suggesting that at least some of the heterogeneity seen in the sucrose gradients was due to aggregation of the mRNA. The final conditions for the formamide fractionation were as follows: the [32P]RNA collected by ethanol precipitation from the second sucrose gradient centrifugation was dissolved in 70% formamide containing 3 mu-EDTA and 3 mMTris, pH 7.4, incubated at 35°C for 30 to 60 min and then centrifuged at 25°C in a The mRNA preparation sucrose gradient (4.6 to 22% w/w) p re p ared in the same solution. exhibited no hyperohromioity above 20°C in this solution, which indicated that it was denatured completely by the formamide. The 3H-labeled rRNA of Xerzopm Zaetis was purified from tissue culture cells (Brown & Weber, 1968a). 3H-labeled rRNA was purified from whole silk glands of B. mori. Larvae at the second day of the fifth instar were injected with 1 mCi each of [3H]uridine and the

SILK

FIBROIN

GENES

FROM

639

MORI

BOdiZBYX

(b) Fraction no.

(a)

FIG. 1. Purification of fibroin 32P-labeled mRNA from the posterior silk gland of B. mod Larvae at the fifth or sixth day of the fifth instar were injected twice with 1 mCi of 3aP0, and ), optical density at 260 nm. RNA was prepared one day later. (--O-O-), Cts/min; ((a) First-round sucrose gradient centrifugation. Total [3aP]RNA was centrifuged in a 15 to 30% sucrose gradient for 11 hr at 22,000 rev./mm and 25°C in a Spinco SW27 rotor. The RNA designated by the bracket was pooled for a second gradient. (b) Second-round sucrose centrifugation. The pooled RNA was centrifuged in a 15 to 30% sucrose gradient for 6,5 hr at 26,000 rev./mm and 25°C in a Spinco SW27 rotor. About 150 pg of X. Zaevia rRNA was added as a marker. The rapidly sedimenting absorbance peak is due to fibroin mRNA. The RNA within the bracket was pooled for the formamide/sucrose gradient step. (c) Fractionation of pooled fibroin mRNA from (b) on a 70% formamide, 4.6 to 22% sucrose gradient for 20 hr at 39,000 rev./min in an SW41 rotor at 25°C. About 150 pg of intact X. Zaewis rRNA was added as the optical density marker. The bracketed [3ZP]RNA was pooled for hybridization studies.

extracted 24 hr later. The radioactive 18 s and 28 s rRNA were purified by a single sucrose gradient centrifugation and the 2 RNA’s combined. Non-radioactive total RNA consisting primarily of ribosomal RNA was purified from carcasses of B. mori and also from ovaries of X. Zae& (Brown & Weber, 19685). (b) Extra&ion and fractionation of DNA Posterior and middle silk gland DNA was prepared by Donnce homogenization of the dissected mature glands in 1 to 2 ml./gland of SSC/Tris (0.15 M-N&~, 0.015 M-sodium

RNA

citrate,

O-05 M-Tris,

pH 7.5) and 0.5%

sodium

incubated at 37°C for 1 hr with 1 mg pronaselml.,

dodecyl

sulfate.

The homogenate

was then

which had been predigested for 30 min at

37°C. After 2 phenol extractions, NaCl was added to 0.3 M and the DNA was precipitated with one vol. of ethanol. The DNA was dissolved in SSC/Tris, particulate glycogen was

pelleted by centrifugation at 40,000 rev./min for 30 min and the solution was incubated at 37°C for 3 hr with 50 rg pancreatic RNase/ml. and 25 units T, RNase/ml. (each heated before use at 80% for 10 min). Following an additional digestion for 1 hr with 0.5 mg pronase/ml. in the presence of 0.5% sodium dodecyl sulfate the DNA was freed of protem by 2 phenol extractions followed by 3 ethanol precipitations and then stored at 4°C in 0.1 x ssc. DNA was prepared in the same way from carcasses (mature larvae from which the silk gland and gut were removed) which had first been shredded in a Sorvall Omnimixer in SSC/Tris. This procedure yields DNA of about 30 million daltons, as determined by analytical band sedimentation (Studier, 1965). DNA preparations from all 3 tissues were sheared to about 2.5 X lo6 daltons at full speed in the Sorvall Omnimixer in SSC/Tris before centrifugation in CsCl or CssS04. Centrifugation was carried out for 2 to 3 days at 25°C in

either the no. 65 fixed-angle

rotor (5 n&/tube)

or the no. 50.1 rotor

33,000 rev./mm. Silver ion was complexed with DNA (0,138 pg Ag&SO,/pg DNA) followed by equilibrium

Davidson, 42

1966; Brown, Wensink & Jordan, 1971).

in the ratio

(20 ml./tube)

of 0.3 Ag/mole

centrifugation

at

DNA-PO,

in Cs,SO* (Jensen &

640

Y. SUZUKI,

L. P. GAGE

AND

D. D. BROWN

The gradients were fractionated and the optical density recorded automatically with a Gilford spectrophotometer. The DNA in each fraction was denatured with alkali, neutralized and adsorbed individually to a Millipore HA filter. The quantity of DNA bound to the filters was determined after hybridization by hydrolyzing the DNA with 1 N-HCl and measuring the absorbance which was released into the supcrnatant (Brown & Webor, 1963~). (c) Hybridization of RNA with $ilter-bound DNA and charactekation of the hybrid8 Radioactive RNA was hybridizod with DNA filters in 4 x SSC, 0% Jr-Tris (pH 75) and 50% formamide (McConoughy, Laird & McCarthy, 1969) at 5O’C for 16 to 48 hr. The filters were washod extensively with 2 x SSC, incubated with pancreatic RNaso (60 &ml.) for 30 to 60 min in 2 x SSC, washed further with 2 x SSC, dried and counted in toluene fluor in a scintillation spectrometer. When hybridized RNA was collected for partial sequence analysis (experiment shown in Fig. 5) the pancreatic RNa.se step was omitt.ed. RNA was eluted from hybrids by heating the filters at IOO’C for 6 min in 0.1 x SSC. The base composition of 32P-labeled mRNA was determined from alkaline hydrolysatcs by paper electrophoresis (Markham & Smith, 1952). Oligonucleot.ides from exhaustive RNaso T, digests were separated according to chain length by DEAE-Sephadox A25 fractionation. Details of both methods and the baso composition and oligonucleotide pattcrn obtained for fibroin mRNA haye bcon published (Suzuki & Brown, 1972).

3. Results (a,) Pur$im-?ion of thefibroin messenger RNA for hybridization Fibroin 32P-labeIed mRNA purified by two sucrose gradient fractionations has been estimated to be greater t,han 80% pure (Suzuki 6 Brown, 1972). In an initial hybridization experiment, such a preparation was mixed with X. Levis “H-labeled rRNA and incubated with filter-bound DNA which had been isolated from mature posterior silk glands and fractionated in a CsCl gradient (Fig. 2). Prefractionation of the DNA according to buoyant density should have separated the fibroin genes (60% G + C) from bulk B. mori DNA (39% G + C) and the genes for rRSA (48% G + C) (Suzuki & Brown, 1972). X. laevis rRNA hybridizes with B. mori rRNA genes, marking their position in the gradient. In this experiment+ [32P]R,KA hybridized coincidentally wit,h 3H-labeled rRxA across the gradient and t,he [32P]RNA isolated from the peak hybrid fraction (no. 8) had essentially the same base composition as rRKA. The contamination of the 32P-labeled mRNA wit.h labeled rRNA was confirmed by demonstrating that unlabeled carcass rRKA competed for tho hybridization of most of the [asP]RNA (Fig. 2). It was presumed that the small amount of [32P]RNA which hybridized in the presence of the competitor might be fibroin mRNA-at least the part which a.ssociated with high-density D’NA. The domination of the hybridization reaction by a small amount of 32P-labeled rRNA contaminant indicated t,hat the genes for rRNA are at least an order of magnitude more abundant in B. mori DNA than is fibroin DNA. Further purification of the 32P-labeled mR?\TA preparations was necessary. The RNA was denatured in formamide and sedimented through a sucrose gradient containing ‘70% formamide. This met,hod is particularly useful for removing rRNA contamination, since most 40 s and 28 s rRNA molecules of B. wi have specific cleavage points which cause these molecules to dissociate during denaturation into fragments which sediment at the rate of 18 s RNA. The same observation has been made for the 28 s rRNA from Hydophora cecropia (Applebaum, Epstein & Wyatt, 1966). Denatured fibroin mRNA sediments as an heterogeneous component with a peak at about

SILK

FIBROIN

GENES

FROM

BOMBYX

i%fORI

641

3000

600

0

0 2

IO

6 Fraction

no.

FIG. 2. Hybridization of CsCl-fractionated B. tori DNA with ctn impure fibroin mRNA prepemtion. The fibroin mRNA ww purified through 2 sucrose gradients [Fig. 1 (b). Two tubes each containing 200 pg of sheared posterior silk gland DNA in CsCl (ne = 1.4005) were centrifuged to equilibrium at 33,000 rev./min in the no. 65 Spinco rotor. The filter-bound DNA from each fraction (3 x lo* cts/min/rg) end of one gradient was hybridized with 1.5 pg fibroin 32P-labeled mRNA/ml. 2 pg X. Zaevis 3H-labeled rRNA/ml. (3.5 X lo5 cts/min/pg). The RNA mixture for the hybridization with DNA of the second gradient w&s the same with the addition of 780 pg unlabeled c&mess saP (cts/min), uncompeted; --O--O--, “H (&s/mm), unRNA/ml. 88 competitor. -a-@--, optical density at 260 mn. s2P (cts/min). competed; (-), competed ; -a-A-,

32 s (Fig. l(c)). The small radioactive peak at 18 s which had a base composition like rRNA attests to the release of 32P-labeled rRNA fragments. The 3aP-ls,beled mRNA which sedimented faster than intact X. laevis 28 s rRNA in formamide (Fig. l(c)) was mixed with 3H-labeled rRNA of X. Zaevisand hybridized to CsCl-fractionated DNA from carcasses (Fig. 3(a)). The formamide step greatly reduced the extent of C3”P]RNA contamination. Hybrids of [3aP]RNA with DNA of high buoyant density were revealed which we tentatively identified as hybrids of iibroin mRNA with its genes. In order to identify unequivocally and to quantitate the very low level hybridization of fibroin mRNA, we sought a technique by which larger amounts of DNA could be frectionated with a greater density separation of fibroin genes from rDNA and bulk DNA. Both criteria were met by complexing the DNA with Ag+ followed by centrifug&ion in Cs,SO, (Jensen & Davidson, 1966). Fibroin genes bind less Ag+ than does rDNA which binds less Ag+ than bulk B. tori DNA. Consequently, their relative buoyant densities are inverted so that the fibroin genes are the least dense of the three kinds of DNA. This is demonstrated by hybridization of formamide-purified 32Plabeled mRNA and 3H-labeled rRNA with DNA fractionated by Ag+/Cs,SO, (Fig. 3(b)). The hybridizations shown in Figure 3(a) and (b) can be compared directly because they were carried out together in one vessel and because the same sheared DNA preparation was used for each. Besides reducing the overlap of rRNA and mRNA hybridization, Ag+/Cs,SO, centrifugation has the further advantage that 100 pg DNA/ml. can be centrifuged in each tube of the 50.1 Spinco rotor (2 mg/tube) without affecting the separation of the fibroin genes. (b) IdentiJcation of the Jibroin messengerRNA-$broin gene hybrid Only small amounts of RNA from a fibroin mRNA preparation actually hybridized with G + C rich DNA. Consequently, it was necessary to show that the RNA in the

642

Y.

SUZUKI,

L.

P. GAGE

AND

D. D. BROWN

Fraction no Fra. 3. Hybridization of formerm ‘de-purified fibroid mRNA with B. mori DNA which had been fractionated in (a) C&l or (b) Ag+ /C&SO+. In each ease, 2 mg of sheared oarc&s8 DNA w&9 centrifuged to equilibrium in the 60.1 Spinco rotor. The immobilized DNA fractions were hybridized together with 0.6 pg 3aP-labeled mRNA/ml. (3.2 x 104 ots/min/~g) and 0.6 pg X. Zaewis 3H-labeled rRNA/ml. -a-•-,33P (ots/min); -- O-- O--, 3H (cts/min); (-), optical density at 260 nm.

hybrids was identical to the input RNA. For this purpose, about 8.5 mg of B. mori DNA was complexed with Ag+ and banded in CssSO,. The DNA pooled from the light side of the gradient was centrifuged again in C&30,, immobilized on filters and hybridized with 32P-labeled mRNA, 3H-labeled rRNA and an excess of unlabeled carcass RNA to dilute 32P-labeled rRNA contaminants (Fig. 4). Essentially all of the [““PIRNA which hybridized did so with a DNA which banded to the light side of the rDNA. The [32P]RNA which was hybridized with DNA of fractions 20 and 21 was melted from the hybrid and its base composition and RNase T, oligonucleotide profile were examined. The base composition (Table 1) revealed the characteristic high G + C content of fibroin mRNA. A more definitive characterization of the RNA which had been recovered from the hybrid was made by digestion with RNase T, and fractionation of the resulting oligonucleotides on DEAE-Sephadex. A large pentanucleotide fraction with only traces of tetra- and hexanucleotide was obtained (Fig. 5) ; this is the characteristic oligonucleotide profile of fibroin mRNA (Suzuki & Brown, 1972). In contrast, RNase T, digests of rRNA from B. tori contain abundant tetra- and hexanucleotides. Consequently, the low level of E3”P]RNA which hybridizes to the DNA of low buoyant density in Ag +/Cs,S04 is fibroin mRNA. (o) Relative abundance of Jibroin genes ilz DNA from the posterior and middle silk glands and the carcass The amount of fibroin genes in the DNA from three tissues of B. mori was compared by the extent to which these DNA’s hybridized to fibroin mRNA. DNA was isolated

SILK

FIBROIN

GENES

rj

FROM

20

BOMBYX

MORI

643

25

Fraction no.

Fro. 4. The enrichment of fibroin genes. About 6.6 mg of c8r0889 DNA WBBcomplexed with silver 8nd banded in CssSO, in 4 tubes of the 60.1 Spinco rotor. The DNA from the light side of eech gmdient w8s pooled (0.96 mg) 8nd recentrifuged in 8 single tube and the DNA in e8ch fraction 8cros8 the gmdient wa9 immobilized on 8 filter. The fmctionated DNA ~8s hybridized with 0.7 H form8midcpurifled 3aP-18beled mRNA/ml. (2.2 x lo4 cts/min/crg), 0.8 pg X. Zaevia sH-labeled rRNA/ml. (3.5 x lo6 cts/min/pg) and 136 ry unlabeled c8rc8ss RNA/ml. -@-•---, 3aP (cts/min); -- 0 -- 0 --, sH (cts/min).

TABLE 1 The base compositions of 32P-lubeled mRNA and of [32P]Rh7A recoveredfrom the hybrid 2’, 3’ Nucleotides

s2P-labeled mRNA hybridization

U G A C G+C t The [s2P)RNA which hybridized gradient shown in Fig. 4.

before

22.7 42,2 17.0 18.1 60.3 with

the DNA

[=I’]RNA recovered from the hybridt 22.1 42.1 14.9 16.3 68.4

of fmctions

20 end 21 of the Ag+/C!s2SOI

from the highly polyploid cells of the posterior silk gland late in the fifth instar when fibroin synthesis had begun and, for comparison, also from the polyploid middle silk gland and from the animal’s carcass. Individual carcass cells contain orders of magnitude less DNA than the polyploid cells of the silk gland. It is not known whether carcass tolls are polyploid but to a lesser extent than silk gland cells. Similar amounts of each DNA were complexed with silver, centrifuged to equilibrium in Cs,SO,, and the DNA in fractions from the light side of the gradients known to cont.ain the fibroin genes was immobilized on filters. The three DNA filter sets were hybridized together in the same vessel with 32P-labeled mRNA, 3H-labeled rRNA of B. mori and excess unlabeled carcass RNA. It is evident in Figure 6 that a similar quantity of radioactive fibroin mRNA hybridized with each DNA. The 32P-labeled mRNA which bound to each of the three DNA’s was summed. The amount of 3H-labeled rRNA which hybridized to each filter assessed the extent to which fibroin genes and rDNA overlapped

644

Y.

SUZUKI,

L.

P. GAGE

AND

D. D. BROWN

0

60 Fraction

90 no.

FIU. 5. DEAE-Sephadex fraction of 3aP.labeled oligonucleotides from a T, RNase digest of szPlabeled mRNA which bad hybridized to B. mori DNA. The ra2P]RNA which hybridized with the DNA of fractions 20 and 21 in the experiment of Fig. 4 was recovered from the hybrid for base composition analysis (Table 1) and the hybridization was repeated with additional 32P-labeled mRNA. Three additional cycles of hybridization and melting of the hybrid were carried out. The RNA/DNA hybrids were not treated with pancreatic RNase. The pooled 32P-labeled mRNA from the hybrids containing 2500 cts/min was digested with RNase T1 plus 200 pg of carrier E. COG soluble RNA. --+-a---, 32P (&/mm) ; (), optical density at 260 nm; (---), NaCl concentration.

50

Posterior gland

Middle gland

Carcass

t

20

26

20 Fraction

26

20

26

no.

FIG. 6. Hybridization of fibroin mRNA with DNA from carcass, and from posterior and middle silk glands. DNA which had been sheared to about 2.5 x lOa daltons was complexed with silver, banded in Cs,S04 and immobilized on filters. Filters containing DNA from the fibroin gene regions of the 3 gradients (11 of 35 fractions) were hybridized together for 48 hr with 0.9 II& formamidepurified fibroin s2P-labeled mRNA/ml. (1.4 X lo4 cts/min/pg), 6.6 pg B. mori 3H-labeled rRNA/ml. and 130 pg unlabeled carcass RNA/ml. Pancreatic RNase-resistant hybrids were counted, and then the quantity of DNA bound to each filter was measured. Typically, about 60% of the DNA which was centrifuged was recovered from the filters after hybridization. In this experiment the fibroin genes from 550 pg of posterior gland DNA, 620 pg of middle gland DNA and 690 pg of carcass DNA --m--e--, 32P (cts/min); --Cl-Cl---, 3H (cts/min). were hybridized.

SILK

FIBROIN

0 2

GENES

02

82

FROM

82

BOMRYX

82

MORI

646

8’

Fraction no.

Fm. 7. Hybridization saturetion of B. mori posterior gland DNA with fibroin mRNA. Portiona of a sheared posterior gland DNA preparation were centrifuged in Ag + /CssS04, and the DNA in each fraction was immobilized on a filter for hybridization. Filters containing the fibroin gene regions were hybridized with about 130 pg unlabeled carcass RNA/ml., between 3 and 6 pg %labeled B. tori rRNA/ml. and the concentration of formamide-purified 3ZP-labeled mRNA in pg/ml. shown in each panel. The sZP-labeled mRNA had a specific activity of 1.3 x 164 cts/min/pg. --o-e---, azP (cts/min); --o--o--, sH (cts/min).

and permitted a correction for 32P-labeled rRNA hybridization. The amount of DNA which had been actually immobilized was measured after the filters had been counted. This experiment was repeated once and in both experiments the amount of 32Plabeled mRNA which hybridized with the three DNA’s was the same within + 10%. We conclude that there is no selective amplification of the DNA homologous to fibroin mRNA in posterior silk gland cells; the fibroin genes are replicated in proportion with the rest of the genome in these highly specialized and polyploid cells. (d) Saturation hybridization of B. mori DNA with 32P-labeled$broin mRNA The fraction of B. mori DNA homologous to fibroin mRNA was determined by a hybridization saturation experiment. Equal portions of a sheared preparation of B. mori posterior gland DNA were centrifuged in Ag +/C&SO, and then the DNA from the fibroin gene region of each gradient was immobilized on a filter set for hybridization. A different concentration of fibroin 32P-labeled mRNA was hybridized with each DNA filter set, along with 3H-labeled rRNA and non-radioactive carcass RNA (Fig. 7). The portion of [32P]RNA bound to each filter set which was true fibroin mRNA hybrid was determined by first subtracting from each fraction as background the radioactivity bound to fraction 9 and then correcting for hybridization of [32P]RNA whioh was not fibroin mRNA. The latter correction was carried out by assuming that all [32P]RNA bound to fraction 1 of each gradient was rRNA or DNA-like RNA. A second correction was made for the amount of [32P]RNA which was not mRNA that bound to each filter. It was assumed that the amount of 3H-labeled rRNA which bound to each filter measured the rDNA and bulk DNA contamination of each filter. By assuming that all [32P]RNA which bound to fraction 1 of each gradient was contaminant, the extent to which contaminating [32P]RNA hybridized to each successive filter (2 through 8) was calculated as a proportion of the [3H]RNA which hybridized to each filter. The amount of DNA actually immobilized on the filters was measured after the filters had been counted. The corrected amount of fibroin mRNA which hybridized

646

Y.

SUZUKI,

L.

P. GAGE

AND

D. D.

BROWN

32P- labeled mRNA (,@/ml.)

Fro. 8. &&ration curve for the hybridization of “aP-labeled mRNA with B. tori DNA. The amount of 3aP-labeled mRNA which hybridized to DNA at each concentr&ion of RNA was calculated from the experiment shown in Fig. 7 along with data from one other similar experiment not shown here.

to B. mori DNA is plotted versuS the concentration of RNA in the hybridization reaction shown in Figure 8. Only O*O022°/0of B. tori DNA hybridizes with fibroin mRNA.

4. Discussion (a) Detection of the Jibroin genes Fibroin mRNA prepared by sucrose gradient centrifugation hybridizes like an rRNA preparation (Fig. 2) although it is at least 80% pure (Suzuki & Brown, 1972). The small amount of contaminating rRNA dominates the hybridization because rDNA is almost two orders of magnitude more abundant than the DNA homologous with fibroin mRNA. B. mori rRNA hybridizes with 0.17% of B. mori DNA (Gage, unpublished results) while fibroin mRNA hybridizes to only 0.0022%. The rRNA contamination was reduced by fractionation of the mRNA under denaturing conditions, which leads to the dissociation of 28 s and 40 s rRNA into fragments which sediment at 18 s. Specificity of the hybridization between fibroin mRNA and its genes was enhanced by prefraotionating the DNA in density gradients. The fibroin genes, which were predicted to have an unusually high G + C content (60%) compared to rDNA (48%) and bulk DNA (38%) (Suzuki & Brown, 1972), have a higher buoyant density in CsCl (Fig. 3(a)). In addition, the fibroin genes bind less Ag+ and therefore band to the light side of the other DNA fractions in Cs,SO, (Fig. 3(b)). The RNA which hybridized with this unusual DNA fraction was identified as fibroin mRNA by its base composition (Table l), and its oligonucleotide profile after digestion with RNase T, (Fig, 5). The latter pattern is characterized by a large fraction of pentanuoleotides and small amounts of tetra- and hexanucleotides (Suzuki & Brown, 1972). Hybridizations were carried out at 12°C below the T, of bulk B. mori DNA and about 20°C below the T, predicted for the fibroin genes. These stringent conditions were designed to reduce the hybridization of any contaminating “DNA-like RNA” and to increase the probability of accurate base-pairing of fibroin mRNA with its genes. We deduce from the structure of 6broin itself and its mRNA (Suzuki & Brown, 1972) that the gene must be internally repetitive so that a mRNA molecule might hybridize in a number of poorly base-paired alignments with a single gene sequence, causing mismatching as well as free RNA ends not duplexed with DNA. The high criterion of hybridization was designed to reduce mismatching in the hybrid, and

SILK

FIBROIN

GENES FROM BOMBYX

MORI

647

RN&se treatment of the hybrid should have eliminated regions of the mRNA which were not duplexed. However, the accuracy with which hybrids were base-paired was not studied. (b) The genesfor silk$broin are not amplijed The concentration of sequences homologous to fibroin mRNA is the same in the DNA of the ctircass, of the highly polyploid cells of the middle silk gland, and of the equally polyploid posterior silk gland where fibroin is synthesized in viva (Fig. 6). Even though the ooncentrstion of mRNA in the incubation was just below saturrttion. the three filter sets of DNA were hybridized together. Determinations made under similar conditions with rRNA have indioated that the hybridization level is related linearly to the amount of homologous DNA when the DNA’s are immobilized to filters and hybridized together in the same reaction mixture (Brown & Weber, 1968a). Polyploidization of the silk gland yields cells with nearly a million-fold more DNA than is contained in a sperm nucleus, and yet the ratio of bulk DNA (or fibroin gene) to ceh mass remains virtually unchanged, because the celluIar DNA content and cell mass increase proportionally during larval growth (Tashiro et al., 1968). Replication of the fibroin genes in proportion with the bulk DNA is consistent with other analyses (Gage, 1971), which suggest that silk gland polyploidization occurs by multiple replications of all sequences in the B. wwri genome. (c) The number of$broin. genes in a haploid complement of DNA An accurate determination of the number of fibroin genes per genome depends upon three numbers (1) the genome size (2) the molecular weight of the mRNA and (3) the fraction of the genome homologous with fibroin mRNA. Preliminary measurement of the DNA content of individual B. mori sperm&ids by E. Rasoh (personal communication) has given a value of about 0.5 pg. Gage (unpublished results) has estimated a genome of about the same size by DNA renaturation kinetics. The size of fibroin mRNA is estimated by its rate of sedimentation relative to X. laevis 28 s and 18 s rRNA (l-5 and 0.7 x lo6 daltons, respectively; Loening, Jones & Birnstiel, 1969) in a formamide/sucrose gradient (Fig. l(c)). Although the mRNA does not sediment as an homogeneous component, thegradients indicate that heterogeneity extends toward the top of the tube. This would be expected if the highest molecular weight fraction was undegraded and the heterogeneity was due to degraded fragments of mRNA. The peak of mRNA in formamide gradients (Fig. l(c)) sediments at about 32 s which is roughly equivalent to a molecular weight of 2 x lo6 daltons. The most recent measurement of the major polypeptide component of fibroin gave a molecular weight of 3-O x lo5 daltons (Tashiro, Otsuki & Shimadzu, 1972). This polypeptide should be encoded by e mRNA molecule of about 4 x lo6 daltons. The hybridization saturation value of 0.0022 y0 is subject to several sources of error. Three separate experiments in which hybridization was carried out at ooncentrations of mRNA greater than 4 pg/ml. gave values within 15% of O-0022%. Errors introduced by internal repetitiveness of the mRNA and its homologous DNA have been discussed above. The amount of fibroin genes may have been underestimated by only scoring RNA which hybridized at the low buoyant density in Ag+/Cs,SO,. To minimize this error the DNA was sheared to a weight-average molecular weight of approximately half gene size (2.5 x lo6 daltons). As can be seen in Figure 3(b), at concentrations of less than 1 ,ugL&/ml. the 32P-labeled mRNA hybridized only very slightly with main band DNA, the main problem being 32P-labeled rRNA contamination.

648

Y.

SUZUKI,

L.

P. GAGE

AND

D. D. BROWN

Although none of the numbers used for this calculation are accurate enough yet to determine the exact number of fibroin genes, and hybridization has not been done with true germ-line DNA (sperm), we calculate from the above numbers that there are no more than three fibroin genes per haploid complement of DNA. If one strand of a single fibroin gene contained 4 x lo6 daltons of DNA this would~constitute about 0.002% of a haploid genome of 05 pg of DNA. (d) Transcriptional and translational “ampli$cation” for Jibroin synthesis A single cell in the posterior silk gland of B. mori synthesizes about 300 pg of fibroin in the last 4 days of the fifth instar (Tashiro et al., 1968). Each giant polyploid cell contains as much as O-2 pg of DNA (Tashiro et al., 1968; Gillot & Daillie, 1968) of which O*OO22o/ocodes for fibroin mRNA. Therefore, each cell contains about lo* fibroin genes whose expression results in the production of 1015 fibroin molecules. During this developmental period fibroin mRNA accumulates until it comprises about 1% of the cellular RNA or about lOlo molecules per cell (Suzuki & Brown, 1972). Since the mRNA is stable (Suzuki & Brown, 1972) each gene produces lo4 mRNA molecules each of whichin turn can translate on the average of lo5 protein molecules in about 4 days. This high translational yield can be achieved if each stable mRNA functions in a polysome containing 50 ribosomes and polymerizes amino acids at the rate of 15 per second per ribosome. In order to prepare for this remarkably efficient translational specialization, each cell need only transcribe its fibroin genes and stabilize the resultant mRNA with the same efhciency that it transcribes its ribosomal genes and stabilizes rRNA. This ean be concluded from the fact that the posterior gland accumulates fibroin mRNA andrRNA to about 1% and 85%, respectively, of its total RNA content, and the rDNA is about 80 times more abundant than the fibroin genes (0.17% and 0.0022% of the total DNA, respectively). The extent to which transcriptional control is exerted in a cell in which tlbroin is not synthesized has not yet been determined. However, the most impressive feat which the posterior gland cells can carry out is the stabilization of fibroin mRNA and the efficient programming of the majority of its ribosomes with the mRNA produced by only O-O022°h of its total DNA. We conclude that control of fibroin synthesis in the posterior silk gland does not involve specific amplification of fibroin genes. Specific gene amplification may be reserved for genes whose final products are RNA’s (such asrRNA). In these cases, the rate of synthesis is limited by the number of genes and by transcriptional controls. However, when a protein such as fibroin is the final product of a gene, instead of changing the number of genes, the range of synthetic rates caused by transcriptional controls is increased enormously by translation of a stable mRNA which has programmed a large fraction of the cell’s ribosomes. We thank Dr Keizo Hayashiya of the Kyoto University of Industrial Arts and Textile Fibres, Kyoto, Japan for kindly supplying artificial diet, and Dr James Vaughan of the Agricultural Research Center at Beltsville, Maryland, U.S.A. who provided US with silkworms several times. We also thank Mrs Etsuko Suzuki for her excellent technioal assistance, and Drs I. Dawid, T. Honjo, P. Lizardi, R. Reeder, K. Sugimoto and Mr R. Stern for critical readings of the manuscript. REFERENCES Applebaum, S. W., Epstein, R. P. & Wyatt, G. R. (1966). J. MoZ. Biol. 21, 29. Brown, D. D. & Dawid, I. B. (1968). Science, 160, 272.

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Brown, D. D. & Weber, C. S. (1968a). J. Mol. Biol. 34, 661. Brown, D. D. & Weber, C. S. (1968b). J. Mol. Btil. 34, 681. Brown, D. D., Wensink, P. C. & Jordan, E. (1971). Proc. Nat. Acad. Sci., Wash. 68, 3175. Dickson, E., Boyd, J. B. & Laird, C. D. (1971). J. MoZ. BioZ. 61, 615. Gage, L. P. (1971). Carnegie Inst. Wash. Year Book, 70, 39. Gall, J. G. (1968). Proc. Nat. Acud. Sci., Wash. 60, 553. Gall, J. G., Cohen, E. H. & Polan, M. L. (1971). Chromosome, 33, 319. Gillot, S. & Da&e, M. J. (1968). C. R. Acad. Sci. Paris, 266, 2295. Jensen, R. H. & Davidson, N. (1966). Biopolymers, 4, 17. Loening, U. E., Jones, K. & Birnstiel, M. L. (1969). J. Mol. BioZ. 45, 353. Lucas, F. & Rudall, K. M. (1968). In Comprehensive Biochemistry, vol. 26, part B, p. 475. New York: American Elsevier. Lucas, F., Shaw, J. T. B. & Smith, S. G. (1957). Biochem. J. 66, 468. Lucas, F., Shaw, J. T. B. & Smith, S. G. (1962). Biochem. J. 83, 164. McConaughy, B. L., Laird, C. D. & McCarthy, B. J. (1969). Biochemistry, 8, 3289. Markham, R. & Smith, J. D. (1952). Biochem. J. 52, 552. Schultz, J. (1965). In Brookhmen Symposium no. 18, Genetic Control of Differentiation, p. 116. New York; Upton. Studier, F. W. (1965). J. Mol. BioZ. 11, 373. Suzuki, Y. & Brown, D. D. (1972). J. Mol. BioZ. 63, 409. Tashiro, Y., Morimoto, T., Matsuura, S. & Nagata, S. (1968). J. Cell BioZ. 38, 574. Tashiro, Y., Otsuki, E. & Shimadzu, T. (1972). Biochim. biophys. Aeta, 257, 198.