Accentuated expression of silk fibroin genes in vivo and in vitro

J. Mol. Biol. (1976) 107, 183-206

Accentuated Expression of Silk Fibroin Genes in Vivo and in Vitro YOSnTA~T SUZUKI AND P~UL E. GIZA

Del~a'rlmeut of Embryology, Carnegie Institution of Washington 115 West University Parkway, Baltimore, Md, 21210, U.S.A. (Received 13 February 1976) Fibroin messenger R N A accumulates in the posterior silk gland of Bombyx mori a t an ahnost constant rate (7 to 10 moleeules/gene per min) throughout the fifth larval instar. During this period ribosomal R N A synthesis decreases from 7 to 0-2 molecules/gene per minute a n d synthesis of heterogeneous R N A also decreases. Thus, fibroin mRIqA comprises a b o u t 1, 3 and 8% of the R N A pulse-labeled for 30 minutes in rive on the second, fourth and seventh day, respectively. Pulselabeling experiments on the seventh d a y of the fifth instar indicate t h a t a single fibroin gene is transcribed at a rate 50-fold higher t h a n an rRIqA gene. I n contrast, fibroin m R N A was not detectable in R N A labeled for 30 minutes during the preceding fotu'th moulting stage. Posterior silk gland nuclei have an extremely ramified structure and a t t a i n a b o u t 4 x 105 ploidy in the late fifth instar. These nuclei have been mechanically fragmented and 90 to 95% of the cytoplasmic contaminants removed. W h e n pulse-labeled for 30 minutes in vivo before nuclear isolation, most of the unlabeled fibroin m R N A a n d about half of the pulse-labeled m R N A were found in the cytoplasmic fraction. In vitro synthesis of t o t a l R N A b y endogeneous R N A polymerase in the disr u p t e d nuclei was almost linear for 30 minutes, and some reinitiation was observed. The in vitro p r o d u c t labeled for 30 minutes yielded a profile similar to t h a t of R N A labeled in vivo when fractionated on a Bio-Gel A50m column. H y b r i d i z ation of the in vitro R N A to B. mori DlqA under conditions which allow transscripts of repetitious sequences as well as fibroin m R N A to hybridize waa completely competed for b y t o t a l posterior gland R N A . B y sequence analysis a b o u t 2% of the in vitro transcripts labeled in 30 minutes were identified as fibroin m R N A of a p p a r e n t full size length. In vitro fibroin m R N A synthesis was abolished b y 0.2 /~g of =-amanitin/ml suggesting t h a t R N A polymerase I I is responsible for fibroin gene transcription. 1. I n t r o d u c t i o n

I n t h i s p a p e r we r e p o r t t h e d e t e c t i o n a n d q u a n t i t a t i o n o f fibroin m e s s e n g e r R N A l a b e l e d b o t h in vivo a n d in i s o l a t e d nuclei for s h o r t t i m e periods. T h e s e results were o b t a i n e d b e c a u s e o f t w o f a v o r i t e c i r c u m s t a n c e s . F i r s t , t h e fibroin gene is p r e s e n t in a r e l a t i v e l y h i g h c o n c e n t r a t i o n in Bombyx mori D N A even t h o u g h i t is p r e s e n t a t o n l y one c o p y p e r g e n o m e (Suzuki et al., 1972 ; G a g e & Manning, 1976). This is d u e t o t h e s m a l l size o f t h e B. mori g e n o m e (0.52 p g ; Gage, 19744; R a s c h , 1974) a n d t h e large size o f t h e fibroin gene itself ( L i z a r d i & B r o w n , 1975). T h u s t h e fibroin gene c o m p r i s e s 0 . 0 0 4 % of t h e g e n o m e (Suzuki et al., 1972), a c o n c e n t r a t i o n t h a t is t w o o r d e r s o f m a g n i t u d e h i g h e r t h a n for t h e m a m m a l i a n g l o b i n gene ( H a r r i s o n et al., 1972). Second, b e c a u s e o f its large size, h i g h G-{-C c o n t e n t , a n d i n t e r n a l l y r e p e t i t i o u s 13

183

184

Y. S U Z U K I A N D P. E. GIZA

n a t u r e fibroin m R N A h a s b e e n i s o l a t e d in p u r e f o r m a n d c a n b e i d e n t i f i e d c h e m i c a l l y b y p a r t i a l sequence a n a l y s i s (Suzuki & Brown, 1972). A p r o c e d u r e has b e e n d e v e l o p e d t o m e a s u r e fibroin m R N A b y a d i r e c t c h e m i c a l a s s a y (Suzuki & Suzuki, 1974). W e h a v e u s e d t h e s e m e t h o d s t o m e a s u r e t h e r a t e o f fibroin m R N A s y n t h e s i s t h r o u g h o u t t h e fifth i n s t a r a n d to c o m p a r e t h a t r a t e t o t h e r a t e o f r R N A synthesis. W e h a v e also m e a s u r e d t h e e n d o g e n o u s s y n t h e s i s o f fibroin m R N A in i s o l a t e d nuclei.

2. M a t e r i a l s a n d M e t h o d s

(a) B. mori eggs and larvae Both diapausing a n d non-diapausing eggs of a hybrid, Gtmka (a Chinese strain, male) • Hoshun (a Japanese strain, female), were purchased from Gunze Co., A y a b e City, K y o t o - f u J a p a n , through Gunze Sangyo Co., Tokyo, a n d the larvae were raised and dissected as described previously (Suzuki & Brown, 1972; Suztfl~i & Suzuki, 1974). (b) Chemicals [3H]uridine, [ZH] or [~-3~P]uridine 5"-triphosphate, and [r-aSP]adenosine 5'-triphosphate were purchased from l~ew E n g l a n d Nuclear, Boston; S-adenosyl-T.-methionine, ~-amanitin, a n d Escherichia coli D N A from Calbiochem; dithiothreitol, actinomycin D, U T P , ATP, CTP, GTP and bovine serum albumin fraction V from Sigma Chemical Co. ; G r a c e ' s insect m e d i u m 194G from Grand I s l a n d Biological Co., G r a n d Island, New Y o r k ; a n d Harleco synthetic resin from A. H. Thomas Co., Philadelphia. Nonidet P40, a non-ionic detergent, was a product of Shell Co. (c) I n vivo labeling and in vitro culture of posterior silk glands Isotopes were introduced in vivo b y intraeoelomie injection (Suzuki & Brown, 1972). F o r organ culture generally a pair of posterior silk glands were dissected from a larva in the cold, rinsed with a large a m o u n t of SCC (SCC is 0-15 ~-sodium chloride, 0"015 ~-sodium citrate, p H 7) followed b y incubation medium, a n d labeled with 1 to 10 mCi [ZH]uridine (spec. act. 17 to 54 Ci/mmol) in 1.0 ml of the Chironomus meditun described b y L a m b e r t & Daneholt (1975) or Grace's insect m e d i u m 194G without supplement. These 2 m e d i a were essentially the same in supporting the proper R N A synthesis of t h e posterior silk gland. These concentrations of labeled uridine were fomld less t h a n saturating. Labeling proceeded for 30 to 90 min a t 25~ Labeling of the R N A with a2PO 4 in organ culture has been lmsuccessful for unknown reasons. (d) Histological preparations of silk glands Silk glands were fixed with formaldehyde, h y d r o l y z e d b y HCI, stained b y the F e u l g e n Schiff reaction, d e h y d r a t e d v e r y slowly, cleared in xylene, a n d m o u n t e d in Harleco synthetic resin (Lillie, 1948).

(e) Isolation of fragmented nuclei The m e t h o d described for liver nuclei (Reeder, 1973) was modified slightly and used for silk gland nuclei. A pair of posterior silk glands were homogenized with 5 strokes of a loose-fitting 1-)ounce homogenizer in 25 ml NP40 medium containing 0.2% Nonidet Pd0 (v/v), 10 mM-Tris (pH 8"0), 5 mM-MgC1 u, 0-5 n ~ - d i t h i o t h r e i t o l . The homogenate was centrifuged for 10 rain a t 1500 g in a swinging rotor 269 in the I n t e r n a t i o n a l centrifuge PR2, a n d the supernate was saved. These procedures were r e p e a t e d once or twice more, and all t h e supernates were combined as the cytoplasmic fraction. The pellet was homogenized in 3 ml NP40 medium, mixed with 12 ml 2.2 ~-sucrose solution containing 10 mM-Tris (pH 8.0), 5 mM-MgC1 u, 0-5 mM-dithiothreitol, layered over 19 ml 2.2 M-sucrose solution with a gentle stir, a n d the nuclei were pelleted b y eentrifugation for 30 rain a t 20,000 revs/min in a SW25.1 rotor. The nuclei were suspended a n d homogenized in m e d i u m containing 25~o (v/v) glycerol, 50 m~-Tris (pH 8"0), 5 ml~-MgC12, 0.1 mM-EDTA, 5 mM-dithiothreitol (Reeder & Roeder, 1972) a n d used for in vitro transcription. Portions

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were used for assays of DNA, R N A a n d protein, and also for morphological observation of the fragmented nuclei after Feulgen staining. (f) R N A synthesis in fragmented nuclei and extraction of the R N A product~ The volume of reaction mixtures was a d j u s t e d to give a D N A concentration of 200 ~g/ml, and generally a 1"0 ml volume was used for the reaction m i x t u r e containing suspension of the nuclei (about 200 ~g DNA) derived from a p a i r of posterior silk glands. T h e ingredients for R N A synthesis described b y Reeder & Roeder (1972) were slightly modified: 12.5~o (v/v) glycerol, 25 mM-Tris (pH 8.0), 5 mM-MgC12, 1 m~-M_uCla, 0.5 mM-EDTA, 150 mM-KC1, 2.5 mM-dithiothreitol, 10 ~M-S-adenosyl-L-methionine, 0"6 m ~ each of GTP, A T P a n d CTP, a n d 0.01 to 0.06 m ~ - [ z H ] U T P or [~-z2P]UTP. The synthesis was done a t 25~ (Marzlu-ff et al., 1973), a n d t e r m i n a t e d b y the addition of 50/zg RNAase-free DNAase/ ml a t 0~ or simply b y the a d d i t i o n of sodium dodecyl sulfate. A f t e r DNAase t r e a t m e n t 1 ~/o sodium dodecyl sulfate a n d 0"5 mg p r e i n c u b a t e d Pronase]ml were added, incubated for 30 min a t 37~ a n d t h e R N A was e x t r a c t e d with phenol s a t u r a t e d with 0.1 ~-Tris base, precipitated with ethanol twice, a n d passed through a 0.9 cm • 60 cm column of Sephadex G25. (g) Bio-Gel column fractionation and RNAase T1 fingerprint analysis of the R1VA These methods have been described previously (Suzuki & Suzuki, 1974), a n d details are given in the Figure legends. (h) Estimation of the purity of fibroin messenger R1VA labeled

with [3H]uridine, [zH]UTP or [~-32P]UTP The m R N A sequence is so characteristic t h a t i t gives a unique oligonucleotide profile after R N A a s e T1 digestion (Suzuki & Brown, 1972; see also Suzuki & Suzuki, 1974; Suzuki, 1976a,b). W h e n the m R N A is labeled with [3H]uridine or [zH]UTP there is a v i r t u a l absence of tetranucleotides as well as oligonucleotides larger t h a n hexanucleotidss in the T1 R N A a s e digest. A t y p i c a l p a t t e r n of di-, tri- a n d pentauucleotides from pure m R N A is shown in Fig. l(b). A_~ increase of tetranucleotides a n d oligonucleotides larger t h a n hexanueleotides in a sample digest means an increase of impurities. The presumed 100% pure profile shown in Fig. l(b) was obtained as follows. The posterior silk gland R N A was labeled with [ZH]uridine for 12 h on the 7th d a y of the 5th instar, fractionated b y a Bio-Gel A50m column (Fig. 2(d)), a n d t h e void volume fraction was pooled as crude fibroin m R N A (which was 90% pure b y itself; Fig. 3(d)). The m R N A was h y b r i d i z e d to B. mori D N A fractionated b y actinomycin D/CsC1 (Fig. l(a)). The R N A hybridized to the fibroin gene region was assumed to be essentially pure, recovered, digested b y R N A a s e T1, a n d t h e digest was fractionated b y a D E A E - S e p h a d e x A25 column (Fig. l(b)). The digest contains 2.5% tetranucleotides a n d 22~/o oligonueleotides larger t h a n hexanucleotides. F o r p u r i t y estimation an artificial calibration curve has been p r e p a r e d using poly(A)contaflfi_ug R N A described below as an artificial contaminant. The posterior glands were pulse-labeled with [3H]uridine for 30 rain on t h e 2nd d a y of t h e 5th instar. The labeled R N A was extracted, fibroin m R N A a n d t R N A were eliminated b y the use of a Bio-Gel A50m column, r R N A was also eliminated b y passage through an oligo(dT)-cellulose column a n d finally poly(A)-containing R N A s which specifically lacked fibroin m R N A were eluted from the column. After R N A a s e T1 digestion this p r e p a r a t i o n revealed 7"5~o tetranucleotides a n d 66% oligonucleotides larger t h a n hexanueleotides (not shown). The p u r i t y (~/o) of a sample of fibroin m R N A was calculated b y t h e following equation: p • 100 ( b - x ) / b - a, where a, b a n d x are tetranucleotide per cent of R N A a s e T1 digests of: a, the purest m R N A (2.5~ ; Fig. l(b)); b, the artificial c o n t a m i n a n t mentioned above (7.5%} ; a n d x, t h e sample to be tested. The result was checked b y comparing t h e a m o u n t of oligonucleotides larger t h a n hexanucleotides in the sample digest with t h a t contributed b y the c o n t a m i n a n t a t the e s t i m a t e d p u r i t y . F o r the samples labeled for 0-5 to 4 h the correction derived from the tetranucleotide levels m a t c h e d very well w i t h the estimate based on larger oligonucleotides. F o r samples labeled for 12 h or longer r R N A

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labeled with [SH]uridine was employed as the standard c o n t a m i n a n t (tetranucleotides; 13.5%) since r R N A was actually the major c o n t a m i n a n t in such preparations of fibroin m R N A (Suzuki e~ a/., 1972; Suzuki & Suzuki, 1974). I n some experiments R N A was labeled with [~-32PJUTP. The RNAase T i fingerprint of suP-labeled pure fibroin m R N A is shown in Fig. 9(a). The sup is transferred to nearestneighbor nucleotides producing a high level of labeled GMP upon T i RNAase digestion since GpG is the most a b u n d a n t nucleotide next to p U in fibroin m R N A (see Suzuki & Brown, 1972). We do n o t expect a b u n d a n t dinucleotides in the digest since they could be derived only from the GpNpGpU sequence (where N is either A, U or C b u t n o t G); this is n o t a major sequence in fibroin m R N A . Therefore pure m R N A should reveal large a m o u n t s of mono-, tri- and pentanucleotides together with an intermediate amotmt of dinucleotides aald traces of tetra- a n d oligonucleotides larger t h a n hexanucleotides (Fig. 9(a)). I n Fig. 9(b) the oligonucleotide profile is shown of high molecular weight R N A isolated from the animal's carcass after a 12-h pulse on the 7th day of the 5th instar. Using this profile a correction for contamination was made b y the following equation. p -----100 (b -- x)/b -- a, where p is purity percent of fibroin m R N A , and a, b a n d x are the percent of tetranucleotides in the RNAase T i digest of pure m R N A (2"0~ of the artificial c o n t a m i n a n t (8"3~/o), a n d of the sample to be tested, respectively. (i) Quantitation of fibroin m R N A and r R N A Labeled or unlabeled R N A was fractionated b y a Bio-Gel A50m column. All fibroin m R N A is assumed to be present in the void volume fraction. The proportion of fibroin m R N A in this fraction was estimated b y the RNAase T i fingerprint method described above and before (SuzLfld & Suzuki, 1974). With this method a sample which contains as low as 5~/o fibroin m R N A can be subjected to the q u a n t i t a t i v e measurements. For r R N A q u a n t i t a t i o n the proportion of R N A in 28 S a n d 18 S r R N A region in a Bio-Gel A50 column was measured, and was taken as r R N A q u a n t i t y without a n y correction. (j) Quantitation of D N A , R N A and protein DNA, R N A and protein were separated by the S c h m i d t - T h a n n h a u s e r method (Schmidt & Thannhauser, 1945) and q u a n t i t a t e d b y the diphenylamine reaction (Giles & Myers, 1965), orcinol reaction (Dische, 1953) and Lowry et al.'s method (Louqcy et al., 1951), using E. cell DNA, B. mori 28 S rRNA, and bovine serum a l b u m i n fraction V as standards respectively. (k) Actinomycin D/CsCl fractionation of B. mori D N A and hybridization The methods described by Lizardi & Brown (1975) a n d Gage & Mamling (1976) have been employed. DNA was sheared to smaller t h a n fibroin gene size b y repeated passage through a 26-gauge syringe-needle. A total of 1 mg actinomycin D was added to 2 mg DNA for one gradient. Refractive index of the gradient was adjusted to 1.384 to 1"388 by the addition of CsC1. Filter hybridization was carried out at 50~ in 50~o formamide/4 • Tris/ EDTAfNaC1 buffer (0.15 ~-sodium chloride, 0.02 M-EDTA, 0.03 M-Tris, p H 7.6). Because of the internally repetitious nature of the fibroin gene, m a x i m u m hybridization with fibroin m R N A was attained by 3 h either using f l t e r hybridization or solution hybridization (Suzuki & Reeder, unpublished work). I f necessary the fibroin gene partially purified in this m a n n e r was used as an assay probe to detect fibroin m R N A sequences in R N A mixtures. Fig. 1 documents the assay method. All R N A preparations were labeled with [aH]uridine. Fig. l(a) shows the hybridization of fibroin m R N A which was 90~ pure. The RNAase T i fingerprint of the R N A which hybridized to the fibroin genes is shown in Fig. l(b). I n Fig. l(c) total posterior gland RNA, which had been labeled for 24 h during t h e 6th day of the 5th instar and known to contain 6~o fibroin m R N A , was hybridized to the DNA. Fibroin m R N A - f i b r o i n gene hybrid is recognized at the expected position and the RNAase T i fingerprint of the hybridized m R N A is shown in Fig. 1(d). Total posterior gland RNA, which had been labeled for 24 h during the 3rd day of the 5th instar and contained 3~/o fibroin rn_~NA, was also hybridized (Fig. l(e)). The fibroin m R N A hybridization is still recognizable (Fig. l(f)). Finally total posterior gland RNA, which had been labeled for 30 rain in the middle of the 2nd day a n d contained only 1~o fibroin mRNA,

ACCENTUATED

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Fro. 1. Hybridization of fibroin m R N A of different purities to partially purified fibroin gene, a n d RNAase T, fingerprint of the hybrid. To 2-2 m g sheared D N A 1.1 m g actinomyein D was added to each gradient, the refractive index was adjusted to 1.3880 with CsC1, a n d the mixtures wore centrifuged a t 33,000 revs/min a t 20~ for 66 h in a Spinco 50.1 fixed-angle rotor. Hybridization was done a t 50~ in 50% formamide/4 • Tris/EDTA]NaC1 buffer for 16 h. I n each panel the actinomycin D/CsC1 gradient is shown on the left a n d the RNAase Tz fingerprint of RNA, which hybridized to fibroin gene region a n d recovered from the bracketed region, on the right. Density of fibroin genes from each panel was 1.510 (a), 1.504 (c), 1.502 (e), a n d 1.504 g/ore 3 (g). Details for R N A sources are given in Materials a n d Methods. (a) A 90% pure fibroin m R N A , 0.13 ~g/ml. (c) Total posterior silk gland R N A which contains 6 % fibroin m R N A , 3.6 Fg/ml. (e) Total posterior silk gland R N A which contains 3 % fibroin m R N A , 20 ~g/ml. (g) Total posterior gland R N A which was pulse-labeled for 30 rain a t the middle of t h e 2nd day of t h e 5th instar a n d contained 1% fibroin m R N A , 13 ~g/ml. - - Q - - Q - - , 3H radioactivity; ( ), A26c monitored b y a Gifford speotrophotometer. The r o m a n numerals in the Figure s t a n d for the chain length of oligonucleotides.

Y. S U Z U K I AND P. E. GIZA

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was hybridized (Fig. l(g)). The hybrid at fractions 18 to 19 was non-fibroin mRNA type, but the shoulder at 20 to 21 gave a T1 fingerprint suggestive.of impure fibroin mRNA (Fig. l(h)). Taken altogether Fig. 1 shows that small amounts of fibroin mRNA can be detected in a mixture of RNAs by the hybridization technique. 3. R e s u l t s (a) Predominant synthesis of fibroin messenger R N A over ribosomal R N A

synthesis during the fifth larval instar We have reported t h a t the amounts of fibroin m R N A and r R N A which accumulate in a posterior silk gland cell by the end of the sixth day of the fifth instar were 0.17 Fg and 3.9 Fg, respectively (Suzuki & Suzuki, 1974). The relative abundance of fibroin m R N A is even greater in later stages of the instar (Table 1). A pair of posterior silk T~LBLE 1

Amounts of fibroin messenger R N A and ribosomal RIVA accumulated in the l~osterior silk glands by the end of seventh day of the fifth larval instar RNA rRNA Fibroin mRNA Total cellular RNA

Amount (~g/cell)

Number of molecules/cellt

Number of molecules/geneS:

4.0 0.22

1.1 • 10is 2.3 • 10~~

1.1 • 104 5.8 x 104

5.0

n.d.

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t Molecular weight of 28 S plus 18 S rRNA is about 2.2 • 106 (Suzuki, unpublished results) and that of fibroin mRNA is 5.7 • 106 (Lizardi e$ al., 1975). The B. mori genome contains about 240 copies of rDNA (Gage, 1974b) and 1 fibroin gene per haploid complement (Suzuki et al., 1972; Gage & Manning, 1976; Lizardi & Brown, 1975). The genome size is about 0.52 pg (Rasch, 1974; Gage, 1974a), the D:NA content per cell is about 0-20 g (see the legend to Table 2), and therefore the ploidy of the cell is about 4 • l0 s. n.d. Not determined. glands from one larva contain about 1000 cells (0no, 1942) and about 5.0 mg of R N A b y the end of seventh day. About 4.4~/o of the R N A is fibroin mRNA, an amount equivalent to 0.22 Fg/cell. Only a shght increase of r R N A occurs during this period. Knowing the molecular weight of the RNAs, the numbers of copies of the fibroin gene and rDNA per haploid complement, and the approximate degree of polyploidy in a nucleus, we can calculate the number of accumulated R N A molecules per gene (Table 1). The number of fibroin m R N A molecules per gene is about fivefold greater than the r R N A molecules per gene (Table 1). I n order to estimate the rates of fibroin m R N A and r R N A accumulation we measured the amounts of these RNAs at various stages of the fifth instar. These data were combined with those pubhshed previously (Suzuki & Suzuki, 1974), and the results are shown in Table 2. D N A content was measured at various stages (see the legend to Table 2) and the number of gene copies was calculated based on the fact t h a t there are about 240 copies of r D N A i(Gage, 1974b) and one copy of fibroin gene per haploid (Suzuki et al., 1972 ; Gage & Manning, 1976; Lizardi & Brown, 1975). The striking features shown in Table 2 are: (1) the ratm of m R N A accumulation is almost constant throughout the stage, whereas that of r R N A changes as much as 30-fold, and (2) m R N A accumulation relative to

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TABLE 2

Rates of accumulation of fibroin messenger R N A and ribosomal R N A during the fifth instar Time intervalst

rRNA

Fibroin mRNA

(molecules/gene per min) Ecdysis--lst day 1st day--2nd day 2nd day--4th day 4th day--6th day 6th day--7th day

1.8 6.6 2.6 0.3 0-2

~ 1.2 10 7.9 7.4 9.3

t Every 24-h period after the 4th ecdysis was designated as 1st day, 2rid day, etc. The quantitation of the RNAs was done at the eedysis, and at the end of 1st, 2nd, 4th, 6th and 7th day. The difference of RNA amounts between 2 time points was divided by the total nwnber of genes and the time elapsed (24 h or 48 h). The number of each gone was calculated from the amount of DNA present at the middle of the 2 time points and the further assumption that each haploid oomplenemt of DNA contains 240 ribosomal RNA genes and 1 fibroin gene. DNA amount at the middle of the each time interval was 35, 65, 160, 200 and 200 ~g/pair of posterior silk glands. gene number is going on at a higher rate than t h a t of r R N A not only at later stages of the instar but at any time of the instar except the first day. The most exaggerated situation is seen on the seventh d a y of the instar. At this stage new r R N A synthesis almost ceases while m R N A accumulation continues actively. The approximate ratio of these rates of 1 : 46 (Table 2) is not accurate because of the difficulty in measuring the rate of r R N A accumulation at this stage. For this reason we carried out the pulse-labeling experiments described in section (b) below. (b) Detection and quantitation of fibroin messenger R N A labeled with short pulses in vivo I t has generally been assumed t h a t in eukaryotes it would be difficult to detect a specific single m R N A in pulse-labeled materials unless one used some sensitive technique such as hybridization. However, the results mentioned above as well as the consideration of the high proportion of the fibroin gene in the genome have prompted us to t r y to detect fibroin m R N A b y a direct chemical method (Suzuki & Suzuki, 1974). Posterior silk gland RNAs were labeled in vivo with [3H]uridine for 0.5, 1.5, 4 or 12 hours on the seventh day of the fifth instar. The RNAs were fractionated b y a Bio-Gel A50m column, and large radioactive peaks were observed at the void volume region of the column (Fig. 2) where mature fibroin m R N A was known to be eluted (Suzuki & Suzuki, 1974). The RNAs in the void volume peaks were digested with RNAase T1, and the digest was fractionated b y a DEAE-Sephadex A25 column (Fig. 3). The RNAs labeled for 0.5, 1.5, 4 and 12 hours were calculated from their oligonucleotide profiles to be 17, 46, 65 and 90% pure, respectively, for fibroin m R N A (Fig. 3). The profiles shown in Figure 3(a) through 3(d) demonstrate the rapid turnover of heterogeneous RNAs compared to the more stable fibroin m R N A . The calculated proportions of fibroin m R N A synthesized and accumulated for different labeling periods in the seventh day of the fifth instar are summarized in the bottom line of

Y. S U Z U K I AND P. E. G I Z A

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60

FroctJon number

Fro. 2. Bio-Gel cohmm fractionati(m of posteri.r silk glaml RNAs pulse-labeled in vlvo on the 7th day of the 5th instar. Each larva was injected with 2.5 mCi [aH]uridine on the 7th day, and incubated at 25~ for 30 min (a), 1.5 h (b), 4 h (e) and 12 h (d). The RNA was extracted and fractionated by a 2.6 e m • 66 cm column of Bio-Gel AS0m. For each fraction 400 drops (5"5 ml) were collected, and a 0"5-ml portion was counted. - - O - - O - - , att radioactivity. The arrows in the Figure indicate the positions of fibroin mRNA, 28 S rRNA, 18 S rRNA and 4 S tRNA recorded by A26omollitoring from left to right, respectively. Table 3. As the time of pulse-labeling increases from 0"5 to 12 hours, the proportion of fibroin m R N A in total labeled R N A increased from 7"5~/o to 270/o indicating the stable nature of the m R N A . F r o m the proportion of fibroin m R N A synthesized in a 4-hour and a 12-hour pulse in the seventh d a y (Table 3) we calculated rates of fibroin m R N A synthesis per fibroin gene. Rates of r R N A synthesis per ribosomal R N A gene were also calculated from the radioaetivities in the r R N A region of Figure 2(e) and (d). The ratio of the rate of fibroin m R N A synthesis to t h a t of r R N A synthesis is 51 for the 4-hour pulse and 52 for the 12-hour pulse. The result indicates t h a t fibroin m R N A is synthesized a b o u t 50 times more rapidly t h a n r R N A . Because the ratios were essentially the same between the two time points we infer t h a t r R N A and m R N A have similar stability during these labeling periods a n d the ratio of transcription for these genes would also be near 50 during shorter labeling times. These results also indicate t h a t the rates of a c c u m u l a t i o n described in Table 2 a p p r o x i m a t e the rates of synthesis since we f o u n d a similar ratio, 46, for the rate of fibroin m R N A accumulation to t h a t of r R N A a c c u m u l a t i o n on t h e seventh d a y of the instar. The same technique has been applied to q u a n t i t a t e newly synthesized fibroin m R N A at various stages of the fifth instar. R N A s were labeled in vivo for 30 minutes with [3H]uridine at the middle of the second, third, fourth a n d sixth d a y of the instar. The results are summarized in Table 3 together with the d a t a on the seventh day. A l t h o u g h during these stages rates of accumulation of fibroin m R N A per gene were

ACCENTUATED 6

EXPRESSION

I I EI~v~

(a)

OF FIBROIN

GENES

191

15

oJ I

0

> .2

6C

~o IZ 0

I]I

aI ~ZV w

(d)

2 3C

w o

0

0

40

20

20

40

Fraction number

FIG. 3. R N A a s e Tz fingerprhlts of h i g h molecular w e i g h t R N A s pulse-labeled in vivo for different time periods on the 7th d a y of the 5th instar. T h e void v o l u m e fractions indicated b y b r a c k e t s in Fig. 2 were recovered, digested w i t h R N A a s e T1, a n d the digests were f r a c t i o n ~ t e d b y DEA_ES e p h a d e x c o l u m n s w i t h a NaCI g r a d i e n t (0.10 M to 0.45 ~) in the presence of 7 u - u r e a . Samples pulse-labeled for 30 rain (a), 1.5 h (b), 4 h (c), a n d 12 h (d) are o b t a i n e d f r o m Fig. l(a), (b), (c) a n d (d), respectively. - - @ - - @ - - , 3H radioactivity. The R o m a n n u m e r a l s in the F i g u r e s t a n d for the chain length of oligonucleotides.

TABLE 3

Proportion of fibroin messenger R1VA to total cellular 1?NA which was synthesized during short pulse periods T i m e of pulse-labeling

(h) Stage

0.5

1.5

4

12

Proportion

(%) Fourth moulting

< 1.7

--

--

<0.5 t

--

--

0.6

Fifth instar 1st d a y

--

2nd d a y

1.2

--

--

1-8~

3rd d a y

2.0

--

--

--

4th day

3.0

--

--

4'6t

6th d a y

4.8

--

--

10t

7th d a y

7.5

12

24

27

T h e stage w a s n a m e d as in the legend to Table 2, a n d pulse-labeling e x p e r i m e n t s were done a t the middle of the day. t T a k e n f r o m Suzuki & Suzuki (1974). - - , N o t determined.

192

Y. S U Z U K I

AND

P. E. G I Z A

almost constant (Table 2), relative proportions of newly synthesized fibroin m R N A to the total R N A labeled were found to be constantly increasing from 1.2~/o on the second day up to 7"5~/oon the seventh day of the instar. (c) Synthesis of fibroin messenger R N A in the posterior silk glands cultured in a chemically defined medium Before initiating in vitro experiments on nuclei or chromatin we tested the ability of synthetic media to support fibroin mRNA synthesis by isolated silk glands. Posterior glands from fifth instar larvae on the seventh day were rinsed extensively and incubated in a chemically defined medium (Lambert & Daneholt, 1975). Incorporation of [3H]uridine into RNA was almost linear for 90 minutes, but then increased only 20% more in the next two hours. The RNAs were labeled for 39 minutes and 90 minutes and fractionated by Bio-Gel A50m columns. Bio-Gel profiles (Fig. not shown) were similar to those found for in vivo labeled RNA (Fig. 2(a) and (b)). The void volume fraction was recovered, digested with RNAase T 1, and the oligonucleotide pattern analyzed. Again the profiles (Fig. not shown) are characteristic of fibroin mRNA which is 15% and 30% pure for the RNA labeled in the 30-minute and the 90-minute pulse, respectively. The calculated abundances of mRNA in the total labeled RNA were 5.7 % and 9.6%, respectively, for the 30-minute and the 90-minute pulse; abundances slightly less than were found in the in vivo experiments (Table 3). The results indicate that isolated glands not only synthesize mRNA in vitro but stabilize it in a manner similar to that which occurs in vivo.

(d) l~'ibroin messenger R N A is not detectable in silk glands which were pulse-labeled during the fourth moulting stage During the feeding stage of the fourth larval instar mRNA accumulates to finally comprise 2% of the total RNA. During the subsequent fourth moulting stage this mRNA disappears and no newly made mRNA could be detected in a 12-hour pulselabel (Suzuki & Suzuki, 1974). Whether this is due to selective degradation of the mRNA or to a shut-off of fibroin gene transcription could not be decided at that time because of the long labeling period. Therefore we examined the RNA synthesized in a shorter pulse-labeling of the glands. Posterior silk glands from 26 larvae in their fourth moulting stage were incubated with [3H]uridine in Grace's medium for 30 minutes at 25~ The RNA was extracted and fractionated by a Bio-Gel A50m column. As shown in Figure 4(a) the labeling profile is similar to that shown in Figure 2(a). The void volume fraction was digested with RNAase T1 and the digest was analyzed by a DEAE-Sephadex column. As shown in Figure 4(b) the oligonucleotide profile has no detectable fibroin mRNA component. We conclude that RNA in the void volume contains less than 5~/o fibroin mRNA, or less than 1.7% of the total RNA synthesized in 30 minutes (Table 3). (e) Morphological observation of the ramified nuclei of the posterior silk gland It has been long known that silk gland nuclei undergo dramatic changes in appearance as the larva matures. However, only selected features have been published; usually schematically in references of limited accessibility (Yamanouchi, 1921; Tanaka, 1928). Therefore we analyzed the in vivo nuclear morphology for later comparison with isolated nuclear fragments.

ACCENTUATED

EXPRESSIO~

200

OF FIBROIN

GENES

193

zI H

b E

I

lOO

g~ _

0

A

j

20

40

60

0

I

--

20

40

(b)

(c) Fraction number

Fro. 4. Bio-Gel fractionation of posterior gland R N A pulse-labeled in viero at the 4th moulting stage, and RNAase TI fingerprint of the void volume fraction. (a) During the middle of 4th moulting stage 26 pairs of the posterior glands were dissected out, and incubated with 5 mCi [3H]uridine in 0.5 ml Grace's insect medium 194G for 30 m i n at 25~ R N A was extracted a n d applied to a 1.5 cm • 56 cm column of Bio-Gel A50m. E a c h 2.7-ml fraction was collected, a n d a 0-5-ml portion was counted. (b) The high molecular weight R N A was obtained from the void volume fraction shown b y the bracket in (a), a portion was digested with RNAase T1, and the digest was fractionated b y a D E A E - S e p h a d e x A25 column. The R o m a n numerals in the Figure s t a n d for the chain length of oligonucleotides. - - 9 9 SH radioactivity.

Silk glands were studied from late embryonic (about 1-5 days before hatching) through the late fifth larval instar stages. No cell division takes place in the silk gland during larval life (0no, 1942) but DNA increases approximately tenfold during each instar (Suzuki & Suzuki, 1974). Figure 5 shows the morphological changes that the posterior silk gland nuclei undergo during larval development. The shape of silk gland nuclei from late embryonic (similar to Fig. 5(a) and not shown here) to early third instar (similar to Fig. 5(b)) is not unusual when compared to nuclei of other eukaryotic cells. However, then the silk gland nucleus becomes elongated occupying a larger portion of the cellular volume, as its ploidy increases. During the middle to late third instar the nuclei begin to ramify at both ends of the elongated nuclear structure (Fig. 5(c)). The process continues through the fourth (Fig. 5(d)) and fifth instars, and culminates in the extremely complex structure shown in Figure 5(f). This nuclear ramification and increase in cellular DNA content appears to represent uniform replication of the entire genome (Gage, 1974b). A single cell in the mature posterior gland has dimensions of about 1.3 m m • 1.4 mm (Fig. 5(f)), contains 0.20 /~g of DNA (see the legend to Table 2) which is equivalent to about 4 • 105 haploid amounts of DNA, and the branched nucleus extends throughout the cytoplasm (Fig. 5(f)). (f) Disruption of the nuclei, and isolation and vharacterization of the fragmented nuclei All preliminary attempts to isolate intact nuclei from later stages have been unsuccessful in our laboratory. Nuclear isolation in earlier stages would be easier, and we know that fibroin genes are also expressed in the third and fourth instars

194

Y. S U Z U K I AND P. E. G I Z A

FIe. 5. Morphological changes of silk gland nuclei during development. They are all posterior glands except 1 middle silk gland (e). (a) First instar (2 days after hatching), (b) second instar (3 days after 1st ecdysis), (c) third instar (4 days after 2nd ecdysis), (d) fourth instar (4 days after 3rd ecdysis), (e) middle silk gland of 4th instar (2 days after 3rd ecdysis), (f) fifth instar (7 days after 4th ecdysis). The bars in the Figure all represent 100/zm. (e) is displayed to show hexagonal cell boundaries clearly.

(Suzuki & Suzuki, 1974}. However, we obtain one order of m a g n i t u d e less D N A as we go back to each preceding instar. Therefore we isolated f r a g m e n t e d nuclear preparations from the fifth instar (Fig. 6). A l t h o u g h the complicated and ramified nuclei (Fig. 5(f)) have been fragmented, the disrupted nuclei still m a i n t a i n some identifiable m o r p h o l o g y (Fig. 6). Furthermore, the D N A extracted from the disrupted nuclei was r a t h e r homogeneous in size, and had an average molecular weight greater t h a n 40 • 106 (Suzuki & Giza, unpublished results). The D N A , R N A and protein composition of nuclear fragments a n d of cytoplasmic

9

~-

-.%:~-:F ~

F

FIG. 6. Structure of fragmented nuclei isolated from a posterior silk gland. The fragments were isolated from a posterior gland on the fom'th day of the fifth instar and stained w i t h F e u l g e n Sehiff reagent. Three typical examples of these are shown. The bar represents 100 ~am.

Y. SUZUKI

196

AND

P. E. GIZA

TABLE 4

Ghemical analyses of fragmented nuclei from posterior silk glands in their fifth instar Fractions

DNA (~g)

RNA (mg)

4th d a y

7th d a y

4th d a y

5.0 0.3 57 104 166

3.2 3.4 51 209 267

2.5 0.21 0.87 0.16 3.7

1st supernate 2nd supernate Sucrose supernate Crude nuclei Total

7th

day

Protein (rag) 4th day 7th day

3.9 0.36 0.27 0.62 5.2

18 0.37 1.9 0.48 21

25 5.9 1.9 1-3 34

A pair of posterior silk glands from one larva in its 4th day or 7th day of the fifth larval instar isolation. The details of the method are described in the text.

were used for the nuclear

fractions is shown in Table 4. In nuclear preparations DNA recovery was about 60 to 80%, while about 90 to 95% of the cellular RNA was eliminated. The nuclear preparation from the seventh day of the instar had a higher RNA to DNA ratio (3.0) than t h a t of the fourth day of the instar (1.5) suggesting t h a t the more complicated the nucleus becomes in its structure, the more difficult it becomes to free it of cytoplasmic contaminants. Whereas these nuclear preparations contain more cytoplasmic contaminants than those reported for other organisms, they have proven useful for the localization of fibroin mRNA, and analysis of in vitro RNA synthesis. In order to determine the distribution of unlabeled and newly synthesized fibroin mRNA, two pairs of posterior silk glands were first pulse-labeled in vivo with [3H]uridine for 30 minutes, mixed with ten pairs of unlabeled glands, and the mixture was subjected to nuclear isolation. RNAs were extracted from (1) the supernates which contain most of the cytoplasmic RNA and only a neglible amount of nuclear DNA (see Table 4), (2) the supernate from the sucrose centrifugation step, and (3) the nuclear preparation which contains most of the nuclear DNA. The RNAs were fractionated by a Bio-Gel AS0m column. Their A26o profiles showed intactness of all three preparations (not shown). The void volume fractions were subjected to RIqAase T 1 digestion for quantitation of newly synthesized and long-lived fibroin mRNA. The results are shown in Table 5. More than 80% of the long-lived mRNA which was identified b y A26o oligonucleotide profile after T1 RlqAase digestion was found in the cytoplasm, and only 8 % in the nuclei. As shown in Table 4 we must admit the possibility of contamination of cytoplasmic RNA into the nuclear preparation. Therefore we conclude t h a t most, if not all, of the long-lived mRNA is localized in the cytoplasmic fraction. The result also indicates that with the current method we can eliminate about 90% of unlabeled fibroin mRNA from the nuclear preparation. The newly synthesized fibroin mRNA was found about equally in the cytoplasmic fraction (47%) and the nuclear fraction (51%). The result suggests t h a t it takes less than 30 minutes to transport fibroin mRNA from the nucleus to the cytoplasm. (g) In vitro R N A synthesis in disrupted nuclei RNA synthesis with disrupted nuclei using the endogenous R N A polymerase was carried out at 25~ (the rearing temperature of the animal). The incorporation of [sH]UTP or [~-32P]UTP was almost linear for 30 minutes, and then slowed down,

ACCENTUATED

EXPRESSION

OF FIBROIN

GENES

197

TABLE 5

Localization of fibroin messenger R N A in cytoplasmic and nuclear fractions

Fractions

Newly synthesized mRNAr

Long-lived unlabeled mRNA:~

47 2 51

84 8 8

(%)

Cytoplasmic sup ~ Sucrose sup b Nuclear pellet ~

(%)

An homogenate of posterior silk glands from the 7th day of the 5th instar was centrifuged 3 times and the combined supernates were designated as cytoplasmic sup ~. Crude pellet was centrifuged through 2.2 M-sucrose, and the supernate b and nuclear pellet ~ were obtained. Posterior silk glands were labeled i n vivo for 30 m i n w i t h [SH]uridine, subjected to the frae. tionation mentioned above, and radioactive fibroin m R N A was q u a n t i t a t e d b y the T1 RNAase fingerprint method. The long-lived, unlabeled m R N A was quantitated by A26o oligonucleotide profile after T 1 RNAase digestion.

but a slight increase was still observed at 120 minutes (not shown). Typical incorporations were about 0.70, 1-2, 1.5 and 1.8 pmol UMP//~g DNA in 15, 30, 60 and 120 minutes, respectively, using disrupted nuclei from the seventh day of the fifth instar. The rates of synthesis with nuclei from the fifth day of the instar were approximately fourfold higher t h a n those of the seventh day. However, all these rates are minimal values because we did not measure the endogenous level of

X" I o

3

x

E

~2 v

> u

5

10

15

2O

Fraclion number

Fro. 7. Hybridization of the in vitro R N A to B. m o t / D N A fractionated by actinomycin D]CsCI and competition of the hybridization b y unlabeled posterior silk gland RNA. The R N A was labeled with 500 ~Ci [~-3~P]UTP (23-7 Ci]mmol) i n vitro for 30 mln in the disrupted nuclei (170 ~g DNA). Portions equivalent to 5% of whole nuclear preparation from a pair of posterior silk glands were hybridized to B. mot/ D N A (200 /~g]gradient) in the presence or absence of competitor at 50~ for 17 h in 50 % formamide/4 • Tris]EDTA]NaC1 buffer. - - 0 - - Q - - , No competitor added; - - O - - O - - , competitor R N A equivalent to 20-fold excess of posterior gland nuclei was added; - - A - - A - - , competitor equivalent to 200-fold excess of posterior gland nuclei was added; ~ ), absorbance at 260 n m monitored with a Gilford spectrophotometer.

198

Y. SUZUKI

AND

P. E. GIZA

nucleoside triphosphate in the nuclear preparations. For characterization the labeled RNA was usually extracted after 30 minutes incubation. RNA transcribed in vitro was hybridized to B. mori DNA fraetionated b y actinomyein D/CsC1 (Fig. 7). The hybridization conditions were such that only transcripts of repetitious sequence, which include fibroin mRNA because of its internal repetitious nature (Suzuki & Brown, 1972), hybridized. As shown in Figure 7 a prominent hybridization peak is seen at fractions 9 to 11. This is presumed to be rRNA since it was competed for by cold rRNA (data not sho~m). A small and heterogeneous peak at fractions 16 to 19 which is the region for fibroin gene (see Fig. 1) was competed for by unlabeled pure fibroin mRNA (see Table 6). The synthesis of fibroin mRNA will be documented in the next section. I t is also sho~wn that virtually all of the counts hybridized were competed for by total posterior gland RNA. For the hybridization we employed in vitro I~NA synthesized by the equivalent of about 5% of the whole nuclei from a pair of posterior glands, whereas the unlabeled RNA equivalent to total cellular RNA from one or ten pairs of glands was used as the competitor. The in vitro RNA preparation contained unlabeled RNA equivalent to 0.5% of total cellular RNA from a pair of posterior glands. Therefore the competitor was in about 200-fold or 2000-fold excess for cytoplasmic RNA and 20-fold or 200-fold excess for nuclear RNA. Thus the results indicate that similar repetitious sequences are transcribed both in vivo and in vitro. The RNA labeled for 30 minutes was fractionated on a Bio-Gel A50m column. Figure 8 shows both the radioactivity profile and absorbance at 260 nm b y the bulk RNA and undigested DNA. More than 55% of the radioactivity was found in the region larger than 28 S under non-denaturing conditions. Comparison of Figure 8 with Figure 2(a) reveals that the radioactivity profiles from Bio-Gel columns are 12

b_ .c__ E

"G

CC

20

40

60

Frocfion number

Fr~. 8. Bio-Gel f r a c t i o n a t i o n o f t h e R N A s y n t h e s i z e d i~ vitro b y d i s r u p t e d n u c l e i in 30 rnin. Nuclei c o n t a i n i n g a b o u t 200 Fg D N A were i n c u b a t e d w i t h 250 FCi [cr (spee. act. 10 Ci/mmol) a n d o t h e r i n g r e d i e n t s in a 1.0-ml vol. a t 25~ for 30 rnin. T h e R N A p r o d u c t s were a p p l i e d to a 2.6 e m • 66 c m c o l u m n o f Bio-Gel A 5 0 m , 5.5-ml f r a c t i o n s were collected a n d 0.5-ml p o r t i o n s were c o u n t e d a f t e r trichloroacetic acid p r e c i p i t a t i o n . - - 0 - - 0 - - , 32p r a d i o a c t i v i t y ors/ m i n • 10 -3 p e r 0.5 rnl; ( -), A26o m o n i t o r e d b y a Gilford s p e c t r o p h o t o m e t e r .

ACCENTUATED E X P R E S S I O N OF F I B R O I N GENES

199

similar for in vitro and in vivo labeled RNA. However, there is a higher proportion of lower molecular weight RNAs labeled in vitro than that labeled in vivo or by organ culture. (h) I n vitro synthesis of fibroin messenger R N A in disrupted nuclei The void volume fraction shown in Figure 8 which contained 14% of the total radioactivity was recovered and a portion was subjected to RNAase T1 digestion for characterization. The sample was estimated to contain about 15% fibroin m R N A (Fig. 9(c)). Therefore, we can conclude t h a t about 2-1% of the total transcripts synthesized in vitro in 30 minutes was fibroin m R N A of apparent full size length. The hybridization assay was used to confirm the presence of m R N A sequences. A portion of the void volume fraction of Figure 8 was hybridized to DNA fractionated by actimomycin D/CsC1, and some radioactivity hybridized to DNA in the fibroin gene region (Fig. 10). The hybrid was recovered from filters 10 to 15, digested with T1 RNAase, and the digest was subjected to a DEAE-Sephadex column (Fig. 9(d)). Figure 9(d) demonstrates clearly the existence of fibroin m R N A sequence in the in vitro RNA, and comparison of Figure 9(c) (15% pure fibroin mRNA) with (d) (40% pure) indicates t h a t the m R N A was selected by the hybridization procedure. Nucleoplasmic R N A polymerase I I (Roeder & Rutter, 1969) is sensitive to low

z 11 9 ~ T m

31

(o)

z ,r x ~

(b)

i

0

9

x i=

x

E

!

0~ 75,

o

(d)

(c)

~oI

I 0 x c E

25

0

20

0

40

Froction

20

40

number

Fro. 9. RNAase 's fingerprints of various RNAs labeled with [~-32P]UTP. (a) Fibroin mRNA from the posterior glands labeled in vivo for 24 h during the 6th day of the 5th instar. (b) High molecular weight RNA (void volume fraction of a Bio-Gel A50m column) from an animal's carcass labeled in vivo for 24 h during the 6th day. (c) A portion of crude fibroin mRNA fraction (the void volume fraction shown by the bracket in Fig. 8) synthesized in vitro in disrupted nuclei. (d) Another portion of the void volume fraction of Fig. 8 was hybridized to fibroin genes (Fig. 10), and purified. - - 0 - - 0 - - , 32p radioactivity. The Roman numerals in the Figure stand for the chain length of oligonucleotides. 14

200

Y. S U Z U K I

AND P. E. GIZA

6 w

O x .C

4

t~

I

I

~5

0

5

I0

15

20

Fraction number

FIe. 10. Hybridization of crude fibroin m R N A synthesized i n vitro to partially purified fibroin genes. JB. mori DNA (2.0 rag) was sheared, actinomyein D (1.0 rag) was added, and the refractive index was adjusted to 1.3842. The mixture was centrifuged at 32,000 revs/min a t 20~ for 66 h in a Spineo 50.2 rotor. The void volume fraction of Fig. 8 was hybridized to the DNA filter in 50% formamide]4 • Tris]EDTA/NaC1 buffer at 50~ for 20 h, and the R N A hybridized to the fibroin gene region shown by the bracket was recovered for RNAase T1 fingerprint analysis {Fig. 9(d)). The hybridization and elution of the hybrid R N A was repeated. - - @ - - @ - - , a2p radioactivity; ( ), A260 monitored b y a Gilford spectrophotometer.

concentrations of cr (Lindell et al., 1970; Kedinger et al., 1970; Schwartz et al., 1974; Weinman et al., 1974) and is probably the polymerase form t h a t is involved in transcription of the nuclear precursors to mRNA (Blatti et al., 1970; Zylber & Penman, 1971; Reeder & Roeder, 1972). Therefore we wanted to know if fibroin mRNA synthesis in the disrupted nuclei is sensitive to low doses of ~-amanitin. RNA synthesis was carried out for 30 minutes in the presence or absence of 0.2 Fg of ~-amanitin/ml. The drug inhibited 21~o of the in vitro RNA synthesis. Each product was hybridized to partially purified fibroin genes in the presence or absence of unlabeled pure fibroin mRNA (Table 6). Fibroin mRNA as defined by labeled RNA TABLE 6

Inhibition of in vitro fibroin messenger R N A synthesis by a low dose of o~-ama~itin

System of R N A synthesis

Complete ~ Complete + ~-amanitin b

Hybridization to partially purified fibroin genes Without cold fibroin With cold fibroin

mRNA (cts]min)

mRNA (cts]min)

474 156

124 112

Nuclei containing 150/~g DNA were incubated with 80 ~Ci [aH]UTP for 30 min in the absence (~) or presence (b) of 0.2 #g cr For assay 2.0 mg B. m e r i DNA was fractionated b y actinomycin D/CsC1, and 10% portions of the fractions containing fibroin genes were used for hybridization. For the hybridization 2.5~/o portions of each reaction mixture (~ or b) were used in the absence or presence of unlabeled fibroin m R N A (5.1 ~g/ml). Hybridization was done at 50~ for 2.5 h in 5 ml 50~o formamide]4 • Tris/EDTA/NaC1 buffer.

ACCENTUATED

EXPRESSION

OF FIBROIN

GENES

201

which hybridized to fibroin DNA and was competed for by excess fibroin mRNA, was not synthesized in the presence of ~-amanitin (Table 6). (i) Ability of disrupted nuclei to initiate R N A synthesis We wanted to test the ability of disrupted nuclei to initiate RNA synthesis in vitro. We chose [7-82P]ATP as a substrate because the 5' end structure of mature fibroin mRNA is known to be mTG(5')ppp(5')AmpUmpCp (Yang eta/., 1976). The incorporation of [y-32P]ATP was carried out for 30 minutes. The RNA was purified as described in the legend to Figure 11 and fractionatcd by a Bio-Gel A50m column (Fig. 11). Portions of fractions 14 to 19 were pooled, and subjected to the following

H

b

2

I

I

>, >

"d

8

Ol.

o

~ 20

40

60

Froction number

Fxo. 11. Fractionation of R N A labeled with [7-32P]ATP in vitro b y the disrupted nuclei. Nuclei containing 150 #g DNA were incubated with 500/~Ci [y-32P]ATP (speo. act. 18.7 Ci/mmol) and other ingredients in a 1.0-ml vol. a t 25~ for 30 min. The R N A was phenol-extracted, precipitated twice with ethanol, passed through a 0.9 cm • 66 cm column of Sephadex G25, precipitated again twice with ethanol, and fractionated b y a 2.6 cm • 66 em colnmn of Bio-Gel A50m. Out of 5.5-ml fractions each 1.0-ml portion was counted after trichloroacetic acid precipitation. - - 0 - - 0 - - , 32p radioactivity cts/min x 10 -3 per ml.

analysis. The sample was digested with RNAase Tz, and the digest was divided into two. One part was directly fractionated by a I)EAE-Sephadex column (Fig. 12(a)). The other was digested further with alkaline phosphatase, and then subjected to a DEAE-Sephadex column (Fig. 12(b)). In Figure 12(a) digests having negative charges of 5.5, 6.5, 7.5 and so on were observed. These are tentatively identified as 32pppApGp, 32pppApXpGp, 82pppApXpXpGp and so on. The small peak having a negative charge of 4-3 in Figure 12(a) was unidentified. The 32p radioactivity shown in Figure 12(a) was mostly sensitive to alkaline phosphatase (Fig. 12(b)). This result excludes the unlikely possibility t h a t a strange structure like A(5')ppp82(5')Gp or

202

Y.

SUZUKI

AND

P. E. GIZA

50

-2

-3 - 4 -5 - 6

(c)

F

0,4 I, f

25

d,f ~

21

0.2

0 I0

-z

0

-~

-4

-5 -6 - 7 ~ . 9

(d)

A :i

~a

o

>

zL~

u

ev

-2

-~ - , * -s -~-7-e-9

I 20 Froct|on

(el ~

.o 0"4

40 number

\

20 Froction

40

o

number

Fzo. 12. Characterization of the in vitro RNA labeled with [F-32p]ATP. Portions of fractions 14 to 19 of Fig. i1 were pooled, E. coli tRNA was added as a carrier, precipitated with ethanol twice, and the precipitate was divided into 2. One was digested with Tz RNAase (a), and the other was digested further with alkaline phosphatase (b). Portions of fractions 20 to 54 of Fig. 11 were pooled, E. cell tRNA was added, and precipitated with ethanol twice. Portions were subjected to digestion with 0.3 ~-KOH (c), pancreatic RNAase (d) and T1 RNAase (e). All the digests were fractionated on D E A E - S e p h a d e x A25 columns with a NaC1 gradient of 0.10 M to 0.40 M in the presence of 7 M-urea. Before application to t h e c o l u m n the digest for (e) w a s n e u t r a l i z e d a n d

carrier oligonucleotides were added, and to the digest for (e) cold ATP was added, as markers. - - 0 - - 0 - - , 32p radioactivity; ( ), A2~o monitored by a Gflson spectrophotometer; ( . . . . . ), NaCI gradient. A(5')ppp32(5')XpGp is the product. Fractions 20 to 54 were also pooled a n d analyzed. Hydrolysis of a portion of the sample b y 0-3 ~ - K O H gave essentially a single peak at a negative charge of 4.3 (Fig. 12(c)), which we t e n t a t i v e l y identify as 32pppAp. Therefore 32p was n o t incorporated into internal positions. Digests h a v i n g negative charges of 5, 6, 7 etc. were obtained b y pancreatic R N A a s e digestion (Fig. 12(d)) t o g e t h e r with an unidentified p e a k having - - 3 . 8 charge. B y T1 R N A a s e digestion we f o u n d digests h a v i n g negative charges of 5.5, 6.5, 7.5 etc. (Fig. 12(e)). There was a small p e a k co-chromatographing with p p p A m a r k e r and a large peak h a v i n g ---4.3 charge which we could n o t identify (Fig. 12(e)). A l t h o u g h there are some unidentified p r o d u c t s in the digests we conclude t h a t the m a j o r i t y of 32pppA was incorporated at t h e 5' end of R N A molecules in t h e void volume fraction, which contained 15%

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fibroin mRNA, as well as in the lower molecular weight region of the Bio-Gel column (Fig. 11). 4. Discussion (a) In vivo synthesis of fibroin messenger R N A The rate of fibroin mRNA synthesis is almost constant throughout the fifth instar (Table 2). The expression of the fibroin gene is regulated nevertheless since mRNA synthesis is not detected during the fourth moulting stage (Fig. 4; Suzuki & Suzuki, 1974; Suzuki, 1976a,b). The relative proportion of newly synthesized fibroin mRNA to total cellular RNA labeled in vivo for 30 minutes or 12 hours was low in the early part of the instar but increased steadily as the instar proceeded (Table 3). This was probably due to the decrease in rRNA and heterogeneous RNA synthesis observed during the fifth instar. The rate of rRNA synthesis dropped about 30-fold between the early and late instar (Table 2). Synthesis of heterogeneous RNAs, which a r e eluted together with fibroin mRNA in the void volume of a Bio-Gel AS0m column, was very active in the early part of the instar and retarded by the late instar (Fig. 3 and Table 3; see also Fig. 5 of Suzuki & Suzuki, 1974). It should be emphasized that except for the first day of the instar the rate of fibroin mRNA synthesis always exceeds the highest rate of rRNA synthesis which was observed during the second day of the instar. Late in the fifth instar fibroin mRNA is being synthesized 50 times more rapidly than rRNA, and it comprises as much as 7.5% of the RNA which is synthesized in vivo in a 30 minute period. This is the highest proportion of transcription ever found for a single mRNA. These results indicate that the late fifth instar is the best stage to analyze fibroin gene transcription. Recent pulse-labeling experiments have shown that fibroin mRNA comprises about 4 and 6% of the total RNAs labeled in 10 minutes on the middle of the seventh and eighth (last) day of the fifth instar, respectively (Suzuki, unpublished work). In the initial report on fibroin mRNA (Suzuki & Brown, 1972) we could not detect fibroin mRNA in the posterior silk glands labeled for 50 minutes on the sixth day of the instar. As shown in Table 3 about 4-8% of the RNA labeled in a 30-minute pulse was identified as fibroin mRNA. Furthermore, by a 50-minute pulse 6.8~/o of the RNA was identified as fihroin mRNA. These discrepancies are due to less sensitive methods used in the initial studies (fractionation of RNA by sucrose gradient centrifugation and base composition analysis of a pooled fraction). The profiles shown in Figure 3(a) through (d) indicate that fibroin mRNA has a stable nature compared to other heterogeneous RNAs and is accumulating throughout the labeling time of 0-5 to 12 hours (Table 3). It was also shown that fibroin mRNA is accumulating throughout the fifth instar (Suzuki & Suzuki, 1974) and culminates in comprising as much as 4"4~/o of the total cellular RNA toward the end of the instar (Table 1). Our previous finding that fibroin mRNA is as stable as rRNA and has a half-life measured in days (Suzuki & Brown, 1972) has been confirmed in this paper. When we labeled posterior gland RNA for 4 and 12 hours on the seventh day of the fifth instar, the ratio of fibroin mRNA synthesized per gene to that of rRNA synthesized per gene was constant for both labeling periods (about 50). It means that fibroin mRNA has almost the same stability as rRNA at least during the 8-hour period. Therefore, a previous suggestion that some pulse-labeled fibroin mRNA might turnover (Suzuki, 1976b) is unlikely. When we calculated the rates of radioactivity

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incorporated into fibroin mRNA]gene per minute from the pulse-labeling experiments shown in Table 3, we did not find a higher rate but a lower rate for a shorter pulse than that for a longer pulse. However, we cannot be definite about this calculation until we measure the pool size of nucleotides in the posterior silk glands. (b) In vitro synthesis of fibroin messenger R N A The proportion of newly synthesized fibroin mRNA to total RNA labeled in vitro for 30 minutes or 90 minutes in organ culture was similar to that found in the in vivo experiment. Therefore the activated state of fibroin genes is being retained even in the isolated posterior silk glands. By isolation of nuclear fragments we were able to eliminate over 90% of the stable cytoplasmic rRNA and fibroin mRNA. However, a nuclear preparation containing about 200 ~g of DNA still contains 10 to 20 ~g of unlabeled fibroin mRNA. This is a great disadvantage; it practically prohibits the use of a probe like complementary DNA synthesized by reverse transeriptase because the cold mRNA greatly reduces the specific activity of fibroin mRNA synthesized in vitro. Fibroin mRNA synthesized during a 30-minute pulse in vivo was found evenly distributed between the cytoplasmic and nuclear fractions. Therefore the time required to transport fibroin mRNA from nucleus to cytoplasm is less than 30 minutes provided leakage of the mRNA did not occur during the nuclear isolation. This is a considerably longer time than that found for transport of histone mRNAs measured in HeLa or L cells (Adesnik & Darnell, 1972; Schochetman & Perry, 1972). Incorporation of [aH]UTP was almost linear in isolated nuclei for 30 minutes at 25~ By hybridization competition experiments we have shown that repetitious sequence transcripts including rRNA and fibroin mRNA are synthesized in vitro, and competed for completely by unlabeled posterior gland RNA. By the T 1 RNAase fingerprint method fibroin mRNA was quantitated in the in vitro products, and the proportion of fibroin mRNA to total transcripts synthesized in a 30-minute pulse w a s estimated at about 2%. This fraction is lower than that made in vivo (7.5%) or in organ culture (5.7%). However, we assayed only the mRNA sequences of apparent full size length which appeared at the void volume fraction of a Bio-Gel AS0m column. With the current method we cannot quantitate incomplete fibroin mRNA molecules. In fact, although this system synthesizes somewhat higher molecular weight RNAs than other systems (Marzluff et al., 1973; Wu & Zubay, 1974; Groner et al., 1975), the in vitro RNA shown in Figure 8 contains a lower proportion of higher molecular weight RNAs than does the in vivo RNA (Fig. 2(a)). This could be due to several factors including premature termination and RNAase activity. It is also possible that processing of RI~A and stabilization of mRNA may be altered greatly in these disrupted nuclei. Yet the proportion 2% for fibroin mRNA is still about two orders of magnitude higher than that of globin mRNA sequences synthesized in vitro (Axcel et al., 1973; Gflmour & Paul, 1973; Steggles et al., 1974). The predominance of fibroin mRNA proportion can be explained partly by its huge molecular weight nature compared to globin mRNA (Gaskill & Kabat, 1971; Williamson et al., 1971) a s well as the high concentration of fibroin gene in the B. mori genome (Suzuki et aI., 1972) compared to globin gene concentration in mammals (Harrison et al., 1972). However, it should be reminded that the constant rate of fibroin mRNA synthesis i s higher than the highest rate of rRNA synthesis in the posterior silk glands. ~urthermore, at the late fifth instar expression of rDNA and genes for heterogeneous RNAs

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are greatly suppressed. Therefore we m a y expect to find regulatory factors for fibroin genes a b u n d a n t l y in this system. Furthermore the observation t h a t the isolated nuclei retain their ability to reinitiate R N A synthesis strengthens the usefulness of the system for i n vitro study of fibroin gene regulation. The fact t h a t fibroin m R N A synthesis is sensitive to 0.2/zg of a-amanitin/ml supports the idea t h a t R N A polymerase I I is responsible for the synthesis of m R N A s and their precursors. This is the first direct evidence t h a t R N A polymerase I I is the form which transcribes structural genes in the living cell. We acknowledge the critical reading of the manuscript by Drs Donald D. Brown, Igor B. Dawid, James D. Ebert, Yasumi Ohshima and Ronald H. Reeder, and occasional technical assistance b y Etsuko Suzuki. REFERENCES Adesnik, M. & Darnell, J. E. (1972). J . Mol. Biol. {}7, 397-406. Axcel, 1~., Cedar, I=L & Felsenfeld, G. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 2029-2032. Blatti, S. P., Ingles, C. J., Lindell, T. J., Morris, P. W., Weaver, R. F., Weinberg, F. & Rutter, W. ft. (1970). Cold Spring Harbor Syrup. Quant. Biol. 35, 649-657. Dische, Z. (1953). J . Biol. Chem. 204, 983-997. Gage, L. P. (1974a). Chromosoma, 45, 27-42. Gage, L. P. (1974b). J . Mol. Biol. 86, 97-108. Gage, L. P. & Manning, R. F. (1976). J . Mol. Biol. 101, 327-348. Gaskill, P. & Kabat, D. (1971). Proc. Nat. Acad. Sci., U.S.A. {}8, 72-75. Giles, K. W. & Myers, A. (1965). Nature (London), 20{}, 93. Gilmour, R. S. & Paul, ft. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3440-3442. Groner, Y., Monroy, G., ffacquet, M. & Hurwitz, J. (1975). Prov. Nat. Aead. Sci., U.S.A. 72, 194-199. Harrison, P. R., Hell, A., Birnie, G. D. & Paul, J. (1972). Nature (London), 289, 219-221. Kedinger, C., Gniazdowski, M., Mandel, J. L., Gissinger, F. & Chambon, P. (1970). Biochem. Biophys. Res. Commun. 38, 165-171. Lambert, B. & Daneholt, B. (1975). In Methods in Cell Biology (Prescott, D., ed.), col. 9, pp. 17-47, Academic Press, New York. Lillie, R. D. (1948). In Histopathologic Technic, pp. 300, The Blakiston Co., Philadelphia. Linden, T. J., Weinberg, F., Morris, P. W., Roeder, R. G. & Rutter, W. J. (1970). Science, 170, 447-449. Lizardi, P. M. & Brown, D. D. (1975). Cell, 4, 207-214. Lizardi, P. M., WiUiamson, R. & Brown, D. D. (1975). Cell, 4, 199-205. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J . Biol. Chem. 198, 265-275. Marzluff, W. F., Murphy, E. C. & Huang, R. C. C. (1973). Biochemistry, 12, 3440-3446. Ono, M. (1942). Bull. Kagoshima Agr. Coll. 14, 123-156. Rasch, E. M. (1974). Chromosoma, 45, 1-26. Reeder, R. H. {1973). J . Mol. Biol. 80, 229-241. Reeder, R. H. & Roeder, R. G. (1972). J . Mol. Biol. 67, 433-441. Roeder, R. G. & Rutter, W. ft. (1969). Nature (London), 224, 234-237. Schmidt, G. & Thannhauser, S. ft. (1945). J . Biol. Chem. 161, 83-89. Schochetman, G. & Perry, R. P. (1972). J . Mol. Biol. 63, 591-596. Schwartz, L. B., Sklar, V. E. F., Jaehning, J. A., Weinman, R. & Roeder, R. G. (1974). J . Biol. Chem. 249, 5889-5897. Steggles, A. W., Wilson, G. N., Kantor, J. A., Pieciano, D. J., Falvey, A. K. & Anderson, W. F. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 1219-1223. Suzuki, Y. (1976a}. In Results and Problems i n Cell Differentiation (Beerman, W. & Grossbach, U., eds), col. 10, Springer-Verlag, Berlin, in the press. Suzuki, Y. (1976b). In Advances in Biophysics (Kotani, M., ed. ), col. 8, pp. 8 3 - 1 1 4 , University Tokyo Press and University Park Press, Tokyo and Baltimore.

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