Molecular characterization of an 18 kb segment of DNA puff C4 of Bradysia hygida (Diptera, sciaridae)

Chromosoma (1995) 103:715-724

CHROMOSOMA 9Springer-Verlag 1995

Molecular characterization of an 18 kb segment of DNA puff C4 of Bradysia hygida (Diptera, Sciaridae) Nadia Monesi 1, M. Aparecida Fernandez 2, Aparecida M. Fontes i, Luiz R. Basso Jr 1, Yoshinobu Nakanishi 3, Bruno Baron 4, Gerard Buttin 4, Maria Luisa Pa~d-Larson ~ I Departamentode Morfologia,Facutdadede Medicinade Ribeir~oPreto, Universidadede S~o Paulo, Ribeir~oPreto, SP 14049-900, Brazil 2 Departamento de Biologia, UniversidadeEstadual de Maring~,PR, Brazil 3 Departmentof Biology,Facultyof PharmaceuticalSciences, KanazawaUniversity,Kanazawa,Japan 4 Units de G6n~tiqueSomatique (URA CNRS 361), Institut Pasteur, 25 rue du Dr. Roux, F-75724 Paris Cddex 15, France Received: 7 July 1994; in revised form: 6 March 1995 / Accepted: 9 March 1995 Abstract. The data presented here are an extension of the molecular characterization of DNA puff C4 of Bradysia hygida. A cDNA related to a gene amplified in this puff and expressed when puff C4 expands was cloned and sequenced. Analysis of the amino acid sequence deduced from the open reading frame present in the cDNA indicate that the encoded protein is secreted and comprises mostly a-helical coiled-coil. An 18 kb genomic segment containing the transcription unit of this gene was also cloned and the structure and expression of the 1.4 kb mRNA was determined. Quantitative slot blot hybridization of DNA complementary to the transcription unit shows that this gene is amplified about 21 times in the salivary gland, confirming data previously obtained. Fragments upstream of the 5' end, and beyond the 3' end, of the gene transcription unit were also analysed and shown to be amplified at least eight and five times, respectively. Based on these data we discuss how amplification could occur at DNA puffs.

Introduction DNA puffs formed in chromosomes of sciarid salivary glands, late in the fourth larval instar, have been directly characterized as sites of developmentally regulated gene amplification and expression (Glover et al. 1981; Pa96Larson et al. 1992; Fontes et al. 1992; Wu et al. 1993). Current evidence indicates that the rationale for gene amplification is the requirement for large amounts of specific gene products (for review see Stark et al. 1990). In the case of DNA puffs, little is known about the products of the amplified genes. Since the timing of DNA puff formation and the detection of its mRNA in the salivary gland correspond at least in part to the period of cocoon weaving, it is reasonable to assume that some of

Genbank accession numbers: U13883, U13892 Edited by: E.R. Schmidt Correspondence to: M.L. Pa~6-Larson

these genes code for fibrous proteins that compose the cocoon. In Rhynchosciara, certain secretory proteins synthesized at the time of puffing have been assigned to particular DNA puffs on the basis of temporal correlations (Winter et al. 1977). Moreover, analysis of the deduced proteins of the Sciara coprophila DNA puff II/A (DiBartolomeis and Gerbi 1989) and Rhynchosciara DNA puff C8 (Frydman et al. 1993) genes indicate that these puffs encode proteins carrying a putative signal sequence for secretion and have a high content of hydrophobic amino acids, compatible with the insoluble nature of the cocoon material. As demonstrated by early autoradiographic and cytophotometric studies in various species of sciarids, amplification in DNA puffs is due to extra DNA replication, specifically at the puff loci (for review see Lara et al. 1991). At least until the end of the larval stage, the amplified sequences accumulate in the gland as shown by quantitative Southern blot hybridization (Pa~6-Larson et al. 1992; Fontes et al. 1992; Wu et al. 1993), and stay bound to the chromosomes as indicated by cytophotometric studies (Rasch 1970; Pa~6-Larson 1982). The amplified DNA seems to be arranged in parallel rather than in tandem, since the genomic DNA restriction patterns of fly and salivary gland at stages either before or after amplification are the same with all probes and restriction enzymes used (Santelli et al. 1991; Pa~6-Larson et al. 1992; Fontes et al. 1992; Wu et al. 1993). Furthermore, quantitative hybridization of two restriction fragments from Bradysia hygida DNA puff C4 showed that they are unequally amplified in the salivary gland (Coelho et al. 1993). One of the fragments that contains part of the gene expressed when puff C4 expands is amplified about eight times more than the other fragment that does not contain sequences expressed in the gland during this puff expansion. This observation is consistent with the idea that gene amplification in DNA puffs follows an onion skin mechanism, whereby the copy number of the gene is amplified by repeated re-initiation of DNA synthesis in the vicinity of the gene whose expression is correlated with puff expansion. Strong evidence support-

716 ing this p r o p o s i t i o n is the r e c e n t i d e n t i f i c a t i o n o f a r e p l i c a t i o n origin in an - 1 kb fragment, l o c a t e d - 2 . 5 kb ups t r e a m o f the gene II/9-1, p r e f e r e n t i a l l y active during a m p l i f i c a t i o n in S. coprophila D N A p u f f I I / g A ( L i a n g et al. 1993). S e v e r a l lines o f e v i d e n c e h a v e e s t a b l i s h e d that a m p l i f i c a t i o n o f c h o r i o n g e n e s in Drosophila f o l l i c u l a r cells o c c u r s v i a an o n i o n s k i n m e c h a n i s m (for r e v i e w see S p r a d l i n g 1987; O r r - W e a v e r 1991). O n e o f the first i n d i c a t i o n s for this was g i v e n b y S p r a d l i n g (1981) w h o d e s c r i b e d the o c c u r r e n c e o f an a m p l i f i c a t i o n g r a d i e n t in the a m p l i f i e d r e g i o n w i t h its m a x i m u m in the c h o r i on g e n e c l u s t e r r e g i o n . I f g e n e a m p l i f i c a t i o n in D N A puffs also f o l l o w s an o n i o n skin m e c h a n i s m , as the d a t a o b t a i n e d in d i f f e r e n t s p e c i e s suggest, the level o f amp l i f i c a t i o n s h o u l d d e c l i n e in the r e g i o n s f l a n k i n g the g e n e s that are e x p r e s s e d d u r i n g p u f f e x p a n s i o n . A l t h o u g h g e n e ( s ) a m p l i f i e d in the D N A puffs I I / 9 A o f S. coprophila ( D i B a r t o l o m e i s and G e r b i 1989) and in p u f f C8 o f Rhynchosciara americana ( F r y d m a n et al. 1993) h a v e b e e n c l o n e d , and g e n o m i c c l o n e s f r o m puffs C8 and C3 o f Rhynchosciara and p u f f I I / 9 A o f Sciara h a v e b e e n m a p p e d ( M i l l a r et al. 1985; L i a n g et al. 1993), the a m p l i f i c a t i o n level o u t s i d e the t r a n s c r i p t i o n units o f t h e s e g e n e s has not b e e n r e p o r t e d . W e d e s c r i b e h e r e the cloning and molecular characterization of a gene amp l i f i e d in B. hygida D N A p u f f C4, w h o s e e x p r e s s i o n c o r r e l a t e s w i t h the e x p a n s i o n o f this p u f f ( P a 9 6 - L a r s o n e t a l . 1992). Q u a n t i t a t i v e slot b l o t h y b r i d i z a t i o n o f D N A c o m p l e m e n t a r y to the t r a n s c r i p t i o n unit s h o w s that this g e n e is a m p l i f i e d a b o u t 21 t i m e s in the salivary g l a n d , c o n f i r m i n g p r e v i o u s d a t a o b t a i n e d b y S o u t h ern b l o t h y b r i d i z a t i o n ( P a q 6 - L a r s o n et al. 1992). F r a g m e n t s f r o m e i t h e r side o f the g e n e w e r e also a n a l y s e d and s h o w n to b e a m p l i f i e d in the gland. T h e f r a g m e n t u p s t r e a m o f the g e n e is a m p l i f i e d at l e a s t e i g h t times, and the f r a g m e n t b e y o n d the 3' and is a m p l i f i e d at l e a s t five times. A l t h o u g h t h e s e v a l u e s r e p r e s e n t u n d e r e s t i m a t e s o f the level o f a m p l i f i c a t i o n t h e y c o n t r i b u t e to the e v i d e n c e that the w h o l e 18 k b s e g m e n t is c o n t a i n e d in an a m p l i f i e d r e g i o n o f D N A p u f f C4. C l o n i n g and c h a r a c t e r i z a t i o n o f f r a g m e n t s b e y o n d this 18 kb segm e n t are still n e e d e d in o r d e r to d e f i n e the structure o f the w h o l e a m p l i c o n .

cDNA library. A cDNA library was prepared using a poly(A)+ RNA fraction purified from salivary gland RNA at the period when DNA puffs are formed (from E7 until about 16 h after E7). Double-stranded cDNAs were synthesized and ligated to )vgtl0 arms using the )~gtl0 cDNA cloning system (Amersham). The ligated DNA was packaged in vitro and used to infect Escherichia coli strain NM514, resulting in 106 plaque forming units. Screening of the library and phage DNA mini-preparations were performed according to Sambrook et al. (1989). Genomic DNA library. A genomic library was prepared using DNA from female flies. To obtain high molecular weight fly DNA, about 800-1000 female flies were pulverized in liquid nitrogen. The powder was ground in 10 ml of homogenizing buffer (10 mM Tris-HC1, pH 7.4, 60 mM NaC1, 10 mM EDTA, 0.15 mM spermidine, 0.5% Triton X-100), at 0 ~ C in a glass homogenizer with a Teflon pestle. After filtration and centrifugation at 5800 g, for 7 min at 4 ~ C, the supernatant was removed and the pellet was resuspended in 10 ml of homogenizing buffer. The homogenization and centrifugation were repeated. The pellet was resuspended in l0 ml of extraction buffer (20 mM Tris-HC1, pH 7.4, 1 mM EDTA, 0.5% SDS, 1 mg/ml proteinase K), followed by incubation for 4 h at 37 ~ C with gentle agitation. NaC1 was added to 0.2 M, followed by two extractions with 1.1 phenol:choroform and one extraction with chloroform. Nucleic acid was ethanol precipitated, and the DNA fibres were collected, washed with 70% ethanol, dried and dissolved in 500 btl of TE (10 mM Tris-HC1, pH 8.0, 1 mM EDTA). This DNA was partially digested with Sau3A, and fragments of about 20 kb were isolated by sucrose gradient centrifugation. The fractions were assayed by electrophoresis in 0.4% agarose gels. The selected DNA was cloned using the lambda dash II/BamHI cloning kit (Stratagene). The ligation mixture used to infect E. coli strain P2392 resulted in 1.8x106 plaque forming units. About 2x105 phages were used in one screening. The positive clones were submitted to two additional screenings in order to obtain pure plaques. Phage screening, DNA mini-preparations and DNA large scale preparations were performed as described in Sambrook et al. (1989). Mapping of the positive clones was done by single, double and triple digestions. Nucleic acid isolation. Salivary gland DNA and RNA extractions were done following the procedure described previously (Pa~6Larson et al. 1992). Briefly, about 200 hand dissected salivary glands were incubated in 2 ml of extraction buffer (20 mM TrisHC1, pH 7.4, 0.5% SDS, 0.5 mg/ml Proteinase K) and gently shaken for 30 rain at room temperature. After phenol extraction the nucleic acid was precipitated with ethanol. The DNA fibers were collected and the RNA remaining in the ethanol was allowed to precipitate overnight at -70 ~ C. DNA extraction from adult flies was performed as described above. The concentrations of salivary gland and fly DNA were evaluated by fluorimetry using bisbenzimide (H33258 from Boehringer). Salivary gland RNA concentrations were estimated by absorbance at 260 nm.

Materials and methods S1 nuclease assay. The procedure used was essentially that deBradysia hygida. Salivary glands of female larvae and adult flies of B. hybida (Diptera, Sciaridae) were used from a laboratory culture kept at 20 ~ C (Sauaia et al. 1971). A description of the life cycle of B. hygida and relevant details of the fourth instar are given in Fig. 1. The salivary glands show three distinct morphological regions (S1, $2 and $3), whose cells contain four chromosomes named A, B, C and X. Chromosome segments that form the DNA puffs C4 and C5, late in the last larval instar, are shown in Fig. 2 (Laicine et al. 1984). The puff C4 is part of a group of DNA puffs (C4,C5 and C7) that appears first, around stage E7. Sixteen hours later, when this group of puffs has regressed, a second group expands. The formation of puffs C4, C5 and C7 begins at the same time, but they reach maximum expansion at different times. As shown in Fig. 2, puff C4 is fully expanded 4 h after puff C5 has reached its maximum expansion.

scribed by Favaloro et al. (1980). About 1 ~g of the 0.3 kb HindIII-EcoRI fragment and 10 btg of total RNA of E7 salivary glands were co-precipitated in ethanol. The pellet was resuspended in 30 gl of hybridization buffer (40 mM PIPES, pH 6.4, 1 mM EDTA, 0.4 M NaC1, 80% formamide). This mixture was incubated for 10 min at 85 ~ C, and then transferred to a 45 ~ C water bath, and incubated for an additional 3 h to favour the formation of RNA/DNA hybrids. After this time, 300 btl of the S1 nuclease buffer (0.28 M NaC1, 0.05 M sodium acetate, pH 4.6, 4.5 mM ZnSO 4 and 20 gg/ml of salmon sperm DNA), containing 100 U of S1 nuclease (Pharmacia) were added to the reaction, and incubated for 1 h at 37 ~ C. The products resistant to S1 nuclease action were subjected to electrophoresis in neutral agarose gels. The gels were blotted onto nitrocellulose and the filter hybridized with the 0.3 kb HindIII-EcoRI fragment.

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Fig. 1. A Stages in the development of Bradysia hygida. Photographs of a larva at the third moult (3rd M), fourth instar female larvae with eyespot patterns E1 to E7 (dorsal view), and at pupal moult (PM, side view). B Time scale at 20 ~ C. The larvae undergo the third moult 12 days after hatching (H). During day 18 after hatching (day 6 of the fourth instar) the larval eyespots start to appear (El). As they change in shape, size, and position, the eyespots provide useful landmarks for staging larval development during the second half of the fourth instar (Gabrusewycz-Garcia 1964). The eyespot pattern E3 coincides with a turning point in development. At this time the larvae abandon food and start spinning a cocoon. In the next 46 h there are striking changes in the

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puffing pattern of the salivary gland chromosomes. The eyespot pattern E7 is a very reliable indicator for predicting the puffing pattern. The precise stage of older larvae is defined in terms of the number of hours that have passed after the pattern E7 was exhibited (for instances, E7+16=16 h after the larvae present eyespot pattern E7). The larvae used in this work were staged by the observation of their morphology and behaviour. About 16 h after E7 the larva stays quietly inside the coccoon, its body is curved, the eyespot has migrated down towards the capsule of the head and the intestine is devoid of food remains. PM, pupal moult; ECD, ecdysis (Laicine et al. 1984)

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Fig. 2. DNA puffs forming at segment 4 and 5 of salivary gland chromosome C. Puff formation is shown at fourth instar stages E3, E5, E7, E7+4, E7+8, E7+12, E7+16 and E7+20 h. The DNA puffs begin their expansion around ES. Four hours after stage E7 puff C4 reaches its maximum expansion and then regresses leav-

ing a compact mass of chromatin typical of this type of puff. Puff C5, also shown in this picture, reaches its maximum expansion in stage E7 and is in regression when puff C4 is fully expanded (Laicine et al. 1984).

Primer extension analysis. An end-labelled oligodeoxynucleotide (5"GGCACTGACACATTGCAATGCA3", positions 58 to 79 in Fig. 4) and 20 mg of total RNA of E7 salivary glands were co-precipitated in ethanol. The pellet was resuspended in 250 mM KC1, 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA. This mixture was incubated for 1 h at 65 ~ C, transferred to room temperature for 20 min, and then submitted to the cDNA first strand synthesis reaction. The reaction product was analysed by electrophoresis on 6% sequencing gels. Dideoxy sequencing reactions of pT7T318U (Pharmacia) using the M13 universal primer served as size markers.

Amersham, UK). In RNA blot experiments, 20 lag of total RNA from larvae was fractionated by electrophoresis in formaldehyde denaturing agarose gels and transferred to nylon membranes (Hybond N, Amersham,UK). Probes were labelled with [o~32p]dCTP (Amersham, UK) using a random primer kit (Random Primers DNA labeling system, Gibco BRL). Hybridization and post-hybridization washes were performed as described in Sambrook et al. (1989).

Southern and RNA blot hybridization. 10 lag of EcoRI digested genomic DNA was subjected to electrophoresis on 0.8% agarose gels run at 50 V in TAE buffer (Sambrook et al. 1989). Subsequently, the DNA was transferred to nylon filters (Hybond-N,

Slot blots. After RNAase treatment, DNA was denatured by boiling for 5 min and kept for 10 min at room temperature in the presence of 0.4 N NaOH. The samples were neutralised by the addition of 0.4 M CH2COO-(NH4)+. Samples were kept on ice until loading. The samples were loaded in a Hybri-slot manifold (BRL) equipped with nylon membranes (Hybond N, Amersham, UK).

718

Sequencing. The DNA was subcloned into the vector pT7T318U (Pharmacia). Overlapping deletions were made with exonuclease III, using the double-stranded Nested Deletion Kit (Pharmacia). Both strands were sequenced completely by the dideoxy sequencing method (Sanger et al. 1977), using the T7 sequencing Kit (Pharmacia). Sequence analysis. Sequence entry and analysis was performed using the PC/GENE v 6.1 (Intelligenetics, Mountain View, Calif.). Amino acid sequence alignments were done using PC/GENE P/ALIGN.

Results Isolation of a cDNA related to the DNA puff C4 gene A cDNA library was constructed from a poly(A) + RNA fraction purified from B. hygida salivary gland RNA at

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Fig. 3A-C. Characterization of the cDNA related to the gene amplified in DNA puff C4. The top panel shows a diagram of the cDNA (second line) aligned with the genomic 4 kb EcoR1 fragment (top line) from puff C4, based on the results of Southern blot hybridization. On the cDNA map, A:0.2 kb and B=l.2 kb subfragments. A Autoradiogram of RNA blots after hybridization with the 0.2 kb cDNA sub-fragment (lane A) and with the 1.2 kb cDNA sub-fragment (lane B). Each lane of the blot contains about 20 gg of total salivary gland RNA from larvae at stages El, E3 and E7. B Autoradiograms of Southern blots after hybridization with the 0.2 kb cDNA sub-fragment (lane A) and with the 1.2 kb cDNA sub-fragment (lane B). Each lane contains about 10 ~g of EcoRI digested DNA of salivary gland at stage E7+16 h. C St nuclease assay using total salivary gland RNA from larvae at stage E7 and the 0.3 EcoRI-HindIII fragment, derived from the 4 kb genomic fragment as shown in the diagram. After S l nuclease digestion, resistant products were submitted to electrophoresis in an agarose gel and analysed by hybridization of the 0.3 kb EcoRIHindIII fragment with a Southern blot of this gel. The arrow points to the protected fragment in lane (+). Lane (-) is a control in which no RNA was added to the hybridization mixture. Lane 0.3 contained the 0.3 kb EcoRI-HindIII fragment only. The strong signal in lane (-) is a smear of the 0.3 kb fragment overloaded in lane 0.3. The size markers in C are a 1 kb ladder (BRL)

the period when DNA puffs are formed (stages E7-E7+16 h). Double-stranded cDNAs were synthesized and ligated into the vector )~ gtl0 using EcoRI synthetic linkers. To select clones related to the gene amplified in puff C4, the cDNA clones obtained were screened with a 32p-labelled 0.3 kb EcoRI-HindIII sub-fragment from a 4 kb genomic clone of puff C4 DNA, which had previously been characterized (Paq6-Larson et al. 1992) and is known to be amplified about 22-fold in the salivary gland. This 0.3 kb EcoRI-HindIII sub-fragment is also the only region in the 4 kb genomic fragment that hybridizes with a 1.4 kb RNA species that is detected in the salivary gland only during the period of DNA puff C4 expansion (Paq6-Larson et al. 1992). The screening of approximately 2.5x103 cDNA clones resulted in 35 positive plaques. The high number of positive clones detected was consistent with the abundance of the 1.4 kb mRNA in the salivary gland during the period of puff C4 expansion. DNA from eight positive clones was purified and the EcoRI digested DNA was analysed by electrophoresis in agarose gels. Three of these clones carried inserts that were cleaved by EcoRI, giving rise to two fragments: one of about 1.2 kb, which was present in all the three clones, and a smaller fragment whose size varied from 0.1 to 0.2 kb. The other five clones analysed had smaller inserts. The clone carrying the largest insert was further characterized by hybridization of RNA and Southern DNA blots. Hybridization of the 1.4 kb cDNA sub-fragments with blots of total RNA from salivary glands detected an mRNA of 1.4 kb in stage E7 when puff C4 expands (Fig. 3A). The hybridization of the EcoRI cDNA subfragments with Southern blots containing EcoRI digested DNA from salivary glands, showed that the 1.2 kb sub-fragment detects a genomic fragment of about 9.0 kb, while the 0.2 kb cDNA sub-fragment is complementary to sequences present in the 4 kb genomic fragment (Fig. 3B). These results show that the 1.4 kb cDNA is related to the gene partially located in the 4 kb EcoRI fragment from puff C4, previously characterized as being amplified and expressed in the salivary gland at the end of the larval stage (Paq6-Larson et al. 1992).

Sequence of the 1.4 kb cDNA The 1.4 kb cDNA described above was subcloned in the phagemid pT7T3 (Pharmacia). A series of overlapping deletions in both strands were made and sequenced using the dideoxy chain termination method. The nucleotide sequence of the transcript is shown in Fig. 4, along with the deduced amino acid sequence of the longest reading frame, which encodes a 450 amino acid peptide. The open reading frame begins at the first ATG codon (at position 22), and ends at the stop codon TAA (at position 1372). A polyadenylation site is found at position 1433, and a stretch of 21 residues of adenine from the poly(A) tail is also present, indicating that the 3" end of the mRNA is complete in this cDNA. The EcoRI site is situated 205 bp downstream from the 5" end, showing that the 5" region of the transcript corresponds to the 0.2

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