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Functional characterization of mfs (2) 31 : a recessive supressor of position effect variegation Burr, Roderick H. L. 1995

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FUNCTIONAL CHARACTERIZATION OF mfs(2)31: A RECESSIVE SUPRESSOR OF POSITION EFFECT VARIEGATION. by RODERICK H.L. BURR B.Sc, McGill University, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLOUMBIA May 1995 © Roderick H.L. Burr, 1995 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of - ^ S O L - P Hr The University of British Columbia Vancouver, Canada Date DE-6 (2/88) 1 1 Abstract The Drosophila melanogaster gene, mfs(2)31, i s a recessive suppressor of p o s i t i o n e f f e c t variegation (PEV) and has two open reading frames (ORFs) putatively derived from a single heterogeneous nuclear RNA (hnRNA). The coding region of Intronic Centrosomal Protein (ICP) i s located i n the f i r s t intron of the u b i q u i t i n 80 amino acid fusion protein (DUb80) trans c r i p t , and i s known to be l i b e r a t e d as a 1.2 Kb polyadenylated mRNA. Antibodies raised against the C-terminal 85 amino acids of ICP were used as a reagent for western b l o t t i n g and i n d i r e c t immunofluorescent st a i n i n g of Drosophila embryos and HeLa c e l l s . ICP was found to c o - l o c a l i z e with p -tubulin i n a T r i t o n X-100® r e s i s t a n t compartment of both the Drosophila embryonic centrosome, and the HeLa c e l l centrosome. ICP was also l o c a l i z e d to small, discrete, T r i t o n X-100® re s i s t a n t 'flecks' within HeLa c e l l n u c l e i i n a pattern suggestive of the nuclear s c a f f o l d . These data suggest that ICP i s involved i n modulating nuclear and cytoplasmic microtubule dynamics throughout the c e l l cycle. I l l Table of Contents Abstract i i Table of Contents i i i L i s t of Tables v i L i s t of Figures v i i INTRODUCTION 1 Positi o n E f f e c t Variegation 1 mfs(2)31 3 C e l l Biology of mfs(2)31... 3 ...and the Centrosome 3 Genetics of mfs(2)31 6 Molecular Biology of mfs(2)31 1 MATERIALS AND METHODS 11 Midi/Maxi Plasmid Prep 11 Double Stranded DNA Sequencing 12 SDS-PAGE 14 Western B l o t t i n g 14 Transfer 14 Antibody Probes 15 Detection 16 iv Batch Purification of Glutathione-S-Transferase (GST) Fusion Proteins 17 Small Scale Purification 17 Large Scale Purifiaction (1 l i t r e ) 18 Colony Hybridization 19 Generation of the pGEX/ICP Fusion Construct 20 Generation of the pGEX/ICP(C85) Fusion Construct 2 1 ELISA 2 3 Production and Analysis of the ICP(C85) Polyclonal Antiserum 24 Removal of GST Specific Antibodies 2 5 A f f i n i t y Purification 2 5 Staining D r o s o p h i l a Embryos with the ICP(C85) Polyclonal Antibody. 2 6 Cell Staining 28 RESULTS 30 pGEX/ICP 30 pGEX/ICP(C85) 3 3 Immunization of Rabbits and Collection of Antisera 42 ICP is Conserved 44 ICP(C85) Antibodies React With a Protein From Divergent Organisms 4 9 V ICP i s Localized to the Drosophila Embryonic Nucleus ICP(C85) Antibody Stains the Centrosome of HeLa C e l l s ICP Co-localizes With p-Tubulin at the Centrosome ICP i s Also Localized Within the Nucleus DISCUSSION 61 Western B l o t t i n g 61 C e l l S t a i n i n g by I n d i r e c t Immunofluorescence 62 mfs(2)31 i s a Complex Locus 63 L e t h a l i t y 64 Minute 64 S t e r i l i t y 65 PEV 66 PEV and the Centrosome '67 PEV and the Nuclear Matrix 68 PEV and DUb80 ' 71 Further Considerations 71 REFERENCES 74 49 52 56 56 v i L i s t of Tables. l . Growth of c e l l s containing pGEX/cDNA-2 clones. 32 2. ELISA to test the r e a c t i v i t y of the immune serum to p u r i f i e d ICP(C85) fusion protein and to p u r i f i e d GST protein. 43 V l l L i s t of Figures. l . Putative transcriptional regulation of mfs(2)31. 8 2. Graphic representation of protein. 3 . Expression and purification analyzed by SDS-PAGE. 4. Time course of GST/ICP(C85) in E. c o l i . ICP and the ICP(C85) fusion 34 of GST and GST/ICP(C85) 37 fusion protein induction 39 5. SDS-PAGE of purified GST/ICP(C85) fusion protein. 41 6. Western blot to show removal of GST specific antibodies. 46 7 . ICP(C85) antibody reacts on western blots with a 30 KDa protein from divergent species. 48 8. Staining of Drosophila embryos with Hoechst 33258 and ICP(C85) antibody. 51 V I 1 1 9. Staining of HeLa c e l l s with ICP(C85) antibody as observed by i n d i r e c t immunofluorescence with a fluorescence microscope. 55 10 . Double l a b e l l i n g of HeLa c e l l s with ICP(C85) antibody (red) and a monoclonal ant i P~tubulin antibody (green). 58 11 . HeLa c e l l s stained with high concentrations of ICP(C85) antibody. 60 1 I N T R O D U C T I O N P o s i t i o n E f f e c t Variegation Position effect variegation (PEV) is a phenomenon observed when normally euchromatic genes are juxtaposed to heterochromatic breakpoints. The best characterized of these effects is the inactivation of the Drosophila white gene in In(l)whitemottled'4 (hereafter vf4) (8) . This inversion of the X chromosome moves the white gene close to the centromeric o -heterochromatin. This rearrangement drastically alters the expression of the white gene in that expression of this reporter is reduced or completely eliminated in some cells but not others. This decision appears to be propagated clonally and is observed as the mottled eye of the adult (For review see 1,6,7,16) . It has been long suggested that this system may be used to assay for non histone chromosomal proteins, regulatory enzymes, or chaperon-like genes involved in the assembly of chromatin (9,10). Most characterized suppressors of PEV act in a genetically dominant fashion. Probably, dominant suppressors encode structural components of chromatin, while recessive mutations w i l l belong to the regulatory hierarchy that controls the spread, timing, and maintenance of centromeric heterochromatin through DNA replication and c e l l division. For many years our lab, in collaboration with Dr. Gunter Reuter, have been screening for suppressors and enhancers of 2 PEV; genes that w i l l , respectively, restore wild type expression, or completely inactivate the white gene in u/"4 of Drosophila. Many such l o c i have been studied but few have been cloned due to the fact that suppressors seem to be refractory to P-element insertion. Su(Var)205 was cloned by generating antibodies to chromatin proteins and probing an expression library. The protein product of this gene, HP1 (Heterochromatin Protein 1) , was found to localize to heterochromatin on polytene chromosome spreads (2). Su(var)3-9 has recently been cloned and is thought to be involved in the process by which chromatin structure, and thus the transcriptional state of genes regulated by chromatin structure, is clonally transmitted through c e l l division (27). Su(var)3-6, now recognized as Protein Phosphatase 1 (PPl), is a serene/threonine phosphatase and is implicated in the regulation of chromatin condensation in interphase cells (28). Another gene, mus209 (mutagen sensitive), recently identified as a recessive suppressor of PEV, was cloned and was found to encode the previously cloned D r o s o p h i l a PCNA, Proliferating Cell Nuclear Antigen, which is conserved within eukaryotes (24). The only haplo-enhancer of PEV yet to be cloned, E(Var)3-93D, i s known to genetically regulate the homeotic genes, presumably via changes in chromatin structure (25). 3 mfs(2)31 mfs(2)31 (male, female s t e r i l e ) i s a recessive suppressor of PEV, and was f i r s t i s o l a t e d by Sandler i n 1977 (3) on the basis of a s t e r i l i t y phenotype. The mutation was mapped c y t o l o g i c a l l y to the 31 region of chromosome 2L and i s c l o s e l y linked to daughterless (da), abnormal oocyte (abo), and the c e l l cycle control gene, cdc2 (29). This region i s narrowed i n polytene chromosome preparations and may be under-replicated or more highly condensed i n chromosomes which undergo polytenization. Our lab has demonstrated that mfs(2)31 i s a strong recessive suppressor of PEV. C e l l biology of mfs(2)31... Lindsley et a l . (1980) (5) reported that male mfs1 homozygotes f a i l to produce viable sperm at 29°C. Light microscopy revealed that these spermatocytes often contain two nuclei, a macronucleus and a micronucleus. Other spermatocytes were seen to contain two centrosomes. They at t r i b u t e d this defect to a f a i l u r e i n the segregation of the centrosomes aft e r duplication implying a defect i n the centrosome i t s e l f or i n the cytoskeletal framework responsible for centrosomal migration. ...and the centrosome. The centrosome of higher eukaryotes i s responsible for the organization of the mitotic spindle and has the properties of both nucleating microtubule growth and anchoring the r e s u l t i n g 4 microtubule array (20; for review see 61). The centrosome consists of a p a i r of cen t r i o l e s , themselves composed of microtubules i n a highly ordered arrangement, surrounded by an electron dense p e r i c e n t r i o l a r material. This p e r i c e n t r i o l a r material i s the center of microtubule nucleation (21). The centrosome i s r e p l i c a t e d i n a semi-conservative manner during S-phase such that one c e n t r i o l e segregates to each of the r e s u l t i n g centrosomes, and i s then r e p l i c a t e d . Mother and daughter centrosomes remain attached u n t i l migration to the opposite poles of the c e l l commences at prophase. This migration i s i t s e l f microtubule based and i s dependant upon microtubule motor proteins found within the centrosome (20) . Assembly of the mitotic spindle also begins at t h i s time but microtubules that w i l l eventually be captured by kinetochores do not invade the nuclear space u n t i l prometaphase. The p o s i t i o n of the centrosomes at prometaphase determines the p o l a r i t y of c e l l d i v i s i o n , a parameter c r i t i c a l for many processes, including development (60). Fungi lack c e n t r i o l e s . The microtubule organizing centre (MTOC) and the spindle pole body (SPB) , are embedded i n the nuclear membrane. Three proteins of the yeast SPB have been i d e n t i f i e d by generating monoclonal antibodies to an SPB enriched f r a c t i o n (55). Also, mutations have been described that a f f e c t the duplication of SPBs and the attachment of nuclear microtubules (22). Morphologically, there are few s i m i l a r i t i e s between the SPB 5 o f f u n g i and the cen t rosome o f h i g h e r e u k a r y o t e s . There has been some q u e s t i o n r a i s e d as t o whe the r the mechanism o f m i c r o t u b u l e n u c l e a t i o n and a t t a c h m e n t i s c o n s e r v e d be tween t h e s e two p h y l a ( 2 2 ) . M u t a t i o n s i n the y - t u b u l i n gene were o r i g i n a l l y i s o l a t e d i n Aspergillus b ecause t h e y s u p p r e s s e d a c o n d i t i o n a l - l e t h a l p-t u b u l i n m u t a t i o n ( 5 6 , 5 7 ) . The Y - t u k u l i n gene has s i n c e been c l o n e d f rom a w ide v a r i e t y o f o r g a n i s m s i n c l u d i n g f u n g i and h i g h e r e u k a r y o t e s and , i n e v e r y c a s e , has been i m m u n o l o g i c a l l y l o c a l i z e d t o the SPB ( 5 8 , 5 9 ) . T h i s e v i d e n c e s u g g e s t s t h a t y-t u b u l i n a i d s i n the a t t a c h m e n t o f m i c r o t u b u l e s and i s a u n i v e r s a l component o f the m i c r o t u b u l e o r g a n i z i n g c e n t e r . The c o n s e r v a t i o n o f t h i s key s t r u c t u r a l component e s t a b l i s h e s a f i r m l i n k be tween f u n g a l SPBs and the c en t r o somes o f h i g h e r e u k a r y o t e s . The cen t rosome p l a y s an i m p o r t a n t r o l e i n t h e c e l l c y c l e . Many p r o t e i n s i m p l i c a t e d i n c o n t r o l o f t h e c e l l c y c l e have been i m m u n o l o g i c a l l y l o c a l i z e d t o t h e cen t rosome i n c l u d i n g p 3 4 c d c 2 ( 6 3 ) , c y c l i n A, and c y c l i n B l ( 6 2 ) , and a r e t h o u g h t t o c o n t r o l m i c r o t u b u l e dynamics v i a p h o s p h o r y l a t i o n ( 6 4 , 6 5 ) . I n Drosophila, c en t rosomes o r g a n i z e the c y t o s k e l e t o n d u r i n g b o t h o o g e n e s i s (37) and embryogenes i s ( 6 7 - 6 9 ) , t h u s a f f e c t i n g the complex o r g a n i z a t i o n o f c e l l s r e q u i r e d f o r the deve lopment o f t h i s m u l t i c e l l u l a r o r g a n i s m . 6 Genetics of mfs(2)31 Presently, four alleles of mfs(2)31 are available: mfs1, the original hypomorphic al l e l e induced by Sandler; mfs2, an all e l e induced by insertion of a P-element in the 5' regulatory region of the gene; and mfs3 and mfs4, alleles induced by EMS mutagenesis (See ref 4). mfs1 homozygotes are characterized by s t e r i l i t y in both sexes, short thin bristles, and about 40% v i a b i l i t y . When measured in a vf4 background, eye pigment levels increase from 5 -10% of wild type to 90% of wild type in both male and female homozygotes. Males and females are f e r t i l e at 22°C and sterile at 29°C. mfs1/mfs2 trans-heterozygotes are completely viable and display the minute b r i s t l e phenotype. PEV is suppressed to a pigment level of about 40% in females and 75% in males with respect to wild type, indicating that the 5' regulatory region of mfs1 may pa r t i a l l y alleviate the reduction of transcription due to the P-element insertion into the regulatory region of mfs2. Male and female trans-heterozygotes are f e r t i l e at 22°C and ste r i l e at 29°C. mfs3 is a recessive lethal and mfs4 is a weak dominant semi-lethal and each are completely lethal over a l l of the other alleles. Revertants of the P-element insertion revert a l l of the phenotypes associated with mfs(2)31: suppression of PEV, s t e r i l i t y , the b r i s t l e phenotype, and lethality. 7 Molecular biology of mfs(2)31. Our lab cloned a 1.9 Kb genomic fragment from the 31 region of chromosome 2 on the basis of the P-element insertion associated with mfs2 (4) . Northern analysis of the transcripts in the area surrounding the P-element insertion revealed a 1.2 Kb transcript that mapped indistinguishably close to the insertion site. No transcripts were detected d i s t a l to the insertion site for 7 Kb. A second transcript 0.7 Kb in length was mapped very close to the 3' end of the 1.2 Kb transcript. The 1.9 Kb genomic fragment was sequenced and found to contain a complete open reading frame (ORF), corresponding to the 1.2 Kb transcript, putatively encoding a 30 KDa protein (hereafter called intronic centrosomal Protein, ICP). Comparison of the deduced amino acid sequence with the data base revealed that the N-terminal has homology to the tubulin binding region of MAP1B (Microtubule Associated Protein) and the Human 70 KDa Ul snRNP. The region of homology forms a long a helix and corresponds to the microtubule binding domain of MAP1B. The C-terminus has homology to mammalian histone HID. 8 Figure 1. Putative transcriptional regulation of mfs(2)31. See text for details. 9 The 1.9 Kb-'genomic fragment also contained a p a r t i a l ORF encoding a u b i g u i t i n monomer which was assumed to be transcribed into the 0.7 Kb message. The cDNA of a u b i q u i t i n fusion protein was cloned and sequenced by Lee, et a l . (1988) (11) and was thought to hybridize to 31E of chromosome 2. The complete sequence of the region was recently published by Barrio, et a l . (1994) (12) and i s gra p h i c a l l y represented i n figure 1. The mfs(2)31 locus i s thought to be i n i t i a l l y transcribed as a 2.0 Kb heterogenous nuclear RNA (hnRNA) containing two introns, the f i r s t , a 1.2 Kb intron containing the complete ICP ORF, and the second, 184 bp. The s p l i c e d t r a n s c r i p t corresponds to the 0.7 Kb DUb80 cDNA sequenced by Lee, et a l . (1988), encoding the u b i q u i t i n fusion protein. Because the 1.2 Kb 'intron' was found to p u r i f y with poly-A + RNA (4), i t i s thought to be l i b e r a t e d from the hnRNA, then i n t e r n a l l y cleaved such that the l a r i a t structure i s removed. The remaining i n t r o n i c RNA i s then thought to be processed into a messenger RNA with a 3' poly-A t a i l and perhaps a 5' cap. Another p o s s i b i l i t y which has yet to be investigated i s that the i n t r o n i c ORF has a separate t r a n s c r i p t i o n i n i t i a t i o n s i t e and thus i s not always co-regulated with the u b i q u i t i n fusion protein. The P-element associated with mfs2 i s inserted 20 bp upstream of the putative t r a n s c r i p t i o n s t a r t s i t e (4) within a GT-rich region common to ribosomal protein genes (12), such as the u b i q u i t i n t a i l protein (30), that i s thought to have some 10 regulatory s i g n i f i c a n c e . I t seems l i k e l y that the P-element i n s e r t i o n down regulates the t r a n s c r i p t i o n of the complete hnRNA and thus prevents expression of both proteins but, because the other mfs (2) 31 a l l e l e s have not been sequenced, i t i s at this time unknown whether the phenotypes associated with mfs(2)31, such as suppression of PEV, s t e r i l i t y , short b r i s t l e s , or l e t h a l i t y are due to a loss of one or both protein products. In order to approach the problem of separating the phenotypes associated with the loss of the two protein products, I have generated a polyclonal antiserum against the C-terminal 85 amino acids of ICP,- and l o c a l i z e d t h i s protein to Tr i t o n X-100® r e s i s t a n t compartments of the centrosome and the nucleus. 11 MATERIALS AND METHODS Midi/Maxi plasmid prep C e l l s containing the plasmid of in t e r e s t were propagated i n 100ml LB media 0/N at 37°C and were harvested by centrifugation at 5000rpm for 10 minutes at 4°C using 30ml Corex tubes i n a Sorval SS34 rotor. The tubes were reloaded a f t e r each spin. The b a c t e r i a l p e l l e t was resuspend i n 2.5ml of 25mM T r i s - C l , pH 8.0, 50mM glucose, lOmM EDTA and incubated for 10 minutes on ice. Then 5.0ml of freshly made 0.2M NaOH, 1% SDS was added and mixed gently but thoroughly. After the c e l l s were lysed, 3.75ml of 3.0M potassium acetate, pH 4,8 was added and mixed to form a viscous, white p r e c i p i t a t e of potassium and SDS/protein complexes. The p r e c i p i t a t e was p e l l e t e d at 8000rpm for 20 minutes. The supernatant was removed to a fresh 3 0ml Corex tube, and 1 volume of equ i l i b r a t e d phenol was added and mixed i n . The phases were separated by centrifugation at 8000rpm for 10 minutes. The aqueous phase was transferred to a fresh 3 0ml Corex tube, and 1 volume of chloroform/isoamyl alcohol (19:1) was added and the solution was vigorously mixed. The phases were separated by centrifugation at 8000rpm for 5 minutes. The aqueous phase was removed to a fresh 30 ml Corex tube, 2 volumes of 100% ethanol were added and mixed thoroughly to p r e c i p i t a t e the nucleic acids. The reaction was put at -20°C for 5 minutes to speed p r e c i p i t a t i o n . The DNA was p e l l e t e d by centrifugation at 8000rpm for 15 minutes and washed i n 1.0ml 70% ethanol. The 12 p e l l e t was c o l l e c t e d by cenntrifugation at 8000rpm for 5 minutes. The p e l l e t was resuspended i n 3 00ul of TE (lOmM T r i s , pH 7.8, ImM EDTA) and transferred to a microcentrifuge tube. RNAse was added to a f i n a l concentration of 0.lmg/ml and incubated at room temperature (RT) for 30 minutes. In order to increase the i o n i c i t y of the solution so that the RNase and other contaminating proteins were prec i p i t a t e d , 0.5 volumes of 5.0M ammonium acetate was added and incubated at -70°C for 10 minutes. The solution was thawed and spun i n a microcentrifuge at maximum rpm for 5 minutes. The supernatant was transferred to a fresh microfuge tube, the nucleic acids were p r e c i p i t a t e d with 2 volumes of 100% ethanol at -20°C for 20 minutes, and p e l l e t e d at 14,000 rpm for 10 minutes. The p e l l e t was allowed to a i r dry. Then resuspended i n 900ul of TE buffer. In order to p r e c i p i t a t e supercoiled plasmid, 600ul of 20% polyethylene g l y c o l 8 0 0 0 , 2. 5M NaCl was added, mixed, and placed on ice for 1 hour. The DNA was p e l l e t e d at max rpm for 10 minutes, washed i n 500ul of 70% ethanol. The p e l l e t was s o l i d i f i e d at max rpm for 5 minutes, and dried under vacuum, and resuspended i n lOOul of TE. In order to check the q u a l i t y and amount of the DNA, l u l was electrophoresed through a 1% agarose minigel and stained with 0.5 ug/ml ethidium bromide. Double stranded DNA sequencing The plasmid of i n t e r e s t was i s o l a t e d by the midi plasmid prep outlined above and 4ug were d i l u t e d to a f i n a l volume of 13 20ul. In order to permanently denature the plasmid, 2ul of 2M NaOH, 2mM EDTA was added and the resulting solution was incubated at 65°C for 5 minutes. The solution was then quenched on ice and 2ul of 2M ammonium acetate and 55ul of 100% ethanol were added and mixed thoroughly. The solution was stored at -20°C for 20 minutes and the DNA was pelleted in a microfuge at 14,000 rpm for 10 minutes. The pellet was then washed with 200ul 70% ethanol, and air dried. The pellet was then resuspended in 7ul of dH20, and l u l (3pmol/ul) of the appropriate primer was added. 2ul of 5X sequencing buffer was added and mixed by pipetting the solution. The primer was allowed to anneal at 37°C for 20 minutes and then RT for 10 minutes. Before i n i t i a t i o n of the labelling reaction, 2.5ul of each ddNTP was aliquoted into separate labelled tubes, concentrated labelling mix was diluted 2:8, and T7 DNA polymerase 1.5ul into 6.5ul TE in preparation for the termination reaction. To the annealing reaction was added: l u l lOOmM DTT, 2ul dilute labelling mix, l u l [o-35S]dATP, and 2ul diluted T7 DNA polymerase. The reaction was incubated at RT for 7 minutes. ddNTPs were prewarmed at 37°C for 3 minutes and 3. 5u.l of the labelling reaction was added to each tube and mixed well. The reaction was incubated at 37°C for 10 minutes and then stopped with 4ul of stop solution (formamide containing 0.25% Bromophenol Blue). The reactions were analyzed by electrophoresis on 7% 14 polyacrylamide denaturing gels. The gels were dried in a Savant gel dryer under vacuum for 2 hours at 80°C and exposed to Kodak XAR film 0/N at RT. S D S - P A G E Sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the BRL Mini-Protean II apparatus. A l l solutions were as found in Sambrook, et a l . (1989) (13) except the acrylamide: bis-acrylamide which mixed 39:1 instead of 29:1. Proteins were loaded and electrophoresed into the stacking gel at 90 V (10 min). The voltage was then switched to 200 V for the remainder of the run, about 1 hour. The gel was either i) stained with Coomassie B r i l l i a n t Blue (45% methanol, 10% acetic acid, 0.25% Coomassie B r i l l i a n t Blue), then destained in 45% methanol, 10% acetic acid or i i ) used to prepare a nitrocellulose membrane for western blotting. Western B l o t t i n g Transfer. SDS-PAGE was performed as described above. The transfer apparatus used was the BRL Trans-Blot SD semi-dry electrophoretic transfer c e l l . Five pieces of Whatman 3mm paper and a piece of nitrocellulose membrane (Amersham, HYBOND-ECL), cut to the size of the gel, were soaked in Dunn carbonate transfer buffer (lOmM NaC03, 3mM Na2C03, 20% methanol, pH 9.9) (23) for 30 min. The gel was not soaked. 15 Throughout the assembly of the gel stack, care was taken to insure that no a i r bubbles were trapped between the layers as th i s would have impaired transfer. The stack was assembled on the platinum electrode as follows: two pieces of soaked f i l t e r paper removing bubbles trapped beneath by r o l l i n g a Pasteur pipette over the paper, the n i t r o c e l l u l o s e f i t t e d exactly to the paper, the gel (mark orientation), and then another three pieces of paper. The cathode was c a r e f u l l y placed onto the stack. The proteins were transferred for 30 min with the voltage l i m i t e d at 22 V (most of the time, the voltage was below 22V); current was l i m i t e d at 5.5 mA/cm2. After transfer the membrane allowed to a i r dry for 1 hour. Antibody probes. Proteins were separated by SDS-PAGE and transferred using previously outlined techniques. N i t r o c e l l u l o s e membranes were placed into a covered p l a s t i c tray that was just larger then the membrane, previously cleaned with 0.1M HC1, and reserved for western b l o t t i n g . The membrane was b r i e f l y re-hydrated i n TBS-T (40mM T r i s - C l , pH 7.6, 137mM NaCl, 0.1% Tween-20), and then blocked for 1 hour i n TBS-T containing 5% low fat milk powder (MP) and 5% heat inactivated f e t a l bovine serum (FBS). The membrane was then washed for 2 X 5 min i n TBS-T to remove excess blocking proteins. Primary ICP(C85) polyclonal antibody, at the indicated d i l u t i o n (from 1 :250 - 1:1 ,250) , was added to 10ml fresh TBS-T 16 containing 0.5% MP, 0.5% FBS, and the membrane was incubated for 1 hour at RT under constant agi t a t i o n . To remove non-specific rabbit antibodies, the membrane was then washed i n TBS-T for 2 X 5 min and 1 X 15 min at RT under agita t i o n . Secondary antibody, horseradish peroxidase conjugated Donkey an t i - r a b b i t (H + L) (Amersham) , was added at the indicated d i l u t i o n (1:1,000 - 1:10,000) to 10ml TBS-T containing 0.5% MP, 0.5% FBS, and the membrane was incubated i n this solution for 1 hour at RT under agitation. To remove excess secondary antibody, the membrane was then washed i n TBS-T for 2 X 5 min and 2 X 15 min at RT under agi t a t i o n . The membrane was then removed from the tray and b r i e f l y placed on a piece of Whatman 3mm paper to remove any excess TBS-T. Detection. Detection was performed using ECL detection reagents (Amersham). The membrane was placed i n a new tray reserved for detection. 2.5ml of detection reagent #2 was evenly d i s t r i b u t e d across the membrane before 2.5ml of detection reagent #1 was added. The reaction was allowed to proceed for 1 min before the membrane was removed, draining excess solution, and wrapped i n Saran Wrap. The membrane was then placed into a f i l m cassette and secured with Scotch tape. In the dark room, Kodak XAR f i l m was 17 exposed to the membrane for 4 seconds to 5 minutes, depending on the strength of the signal, and developed i n a Kodak M3 5A X-Omat processor. Batch p u r i f i c a t i o n of glutathione-S-transferase (GST) fusion proteins. Small scale p u r i f i c a t i o n . Cultures containing the of clone of in t e r e s t were propagated 0/N, d i l u t e d 1/10 i n fresh medium, and allowed to grow for 1.5 hours. Expression was induced by adding IPTG to 0.2mM and allowing growth to continue for 1 hour. C e l l s were harvested by centrifugation at 14,000 rpm i n a microcentrifuge for 2 min and the b a c t e r i a l p e l l e t was resuspended i n 300ul of MTPBS (150mMNaCl, 16mM Na2HP04, 4mM NaH2P04) containing 2% Trit o n X-100®. The c e l l s were sonicated for 2 x 30 seconds on highest setting. P a r ticulate debris was removed by centrifugation at 14,000 rpm i n a microcentrifuge for 5 minutes. The supernatant was removed and 50ul of a 50% suspension of preswelled s-linked glutathione agarose beads (Sigma, Cat# G-4510) were added and incubated at RT for 5 min. The beads were p e l l e t e d for 3 0 seconds i n a microcentrifuge and washed 3X i n 300ul MTPBS. The f i n a l p e l l e t of beads was either i) b o i l e d for 5 minutes i n SDS-PAGE loading buffer, and then run on a 15% SDS-PAGE, or i i ) incubated at RT for 2 minutes i n 50ul of 15mM reduced glutathione, 50mM T r i s - C l , pH 7.6, p e l l e t e d by 18 centrifugation, and the supernatant transferred to a clean tube. The last step was repeated and the fractions analyzed by SDS-PAGE. Large scale p u r i f i c a t i o n (1 l i t r e ) . A single colony was picked and propagated for 4-5 hours at 37°C, shaking, in 5ml liquid medium containing 200ug/ml amp. The cells were then diluted into 2 x 50ml of medium containing 200ug/ml amp and allowed to grow 0/N at 37°C, shaking. Each 50ml culture was added to 450ml of medium containing 200ug/ml amp for a fin a l volume of 2 x 500ml and allow to grow for 1.5 hours. IPTG was added to 0.2mM and the cultures were induced at 37°C for 4 hours. Cells were harvested by centrifugation in 250ml tubes for 5 min at 5000Xg, resuspended in 3.5ml MTPBS containing 2% Triton X-100®, transferred to 3 0ml Oakridge centrifuge tubes, and sonicated at highest setting for 2 x 40 seconds and 1 x 20 seconds, allowing the protein solution to cool on ice between each sonication. Cellular debris were removed by centrifugation at 10,000Xg for at least 10 minutes, and the supernatants were pooled to 2 fresh Oakridge tubes. To bind GST fusion protein to the beads, each pooled supernatant was incubated at RT for 5 minutes with 0.6ml of a 50% suspension of preswelled s-linked glutathione agarose beads (ie. 1.2ml/l). To wash away contaminating bacterial proteins, 20ml of ice cold MTPBS was added to each tube and the beads were pelleted by centrifugation at 500Xg for 19 10 seconds. The p e l l e t was then washed 2X with 20ml MTPBS, resuspended i n 1.0ml MTPBS, and transferred to two 1.5ml microcentrifuge tubes. The beads were then p e l l e t e d by a b r i e f spin i n a microcentrifuge and washed with 1.0ml MTPBS. In order to elute the desired protein from the beads, 0.6ml of 15mM reduced glutathione, 50mM T r i s - C l , pH 7.6 was added to each tube (ie. 1.2ml/l), and the r e s u l t i n g suspension was incubated at RT for 2 min. This step was repeated 2X and each f r a c t i o n was analyzed by SDS-PAGE. Protein concentration was estimated by o p t i c a l density at 280nm. For GST, 1 OD280= 0.5mg/ml. The protein containing fractions were stored at -70°C. Colony hy b r i d i z a t i o n . Colonies were allowed to grow O/N at 37°C. A HYBOND-N nylon c i r c l e (Amersham) was hydrated with dH20 and placed over the colonies. A l l a i r bubbles were smoothed out with a bent Pasteur pipette and the membrane was allowed to s i t for 5 min. The membrane was then removed and placed onto Saran Wrap upon which 1.0ml of 0.5M NaOH had previously been spotted. It was made c e r t a i n that the solution completely soaked the membrane. This lysis/denaturation step was repeated one more time. The membrane was then twice placed onto Saran Wrap with 1.0ml of T r i s - C l , pH 7.5 i n order to neutralize the NaOH, and then transferred to another piece of Saran Wrap with 1.0ml of 1.0M T r i s - C l , pH 7.5, 1.5M NaCl. The membrane was then placed on a 20 piece of Whatman 3mm paper and allowed to air dry. Probe was labelled with [a-32P]dATP using the Boeringer Manheim Random Priming k i t according to the manufacturers instructions. Briefly, lOOng of probe DNA was diluted to 7ul with dH20, boiled for 10 min to denature the DNA, and quenched on ice. To the template was added 2ul of random prime mix (random hexamers and buffer) , 2ul of dNTPs (no 'cold' dATP) , 5ul of [ec-32P]dATP, l u l of Klenow fragment (2 U/ul), and brought to 20ul with dH20. The reaction was allowed to proceed for 1 hour at 37°C. Probe DNA was then par t i a l l y purified on a Sephadex G-50 column, denatured by boiling for 5 min, and quenched on ice in preparation for hybridization. The membrane was then pre-hybridized in 10ml 5X SSC, 5X Denhardts solution, 0.5% SDS for 1 hour at 64°C then probe was added directly and hybridized for 14 hours at 64°C. The membrane was then washed twice in 2X SSC, 0.1% SDS for 10 min at RT. It was then washed once in 0.5X SSC, 0.1% SDS and once in 0.3X SSC, 0.1% SDS both for 30 min at 68°C. The membrane was then autoradiographed at -7 0°C with enhancing screens for 3 hours. Generation of the pGEX/ICP Fusion Construct. pGEX-lN DNA (Pharmacia) (19) was prepared as per the midiprep protocol, linearized with Eco RI, de-phosphorolated with 24 U of calf intestinal alkaline phosphatase for 1 hour at 21 37°, phenol extracted with 1 volume of phenol, chloroform extracted with 1 volume of chloroform, and ethanol p r e c i p i t a t e d with 2 volumes of 100% ethanol. cDNA-2, the shortest cDNA containing the whole ICP open reading frame, was cut out of the pUC19 vector with Eco RI, gel p u r i f i e d with the Qiaex gel p u r i f i c a t i o n k i t (Qiagen) as per the manufacturers instructions, and resuspended i n TE buffer. lOOng of de-phosphorolated pGEX-lN/RI was mixed with 25ng 'of the cDNA-2 fragment and 1 U of ligase i n a f i n a l volume of 20ul for 16 hours at 18°C. DH5a competent c e l l s were transformed with 5ul of the l i g a t i o n mixture and plated on LB-agar plates containing lOOug/ml Amp and allowed to grow O/N. Approximately 50 of the r e s u l t i n g colonies were transferred with s t e r i l e toothpicks to a new LB-Amp plate and inserts were i d e n t i f i e d by colony hybridization using the cDNA-2 fragment as a probe. To determine i f the orientation and fusion point were correct, four cDNA-2 p o s i t i v e s were picked and sequenced using the ICP 3'-3 primer which i s in t e r n a l to the cDNA-2 fragment. Generation of the pGEX/ICP(C85) Fusion Construct. pGEX/ICP fusion construct was prepared by the midiprep protocol and 5ug was digested with 20U of Nco I and 10U Bam HI i n 30ul of 'Buffer B' (Boeringer Manheim) for 1 hour at 37°C. Nco I has 50% a c t i v i t y i n 'Buffer B'. The reaction was pr e c i p i t a t e d with 2 volumes of 100% ethanol and the p e l l e t was washed with 70% ethanol. Without further p u r i f i c a t i o n , the 22 p e l l e t was resuspended i n 20ul of SI nuclease buffer (33mM NaOAc, pH 4.5, 50mM NaCl, 0.03mM ZnS04) containing 0.8 U SI nuclease (diluted i n dH20 from the 400 U/ul stock (Boeringer Manheim) ) and incubated at 37°C for 10 minutes. The reaction was stopped by d i l u t i o n to 50ul with TE buffer and extraction with 50ul CHG13. The resultant aqueous phase was transferred to a new microfuge tube, p r e c i p i t a t e d with 2 volumes of 100% ethanol and the p e l l e t washed with 70% ethanol. The p e l l e t was resuspended i n 17ul of TE buffer and 2ul was removed for analysis. To the r e s u l t i n g 15ul were added 2ul of 10X l i g a t i o n buffer (10X = 500mM T r i s - C l , pH 7.5, lOOmM MgCl2, 90mM DTT) , 2ul 5mM ATP ( L i + s a l t , Boeringer Manheim), and 1U l i g a s e for a f i n a l volume of 20ul. This reaction was incubated at 14°C for 20 hours. 5ul of the l i g a t i o n mixture was used to transform DH5a competent c e l l s (BRL) which were plated on LB-agar containing lOOug/ml Amp and allowed to grow O/N at 37°C. Individual colonies were screened for the presence of the deletion by i s o l a t i o n of plasmid DNA, digestion with Eco RI, and agarose gel electrophoresis. Generation of the deletion eliminated one of the Eco RI s i t e s present on the f u l l length construct thus, i f the deletion was correct, the plasmid should be l i n e a r i z e d by Eco RI and have a molecular weight of 5.3 Kb as compared to the pGEX vector, 4.995 Kb. Putative deletions were screened by expression. C e l l s were allowed to grow overnight at 37°C i n 3ml LB l i q u i d medium, 23 induced with 0. ImM IPTG for 1 hour at 37°C, harvested by centrifugation at 14,000 rpm i n a microcentrifuge for 2 min., lysed by b o i l i n g i n IX SDS-PAGE loading buffer for 5 min., and analyzed by comparison to both uninduced c e l l s and a pGEX-lN control by SDS-PAGE. Clones displaying the c h a r a c t e r i s t i c s of the fusion protein were i s o l a t e d by the midiprep protocol and sequenced using the ICP 3'-3 i n t e r n a l primer (4). The sequence at the breakpoint was predicted to be 5'...CCA AAA TCG GGT CTG CGC...3' i n the d i r e c t i o n of t r a n s l a t i o n . Bold characters indicate the vector while normal characters indicate the s t a r t of the C-terminal 85 amino acids of ICP. ELISA 2ug of protein was placed i n m i c r o t i t r e plate wells i n a f i n a l volume of 200ul of coating buffer (15mM Na2C03, 3 5mM NaHC03, 0.02% NaN3) and adsorbed O/N at 4°C. Excess protein was removed the wells were washed 3X with 200ul of PBS-T (Ab-PBS, 0.5% Tween-20) . The wells were blocked with 3% BSA i n PBS-T for 1 hour at 37°C. Serum was d i l u t e d i n PBS-T from 1:102 - 1:10s to a f i n a l volume of 200ul, incubated for 1 hour at 37°C then washed 3X with PBS-T. Alkaline phosphatase conjugated goat a n t i - r a b b i t IgG (H + L) (BRL) was d i l u t e d 1:4,000 i n PBS-T to a f i n a l volume of 200ul, added to each well, and incubated at 37°C for 1 hour. The wells were washed 3X with PBS-T and IX with DEA buffer (9.7% diethanolamine (v/v), 0.5mM MgCl2, 0.02% NaN3, pH 9.8 with HC1). The substrate used was p-nitrophenyl phosphate (p-NPP). The substrate was added at a concentration of 1 mg/ml in DEA buffer and incubated at 37°C for 3 0 min. Optical density at 405nm was determined i n a mi c r o t i t r e plate reader. Control wells, incubated without any serum, were included with each experiment to control for non-specific binding of the secondary antibody. Production and Analysis of the 1CP(C85) Polyclonal Antiserum. A pre bleed (10ml) was taken from each of two rabbits. Serum was i s o l a t e d by allowing the blood samples to c l o t at 37°C for 30 min. then storing the samples O/N at 4°C. Blood c l o t s were dislodged from the edges of the tubes with a Pasteur pipette, and the c e l l s were p e l l e t e d at 12,000Xg for 10 min. NaN3 was added to the pre-immune serum to a f i n a l concentration of 0.02% and 1ml was removed to a microfuge tube and stored at 4°C for analysis; the remainder was stored at -20°C. 190ug of p u r i f i e d GST/ICP(C85) fusion p r o t e i n i n a f i n a l volume of lOOul MTPBS was emulsified with lOOul of TiterMax adjuvant (Vaxcel, Inc., Georgia) by the one syringe, blunt needle method according to the manufacturers i n s t r u c t i o n s . This emulsion was used to immunize 2 rabbits by i n j e c t i n g 50ul into each hind quadricep. A 2 week test bleed was taken to check the effectiveness of the i n i t i a l immunization. Serum was i s o l a t e d and tested for the presence of anti-GST/ICP(C85) IgG by ELISA. The rabbits were boosted twice at 3 week in t e r v a l s by 25 intravenous i n j e c t i o n of 380ug of (soluble) GST/ICP(C85) fusion protein i n 250ul of MTPBS. Three weeks a f t e r the f i n a l boost, the rabbits were s a c r i f i c e d and 350ml of immunoreactive serum was taken from each. Removal of GST S p e c i f i c Antibodies. 41 of culture containing pGEX-lN were induced with IPTG and about 20mg of GST protein was p u r i f i e d on a t o t a l of 20ml of a 50% s l u r r y of s-linked glutathione beads by the protocol outlined above. Instead of eluting the protein from the beads with free glutathione, GST protein was l e f t bound to the beads. The beads were allowed to s e t t l e to a f i n a l volume of 10ml and s p l i t into 4 aliquots of 2.5ml. Serum (10ml) was added to the f i r s t 2.5ml aliquot of beads and agitated at RT for 2 hours. The beads were then allowed to s e t t l e , the serum was removed to the second aliquot of beads and agitated for 4 hours. This procedure was repeated twice more but incubation times were increased to 20 and 22 hours respectively. A f f i n i t y P u r i f i c a t i o n 120 ug of GST/ICP(C85) fusion protein was separated by SDS-PAGE with a preparative comb, and transferred onto a n i t r o c e l l u l o s e f i l t e r by standard techniques (see above). Protein was stained with Ponceau-S and the s t r i p of membrane to 26 which the protein was bound was cut out and incubated with serum from which GST specific antibodies had been removed diluted 1:10 in TBS-T O/N at 4°C. The f i l t e r was then washed with 3 X 10ml of TBS-T for 10 min each. Remaining antibodies were then eluted from the f i l t e r with lOOmM glycine, pH 2.5, for 10 min at RT. The f i l t e r s were then removed and eluted a second time. The antibody containing solution was neutralized with 1/10 vol of 1M Tris-Cl, pH 7.25. In order to test the resulting activity, a western blot to a small amount of fusion protein was performed with each fraction. Staining Drosophila Embryos with the ICP(C85) Polyclonal Antibody. Wild type flys (Oregan R) were transferred to laying bottles and allowed to lay eggs on laying trays containing 5% acetic acid, 5% ethanol, and 2% agar. Embryos were washed with dH20 on to stainless steel mesh, rinsed thoroughly with dH20, dechorionated in 100% bleach for 3-5 minutes, and then rinsed again with dH20. Embryos were then removed with a paintbrush to a 1.5ml eppendorf tube containing 700ul heptane. Formaldehyde (700ul, 37%) was added and the tubes were placed on a rotator for 10 min at RT. After fixation, the lower aqueous phase was removed with a Pasteur pipette and 700ul of 95% methanol, 5% 0.5M EGTA (v/v) was added. The tubes were then vortexed at high speed for 20 seconds to remove the embryonic v i t e l l i n e membranes. 27 Devi t e l l ineized embryos s e t t l e d to the bottom of the tube and everything else, including embryos remaining at the interface, was removed. The remaining embryos were washed once with methanol/EGTA and then incubated i n methanol/EGTA for 2 hours at RT or 0/N at 4°C. Embryos were either re-hydrated immediately or stored at 4°C i n methanol/EGTA. Embryos were re-hydrated by washing for 3 X 5 min i n TBSBT (lOOmM NaCl, 40mM T r i s - C l , pH 7.3, 0.2% BSA, 1% T r i t o n X-100®) at RT. In order to expose the epitopes recognized by the ICP(C85) polyclonal antibody, the embryos were incubated i n 0.1% trypsin, 20mM T r i s - C l , pH 7.8 for f i v e minutes at RT and then washed 3 X 2 min i n TBSBT. The embryos were then incubated on the rotator i n the ICP(C85) primary antibody, d i l u t e d 1:500 i n TBSBT containing 5% FBS as a blocker, for 3 hours at RT or O/N at 4°C. The primary antibody was then washed from the embryos with TBSBT for 2 X 2 min, and then rotating with at least 4 changes of TBSBT over 3 hours at RT or O/N at 4°C. The secondary antibody used was Texas Red conjugated Goat ant i - r a b b i t Ig (H+L) (Jackson Immunoresearch Laboratories) d i l u t e d 1:50,000 i n TBSBT containing 5% FBS as a blocker. Embryos were incubated at RT for 1.5 hours or O/N at 4°C on the rotator, washed 2 X 2 min, and then with at lea s t 4 changes of TBSBT over 3 hours. Embryonic DNA was stained with 0. lug/ml Hoechst 33258 for 5 min i n TBSBT. The embryos were then mounted i n 80% g l y c e r o l and observed 2 8 under o i l emersion with a 100X objective on a Zeiss Axiophot microscope using rhodamine f i l t e r s . Pictures were taken on either Kodak T-Max ASA 400 f i l m (black and white) or Fugichrome ASA 400 s l i d e f i l m (colour). C e l l Staining HeLa c e l l s were propagated at 37°C under 4.5% C02, i n Dulbecco's modified Eagle's medium (Sigma) with 10% f e t a l bovine serum. Aliquots of approximately 104 c e l l s were transferred, each i n a f i n a l volume of 400ul, to 8 well chamber s l i d e s (Lab-Tek, Cat #4808) and allowed to attach to the glass surface of the s l i d e for 24 hours. The c e l l s were then fed with fresh media and allowed to grow for a further 24 hours. The c e l l s were then fi x e d i n 3.7% paraformaldehyde i n PBS for 10 min. Permeablization of the fi x e d c e l l s was achieved by incubation i n TBSBT (containing 1.0% BSA instead of 0.1%) for at least 10 min. C e l l s were then incubated i n 0.025% trypsin, 40mM T r i s - C l , pH 7.8 for 30 seconds. Although s t r i c t l y unnecessary, th i s step increased the signal to background r a t i o considerably. Excessive t r y p s i n i z a t i o n resulted i n detachment of the c e l l s from the glass s l i d e s upon mounting. The t r y p s i n was removed and then quenched by addition of TBSBT and the c e l l s were washed 3X 1 minute with TBSBT. C e l l s were incubated i n primary ICP(C85) antibody was added at a d i l u t i o n of 1:100 i n TBSBT (1.0% BSA) for 3 0 min, then washed 3 X 3 min with TBSBT. Secondary antibody (TRSC 2 9 conjugated Goat anti-Rabbit, Jackson) was added to a f i n a l d i l u t i o n of 1:200 and allowed to bind for 3 0 min. Excess secondary was removed by washing 4 X 3 min with TBSBT (1.0% BSA), mounted i n 80% gly c e r o l , and observed under o i l emersion on a Zeiss Axiophot microscope with a 100X objective using rhodamine f i l t e r s or a MRC 600 series confocal microscope with a 60X objective and a 10X Zoom function. For double l a b e l l i n g , c e l l s were incubated with an t i P~ tubulin monoclonal antibodies (Cedar Lane, CLT 9003) at a d i l u t i o n of 1:100 i n the same solution as a n t i ICP(C85) antibodies. The secondary used was FITC conjugated Goat a n t i -Mouse IgG at a d i l u t i o n of 1:200. To control for cross-r e a c t i v i t y , some c e l l s were incubated with both secondary antibodies, but only one or the other primary. No major cross-r e a c t i v i t y was observed; these c r o s s - r e a c t i v i t y controls were observed to be e s s e n t i a l l y s i m i l a r to single l a b e l l i n g experiments or straight secondary controls. 30 R E S U L T S It was necessary to isolate large amounts of the purified protein in order to generate a polyclonal antiserum to ICP. The pGEX expression plasmid has a copy of the Schistosoma japonicum glutathione-S-transferase gene under the control of the IPTG inducible tac promoter (19). Using recombinant DNA technology, an open reading frame of interest can be introduced into the pGEX multiple cloning site (MCS) in the reading frame of the GST gene and thus fused to the C-terminus. IPTG w i l l thus induce high levels of expression of the ORF of interest as a fusion protein with GST. GST protein can be easily purified by a f f i n i t y chromatography to reduced glutathione attached to a solid support, and then eluted using free reduced glutathione. The addition of further amino acids to the C-terminus does not inhibit the glutathione binding activity of GST thus soluble fusion proteins can be purified in the same manner. p G E X / I C P The complete ORF of ICP was introduced in frame into the MCS of pGEX-lN and the fusion point sequenced as outlined in the materials and methods. One clone was chosen for expression analysis. The expected molecular weight of the fusion protein was about 57KDa (27.5KDa for GST plus 30KDa for ICP), but upon induction with IPTG and analysis by SDS-PAGE, no new band was 31 seen to migrate at thi s p o s i t i o n (data not shown). Expression at 3 0°C instead of 37°C produced s i m i l a r r e s u l t s . To investigate whether or not small quantities of the fusion protein was preventing i t s own expression, the o p t i c a l density at 600nm was measured over a period of 3 hours aft e r induction with IPTG. This was done for cultures containing clones with cDNA-2 i n the correct o r i e n t a t i o n and frame (cDNA-2(+)), or clones with cDNA-2 i n the opposite orientation (antisense; cDNA-2(-)) as a control for overexpression of the RNA; i t was possible that the RNA was the molecule responsible for the lack of expression. Table 1 demonstrates that induction of the fusion protein stopped c e l l growth completely before the 60 minute time point while overexpression of the antisense RNA merely slowed c e l l growth. 32 Table 1 . Growth of cells containing pGEX/cDNA-2 clones. The optical density at 600nm was determined at three time points in order to follow c e l l growth. Cultures containing the in frame (cDNA-2(+)) or antisence (cDNA-2(-)) clone were allowed to grow in the presence ( + ) or absence (-) of IPTG for the indicated times. Clone cDNA-2(+) cDNA-2(-) Time (min) IPTG + IPTG IPTG + IPTG 0 0.31 0.31 0.24 0.24 60 0.55 0.45 0.47 0.42 180 0.83 0.46 0.82 0.62 33 p G E X / I C P ( C 8 5 ) Because the f u l l length protein was not amenable to high l e v e l expression, another approach was taken. A deletion of the o r i g i n a l pGEX/ICP construct was made i n which the C-terminal 85 amino acids of ICP was fused i n frame onto the C-terminus of the GST (See f i g . 2) . It was reasoned that even though an undetectable amount of the f u l l length protein prevented further c e l l growth, a small portion of the protein might not have the same e f f e c t . 34 0 100 200 259 1 I I I ICP N - MAP1B Homology | HistoneHID Homology | - C I Fusion Breakpoint GST/ N - 1 Glutathione-S-Transferase I ICP - C ICP(C85) 1 ; 1 1 Figure 2. Graphic representation of ICP and the GST/ICP(C85) fusion protein. For d e t a i l s of the' construction of the GST/ICP(C85) fusion protein, see Materials and Methods. 35 SDS-PAGE analysis of c e l l s containing t h i s construct and induced with IPTG revealed an intensely staining band migrating at the predicted molecular weight of the fusion protein, 36.7 KDa, that was not present i n uninduced c e l l s ( f i g . 3). This new protein was determined to be soluble and p u r i f i a b l e by a f f i n i t y chromatography to reduced glutathione and thus was construed to be the protein product of the fusion construct. A time course of expression at 37°C was performed to determine at what time maximum expression was induced ( f i g . 4) . Steady state le v e l s of the fusion protein continued to increase up to 4 hours post-induction. P u r i f i c a t i o n of the fusion protein was then scaled up i n order to i s o l a t e enough protein to immunize rabbits for the production of polyclonal antibodies. P u r i f i e d protein was analyzed by SDS-PAGE to check for the presence of impurities ( f i g . 5). The only notable impurity, about 5% of t o t a l protein, was a breakdown product of the fusion protein that migrated at a molecular weight s i m i l a r to that of the GST control. 36 Figure 3. Expression and p u r i f i c a t i o n of GST and GST/ICP(C85) analyzed by SDS-PAGE. Lane 1: t o t a l c e l l lysate of E. c o l i containing pGEX-lN, un-induced. Lane 2: E. c o l i containing pGEX-lN, induced with 0.2mM IPTG. Lane 3: soluble GST protein p u r i f i e d with glutathione-agarose beads. Lanes 4,7: t o t a l c e l l lysates of E. c o l i containing independently i s o l a t e d pGEX/ICP(C85) fusion constructs, un-induced. Lanes 5,8: same as lanes 4,7 but induced with 0.2mM IPTG. Lanes 6,9: p u r i f i e d GST/ICP(C85) fusion protein. 37 1 2 3 4 5 6 7 8 9 GST/ICP(C85). GST 38 Figure 4. Time course of GST/ICP(C85) fusion protein induction in E. c o l i . Lane 1: un-induced ce l l s . Lanes 2, 3, 4, and 5: an equal volume of cells taken at 1 hour, 2 hours, 3 hours, and 4 hours post-induction with IPTG (respectively). 3 9 40 Figure 5. SDS-PAGE of p u r i f i e d GST/ICP(C85) fusion protein. Lane 1: p u r i f i e d GST protein. Lane 2: GST/ICP(C85) fusion protein from a small scale p u r i f i c a t i o n . Lanes 3,4: f i r s t e l u t i o n of glutathione-agarose beads with free glutathione. Lanes 5,6: second e l u t i o n with free glutathione. Note the lower molecular weight breakdown product observed i n lane 3. Molecular weight markers (MW) are given i n KDa. 4 1 18.1 15.4 42 immunization of Rabbits and C o l l e c t i o n of Antisera. P u r i f i e d protein was emulsified with TiterMax adjuvant and injected into rabbits. At two weeks post-immunization, a test bleed was taken to determine the i n i t i a l immune reaction to the fusion protein. ELISA analysis of test antiserum versus pre-immune serum revealed that, at a d i l u t i o n of 1:100,000, the test serum had a more than 3-fold greater r e a c t i v i t y to the fusion protein than the pre-immune serum. After two booster injections, the polyclonal antiserum was c o l l e c t e d and re-tested by ELISA. Table 2 shows that, at a 106 d i l u t i o n , the immune serum i s f i v e f o l d more reactive to the fusion protein than to the GST c a r r i e r . The immune serum was also tested for r e a c t i v i t y to equal amounts of the fusion protein and GST by western b l o t analysis ( f i g . 6). This demonstrated that the immune serum was reactive to both the fusion protein and the GST c a r r i e r a f t e r western transfer. In order to remove GST s p e c i f i c antibodies, 10ml of the serum was adsorbed to 20mg of immobilized GST protein (See Materials and Methods). A small amount of t h i s adsorbed serum was then a f f i n i t y p u r i f i e d against the fusion protein. This a f f i n i t y p u r i f i e d antibody was used for further experiments. Figure 6 also shows a r e p l i c a t e b l o t probed with the a f f i n i t y p u r i f i e d antibodies from which GST s p e c i f i c antibodies have been removed. 43 Table 2. ELISA to test the r e a c t i v i t y of the immune serum to p u r i f i e d fusion protein and to p u r i f i e d GST protein. Values indicate o p t i c a l density at 405nm. A l l values have been normalized to buffer blanks and represent the average of two experiments. Pre-immune Serum Immune Serum D i l u t i o n Factor GST GST/ ICP(C85) GST GST/ ICP(C85) 105 0.012 0.017 0.113 0.938 106 0.000 0.039 0. 022 0.110 44 ICP i s Conserved. Sequencing of yeast chromosome V has revealed an open reading frame that codes for a protein with 62.5% amino acid identity over the f u l l length of the predicted protein sequence of ICP (69). It is unknown to me whether any mutations that affect the function or expression of the protein exist in yeast. A partial H. sapiens cDNA was also recently entered into the data base that has 71% identity over 160 nt with the 3' end of the 1.2 Kb transcript (EMBL acc# Z28709). 45 Figure 6 . Western b l o t to show removal of GST s p e c i f i c antibodies. Lanes 1,2: 250ng of GST/ICP(C85) fusion protein (lane 1) and 250ng GST (lane 2) probed with raw serum. Lanes 3,4: i d e n t i c a l with 1 and 2 except they have been probed with serum from which GST s p e c i f i c antibodies have been removed. 46 GST/ICP(C85) GST 47 Figure 7 . ICP(C85) antibody reacts on western blo t s with a 30 KDa protein from divergent species. 7.1 shows a western b l o t of t o t a l Drosophila KC c e l l lysates from cultures that had not reached confluence. 7.2 shows a western b l o t of J77A mouse macrophage c e l l s that had not reached confluence. 7.3 shows a western b l o t of HeLa c e l l s that were allowed to grow to confluence; note the absence of the 30 KDa, 18 KDa, and 20 KDa species, but the presence of the high molecular weight species (approx. 75 KDa). These western blots were probed with a f f i n i t y p u r i f i e d antibody at a d i l u t i o n of 1:250. 48 7.1 7.2 7.3 43.3 28.3 Kc Mouse Hela 4 9 ICP(C85) Antibodies React With a 30 KDa Protein From Divergent Organisms. Western blots of t o t a l proteins from Drosophila KC c e l l s , mouse J77A transformed macrophage c e l l s , and HeLa c e l l s , a human immortalized f i b r o b l a s t c e l l l i n e , probed with the ICP(C85) antibody a l l display two strongly reactive bands that migrate at a s i m i l a r molecular weight during SDS-PAGE (Fig 7). The r e l a t i v e i n t e n s i t y of these bands was observed to vary with respect to each other from sample to sample. Two intensely staining bands running at approximately 18 and 2 0 KDa were also observed on western b l o t s from every organism tested but were not investigated further. ICP i s Localized to the Drosophila Embryonic Nucleus. Lethal phase analysis of mfs(2)31 mutants indicated that l e t h a l i t y occurs i n embryonic and l a r v a l stages ( 4 ) . Because the major 3 0 KDa species of ICP i s not detectable by western b l o t t i n g at the l a r v a l stage, but i s detectable i n pre-blastoderm embryos (data not shown), i t i s l i k e l y that the primary defect causing l e t h a l i t y occurs during embryogenesis. In order to investigate t h i s phenomenon, whole mount embryos were fixed, l i g h t l y digested with trypsin, stained by i n d i r e c t immunofluorescence with ICP ( C85 ) antibodies, then counterstained with Hoechst 3 3 2 5 8 , a fluorescent DNA binding dye. Because Hoechst 3 3 2 5 8 stains A-T r i c h DNA p r e f e r e n t i a l l y , low concentrations of t h i s i n t e r c a l a t i n g dye w i l l p r e f e r e n t i a l l y 50 Figure 8. Staining of Drosophila embryos with Hoechst 33258 and ICP(C85) antibody. Figure 8.1: embryo stained with the DNA s p e c i f i c fluorescent dye, Hoechst 33258. 8.2: i d e n t i c a l f i e l d as 8.1, but stained with ICP(C85) antibody. 8.3: double exposure of Hoechst 33258 (blue) and ICP(C85) antibody (red). Note that the signal from ICP(C85) antibody overlaps the border of the nucleus as defined by Hoechst 33258 staining. • 8.1 Hoechs t 3 3 2 5 8 8.2 ICP (C85 ) 52 s t a i n centromeric r e p e t i t i v e elements that have a high A-T content. This i s observed i n interphase embryonic nu c l e i as bright fluorescent dots within a background of l i g h t l y staining unique sequence DNA ( f i g . 8.1). The area of l i g h t staining roughly defines the boundaries of each nucleus. Indirect immunofluorescence with ICP(C85) antibodies, but not pre-immune serum or secondary controls, revealed very small, intensely staining spots that roughly corresponded to the location of Hoechst 33258 p r e f e r e n t i a l staining, but overlapped the nuclear membrane (see Fig. 8.3). Without digestion with trypsin, no staining was observed i n d i c a t i n g that the epitopes recognized by the ICP(C85) antibody are buried within a proteinaceous structure that i s susceptible to trypsin digestion. The epitope was also found to be r e s i s t a n t to s o l u b l i z a t i o n with T r i t o n X-100®. These data indicate that ICP i s l o c a l i z e d to the a p i c a l t i p of the nucleus which corresponds to the l o c a t i o n of the centromeric heterochromatin, the centrosome, and the apparatus that holds these two large structures i n place. ICP(C85) Antibody Stains the Centrosome of HeLa C e l l s . To test i f l o c a l i z a t i o n within the c e l l and thus, perhaps, function, i s also conserved, HeLa c e l l s were fixed, treated with t r i t o n X-100, and stained with the ICP(C85) antibody. Figure 9 shows that the ICP(C85) antibody stains a highly condensed structure at the d i s t a l t i p of a growing chain of c e l l s (9.1), 53 that t h i s structure r e p l i c a t e s (9.2), the p a i r separates (9.3), and comes to rest at the opposite poles of the c e l l (9.4) . This data i s consistent with the hypothesis that the structure stained by the ICP(C85) antibody i s the centrosome. The observed d i f f u s e nuclear s t a i n i s also present i n secondary controls (preparations incubated without primary ICP(C85) antibody) and thus may not be due to s p e c i f i c binding of the ICP(C85) antibody to ICP (data not shown), but the question remains as to whether there i s a change i n the i n t e n s i t y of the signal between experimental preparations and secondary controls. Some experimental preparations showed vari a t i o n s of i n t e n s i t y within i n d i v i d u a l n u c l e i , but again i t i s unknown whether this' observation was due to compartmentalization of ICP within the nucleus, or was an a r t i f a c t of the s o l u a b l i z a t i o n or t r y p s i n i z a t i o n procedures. 54 Figure 9. S t a i n i n g o f HeLa c e l l s w i t h ICP(C85) a n t i b o d y as o b s e r v e d by i n d i r e c t immunofluorescence w i t h a f l u o r e s c e n c e m i c r o s c o p e . See t e x t f o r d e t a i l s . 55 9.1 9.2 9.3 9 .4 56 ICP c o - l o c a l i z e s with P ~tubulin at the Centrosome. Double l a b e l l i n g experiments with both ICP(C85) antibody and a monoclonal an t i p-tubulin antibody ( f i g . 10) show co-l o c a l i z a t i o n of ICP and p-tubulin. Although not f u l l y characterized, I should mention here that s t a i n i n g of the centrosome with ICP(C85) antibodies by this technique i s observed to be c e l l cycle s p e c i f i c . To c i t e an example, no centrosomal staining i s observed while the c e l l i s at metaphase; c e l l s with a f u l l y formed spindle apparatus, as revealed by p-tubulin staining, displayed no staining of the centrosome with ICP(C85) antibodies (data not shown). ICP i s Also Localized Within the Nucleus. Upon incubating HeLa c e l l s with high concentrations of both primary and secondary antibodies (1:100 and 1:1,000 vs. 1:1,000 and 1:10,000), a second staining pattern was observed. Many small, intense, T r i t o n X-100® r e s i s t a n t 'flecks' were observed within the nu c l e i of post-mitotic c e l l s ( f i g . 11). This staining pattern was not observed i n a l l post-mitotic c e l l s thus i s presumably c e l l cycle s p e c i f i c . The exact timing of the appearance and disappearance of thi s pattern has not been investigated further. 57 Figure 10. Double labelling of HeLa cells with ICP(C85) antibody (red) and a monoclonal anti p-tubulin antibody (green). 10.1, 10.4: HeLa cells stained with ICP(C85) antibody. 10.2, 10.5: identical fields (respectively) stained with anti p-tubulin antibody. 10.3, 10.6: images from the two antibodies superimposed. Note strong yellow signal at areas of overlap. Images were collected on a MRC 600 series confocal laser-scanning microscope, and processed with Adobe Photoshop image analysis software. 10.1 10.3 » 59 Figure 11. HeLa c e l l s stained with high concentrations of ICP(C85) antibody. 11.1: single 0.2 urn s l i c e of a post-mitotic doublet. Note centrosomal staining between daughter nuclei, cytoplasmic 'blebs', and punctate staining pattern within each daughter nucleus. 11.2: projection of multiple s l i c e s to form a pseudo- 3-dimensional image of post-mitotic HeLa c e l l s . Note fibrous nature of the staining pattern emanating from the region of the centrosome. 60 61 DISCUSSION In order to study the effect of mutations at the mfs(2)31 locus at the molecular level, a polyclonal antiserum was generated against a fusion of the glutathione-S-transferase (GST) and the. C-terminal 85 amino acids of Intronic Centrosomal Protein (ICP). GST specific antibodies were removed by adsorption to immobilized GST protein and ICP(C85) antibodies were a f f i n i t y purified. ICP(C85) antibodies were highly reactive both on western blots and as a reagent for indirect immunofluorescence of fixed c e l l s . Western B l o t t i n g . Probing western blots of total proteins from divergent organisms with a f f i n i t y purified anti ICP(C85) antibodies revealed that at least two proteins that strongly cross react with the ICP(C85) antibody exist in the mitotic c e l l . These two reactive species appear as a doublet with a molecular weight of approximately 3 0 KDa, the predicted molecular weight of ICP as deduced from the DNA sequence (4). This demonstrates that the ICP ORF i s translated into a protein product. One of these species may be the product of a post translational modification but i t cannot be determined by simple western blotting which species this is or whether i t has any functional significance. Two strongly reactive bands were also observed with apparent molecular weights of approximately 18 and 20 KDa. It 62 is unknown at this time whether these bands are due to strong cross-reactivity of the ICP(C85) antibody with a novel protein that has a similar epitope (s) to ICP, or i f the two bands correspond to C-terminal proteolytic products of ICP. It is possible that ICP is translated and then, in order to function properly, is proteolyticly cleaved into smaller fragments. Western blots also show a variety of less intensely staining bands at higher molecular weights; for example, there is a protein of about 37 KDa that was found to be enriched in proteins isolated from D r o s o p h i l a testes (data not shown). Although structural characterization of each of these reactive species and correlation of each species with a specific function or location within the c e l l is required for a complete understanding of ICP, these higher molecular weight species w i l l not be further discussed in this thesis. C e l l Staining by Indirect Immunofluorescence. To investigate the localization of ICP within the c e l l , ICP(C85) antibodies were used as a reagent for indirect immunofluorescence. The staining of D r o s o p h i l a embryos with the fluorescent, DNA specific dye, Hoechst 33258, in conjunction with the ICP(C85) antibody demonstrated that ICP i s located at the apical tip of the c e l l in a position consistent with the location of the centrosome, the apparatus that anchors the centrosome to the nuclear membrane, and the centromeric heterochromatin. 63 In order to f a c i l i t a t e interpretation of this staining pattern, and to determine whether the localization of ICP was conserved to human cells, HeLa cells were stained with the ICP(C85) antibody and with low concentrations of a anti 0-tubulin monoclonal antibody. ICP was found to co-localize with P-tubulin in a Triton X-100® resistant compartment of the centrosome. Higher concentrations of the ICP(C85) antibody revealed a second staining pattern within the nucleus; the nuclei of post-mitotic HeLa cells were seen to contain many small, Triton X-100® resistant 'flecks' that was sometimes suggestive of a fibrous network within the nucleus (see fi g . 11) . The possible significance of these staining patterns is discussed below. mfs(2)31 i s a Complex Locus. The mfs (2) 31 locus is comprised of two ORFs, ICP and DUb80, and has four distinct recessive phenotypes: suppression of PEV, male and female s t e r i l i t y , short, thin bristles, and, for strong mutations, complete lethality (4). At the present time the only mfs(2)31 mutant a l l e l e that has been sequenced is mfs2, the al l e l e caused by the insertion of a P-element in the 5' regulatory region that is thought to down regulate transcription of both ORFs. The three other mutant alleles of mfs (2) 31, mfs1, mfs3, and mfs4, have not been sequenced, thus no correlations can be made between changes at the molecular level, perhaps in one or the other ORF, and the 64 phenotypes observed. However with the data presented here, and the known function of DUb80, i t is now possible to speculate on the possible cause of each of these phenotypes. L e t h a l i t y The lethality phenotype could be caused by a loss of either or both of the proteins. ICP is present in the centrosome, correct function of which is required for c e l l division. Barrio, et a l . (1994) (12) have reported that the steady state level of DUb80 mRNA is much reduced in stationary phase SH2 cells as compared to in cultures which are rapidly dividing. Assuming that ICP is co-regulated with DUb80, this regulation may also be true of ICP. The western blot data show severe changes in the steady state level of ICP protein found in log phase versus stationary phase cells (fig 7). It is probable that expression of both protein products is required for c e l l division. Loss of either the ubiquitin fusion protein or ICP would most lik e l y arrest c e l l division thus preventing correct development of a multicellular organism such as Drosophila. Minute The b r i s t l e phenotype could be caused by a loss of the DUb80 protein product. It is known that the 80 amino acid t a i l moiety of the ubiquitin fusion protein i s a ribosomal protein 65 (30) and i s implicated in protein synthesis. Correct b r i s t l e formation is known to be dependant on high levels of protein synthesis, and other mutations with the small b r i s t l e phenotype (known as Minutes) have been previously characterized as mutations in ribosomal proteins (31,32). These data strongly indicate a role for the DUb80 protein product in brist l e formation. This hypothesis precludes the involvement of ICP in the process of br i s t l e formation. S t e r i l i t y The s t e r i l i t y phenotype was investigated in different ways by both Lindsley (1980) (5) and Whitehead (1993) (4). Spermatogenesis in mfs1 homozygotes was characterized by incorrect migration of the centrosome and faults in the reformation of the two daughter nuclei after meiosis; some cells contained two centrosomes and some contained two nuclei, a macronucleus and a micronucleus (5) . Female s t e r i l i t y i s thought to be due to a breakdown of vitellogenesis (4), the process by which yolk proteins are deposited in the developing egg (33). These phenotypes associated with male s t e r i l i t y can be explained by a mutation affecting the expression of ICP. Partial or complete loss of ICP, a centrosomal protein, may prevent attachment of the centrosome to the nuclear membrane, the anchoring of the centrosome to the cytoskeleton that i s required for migration of the centrosome to the opposite pole of 66 the c e l l d u r i n g p r o p h a s e , o r p e r h a p s the c o r r e c t a s s e m b l y o f the d a u g h t e r c en t r osome a t c e n t r o s o m a l d u p l i c a t i o n . I t i s p o s s i b l e t h a t m i s s - s e g r e g a t i o n o f the n u c l e o p l a s m d u r i n g m e i o s i s i s a s e c o n d a r y consequence o f t h e i n c o r r e c t m i g r a t i o n o f the c en t rosome b u t a more l i k e l y a l t e r n a t i v e i s t h a t the s m a l l ' f l e c k s ' w i t h i n the n u c l e u s i n d i c a t e a i n t r a n u c l e a r f u n c t i o n o f ICP , d i s c u s s e d i n more d e t a i l b e l ow , t h a t i s r e q u i r e d f o r the c o r r e c t s e g r e g a t i o n o f n u c l e a r m a t e r i a l . Oogenes i s i s known t o i n v o l v e a t i g h t l y o r c h e s t r a t e d m i g r a t i o n o f p r e - n u r s e c e l l c e n t r i o l e s t o the d e v e l o p i n g o o c y t e and a commensurate r e o r g a n i z a t i o n o f t h e c y t o s k e l e t o n (34-37) b u t i t i s unknown a t t h i s t ime whe the r t h i s phenomenon i s a f f e c t e d i n mfs(2)31 mu tan t s o r i f i n c o r r e c t a r rangement o f the c y t o s k e l e t o n o f the d e v e l o p i n g o o c y t e w o u l d have an e f f e c t on the p r o c e s s o f v i t e l l o g e n e s i s . A l t e r n a t i v e l y , l o s s o f the DUb80 ORF c o u l d be s l o w i n g v i t e l l o g e n e s i s v i a a d i s r u p t i o n o f the p r o t e i n s y n t h e s i s m a c h i n e r y . P E V P o s i t i o n e f f e c t v a r i e g a t i o n i s a b i o l o g i c a l phenomenon t h a t ou r l a b u s e s t o a s s a y f o r genes i n v o l v e d i n the f o r m a t i o n and m a i n t e n a n c e o f c h r o m a t i n s t r u c t u r e . Because o f the s e n s i t i v i t y o f t h i s a s s a y , any p e r t u r b a t i o n i n the c e l l w h i l e c h r o m a t i n i s i n t h e p r o c e s s o f f o r m i n g o r w h i l e p r e - f o r m e d c h r o m a t i n s t r u c t u r e s a r e c l o n a l l y t r a n s m i t t e d w i l l be m a n i f e s t e d as a 67 change in the expression of the white gene. Discussed below are some of the p o s s i b i l i t i e s of how mutations in mfs(2)31 suppress PEV. PEV and the Centrosome. The co-localization of ICP with p-tubulin at the centrosome suggests that ICP may be in part responsible for the correct segregation of chromatin during mitosis and meiosis. After meiosis has occurred, D r o s o p h i l a chromosomes are known to take a specific conformation within the daughter nuclei. The chromosomes take on the Rabl confirmation during embryogenesis (39) with the centromeres at one end of the c e l l and the telomeres at the other, then later in development, move to specific spacial domains (40). This kind of spatial organization is known to be a feature common to a l l eukaryotic cells thus far studied (40-43). Failure of the centrosome to duplicate, attach to the nuclear membrane, or nucleate and anchor spindle microtubules in a correct manner may affect centrosomal migration or formation of the mitotic spindle, each of which is l i k e l y to be required for correct positioning of chromosomes within daughter nuclei and correct attachment of chromosomes to the nuclear membrane. If specific positioning of genes within the daughter nuclei is required for transcriptional activity, faulty positional cues w i l l manifest themselves as changes in gene expression and may result in a phenotype such as suppression of PEV. 68 PEV and the Nuclear Matrix. In conjunction with the centrosomal localization, ICP could also function as part of the nuclear matrix, the non-chromatin, proteinaceous, fibrous network responsible for maintaining the three dimensional structure of the nucleus (44,45) . The nuclear matrix was originally defined as what was l e f t after enzymatic digestion of nucleic acids, soluablization of proteins with detergents, and high salt (2.0M NaCl) washes (50). Functionally, the nuclear matrix has previously been implicated in the regulation of DNA replication (53,70), regulation of transcription (46,47) and RNA splicing (49). The irregular, punctate nuclear staining pattern of ICP observed in post mitotic HeLa cells (fig. 11) i s suggestive of the association of ICP with a fibrous network within the nucleus. If ICP is s t r i c t l y associated with microtubules or microtubule based structures such as the centrosome, the 'flecks' may indicate sites at which intranuclear microtubules are anchored or non-covalently cross-linked with other nuclear proteins (ICP is not predicted to contain any cystine residues). This hypothesis predicts that ICP functions by binding to microtubules either directly, or via protein-protein interaction with other microtubule associated proteins. The localization of ICP to the Triton X-100® resistant 'flecks' within the post-mitotic nucleus may provide some insight into the mechanism by which hypomorphic mutations at mfs(2)31 suppress PEV. PEV is 6 9 extremely sen s i t i v e to the le v e l s of chromatin components, such as the histones (53), as well as how these components are post t r a n s l a t i o n a l l y modified. I t has been previously demonstrated that the human histone genes are t r a n s c r i p t i o n a l l y regulated i n part by components of the nuclear matrix (46). The d i s t a l promoter contains a matrix attachment region (MAR) (51) that i s proposed to l o c a l i z e and concentrate trans-acting regulatory factors to the histone promoter. If mutations i n mfs(2)31 disrupt the nuclear matrix i n such a way as to p a r t i a l l y de-regulate chromatin genes, such as the histones or other non-histone chromosomal proteins, t h i s would cause suppression of PEV. Chromatin modifying proteins are also known to be associated with the nuclear matrix. Recent biochemical studies have l o c a l i z e d histone deacetylase a c t i v i t y to the nuclear matrix f r a c t i o n of nu c l e i i s o l a t e d from chicken immature erythrocytes (48). The acetylation state of histones i s associated with control of gene expression and i s the r e s u l t of a dynamic equilibrium between histone acetyltransferases and histone deacetylases. Mutations i n Suvar(2)1, a strong dominant suppressor of PEV, cause hyperacetylation of histone H4 i n Drosophila (38). Though no histone deacetylase gene has yet to be cloned from any organism, because of the hyperacetylation phenotype, Suvar(2)1 i s a strong candidate for the Drosophila histone H4 deacetylase 70 gene, the protein product of which may be associated with the nuclear matrix. Mutations i n mfs (2) 31 may somehow, v i a disruption of the nuclear matrix, cause histone deacetylase a c t i v i t y to be reduced or improperly l o c a l i z e d thus suppressing PEV. Regulation of gene expression by chromatin structure i s known to be c l o n a l l y i nherited (16). As DNA i s replicated, the structure of the e x i s t i n g chromatin must be copied on to the new DNA strand. While the d e t a i l s of how the protein configuration that regulates gene expression i s maintained through r e p l i c a t i o n remain elusive, i t i s clear that the nuclear matrix i s involved i n t h i s process; the nuclear matrix i s required to organize the more than 50,000 replicons found i n the mammalian genome i n to 10-100 cl u s t e r s coined replisomes (53,54). It may be that the nuclear matrix i s d i r e c t l y or i n d i r e c t l y responsible for l o c a l i z i n g new chromatin components to the replisome i n such a way that the regulatory configuration i s maintained. ICP may be associated with replisomes. The intranuclear staining pattern of 'flecks' within the nucleus i s detected only i n post-mitotic c e l l s , which are probably eit h e r i n Gx- or S-phase. Suppression of PEV i n mutant mfs(2)31 c e l l s may be due to incorrect chromatin r e p l i c a t i o n . P r o l i f e r a t i n g C e l l Nuclear Antigen, PCNA, i s associated with the r e p l i c a t i n g fork during DNA r e p l i c a t i o n (need ref) and although i t has yet to be d i r e c t l y tested, i s probably associated with the replisome. In Drosophila, PCNA i s encoded 71 by the gene mus209, which when mutated causes a mutagen s e n s i t i v i t y phenotype and suppresses PEV (24). This demonstrates that some genes involved i n r e p l i c a t i o n are suppressors of PEV and lends support to the idea of ICP involvement i n the process of DNA r e p l i c a t i o n . PEV and DUb80. The DUb80 ORF encodes a u b i q u i t i n fusion protein, the t a i l moiety of which i s implicated i n ribosome biogenesis. Down regulation of DUb80 i s sure to slow down the rate of protein synthesis, which i n turn w i l l l i m i t the number of protein monomers available to b u i l d the nucleoprotein complex that i s heterochromatin. PEV i s an extremely sen s i t i v e assay for proteins a f f e c t i n g chromatin assembly. Because of this , either protein could regulate chromatin assembly far upstream of the actual process and s t i l l have a great e f f e c t on the gross phenotype. Further Considerations. The functional characterization of mfs (2)31 i s far from complete; many questions remain unanswered. Eluci d a t i o n of the regulatory mechanism governing t r a n s c r i p t i o n of the mfs(2)31 locus may be of key importance to understanding the function of each of the two ORFs associated with mfs (2)31. The finding that the mfs(2)31 t r a n s c r i p t i s 1 p o l y c i s t r o n i c ' , or has a 'gene-within-a-gene' structure may 72 i n d i c a t e c o - o r d i n a t e e x p r e s s i o n o f t h e s e two i n d e p e n d e n t l y t r a n s l a t e d p r o t e i n s and may s i g n i f y t h a t b o t h p r o t e i n s a r e i n v o l v e d i n a s i m i l a r c e l l u l a r p r o c e s s . The genomic s t r u c t u r e o f the mfs(2)31 l o c u s does n o t appear t o be c o n s e r v e d t o y e a s t (see 69), so t h i s form of t r a n s c r i p t i o n a l r e g u l a t i o n may be a u n i q u e e v o l u t i o n a r y event, a s s o c i a t e d s t r i c t l y w i t h Drosophila. I f b o t h p r o t e i n s a r e i n d e e d r e q u i r e d f o r the same p r o c e s s , y e a s t has e v o l v e d a d i s t i n c t mechanism f o r c o - r e g u l a t i o n . The f o u r mutant a l l e l e s o f mfs(2)31 r e q u i r e c h a r a c t e r i z a t i o n a t the m o l e c u l a r l e v e l . The e x a c t changes i n the DNA sequence t h a t r e s u l t i n the mutant phenotypes need t o be d e t e r m i n e d . G e n e t i c d i s s e c t i o n o f the l o c u s i n t o d i s c r e t e domains by t r a n s f o r m - t o - r e s c u e e x p e r i m e n t s w i l l a l l o w f o r m a l e v a l u a t i o n o f t h e phenotypes. T h i s work d e s c r i b e s the g e n e r a t i o n o f a p o l y c l o n a l a n t i s e r u m a g a i n s t the C - t e r m i n a l 85 amino a c i d s o f ICP. The g e n e r a t i o n o f e i t h e r mono- o r p o l y c l o n a l a n t i b o d i e s t o the N-t e r m i n u s o f ICP i s r e q u i r e d t o c o n f i r m the c e l l s t a i n i n g r e s u l t s and w i l l f a c i l i t a t e t he assignment o f i n d i v i d u a l r e a c t i v e s p e c i e s , as d e t e c t e d by w e s t e r n b l o t t i n g , t o s p e c i f i c f u n c t i o n s and/or l o c a t i o n s w i t h i n the c e l l . F u r t h e r c h a r a c t e r i z a t i o n of ICP f u n c t i o n w i t h i n the centrosome w i l l r e q u i r e p u r i f i c a t i o n o f centrosomes and immuno-g o l d l a b e l l i n g i n p r e p a r a t i o n f o r e l e c t r o n m i c r o s c o p y . F r a c t i o n a t i o n o f i s o l a t e d n u c l e i i n t o m a t r i x and n o n - m a t r i x 73 components using the ICP(C85) antibody to probe for ICP content w i l l allow biochemical characterization of the nuclear ICP species. A determination of what portion of ICP i s required for nuclear import may also further functional characterization. The c e l l cycle s p e c i f i c i t y of ICP expression and l o c a l i z a t i o n also needs to be addressed. ICP could prove to be a molecular marker for c e l l s that have undergone transformation to a cancer phenotype. ICP could be involved i n checkpoint regulation - how c e l l s determine i f , to c i t e two examples, a l l the chromatin has been c o r r e c t l y replicated, or i f the centrosomes are i n the correct orientation for c e l l d i v i s i o n . Immunoprecipitation of ICP protein complexes or in vivo analysis using the yeast two-hybrid system may allow i d e n t i f i c a t i o n of s p e c i f i c proteins that d i r e c t l y i n t e r a c t with ICP. 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