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MAPPING OF MONOCLONAL ANTIBODY EPITOPES IN THE NUCLEOLAR PROTEIN FIBRILIARIN (B-36) OF PHYSARUM

POL YCEPHALUM. Mark E. Christensen 1 and Nitesh Banker z.

School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri, 64110, USA; and ZDepartment of Biology, Texas A & M University, College Station, Texas 77843, USA. 1Corresponding author. ABSTRACT

We have mapped the epitopes for nine monoclonal antibodies raised against the nucleolar protein fibrillarin of the slime mold Physarum polycephalum. This has been done using a combination of specific chemical and enzymatic cleavage, Western blotting and partial sequencing of fragments. Cleavage with cyanogen bromide reveals four prominent methionine cleavage sites within the protein. Western blotting shows that none of the monoclonal antibody epitopes are dependent on long range interactions. Eight highly-conserved epitopes are clustered in the carboxy terminal half of the protein, while a single less-conserved epitope (for monoclonal antibody PIG12) is located at the amino terminus and appears to lie within the GIy/DMA/Phe domain. INTRODUCTION

Fibrillarin is a highly-conserved 34 kD protein (Guiltinan et al., 1988) associated with the dense fibrillar component of the eukaryotic nucleolus (Ochs et al., 1985; Pierron et al., 1989), the region enriched in nascent pre-rRNA (Scheer and Benavente, 1990). The protein was originally found and termed B-36 in the slime mold Physarum polycephalum (Christensen et al., 1977). Fibrillarin shares certain structural domains with other nucleolar (nucleofin) and nucleoplasmic (hnRNP protein AI) RNA-binding proteins, most notably a region rich in glycine, dimethylarginine, and phenylalanine (termed the GIy/DMA/Phe domain) at its amino terminus (Christensen and Fuxa, 1988; Ghisolfi et al., 1992). Sequence analysis of cDNAs has also suggested that fibrillarin possesses an "RNP-CS" RNA-binding domain (Lapeyre et al., 1990). Consistent with its predicted role in nucleolar RNA binding, fibrillarin has been shown to be associated with U3 snRNA (Parker and Steitz, 1987); together they function in an early rRNA processing event in the 5'ETS region of nascent pre-rRNA (Kass et al., 1990). In order

to

examine the domains of fibrillarin which participate in specific

0309-1651/921111119-13/$08.00]0

© 1992 Academic Press Ltd

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interactions, a panel of monoclonal antibodies (MAbs) has been developed against Physarum fibrillarin (Christensen et al., 1986). Here we report the mapping of epitopes for nine MAbs using a chemical and enzymatic cleavage strategy. MATERIALS AND METHODS lmmunoaffinity and gel purification of fibrillarin: Fibrillarin used in the cleavage reactions was purified using a three-step procedure: First is preparation of the $2 nuclear extract enriched in fibrillarin. Briefly, isolated nuclei (Christensen et aL, 1986) from five ml of packed plasmodia were washed briefly with five ml of STM (0.1 M NaCI, 10 mM "Iris, pH 8.0, 1.0 mM MgCI2) and centrifuged at 1000 x G. The supernatant designated $1 was discarded. The nuclear pellet was gently resuspended in 2.5 ml of TE buffer (10 mM Tris, pH 8, 5 mM EDTA) and incubated on ice for 10 rain with occasional gentle mixing by hand tapping. The suspension was centrifuged at 4000 x G and the supernatant, designated the $2 nuclear extract, was carefully removed with an automatic pipetter and placed in a clean tube on ice, ready for immediate use in the next step. Second is immunoaffinity chromatography using a one ml column of Sepharose 4B beads (Sigma Chemical Co., St. Louis) to which the fibrinarin MAb P2G3 is bound. Prior to addition to the column, the NaC1 concentration of the $2 extract was raised to 0.5 M by the addition of a 2 M NaC1 stock. The salt-treated extract was added to the Sepharose 4B-MAb P2G3 beads in a polypropylene tube and incubated at 4°C for 3-4 hr with 360 ° rotation. The beads were batch washed twice with 10 ml of Tris- Buffered Saline (TBS-0.5 M NaCI, 10 mM Tris, pH 7.5) and transferred to a Bio-Rad 5 ml Econo-column. Washing was continued with an additional 15 ml of TBS at a flow rate of 0.25-0.5 ml/min. Bound fibrillarin was then eluted with 3-5 ml of 4 M guanidine hydrochloride. The eluate was dialysed against 0.1% SDS and lyophflized to dryness. The dried protein was resolubilized in 300 I~1 of deionized water, giving a final SDS concentration of 1.0%. A typical purification yielded roughly a 75% pure preparation containing approximately 25 I~g of fibrillarin. This preparation was not of sufficient purity to conduct cleavage experiments. In order to obtain highly purified fibrillarin, a third step involving gel purification was done prior to cleavage. Fibrillarin (10-25 I~g)was electrophoresed in a single wide lane of a 12.5% polyacrylamide SDS gel (see details below) and stained for 30 min with Coomassie Blue to identify the band. The band was excised from the gel in a pure form to use in subsequent cleavage experiments. Cyanogen bromide cleavage: The method used was adapted from a procedure originally described by Pepinsky (1983). Gel slices containing fibrillarin, cut from 0.5 mm thick polyacrylamide gels in which immunoaffinity-purified protein (see above) had been electrophoresed, were incubated at room temperature for

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11 21

1 hr in 600 mg/ml cyanogen bromide in 70% formic acid, 0.1 N HCI, 1% 2-mercaptoethanol. Following incubation, the slices were washed twice with deionized water, once with 0.25 M Tris-phosphate, pH 6.8, and finally with SDS Sample Buffer (0.1% SDS, 0.25 M sucrose, 10 mM sodium phosphate, Ph 7.5, 1% 2-mercaptoethanol). Each wash was done for 5 rain. The gel slices were placed back into sample wells of a 0.75 mm thick polyacrylamide SDS tricine gel (see below). N-chlorosuccinimide cleavage: N-chlorosuccinimide cleavage was carried out in gel slices according to procedures described previously (Lischwe and Sung, 1977). Gel slices were incubated for 30-60 rain in a solution of 40 mM N-chlorosuccinimide, 1% urea in 50% acetic acid. Slices were subsequently washed four times in SDS Sample Buffer and loaded into a 0.75 mm thick SDS polyacrylamide tricine gel for high resolution separation of cleavage products. "I'D~sin cleavage: Immunoaffinity-purified fibrillarin was digested with trypsin (Sigma Type XIII) at a fibriUarin:trypsin (wt:wt) ratio of 20:1. A typical 25 !~1 reaction containing 10 ttg of fibrillarin had a final concentration of trypsin of 0.02 mg/ml. Reactions were carried out at room temperature for the lengths of time specified in the figure. Following completion of reactions, the digested material was loaded directly onto a polyacrylamide SDS tricine gel. SDS polyacrylamide gel electrophoresis and Western Blotting. For gel purification of fibrillarin, 12.5% polyacrylamide SDS gels, 0.5 mm thick, were run (LeStourgeon and Beyer, 1977). For high resolution separation and analysis of cleavage products down to 1 kDa in size, tricine-containing gels were used, consisting of a two-layer separating gel (16% overlaid with 10% polyacrylamide) as described by Schagger and yon Jagow (1987). These gels were routinely made at a thickness of 0.75 mm to accommodate the 0.5 mm thick gel slices used in the cleavage reactions. Gels were stained with Serva Blue G. To test the MAb reactivity of digestion products, electrophoresed digestion fragments were transferred to lmmobilou (Millipore Corp., Bedford, Mass., USA) using the 20% methanol, tris-glycine buffer of Towbin et al. (1979) to which 03% SDS had been added. Transfer was for 2.5 hrs at 0.5 amps. For direct visualization of transferred bands the lmmobilon strips were stained with Serva Blue G in 50% methanol, 10% acetic acid and destained with 50% methanol, 10% acetic acid. For MAb reactions, replica strips were blocked with 3% gelatin in TBS (0.5 M NaCI, 10raM Tris, pI-I 7.5) and incubated overnight in MAb supernatants diluted 10- fold in 1% gelatin in TBS. The strips were rinsed with distilled water, washed twice with TBS, 10 rain each time, and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) diluted 5000-fold in 1% gelatin in TBS. Following a distilled water rinse and two ten-minute washes with TBS, the strips were developed by addition of a solution containing 66 ttl each of the NBT substrate stock (50 mg/ml NBT [Bio-Rad) in 70% dimethylformamide) and the BCIP substrate

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stock (25 mg/ml of BCIP [Bio-Rad] in 100% dimethylformamide) per 10 ml of alkaline phosphatase buffer (100 mM "Iris, pH 9.5, 100 mM NaCI, 5 mM MgCh). Development times were from 5-30 rain. Development was stopped by rinsing the strips in distilled water and drying under a heat lamp. All steps were carried out at room temperature with gentle agitation. Sequencing of cyanogen bromide fragments: Cyanogen bromide fragments to be sequenced were transferred to Immobilon and stained with Coomassie Blue. Following destaining, the transfers were air dried and the band(s) of interest cut out with a scalpel. The Immobilon-bound fragmentswere sequenced directly using an Applied Biosystems 470A Sequencer together with an Applied Biosystems 120A On-Line-PTH Analyzer. Sequencing was performed by the Texas Agricultural Experiment Station's Biotechnology Support Laboratory located on the Texas A & M University campus, College Station, Texas. RESULTS Mapping of monoclonal antibodies using cyanogen bromide cleavage. Based upon previous amino acid composition analysis (LeStourgeon et al., 1977) Physarum fibrillarin was estimated to have 4-5 methionine residues. Cleavage with the methionine-specific'reagent cyanogen bromide should therefore yield a manageable number of fragments whose positions in the intact protein could be deduced from their reaction patterns with the panel of MAbs. In so doing, the positions of the MAb epitopes within the protein could also be determined. Gel slices containing electrophoretieally-purified fibrillarin were incubated with cyanogen bromide and the cleavage products rerun in high resolution gels and tested with the individual MAbs in Western blots. Fourteen fragments were reproducibly observed and designated CB-1 through CB- 14 (Figure lc). All of the fxagments, except for CB-13 and CB-14, contain one or more MAb epitopes as judged by their detection in a Western blot using a mixture of all nine MAbs (Figure ld). In order to test whether these fragments are all due to bona fide methionine cleavage, replica samples of fibrillarin were treated with the formic acid solvent in the absence of cyanogen bromide (Figure lf) or with cyanogen bromide under non-reducing conditions (Figure le), which can promote cleavage at eysteine residues. None of the fragments are due to acid cleavage alone, while CB-5 appears to be due to a cleavage at eysteine. All other fragments are assumed to be due to cleavage at methionine residues. The fourteen fragments obtained represent a collection of both partial and complete digestion products. Cleavage with higher concentrations of cyanogen bromide or for longer periods of time did not alter the pattern shown in Figure le and it was therefore necessary to work with the incomplete digestion products.

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1123 ¢J

E~ tg~

66453629-

1714.3-

6.2-

.:"

2.5-

a

b

c

d

e

f

Figure 1. Eiectrophoretic analysis of cyanogen bromide cleavage products of fibrillarin. Gel purified fibrillarin (approximately 5 I~g/slice) was digested with cyanogen bromide (CNBr) as described in Materials and Methods and rerun in a 10%/16% polyacrylamide tricine gel. Separated samples were either stained with Serva Blue G (lanes a-c) or transferred to Immobflon and tested with a mixture of all nine MAbs (lanes d-f). The cyanogen bromide cleavage products are designated as CB-1 through CB-14. The standard cleavage reaction contained 2-mercaptoethanol (+ME, lanes c and d). To test for possible cleavage at non-methionine sites, cleavage was also performed in the absence of mercaptoethanol (-ME, lane e) and in the absence of both cyanogen bromide and mercaptoethanol, i.e. in the presence of the formic acid solvent alone (lane f). High (HMW) and low (LMW) molecular weight markers (Sigma Chemical Co., St. Louis, USA) were run in the same tricine gel (lanes a and b). Replica Western blots of cyanogen bromide-digested fibrillarin were tested individually with nine MAbs (Figure 2). The reaction patterns were complex, but reproducible and are summarized in Table 1. Sequencing of the amino termini of several of the fragments (Table 2) gave important information for mapping the cyanogen bromide fragments within the intact protein. First, for fragment CB-10, two amino acids were recovered during each cycle indicating that it was composed of two fragments of nearly identical gel mobility. Furthermore, one of these fragments, designated CB-10a, had a sequence identical to the amino terminus of intact fibrillarin (Table 2) and was therefore derived from that position during cleavage. Secondly, since the amino terminal

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sequence of intact fibrillarin was known (Christensen and Fuxa, 1988), it was possible to deduce the sequence of the other fragment, designated CB-10b (Table 2). Sequencing revealed that fragment CB-8 was also derived from the amino terminus of intact fibrillarin. Since CB-8 is larger (15kDa) than CB-10a (9kDa) and both come from the amino terminus of fibrillarin, they must have a direct precursor-product relationship. Furthermore, since CB-8 was found to contain only a single epitope, that for MAb P1G12 (see Table 1), the reaction of this MAb with "CB-10" can be assigned to CB-10a, while the reactions of all the other CB-10 positive MAbs can be assigned to fragment CB-10b (Table 1).

1 B-36-

CB-234-

~a,.

6-

7-

~

9q0-

+.~

8-

1112-

" , ~.,"

a

b

c

d

e

f

g

h

i

j

Figure 2. Western blot analysis of cyanogen bromide fragments. Ten replicate gel slices containing purified fibrillarin were prepared as described in Materials and Methods and cleaved with cyanogen bromide. Each was then re-electrophoresed'in a separate well in a tricine gel, transferred to lmmobilon, and either stained with Coomassie Blue (lane a) or tested with one of the MAbs (lanes b-j). The MAb used in each case is indicated at the top of the lane. The positions of the cyanogen bromide cleavage products are shown to the left, in addition to that for uncut fibrillarin (B-36). On the basis of these assignments an epitope map has been derived which is consistent with the presence of four methionine residues positioned as indicated (Figure 3). The epitope for PIG12 is located near the amino terminus and is separated by a large gap from the other epitopes.

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TABLE 1. Reaction patterns of monocional antibodies with cyanogen bromide fragments of Physarum flbrillarin. Fragment

Size

MonoclonalAntibody: PIDIO

CB-I CB-2 CB-3 CB-4 CB-5 CB-6 CB-7 CB-8 CB-9 CB-IOa CB-IOb CB-11 CB-12

34kD 30 27 24 20.5 20 15.5 15 10.5 9 9 7 4.5

+ ++ +++ +++ + ++ +++ +/ . +++ . +/+

PID11

P2D7

+ + ++ +++ + ++ ~

P2BII

+ + +++ +++ + ++

+ + ++ + + +

;:::

::::

++++

. ++++

. ++ . ++ + -

. +

P2GI

PIE8

P2G3

+ + + + + +

+ + +++ +++ + +4+ ++++ . ::~

++ ++ +++ :~:: ++ ++ +/-

+ + ++ +++ + + +/-

+/-

+/"

++++

+++

+

+

+++

+++ .

+ -

P3F11

+ ++ ++++ +/-

++ .

::~:= + +++

PIGI'2

++ -

TABLE 2. Amino terminal sequences of fibrillarin cyanogen bromide fragments. Fragment

Sequence

CB-7 CB-8 CB-9 CB-IOa CB-IOb CB-12 FIBRILLARIN

xxxPGVFIEKGKEDALVTRNxxxLEA xFEGxGxF VEVNGEKKEYRVxNPF xFEGxGGFGGxGGxDxGGxxxGGF VDVLFADVANPDNAXIFALNAE xxxxxxl DSVAPAEV FEGRGGFGGRGGGDRGGRGxGGFGGG

Fragments CB-7, CB-9 and CB-11 are sequential precursor/products which share a cluster of epitopes for MAbs PID10, P2B11, P2D7, and P2G1 near the center of the protein. The epitopes for MAbs P1E8 and PID11 lie on the carboxy terminal side of the central cluster, in a region of overlap of fragments CB-7/CB-9 and CB-10b. Finally the epitopes for MAbs P2G3 and P3Fll lie closest to the C-terminus of the protein, downstream of the final methionine residue. Cleavage at a single tryptophan separates the amino-terminal epitope PIG12 from the carboxy-terminal epitope cluster:. To further confirm the epitope map given in Figure 3, cleavage with a reagent with a different specificity was done. Under acid/urea conditions N-chlorosuccinimide has been shown to cleave at tryptophan residues (Lischwe and Sung, 1977). Using N-chlorosuccinimide cleavage of purified fibrillarin in gel slices, digestion products were tested with the panel of MAbs. The results show that N-chlorosuccinimide cleaves fibrmarin into two major fragments of roughly 17 and 21 kDa (data not shown) suggesting that the protein contains a single tryptophan residue somewhere near the middle. Reaction with the MAbs clearly shows that the epitope for PIG12 is the only one present in the 17 kDa fragment, while the 21 kDa

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1126

Trp )

NHa

M

+

L

M

+

I

M

+

M

+ ,,

COOH

t~

CB-8

!

t CB-10a I CB-7 m CB-9 ) CB-10b |

CB-II

:

I CB-12

'i

Figure 3. Cleavage and epitope map of Physarum fibrillarin. Intact fibrillarin is depicted by the line at the top of the diagram. The positions of the four methionine cleavage sites are marked (M). The major cyanogen bromide fragments obtained experimentally are indicated by the bars below. The locations of the epitopes for the nine MAbs used in this study are indicated by the asterisks. The single tryptophan residue cleaved by N-chlorosuccinimide is indicated (Trp) as is the amino terminal trypsin-resistant fragment (+). fragment contains all the remaining epitopes. The 17 kDa fragment therefore represents the amino terminal portion of fibrillarin. Since this fragment is slightly larger than the amino terminal cyanogen bromide fragment CB-8 (15 kDa), the tryptophan residue must lie between the 2nd methionine and the MAb epitope cluster (Figure 3). The amino-terminal Gly/DMA/Phe domain is trypsin resistant. Fibrillarin possesses an approximately 8G-residue domain at its amino terminus that is rich in glycine, arginine and phenylalanine (Axis and Blobel, 1991). Within this domain the arginine residues are at least partially methylated and no lysine residues have been observed. It is therefore predicted, assuming methylation of arginine is complete and blocks the action of trypsin, that extensive digestion of purified fibrillarin with trypsin should produce a trypsin-resistant fragment representing the amino terminal Gly/DMA/Phe domain. To test this, immunoaffinity-purifiedfibrillarin was incubated with trypsin for 6 or 18 hours at room temperature. Subsequent gel analysis revealed a single, approximately 8 kDa band with Coomassie staining (Figure 4b,c). Trypsin at the same concentration used in the digestion, does not produce any detectable bands (Figure 4d). If the 8 kDa band is excised from the gel, rerun in a second gel and then transferred to Immobilon for Western blot analysis, the fragment is found to contain the PIG12 epitope (Figure 4g). In contrast, MAb P2Bll,

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representative of the fibrillarin multiple-epitope cluster, does not react (Figure 4h). The simplest interpretation is that the fragment is derived from the amino terminus of fibrillarin and is trypsin resistant due to complete methylation of the arginine residues. B-36 #Trypsm

E =

P.

,:: $"

a

b

c

d

A

e

f

g

h

B

Figure 4. Analysis of trypsin cleavage of fibrillarin. Panel A: lmmunoaffinity purified fibrillarin (B-36) was prepared in three equal aliquots of 3.5 ttg each. One aliquot, was left untreated, while the remaining two were digested with 0.175 ttg trypsin for 6 and 18 hours, respectively, at room temperature. Following incubation, the samples were electrophoresed in a tricine gel as described in Materials and Methods and stained with Serva Blue G. Lane a, untreated fibrillarin; lane b, fibrillarin digested with trypsin for 6 hrs; lane c, fibriUarin digested with trypsin overnight; lane d, trypsin alone, same amount as used in fibrillarin digestion and incubated overnight; lane e, trypsin alone, ten-fold greater amount loaded in order to see possible self-digestion products; lane f, low molecular weight markers. Panel B: The two trypsin-resistant fragments visualized by Serva Blue G staining in lanes b and c of Panel A (denoted by an asterisk) were cut from the initial gel, rerun in separate wells in a second tricine gel, and transferred to Immobilon. The two lmmobilon strips were tested either with MAb P1G12 lane or P2Bll lane h.

DISCUSSION This work reports the mapping of monoclonal antibody binding sites within the

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nucleolar protein fibrillarin of Physarum polycephalum. This has been accomplished using a panel of nine MAbs generated previously against fibrillarin together with cyanogen bromide cleavage, Western blotting, and sequencing to construct simultaneously a peptide cleavage and MAb epitope map (Figure 3). The data are consistent with four methionine cleavage sites as indicated. Since these sites are defined by the assigned positions of fragments CB-7, CB-9, CB-10a, and CB-12, the proposed map is confirmed by comparison of the relative positions of the amino terminal sequences of these fragments to the homologous regions in fibrillarin sequences recently published for yeast (Sehimmang et al., 1989; Henriquez et al., 1990), frog (Lapeyre et al., 1990), and human (Aris and Blobel, 1991). Of the nine MAbs examined, eight recognize epitopes which are clustered in the earboxy terminal half of the protein. Among these eight MAbs at least five distinct epitopes are represented (Gufltinan et al., 1988) and appear to be clustered in a region of the protein that is highly immunogenie. The single epitope that is not in this cluster is that for MAb P1G12, located close to the amino terminus. This distribution parallels the pattern of conservation of the epitopes within fibriUarin homologues from a variety of eukaryotes (Christensen et al., 1986; Guiltinan et al., 1988). That is, the MAbs clustered in the carboxy half are both highly-conserved in and unique to fibrillarin. In contrast, the amino terminal epitope recognized by MAb PIG12 is often not conserved in the fibrillarin of a given species, but is often observed in other proteins. This correlation suggests that the immunogenic carboxy terminal half of fibrillarin may also be the more highly-conserved portion of the protein. This has been confirmed by recent eDNA sequence data for yeast (Sehimmang et al., 1989), frog (Lapeyre et al., 1990), and human (Aris and Blobel, 1991) fibrillarins. Trypsin cleavage indicated that the epitope for PIG12 lies within the f~st 8 kDa of the amino terminus of Physarum fibrillarin. The question arises as to whether this epitope is within the GIy/DMA/Phe domain. Insight into the answer is derived from examining possible trypsin cleavage sites adjacent to the Gly/DMA/Phe domain. The published sequences for yeast, frog and human fibrillarins reveal lengths for the Gly/DMA/Phe domain of 78-86 amino acids with no internal trypsin cleavage sites (assuming that all arginine residues are methylated). Each has a conserved lysine residue at the next position downstream of the GIy/DMA/Phe domain. Trypsin cleavage at this lysine residue would generate calculated amino terminal trypsin-resistant fragment sizes ranging from 8.0-9.0 kDa. These values are consistent with the interpretation that the 8 kDa trypsin-resistant fragment derived from Physarum fibrillarin is due to cleavage at the same conserved lysine residue immediately downstream of the GIy~MA/Phe domain. If so, the P1G12 epitope must lie within the Gly/DMA/Phe domain itself. The location of the P1G12 epitope within the Gly/DMA/Phe domain has important implications for several reasons. First, as a domain-specific probe, the P1G12 MAb will be useful in

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investigating the structural and functional properties of the Gly/DMA/Phe domain in protein, RNA or protein/protein interactions made by fibrillarin. Second, other proteins which have been observed to cross-react specificallywith this MAb may possess the same domain (Guiltinan et al., 1988). Many RNP-associated proteins possess a related RNA-binding domain referred to as the RNP consensus sequence or RNP-CS (Bandziulis et al., 1989). In con~paring the frog fibrillarin sequence to the RNP-CS sequences of snRNP proteins UIA, U1B and U1-70K, Lapeyre et al. (1990), have reported a putative RNP-CS-like domain. This 90 amino acid long domain is located from residues 140-230 and corresponds closely to the Physarum cyanogen bromide fragment CB-11. This region contains the epitopes for MAbs PID10, P2G1, P2Bll and P2D7 and constitutes the most immunogenic region of the protein. With this information it should be possible to directly test the RNA binding capacity of this region, both in the intact protein and in isolated fragments. Furthermore, our results with N-chlorosuccinimide indicate cleavage at a single tryptophan residue which maps to a position just preceding the CB-11 epitope cluster (Figure 3). This cleavage divides the protein into a 17 kDa amino terminal fragment containing the Gly/DMA/Phe domain (and some downstream sequence as well) and a 21 kDa carboxy terminal fragment containing the putative RNP-CS domain. ACKNOWLEDGEMENTS The authors wish to acknowledge the excellent technical assistance of Patricia Ainsworth who conducted the immunoaffinitypurification of the B-36 protein used in this study. We also acknowledge the high quality protein sequencing done by the Biotechnology Support Laboratory at Texas A & M University, Dr. Timothy Hayes, director. This work was supported by a grant from The National Science Foundation (DCB-8702390) to MEC.

REFERENCES Aris, J.P., and Blobel, G. (1991). cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune sera. Proc. Natl. Acad. Sci. USA 88, 931-935. Bandziulis, R. J., Swanson, M. S., and Dreyfuss, G. (1989). RNA-binding proteins as developmental regulators. Genes Develop. 3, 431-437. Christensen, M. E., and Fuxa, IL P. (1988). The nucleolar protein, B-36, contains a glycine and dimethylarginine-rich sequence conserved in several other nuclear RNA-binding proteins. Biochem. Biophys. Res. Commun. 155, 1278-1283.

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Christensen, M.E., Beyer, A.L., Walker B.W., and LeStourgeon, W.M. (1977). Identification of NG,N°-dimethylarginine in a nuclear protein from the lower eukaryote Physarum polycephalum homologous to the major proteins of mammalian 40S ribonucleoprotein particles. Biochem. Biophys. Res. Commun. 74, 621-629. Christensen, M. E., Moloo, J., Swischuk, J. L., and Schelling, M. E. (1986). Characterization of the nudeolar protein, B-36, using monoclonal antibodies. Exp. Cell Res. 166, 77-93. Ghisolfi, L., Joseph, G., Amalric, F., and Erard, M. (1992). The glycine-rich domain of nucleolin has an unusual supersecondary structure responsible for its RNA helix-destabilizing properties. J. Biol. Chem. 267, 2955-2959. Guiltinan, M. J., Schelling, M. E., Ehtesham, N. Z., Thomas, J. C., and Christensen, M. E. (1988). The nucleolar RNA-binding protein B-36 is highly conserved among plants. Eur. J. Cell Biol. 46, 547-553. Henriquez, It., Blobel, G., and Aris, J. P. (1990). Isolation and sequencing of NOP1. A yeast gene encoding a nucleolar protein homologous to a human autoimmune antigen. J. Biol. Chem. 265, 2209-2215. Kass, S., Tyc, K., Steitz, J. A., and Soilner-Webb, B. (1990). The U3 small nucleolar ribonucleoprotein functions in the first step of pre-ribosomal RNA processing. Cell 60, 897-908. Lapeyre, B., Mariottini, P., Mathieu, C., Ferrer, P., Amaldi, F., Amalric, F., and Caizergues-Ferrer, M. (1990). Molecular cloning ofXenopus fibrillarin, a conserved U3 small nuclear ribonucleoprotein recognized by antisera from humans with autoimmune disease. Mol. Cell. Biol. 10, 430-434. LeStourgeon, W. M., and Beyer, A. L. (1977). The rapid isolation, high resolution electrophoretic characterization, and purification of nuclear proteins, in: Stein, G., Kleinsmith, L., and Prescott, D.,(eds) Methods in Cell Biology. Vo1.16; pp.387-406, New York: Academic Press. LeStourgeon, W. M., Beyer, A. L., Christensen, M. E., Walker, B. W., Poupore, S. M., and Daniels, L. P. (1977). The packaging of core hnRNP part,ides and the maintenance of proliferative cell states. Cold Spring Harbor Symp. Quant. Biol. 42, 885-898. Lischwe, M. A., and Sung, M. T. (1977). The use of N-chlorosuccinimide/urea for the selective cleavage of tryptophanyl peptide bonds in proteins. J. Biol. Chem. 2$2, 4976-4980. Ochs, IL L., Lischwe, M. A., Spohn, W. H., and Busch, H. (1985). Fibrillarin: A new protein of the nucleolus identified by autoimmune sera. Biol. Cell 54, 122-134. Parker, IL A., and Steitz, J. A. (1987). Structural analyses of the human U3 ribonucleoprotein particle reveal a conserved sequence available for base pairing with pre-rRNA. Mol. Cell. Biol. 7, 2899-2913. Pepinsky, 1L B. (1983). Localization of lipid-protein and protein-protein interactions within the routine retrovirus gag precursor by a novel peptide mapping technique. J. Biol. Chem. 258, 11229-11235.

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Pierron, G., Pedron, J., Schelling, M., and Christensen, M. (1989). Immunoelectron microscopic localization of the nucleolar protein B-36 (fibrillarin) during the cell cycle ofPhysarumpolycephalum. Biol. Cell 65, 119-126. Schagger, H., and von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-poly acrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368-379. Scheer, U., and Benavente, R. (1990). Functional and dynamic aspects of the mammalian nucleolus. BioEssays 12, 14-21. Schimmang, T., Tollervey, D., Kern, H., Frank, IL, and Hurt, E.C. (1989). A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J. 8, 4015-4024. Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 46, 4350-4354.