Two DIF-inducible, prestalk-specific mRNAs of Dictyostelium encode
extracellular matrix proteins of the slug
STUART J. McROBBIE1, KEITH A. JERMYN1, KAREN DUFFY1, KENNETH BLIGHT2
and JEFFREY G. WILLIAMS1*1 Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Herts EN6 3LD, UK2Imperial Cancer Research Fund, Electron Microscopy Unit, Lincoln's Inn Fields, London WC2A 3PX, UK
* To whom reprint requests should be sent
Summary
The migratory slug of Dictyostelium discoideum issurrounded by, and continuously synthesizes, anextracellular protein-cellulose matrix known as theslime sheath which is deposited on the substratum as atrail marking the slug's progress. We show that thestalk-specific proteins, ST310 and ST430, are exclus-ively located in the slime sheath and trail and thatfusion genes, containing upstream sequences from thecognate genes, direct correct mRNA accumulationduring development and correct localization of thefusion protein.
Immunoelectron microscopy shows the ST310 andST430 proteins to be present throughout the entirethickness of the slime sheath and almost totally absentfrom the cells of the slug. The genes that encode theST310 and ST430 polypeptides are inducible by DIF, astalk-specific inducing agent, and the mRNAs are
highly enriched in prestalk over prespore cells. Theproduction of these extracellular proteins by prestalkcells suggests that, in a manner somewhat analogous tothat of extracellular matrix proteins of higher eu-karyotes, the anterior region of the slug may beresponsible for the continuous deposition of a track,along which the slug cells migrate. In the matureculminant, the ST310, and possibly the ST430, proteinform part of the stalk tube and stalk cell wall.Therefore, the results also show that there are proteinscommon to both slime trail and stalk tube, indicating apossible precursor-product relationship betweenthese chemically similar integuments.
The slug of the cellular slime mould Dictyosteliumdiscoideum is formed as a result of the aggregation ofindividual amoebae in response to pulsatile emissionsof cAMP from cells at a signalling centre. Theanterior one-fifth of the slug is composed of prestalkcells and the posterior four-fifths is predominantlycomposed of prespore cells but contains a minoritypopulation of cells with prestalk properties, termedanterior-like cells (Sternfeld & David, 1982). The slugis enveloped by a thin (10-50 ran) sheath of proteinand cellulose, which is laid down in a continuousfashion during slug migration (Loomis, 1972; Farns-worth & Loomis, 1975). The slime sheath acts as anextracellular matrix upon which the slug deposits
proteins which have been proposed to play a role inslug traction. Monoclonal antibodies against theseproteins stain characteristic 'footprint' patternswithin the trail (Vardy et al. 1986). At culmination,prestalk cells from the anterior region of the slugmigrate through the prespore zone within a structureknown as the stalk tube, the direct precursor of therigid extracellular matrix of the mature stalk (Georgeet al. 1972). Entry of cells into the stalk tube isbelieved to be the point at which prestalk cellsbecome irreversibly committed to their fate as deadvacuolated stalk cells (Farnsworth, 1974).
The pDd56 and pDd63 cDNA clones derive frommRNAs that respectively encode the ST310 andST430 stalk-specific proteins (McRobbie et al. 1988).These proteins are predominantly composed of a
276 S. J. McRobbie and others
highly conserved 24 amino acid repeat (Williams et al.1987; Ceccarelli et al. 1987). Such a repetitive struc-ture is a feature common to a number of extracellularpresumptive structural proteins of lower eukaryotes(Petersen et al. 1983; Ozaki et al. 1983; Kochan et al.1986; Prat et al. 1986). We have shown, by immuno-electron-microscopy, that ST310, and probablyST430, are located within the stalk tube and extracel-lular matrix that envelopes stalk cells of the matureculminant (McRobbie et al. 1988). The ST310 andST430 mRNAs are induced in response to DIF, theputative stalk-specific morphogen of Dictyostelium.The two messages appear at the tipped aggregatestage of development, the ST430 mRNA is at maxi-mal concentration in the slug and the ST310 messagepeaks during culmination. They are highly enrichedin, and possibly specific to, prestalk cells (Jermyn etal. 1987; Williams et al. 1987). We have now examinedthe expression and localization of these proteins atthe slug stage of development and show that they arelocalized in the slime sheath and slime trail.
Materials and methods
Growth, development and transformation of cellsD. discoideumStrain Ax-2 was grown axenically in suspension as de-scribed by Watts & Ashworth (1970). Strain V12M2 wasgrown in association with Klebsiella aerogenes on SMnutrient agar (Sussman, 1966). Cells were harvested andwashed by centrifugation in 20mM-KH2PO4/K2HPO4pH6-0. Developmental time courses were carried out onMillipore filters (Sussman, 1966). For migrating slugs, cellswere developed as single streaks of approximately 109 cellsper ml on 2 % water agar. These plates were kept moist anddark, except for a unidirectional point light source andincubated for 24-72 h. Percoll gradient separation of pre-stalk and prespore cells (Ratner & Borth, 1983) is describedelsewhere (Jermyn et al. 1987). The transformation ofDictyostelium Ax-2 cells was performed using a modifi-cation (Early & Williams, 1987) of the method of Nellen etal. (1984).
Gel electrophoresis, immunoblotting and NortherntransferSodium dodecyl sulphate-polyacrylamide gel electrophor-esis and electrophoretic transfer of proteins to nitrocellu-lose are described in detail by McRobbie et al. (1988).Northern transfer conditions are described by Williams etal. (1987) and the details of oligonucleotide hybridizationare given by Ceccarelli et al. (1987).
Preparation of slime trails and slugs forimmunostainingWhen exposing the lower surfaces of trails, plugs of agarwith adherent trails were removed from water agar platesusing a cork borer. The agar plugs obtained were then
placed slime trail downwards on poly-L-lysine subbedmultiwell slides. The upper surfaces of the trails adhered tothe slide leaving the lower surfaces exposed. When expos-ing the upper surfaces of trails, the method of Vardy et al.(1986) was followed. All trails were used unfixed. Antibodyincubations and washes were as follows. Slides were incu-bated under drops of the primary antibody, diluted 1:100 inphosphate-buffered saline (PSBA), in a moist chamber for30min at room temperature. The specimens were thenwashed extensively with PBS A for 30 min. Drops of rhoda-mine-conjugated rabbit anti-mouse immunoglobulin, orfluorescein-conjugated pig anti-rabbit immunoglobulin,diluted 1:30 in PBSA were then placed on the trails for30min at room temperature. The specimens were againwashed with PBSA for 30 min before being mounted inGelvatol. For double-labelling experiments using a mono-clonal antibody and a rabbit polyclonal antiserum thefollowing sequence was adopted: simultaneous incubationwith 1:100 dilutions of both primary antibodies; wash;simultaneous incubation with 1:30 dilutions of fluorescein-conjugated pig anti-rabbit and biotin-conjugated sheepanti-mouse immunoglobulins; wash; 1:30 dilution of strep-tavidin-Texas red; wash and mount in Gelvatol. Thepreparations were examined under a fluorescence phase-contrast microscope. We describe the immunoelectronmicroscopy procedure elsewhere (McRobbie et al. 1988).
Results
The ST310 and ST430 proteins are expressed prior toculmination and are located in the slime sheathThe ST310 protein was first detected by Morrissey etal. (1984) on two-dimensional gels and is recognizedby JAbl, a monoclonal antibody raised against pro-teins of the stalk which resist solubilization by non-ionic detergents (Wallace et al. 1984). Both theseprevious studies showed that ST310 was stalk-cell-specific but the protein was not detected at stages ofdevelopment earlier than culmination. Using theJAbl monoclonal antibody to probe Western blots,we detect high levels of ST310 at the standing fingerand slug stages of development and can even detect asmall amount of the protein in tipped aggregates(Fig. 1A). There is no obvious explanation for thedisparity between our results and those obtainedpreviously (Morrissey et al. 1984; Wallace et al. 1984)but we have confirmed them, using a polyclonalantiserum recognizing both ST310 and the relatedST430 protein (data not shown). Our data are alsoconsistent with the ST310 mRNA accumulation pat-tern. The message is present at maximal concen-tration early during culmination but first appears atthe tipped aggregate stage. (Jermyn et al. 1987).
The ST310 protein is enriched in microdissectedprestalk regions from slugs and the protein alsoappears to be reasonably abundant in posterior pres-pore fractions (Fig. IB). However, unlike its
Extracellular matrix proteins of the Dictyostelium slug 277
B
ii
Tip
Finger
Slug
Early culm
Late culm
Fruit
Slug
Front
Back
Light
Heavy
Trail
Stalk
Light
Heavy
Trail
Stalkw
ST430
Fig. 1. Immunoblot analysis of protein preparations fromV12M2 cells. Proteins were run on 7-5 % gels followed byimmunoblotting. Blots A and B were probed with 1:250JAbl and blot C was probed with 1:200 JAb2. Bindingwas detected autoradiographically, blot C was exposedsuch that only ST430 (which JAb2 reacts most stronglywith) was detected. (A) Time course of V12M2development (35 fig protein per lane) tipped aggregateswere of 10 h of development, standing fingers 12-5 h, slugs16 h, early culmination 19 h, late culmination 20-5 h, andfruiting bodies 23-5 h. (B,C) Separation of cells fromslugs into front (prestalk) or back (prespore) fractions bymicrodissection and light (prestalk) or heavy (prespore)fractions by Percoll gradient centrifugation (Ratner &Borth, 1983). Trails were prepared as described by Grantet al. (1985). Loads were 30 fi% protein per lane except forthe stalk preparation which was 20 j/g.
ST310
mRNA, which is highly enriched in Percoll-gradient-fractionated prestalk cells (Jermyn et al. 1987), theprotein cannot be detected at all in either gradient-fractionated prestalk or prespore cells (Fig. IB). Oneexplanation for such a distribution is that the proteinis present in the slime sheath, the structure surround-ing the entire migratory slug. The protein would bepresent in microdissected slug fractions but would belost when cells are dissaggregated prior to gradientcentrifugation. In confirmation of this hypothesis, wefind ST310 in Western blots of proteins isolated fromslime trails deposited by migrating slugs (Fig. IB).
The developmental time course of accumulation ofthe ST430 protein has previously been examined insome detail (Morrissey et al. 1984; Wallace et al.1984). The polypeptide was shown to be present inslugs (in both microdissected cell fractions) and allsubsequent stages of the life cycle. We have con-firmed these results using both a monoclonal antibody(JAb2, Wallace et al. 1984) and a polyclonal anti-serum (STAb56, McRobbie et al. 1988) which recog-nize both ST310 and ST430 (data not shown). TheST430 protein has been claimed to be present onPercoll-gradient-fractionated prestalk and anterior-like cells (Devine & Loomis, 1985). However, like theST310 protein, we find that ST430 is absent fromPercoll-gradient-fractionated prestalk and presporecells but present in slime trails (Fig. 1C).
Immunofiuorescence microscopy confirms thepresence of ST310 in slime trails. On trails preparedwith their lower surfaces accessible, JAbl, whichstains ST310, displays diffuse staining of the trails andof cell outlines and debris left by migrating slugs.There is also frequent staining of footprint-like im-pressions in these trails, resembling the outlines ofslugs. The leading edges of these footprints are oftenvery strongly stained (Fig. 2). Monoclonal and poly-clonal antisera, which recognize both ST310 andST430, give a similar result to that seen using JAbl(data not shown). This pattern of staining is similar tothat reported for putative slug traction proteins(Vardy et al. 1986).
The 5' proximal regions of the ST310 and ST430genes direct correctly regulated expression of thecognate mRNAs and fusion proteinsThe pDd56-10 construct (Ceccarelli et al. 1987) con-tains 1-7 kb of upstream sequence and 500 nucleotidesof the coding region of pDd56. This fragment is fusedto the 3' proximal portion of the pDdlO gene in thevector pBlOTPIO (Early & Williams, 1987). Upontransformation into Dictyostelium, this gene directscorrect temporal regulation of the fusion mRNA(Ceccarelli etal. 1987). We have introduced, betweenthe two gene fragments in the construct, an oligonuc-leotide coding for an 11 amino acid epitope of the
278 5. /. McRobbie and others
Fig. 2. Localization of ST310 by immunofluorescencemicroscopy. V12M2 slime trails were probed with 1:100JAbl ascites (A,B) or 1:100 control ascites (C). x40.
human c-myc protein. This epitope is recognized bythe monoclonal antibody 9E10 (Evan et al. 1985;Munro & Pelham, 1986). We can, therefore, detectexpression of the fusion mRNA and fusion protein byprobing Northern blots with the ohgonucleotide andby immunostaining for the c-myc epitope. The North-ern transfer data presented in Fig. 3A show thataccumulation of the fusion mRNA is confined to
prestalk cells. Slime trails from slugs prepared fromtransformed cells and stained with the 9E10 antibody,show an identical pattern of staining to trails stainedwith JAbl (Fig. 3B). Again the footprints are themost outstanding feature, with the leading edgesbeing particularly heavily labelled. The slime trailsshown had their upper surfaces exposed but identicalresults were obtained on trails prepared with lowersurfaces exposed (data not shown).
Since no specific antibodies are available for theST430 protein, a similar approach was used to gener-ate an ST430 fusion protein containing the humanc-myc epitope (Fig. 4). The construct contains 2-2 kbof upstream sequence and 4-9 kb of coding regionfrom pDd63 fused to the 3' proximal sequence of thepDdlO gene in the vector pBlOTPl. The c-mycoligonucleotide was inserted in frame between thepDd63 and pDdlO gene fragments. When trans-formed into Dictyostelium, the gene directs correcttemporal regulation of the fusion mRNA, as detectedon Northern blots probed with the oUgonucleotide(Fig. 5A). This pattern of mRNA accumulation isidentical to that reported for the endogenous gene(Williams et al. 1987). The lower surfaces of slimetrails, prepared from slugs generated using trans-formed cells and stained with the human c-mycantibody, show heavy staining of the trail and foot-prints within the trail (Fig. 5B). The ST430 genecontains a C-terminal region which diverges from thesimple 24 amino acid repeat which constitutes themajor part of the coding region. This extremeC-terminus has a very glycine-rich region of unknownfunction (Williams et al. 1987). The construct we haveused lacks this region and we cannot therefore ruleout the possibility that this leads to its appearance intrails. However, the demonstration by Western blot-ting that the endogenous ST430 protein is present inslime trails (Fig. 1C), coupled with the localizationdata from the fusion gene, suggests very strongly thatthe protein is indeed genuinely localized to the slimetrail and footprints that we observe.
Many other proteins are localized to footprints in theslime trailBecause we find the ST310 and ST430 proteins to belocalized in footprints and because such proteins havebeen proposed to aid adhesion of cells during slugmigration (Vardy et al. 1986; Breen et al. 1987), wehave investigated these structures further. We haveused several antibodies to test the specificity ofprotein localization in these footprints. Every anti-body we have used stains footprints in slime trails(Fig. 6). These include the monoclonal MUD1 whichrecognizes a 32X103 Mr prespore-specific cell surfaceprotein (Gregg et al. 1982), monoclonal antibody rsa3.1 which binds to 47 and 52x 103 MT prestalk-specific
Extracellular matrix proteins of the Dictyostelium slug 279
pDd56-10-c-myc Fig. 3. Panel A: Northern transfer
analysis of the spatial distribution of thepDd56-10 c-myc fusion mRNA. Totalcellular RNA was extracted from Percollgradient purified prestalk (PST) andprespore (PSP) cells from slugs preparedfrom amoebae transformed with thepDd56-10c-w_yc fusion gene. The RNAwas electrophoresed through a denaturinggel, transferred to nitrocellulose anddetected by hybridization with a syntheticoligonucleotide. Panel B: Localization ofpDd56-10 c-myc fusion protein byimmunofluorescence microscopy. Slimetrails from pDd5&10c-myc transformants(a,b) and control Ax-2 cells (c) wereprobed with 1:100 9E10 immunoglobulin.xl5.
cell surface polypeptides (Barclay & Smith, 1986)monoclonal 41-71-21 which was raised against the celladhesion molecule contact site A, (Bertholdt et al.1985; Bozzaro & Merkl, 1985) and a polyclonalantiserum which recognizes intracellular antigens as-sociated with prespore vesicles (Ikeda & Takeuchi,1971). Thus intracellular, as well as cell surface,proteins become localized in footprints. In addition tothose shown in Fig. 6, we have obtained similarresults (data not shown) with MUD9, a monoclonalthat labels prestalk cells (Krefft et al. 1985), mono-clonal rsa 4.2 which shows no cell-type specificity(Barclay & Smith, 1986) and MUD51, a monoclonal
Fig. 4. The structure of the pDd63-10c-rnyc gene.(A) The pBlOTPIO (Ceccarelli et al. 1987), a derivative ofpBlOTPl (Early & Williams, 1987), contains a synthetic'marker' oligonucleotide of 33 residues which encodes theepitope for the c-myc antigen. pBlOTPIO was cleavedwith BamHl, end-filled using the Klenow fragment ofDNA polymerase 1, and then cleaved with HindlU. AHindlll-Pvull fragment of 7-5 kb from a genomic cloneof pDd63 was then inserted. (B) The Hindlll-Pvullfragment contains 2-2 kb of sequence from the Sau3A site5' to the ATG initiation codon fo the pDd63 gene.Polyadenylation signals are provided by pDdlO (Early &Williams, 1987) to give a fusion mRNA of approximately5-9 kb in length.
antibody recognizing a slime-sheath-specific antigen(Williams etal. 1984). Furthermore, double-labellingexperiments (data not shown) reveal that footprintsstained by these antibodies are coincident with thoselabelled by MUD50, a monoclonal antibody claimedto recognize proteins involved in slug traction (Vardyetal. 1986).
0/14400
pDd63-10 c-myc14-4 kb ATG
-2200
pDd63
amp
Neo
CP1
Pvull/BamHl
pDd63-10 c-myc fusion gene
pDdlO
0Sau3A
I
-1900Cap site -2200
I ATG
pDd63 pDdlO-7100 -7790c-myc poly A-myc pol;
280 S. J. McRobbie and others
Veg 19h 20h 22h
pDd63-10-c-myc
Fig. 5. Panel A: Northern transfer analysis of the developmental accumulation of the pDd63-10 c-myc fusion mRNA inDictyostelium transformants. Total cellular RNA was extracted from vegetative cells (veg), slugs (19h), and early andlate culminants (20 and 22 h). The RNA was electrophoresed through a denaturing gel, transferred to nitrocellulose anddetected by hybridization with a synthetic oligonucleotide. Panel B: Localization of pDd63-10 c-myc fusion protein byimmunofluorescence microscopy. Slime trails from pDd63-10 c-myc transformants were probed with 1:100 9E10immunoglobulin. x80.
The observation that antibodies to intracellularproteins stain footprints in the slime trail led us toquestion whether the ST310 and ST430 proteins aregenuine components of the extracellular matrix. Theslime trail is formed by deposition of the slime sheathwhich invests the slug and it is difficult to preserveduring fixation for light microscopy (Gregg et al.1982). Despite the use of a variety of fixatives, wehave been unable to visualize any stained material onthe surface of the slug. However, we are able tovisualize ST310 and ST430 proteins in the slimesheath by immunoelectron microscopy of slug sec-tions. We have used a polyclonal antiserum (data notshown) and a monoclonal antibody both of whichreact with ST310 and ST430. The sheath is fairly wellpreserved and seems to be composed of fibrillarmaterial and the proteins appear to be associated with
these fibrils and distributed throughout the entirethickness of the sheath (Fig. 7).
Discussion
We have shown that the ST310 and ST430 polypep-tides are present in the slime sheath and slime trails ofDictyostelium slugs. The ST310 protein is located in adiffuse fashion throughout the trail and in regions oflocal high concentration which have been termed'footprints' (Vardy et al. 1986). We also show that1-7 kb of upstream sequence from the ST310 gene,and 2-5 kb from the ST430 gene, contain the necess-ary regulatory elements to direct correct develop-mental expression of their respective fusion mRNAs.The ST310 fusion protein is localized in footprints inthe slime trail, suggesting that the signals necessary
Extracellular matrix proteins of the Dictyostelium slug 281
Fig. 6. Localization of various antigens by immunofluorescence microscopy. V12M2 slime trails (lower surfacesaccessible) were probed with 1:100 dilutions of (A) MUD1 ascites, (B) rsa3.1 culture supernatant, (C) 41-71-21immunoglobulin and (D) antisenim against prespore vesicles. x40.
for correct localization are contained within theN-terminal one-quarter of the protein. Using animmunologically marked ST430 fusion protein, wehave shown that this protein is also localized infootprints, although it appears to be more generallydistributed throughout the slime trail than the ST310protein.
A class of proteins that also form footprint patternsin trails has recently been described by Vardy et al.(1986). Despite the presence of these polypeptides onposterior prespore cells (Williams et al. 1984) theyhave been proposed to be traction proteins secretedby the anterior cells of the slug to aid cellularadhesion to the substratum during slug locomotion(Vardy etal. 1986; Breen etal. 1987). Our finding thatmany proteins, including known intracellular anti-gens, are found in footprints indicates that cautionmust be exercised in interpreting such results. Thefootprints may be caused by cell lysis and the sub-sequent localized deposition of all membrane-associ-ated cellular proteins. If cell lysis was the result ofslippage during traction, then the footprints couldprovide valuable information about the mechanism ofslug migration. However, the presence of a particularprotein does not prove a direct role for that protein in
slug locomotion. In this context, it is important topoint out that the ST310 and ST430 polypeptides aretotally extracellular proteins and, as such, their local-ization in the slime sheath is likely to be of signifi-cance.
In the mature culminant, the ST310, and possiblythe ST430, protein form part of the stalk tube andstalk cell wall (McRobbie et al. 1988). These are therigid, cellulose-containing, structures that invest ma-ture stalk cells (George et al. 1972; Freeze & Loomis,1978). Our demonstration of a shared protein com-ponent suggests a very close relationship between thestalk tube and slime sheath, confirming previoussuggestions for such a link based upon their similarchemical composition (Freeze & Loomis, 1977,1978).It will be of interest to determine whether themorphogenetic changes occurring at culmination leadto a direct transition of matrix proteins from slimesheath into stalk tube and stalk cell wall as suggestedby early ultrastructural studies (George et al. 1972).
The ST310 and ST430 mRNA sequences are,within the limits of available cell purification pro-cedures, completely specific in their localization toprestalk cells (Williams et al. 1987; Jermyn et al. 1987).In this they differ from previously described prestalk
282 S. J. McRobbie and others
mRNA markers, which are present in both presporeand prestalk cells (Jermyn et al. 1987). They alsodiffer in being present in both prestalk and stalk cells.The demonstration that they perform a functionspecific to the slug stage, presumably in maintainingthe integrity of the slime sheath, helps to increaseconfidence that they are authentic markers of themulticellular stage of development. Other prestalkmarkers are first expressed much earlier, duringcellular aggregation, and probably become enrichedin prestalk cells by selective degradation in presporecells (Tasaka et al. 1983; Krefft et al. 1985).
The deposition by prestalk cells of the ST310 andST430 polypeptides may indicate an additional rolefor these proteins analogous to that of the Discoidin 1proteins earlier in development. These three proteinsare believed to mediate cell-substratum attachmentby directing aggregating cells into defined streams
Fig. 7. Localization of ST310 and ST430by immunoelectron microscopy. Sectionsof V12M2 slugs were probed with 1:1000121Ab ascites (a monoclonal antibodyrecognizing both proteins; McRobbie,unpublished data). Binding was detectedby goat anti-mouse immunoglobulinsconjugated to 15 nm colloidal gold.Sheath material is indicated by arrows,x 19 440.
(Springer et al. 1984), a role very similar to that offibronectin during neural crest cell migration (Rova-sio et al. 1983). There is no apparent homologue ofthe cell-binding site which is found in the Discoidinsand in fibronectin (Springer et al. 1984). They do,however, resemble fibronectin and other extracellu-lar proteins of lower eukaryotes (Petersen et al. 1983;Ozaki et al. 1983; Kochan et al. 1986; Prat et al. 1986),in being composed of a reiterated sequence. Theability, readily to generate mutants within the genes(De Lozzane & Spudich, 1987; Witke et al. 1987),should allow a combined genetic and biochemicalapproach to the determination of their precise role inslug integrity and motility.
We wish to thank the following for their gifts of anti-bodies. Gerard Evan (9E10), Gerry Weeks (121Ab), Mar-ianne Krefft (MUD1, MUD9), Gunther Gerisch (41-71-
Extracellular matrix proteins of the Dictyostelium slug 283
21), Steve Barclay (rsa 4.2, rsa 3.1), Keith Williams and LizSmith (MUD50 and MUD51) and Rob Kay (anti-presporevesicles). We are grateful to Rita Tilly for her help andadvice on electron microscopy.
References
BARCLAY, S. L. & SMITH, A. M. (1986). Identification andanalysis of the regulation of a prestalk cell-surfaceantigen of Dictyostelium discoideum. Differentiation 33,101-110.
BERTHOLDT, G., STADLER, J., BOZZARO, S., FICHTNER, B.& GERISCH, G. (1985). Carbohydrate and otherepitopes of the contact site A glycoprotein ofDictyostelium discoideum as characterised bymonoclonal antibodies. Cell Differ. 16, 187-202.
BOZZARO, S. & MERKL, R. (1985). Monoclonal antibodiesagainst Dictyostelium plasma membranes: their bindingto simple sugars. Cell Differ. 17, 83-94.
BREEN, E. J., VARDY, P. H. & WILLIAMS, K. L. (1987).Movement of the multicellular slug stage ofDictyostelium discoideum: an analytical approach.Development 101, 313-321.
CECCARELLI, A., MCROBBIE, S. J., JERMYN, K. A., DUFFY,K., EARLY, A. & WILLIAMS, J. G. (1987). Structuraland functional characterization of a Dictyostelium geneencoding a DEF inducible, prestalk-enriched mRNAsequence. Nucleic Acids Research 15, 7463-7476.
DELOZANNE, A. & SPUDICH, J. A. (1987). Disruption ofthe Dictyostelium myosin heavy chain gene byhomologous recombination. Science 236, 1086-1091.
DEVINE, K. M. & LOOMIS, W. F. (1985). Molecularcharacterization of anterior like cells in Dictyosteliumdiscoideum. Devi Biol. 107, 364-372.
EARLY, A. & WILLIAMS, J. G. (1987). Two vectors whichfacilitate gene manipulation and a simplifiedtransformation procedure for Dictyostelium discoideum.Gene 59, 99-106.
EVAN, G. I., LEWIS, G. K., RAMSEY, G. & BISHOP, J. M.(1985). Isolation of monoclonal antibodies specific forthe human c-myc proto-oncogene product. Molec. Cell.Biol. 5, 3610-3616.
FARNSWORTH, P. (1974). Experimentally inducedaberrations in the pattern of differentiation in thecellular slime mould Dictyostelium discoideum. J.Embryol. exp. Morph. 31, 435-451.
FARNSWORTH, P. & LOOMIS, W. F. (1974). A barrier todiffusion in pseudoplasmodia of Dictyosteliumdiscoideum. Devi Biol. 41, 77-83.
FARNSWORTH, P. & LOOMIS, W. F. (1975). A gradient inthe thickness of the surface sheath in pseudoplasmodiaof Dictyostelium discoideum. Devi Biol. 46, 349-357.
FREEZE, H. & LOOMIS, W. F. (1977). Isolation andcharacterization of a component of the surface sheathof Dictyostelium discoideum. J. biol. Chem. 252,820-824.
FREEZE, H. & LOOMIS, W. F. (1978). Chemical analysis ofstalk components of Dictyostelium discoideum.Biochim. Biophys. Acta. 539, 529-537.
GEORGE, R. P., HOHL, H. R. & RAPER, K. B. (1972).
Ultrastructural development of stalk-producing cells inDictyostelium discoideum. J. gen. Microbiol. 70,477-489.
GRANT, W. N., WELKER, D. L. & WILLIAMS, K. L.(1985). A polymorphic, prespore-specific cell-surfaceglycoprotein is present in the extracellular matrix ofDictyostelium discoideum. Molec. Cell. Biol. 5,2559-2566.
GREGG, J. H., KREFFT, M., FLAAS-KRAUS, A. & WILLIAMS,K. L. (1982). Antigenic differences detected betweenprespore cells of Dictyostelium discoideum andDictyostelium mucoroides using monoclonal antibodies.Expl Cell. Res. 142, 229-233.
IKEDA, T. & TAKEUCHI, I. (1971). Isolation andcharacterization of a prespore specific structure of thecellular slime mould Dictyostelium discoideum.Develop. Growth and Differ. 13, 221-229.
JERMYN, K. A., BERKS, M., KAY, R. R. & WILLIAMS, J.G. (1987). Two distinct classes of prestalk-enrichedmRNA sequences in Dictyostelium discoideum.Development 100, 745-755.
KOCHAN, J., PERKINS, M. & RAVETCH, J. V. (1986). Atandemly repeated sequence determines the bindingdomain for an erythrocyte receptor binding protein ofP. falciparum. Cell 44, 689-696.
KREFFT, M., VOET, L., GREGG, J. H. & WILLIAMS, K. L.(1985). Use of a monoclonal antibody recognizing a cellsurface determinant to distinguish prestalk andprespore cells of Dictyostelium discoideum slugs. J.Embryol. exp. Morph. 88, 15-24.
LOOMIS, W. F. (1972). Role of the surface sheath in thecontrol of morphogenesis in Dictyostelium discoideum.Nature New Biol. 240, 6-9.
MACWILLIAMS, H. K. & BONNER, J. T. (1979). Theprestalk prespore pattern in cellular slime molds.Differentiation 14, 1-22.
MCROBBIE, S. J., TILLY, R., BLIGHT, K., CECCARELLI, A.& WILLIAMS, J. G. (1988). Identification andlocalization of proteins encoded by two DIF-induciblegenes. Devi Biol. 125, 59-66.
MORRISSEY, J. H., DEVINE, K. M. & LOOMIS, W. F.(1984). The timing of cell-type specific differentiation inDictyostelium discoideum. Devi Biol. 103, 414-424.
MUNRO, S. & PELHAM, H. R. B. (1986). Identity with78kd glucose-regulated protein and immunoglobulinheavy chain binding protein. Cell 46, 291-300.
NELLEN, W., SILAN, C. & FIRTEL, R. A. (1984). DNAmediated transformation in Dictyostelium discoideum:Regulated expression of an actin gene fusion. Molec.Cell. Biol. 4, 2890-2898.
OZAKI, L. S., SVEC, R., NUSSENZWEIG, R. S.,NUSSENZWEIG, V. & GODSON, G. N. (1983). Structureof the Plasmodium knowlsei gene coding for thecircumsporozoite protein. Cell 34, 815-822.
PETERSEN, T. E., THOGESEN, H. C , SKORTESGAARD, K.,VIVE-PEDERSON, K., SCOTTRUP-JENSEN, L. &MAGNUSSON, S. (1983). Partial primary structure ofbovine plasma fibronectin, three types of internalhomology. Proc. natn. Acad. Sci. U.S.A. 80, 137-141.
PRAT, A., KATINKA, M., CARON, F. & MEYER, E. (1986).Nucleotide sequence of the Paramecium primaurelia G
284 S. J. McRobbie and others
surface protein: a huge protein with a highly periodicstructure. J. moke. Biol. 189, 47-60.
RATNER, D. & BORTH, W. (1983). Comparison ofdifferentiating Dictyostelium discoideum cell typesseparated by an improved method of density gradientcentrifugation. Expl Cell. Res. 143, 1-13.
ROVASIO, R. A., DELOUVEE, A., YAMADA, K. M., TIMPL,
R. & THIERY, J. P. (1983). Neural crest cell migration:Requirements for exogenous fibronectin and high celldensity. /. Cell Biol. 96, 462-473.
SPRINGER, W. R., COOPER, D. N. W. & BARONDES, S. H.
(1984). Dicsoidin 1 is implicated in cell-substratumattachment and ordered cell migration of Dictyosteliumdiscoideum and resembles fibronectin. Cell 39,557-564.
STERNFELD, J. & DAVID, C. N. (1982). Fate andregulation of anterior-like cells in Dictyostelium slugs.Devi. Biol., 93, 111-118.
SUSSMAN, M. (1966). Biochemical and genetic methods inthe study of cellular slime mold development. Methodsin Cell Physiol. 2, 397-410.
TASAKA, M., NOCE, T. & TAKEUCHI, I. (1983). Prestalkand prespore differentiation in Dictyostelium asdetected by cell type-specific monoclonal antibodies.Proc. natn. Acad. Sci. U.S.A. 80, 5340-5344.
VARDY, P. H., FISHER, L. R., SMITH, E. & WILLIAMS, K.L. (1986). Traction proteins in the extracellular matrixof Dictyostelium discoideum slugs. Nature, Lond. 320,526-529.
WALLACE, J. S., MORRJSSEY, J. H. & NEWELL, P. C.(1984). Monoclonal antibodies for stalk differentiationin Dictyostelium discoideum. Cell Differ. 14, 205-211.
WATTS, D. J. & ASHWORTH, J. M. (1970). Growth ofmyxamoebae of the cellular slime mould Dictyosteliumdiscoideum. Biochem. J. 119, 171-174.
WILLIAMS, J. G., CECCARELLI, A., MCROBBIE, S.,MAHBUBANI, H., KAY, R. R., EARLY, A., BERKS, M. &JERMYN, K. A. (1987). Direct induction ofDictyostelium prestalk gene expression by DIF providesevidence that DIF is a morphogen. Cell 49, 185-192.
WILLIAMS, K. L., GRANT, W. N., KREFFT, M., VOET, L. &WELKER, D. L. (1984). Analysis of cell surface andextracellular matrix antigens in D. discoideum patternformation. U.C.L.A. Symp. Mol. Cell. Biol. 19, 75-90.
WITKE, W., NELLEN, W. & NOEGEL, A. (1987).Homologous recombination in the Dictyostelium a-actinum gene leads to an altered mRNA and lack ofthe protein. EMBO J. 6, 4143-4148.
![Dif In Dif Slides.ppt [Repaired] - Finance Department](https://static.documents.pub/doc/80x56/61b1d68f6b0e604114161860/dif-in-dif-repaired-finance-department.jpg)
![CPT TM CYBER-LAB DIF*±]E3ßåÞZI** ... · CPT TM CYBER-LAB DIF*±]E3ßåÞZI**](https://static.documents.pub/doc/80x56/5e07447e09a007101859965b/cpt-tm-cyber-lab-dife3zi-cpt-tm-cyber-lab-dife3zi-.jpg)
