METHODS 22, 299–306 (2000)

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Genetic Techniques for Enhancing Biochemical andStructural Characterization of DictyosteliumMyosin II

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oi:10.1006/meth.2000.1082, available online at http://www.idealibrary.com on

Bruce Patterson

Department of Molecular and Cell Biology, LSS525, University of Arizona, Tucson, Arizona 85721

Myosin mutants and their suppressors can provide informa-tion about conformational states of the myosin motor andtheir biochemical properties. Appropriate mutations can giverise to motors that arrest or overoccupy otherwise inaccessiblestates in the motor cycle. Intragenic (in the same gene) sup-pressor mutations that counteract mutations of known prop-erties represent “fixes” or counters to the defect of the startingmutation and thus contain information about driving transi-tions or stabilizing states of the motor. Due to its variety ofmyosin-dependent phenotypes, Dictyostelium is a powerfultool for the identification of conditional mutants as well asselection of large numbers of intragenic revertants of a mutantof interest. Techniques are presented that allow isolation andidentification of cold-sensitive myosin mutants in Dictyoste-lium as well as facile selection of revertants and identificationof their suppressing mutation. © 2000 Academic Press

Molecular machines present unique challenges tothose seeking to unlock their secrets. Their occu-pancy of a series of vital but short-lived conforma-tions renders their structural study difficult at best.Motors whose jobs include moving filaments (ormoving along them) add another layer of difficulty inthat cocrystals of motor and filament currently lieoutside of the realm of the possible. Unfortunatelythese structures are central to understanding howthe motor actually functions. To add another

1 To whom correspondence should be addressed. E-mail:patterso@u.arizona.edu.

1046-2023/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

weapon to the arsenal of myosin mystery seekers,we have developed a series of genetic tools in Dic-tyostelium to achieve several goals: (1) to isolatemutants that allow study of motor states too ephem-eral to be stably observed and characterized in thewild type motor, (2) to isolate and characterize in-tragenic revertants to gain insights into criticalstructures and events in the motor’s cycle. In allcases, our goal has been to refine these techniques tothe point that they are readily accessible to labora-tories and investigators for whom genetic tech-niques are not normally considered to be part of theexperimental approach.

AN EXAMPLE: THE G680V MUTATION ANDITS SUPPRESSORS

The G680V mutation of Dictyostelium myosin wasidentified as a cold-sensitive mutant in the screendescribed below (1). Its characterization has vali-dated the utility of the approaches described here.Biochemical characterization of the mutant high-lighted five key differences versus the wild-type mo-tor: (1) mutants moved actin filaments slowly invitro, (2) G680V motors acted as “brakes” on wild-type motors even when mixed at ratios of 1 G680Vmotor:10 wild-type motors, (3) it had a lowered basalATPase, (4) it exhibited extended strong binding toactin even in the presence of ATP, and (5) the strong

binding in the presence of ATP was resistant to KClin a manner unlike that of wild-type or G680V mu-

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tant motors in the absence of nucleotide or the pres-ence of ADP (2). These phenotypes led us to inferthat the G680V motor extended an early actin-bound state of the motor, presumably just prior to P i

(inorganic phosphate) release.The isolation of suppressors of the G680V mutant,

again using techniques detailed herein, substanti-ated and extended the conclusions drawn from char-acterization of the G680V mutant. The majority ofthe suppressors (characterized in the absence of theG680V mutation) had several features in common,most conspicuously dramatically enhanced basalATPase rates (14). This finding is perfectly comple-mentary to the proposed defect of G680V: if theG680V motors “seized up” because of an enhancedbarrier to P i release, we would anticipate that coun-eracting the defect would require facilitation of P i

release. Since P i release is the rate-limiting step inhe basal ATPase, enhanced P i release would man-

ifest as a raised basal ATPase, precisely the conse-quence we observe.

The suppressors not only confirmed the model forthe defect, they provided structural clues to themechanism (see Fig. 1). The suppressors were dis-tinctly clustered in a region tangential to helix 466–

FIG. 1. Location of G680V and some of its suppressors in amyosin crystal structure. The view is from the actin binding sitestoward the light chains. Shown is Smith and Rayment’s (13)S1.Dc-ADP z vanadate structure. The “camshaft” helix (aminocids 466–496) is shown. Helix 669–691 is shown at the right,ith G680V (shown as valine to enhance visibility) in black. ADP

s at the top in stick form, while the vanadate is space-filled inark gray. The “backdoor” residues, R238 and E459, are dark

ray stick figures. Positions of selected G680V suppressors arehown as light gray, space-filled representations. Only sidehains are shown.

496, which we have dubbed the Camshaft in recog-nition of its central role in determining andreporting the conformation of the motor (3). PositionG680 also impinges on the Camshaft, but at a pointfurther toward the C terminus of the helix and ro-tated about 120° compared with the cluster. Almostall G680V suppressors increased the volume of hy-drophobic residues. We have proposed that thestructural consequences of the G680V mutant andits suppressors are opposing alterations in the abil-ity of the Camshaft to adopt different rotary posi-tions observed in the crystal structure. In one posi-tion (which we argue would be favored by the G680Vmutation), the Camshaft lies in a position thatwould shut the “backdoor” route of P i egress sug-gested by Yount et al. (4), while the suppressorswould favor a “backdoor open” position, thereby fa-cilitating P i release. While these conjectures haveyet to be confirmed by direct structural observation,they further illustrate the utility of acquiring largenumbers of genetic suppressors of interesting mu-tants.

GENETIC HANDLES FOR MANIPULATING THEMOTOR: MYOSIN-DEPENDENT PHENOTYPESIN Dictyostelium

Aside from its useful features as a vat for produc-ing quantities of normal and mutant myosin II pro-tein (including S1-like molecules) (5), Dictyosteliumbrings a fearsome array of myosin-dependent biolog-ical behaviors to the table. Since these form thefoundation for a genetic approach, they are brieflyintroduced here. The “normal” biology of Dictyoste-lium discoideum is as a bacteria-consuming ame-boid living and dividing in leaf litter. On starvation,cells emit cAMP signals and undergo an aggregationprocess. Groups of cells briefly adopt a communallifestyle, forming a “slug” that migrates towardlight. This ends with the construction of a fruitingbody consisting of a stalk of stiffened dead cellssupporting a mass of spores derived from individualameba.

The first (and most widely anticipated) phenotypeof Dictyostelium cells lacking the myosin II gene is afailure of cytokinesis (6, 7). In suspension culture,this manifests as progressively larger cells thateventually lyse. Surprisingly, these cells can be

readily propagated on a plastic surface covered withliquid growth medium. Here, the cells increase in

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301GENETIC TECHNIQUES ENABLING MYOSIN CHARACTERIZATION

number by pulling themselves into nucleated pieces,a process they undergo sufficiently efficiently thatafter a period of adaptation, it is difficult or impos-sible to visually distinguish wild-type from myosinnull cells growing on a surface. Thus absence ofmyosin represents a conditionally lethal phenotype:in suspension, myosin II is an essential molecule,whereas on a surface it is wholly dispensable. Asurprising “nonphenotype” of myosin-less Dictyoste-lium cells is their motility: while differences in effi-ciency can be observed, the cells move at reasonablespeeds and are capable of chemotaxis.

Another role played by myosin II in Dictyosteliumis the generation and maintenance of cortical ten-sion. Cortical tension arises from the meshwork ofmembrane-associated actin and myosin in the cor-tex. In the absence of myosin, Dictyostelium cells arerendered more pliable (8). An interesting phenome-non has been observed for wild-type cells exposed tosmall amounts of oxidative phosphorylation inhibi-tors such as azide: cells quickly lose adhesion to thesubstrate. This does not occur with myosin nullcells, which remain firmly attached to substrate inthe presence of azide. These behaviors represent amajor tool for isolation of conditional mutants.

A third myosin-dependent phenotype can be ob-served on bacterial lawns. Since Dictyostelium cellsre slowly motile and consume bacteria, they willlear plaques on bacterial lawns much as bacterio-hage do. The striking difference is that there is noimit to the growth of a Dictyostelium plaque. Foreasons that are not fully clear, the rate of plaquexpansion of Dictyostelium cells is dependent on my-sin function. This does not appear to arise fromeneralized failure of motility or an inability to con-ume bacteria. One possibility is that it reflects aequirement for myosin in generation of force or cellhape required to penetrate the mucilaginous bac-erial lawn. In any event, this behavior provides anmportant handle on myosin function.

The final gross defect observed in Dictyosteliumells lacking myosin is an inability to construct fruit-ng bodies. Cells undergo more or less normal aggre-ation to form mounds of cells, but critical sortingvents and force generation required to erect thetalk do not occur (9).

olecular Genetic Tools

It is worth pointing out that there are a variety ofolecular genetic tools and features employed in the

eneration and analysis of myosin II mutants inictyostelium (10). These are detailed below, but

onsist primarily of a haploid genome, reasonablyfficient transformation (by electroporation) withlasmid DNA, extrachromosomal plasmid vectors,nd homologous recombination between exog-nously added DNAs and the chromosome or onenother. The ability of added DNA to homologouslyecombine with simultaneously introduced plasmidNAs mimics that found in yeast in that the fre-uency of recombination is significantly enhancedy introducing a cut or gap in the target plasmid inregion that is overlapped by the “repairing” piece.

DESCRIPTION OF METHODS

Breaking the Motor: Selecting for ConditionallyDefective Myosin Alleles

To address the challenge of identifying mutantmotors that facilitated study of myosin’s short-livedstates, we developed a technique that allows positiveselection of rapid-effect cold-sensitive mutants (11).Our rationale in seeking this class of mutants wasstraightforward: we were seeking to “interrupt” theworkings of the motor to allow experimental accessto short-lived states. By identifying motors that ex-hibited a dependence on exogenous thermal energyfor successful completion of the cycle, we would begenerating mutants that stalled or arrested at lowertemperatures. Novel characteristics exhibited bythese mutant motors at low temperatures wouldpotentially represent sightings of steps performedtoo quickly to be detected in wild type.

The technique we employ relies on the observationthat wild-type Dictyostelium cells are released fromPetri dishes on exposure to low levels (1.5–5 mM) ofsodium azide, whereas the majority of myosin nullcells remain adherent under these conditions (8, 11).To identify cold-sensitive mutants, we apply alter-nating rounds of selection, harvesting cells thatwere released by azide treatment (wild-type behav-ior) at 26°C but remained adherent on azide treat-ment at low temperature (13°C). This procedure isnot 100% efficacious, so multiple rounds of eachregimen are required to achieve a largely pure mu-tant population from a mutagenized group.

The following is a typical protocol for isolation ofcold-sensitive myosin mutants in Dictyostelium. Theprotocol is for isolating mutants from a population ofmutagenized cells; the procedure should be far more

efficient if cold-sensitive mutants were sought aftertargeted mutagenesis of the myosin gene followed by

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introduction into myosin null cells. Cells are shiftedto appropriate temperature only at the time of theazide application in order to ensure that mutantsare “rapid onset,” thus avoiding “boring” mutationsthat alter synthesis or stability of the myosin motor.

1. Allow cells to recover from mutagenesis (3–5days).

2. Grow cells overnight at 26°C in six-well Petriplates. (Density should remain below 2–5 3 106; ifhe cells become too dense the azide is ineffectual.urther, the cells require at least 12 h to recoverzide sensitivity after achieving excess density.)3. Prechill cells by placing plates in 13°C incuba-

or for 10 min.4. Replace growth medium with 1.5 ml 13°Cash Buffer (1 vol HL-5:9 vol Starvation Buffer (50M Mes, pH 6.8, 2 mM MgCl2, 0.2 mM CaCl2))

containing 2 mM sodium azide.5. Move Petri plates to a nutator for 5 min (gentle

rocking motion and movement of the air–mediuminterface over the cells enhance release).

6. Aspirate off liquid and replace with HL-5. Re-suspend cells. Return Petri plates to 26°C.

7. Once cells have again reached 1–2.5 3 106

cells/ml, grow overnight at 13°C.8. Prewarm plates by placing in 26°C incubator

for 10 min.9. Replace growth medium with 1.5 ml 26°C

Wash Buffer containing 2 mM sodium azide.10. Move Petri plates to a nutator for 5 min (gen-

tle rocking motion and movement of the air–mediuminterface over the cells enhance release).

11. Gently harvest 1 ml of medium with Pipet-man.

12. Recover cells by gently spinning recoveredmedium.

13. Gently resuspend cells and move to fresh six-well Petri dish.

14. Repeat steps 2–6 and 7–13 periodically (ascells achieve densities of 1–5 3 106).

15. When .25% of the cells in a given populationwash off at 26°C and the remaining cells are adher-ent at 13°C, plate serial dilutions onto bacteriallawns. Grow at 26°C for 2–4 days (to allow rapidgrowth of single colonies) followed by growth for 1week at 13°C. Colonies that do not give rise to wild-type fruiting bodies are candidates for cold-sensitivemyosin mutants. They should be retested for azidebehaviors. Those that pass can be sequenced; . 80%

of these should contain mutations in the myosinheavy chain gene.

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Notes. The azide response is somewhat finicky,depending critically on the cell density. Differentbatches of cells may also demand different concen-trations of azide for optimal response; the strainused should be calibrated from 1 to 5 mM and theminimal amount of azide required to achievewashoff of the majority of cells at 13°C should beemployed. Our current recipe for HL-5 is (per liter):7.15 g oxoid special peptone, 7.15 g oxoid proteosepeptone, 15.4 g glucose, 0.485 g KH2PO4, 1.28 gNa2HPO4. Adjust pH to 6.95 prior to autoclaving.We generally supplement HL-5 with 0.1 vol FMMedium (Life Technologies, Rockville, MD) madeper the manufacturer’s instructions.

Biological Quantification of Myosin Function andRevertant Isolation

Isolation of revertants is vastly easier and lesstedious than the isolation of cold-sensitive mutants.We have been able to isolate revertants for everycold-sensitive mutant that confers a significant im-pairment of plaque growth on a bacterial lawn. Fur-ther, to date we have had excellent success isolatingrevertants for several mutants that were not gener-ted in the cs mutant screen. However, it should beoted that not every mutation can be reverted; one isest advised to start with a defect strong enough toonfer a biological phenotype (see below) but thatas detectable function biochemically or does notrastically alter an absolutely conserved amino acid.onetheless, we have reverted several mutants ofbsolutely conserved residues, and the facile naturef the procedure allows one to “go for it” regardless oferceived probability of success.The keystone to the revertant isolation procedure

s the behavior of Dictyostelium cells on bacterialawns. Bacteria are a natural substrate for Dictyo-telium in the wild. The combination of consumption

of bacteria with innate motility of Dictyosteliumamoebas results in rapid expansion of plaques. Fur-ther, the plaques themselves remain nearly circular,allowing for precise quantitation of plaque expan-sion rates. Dictyostelium cells lacking myosin cantill consume bacteria and give rise to clearinglaques, but at a significantly reduced rate. Thisifference in properties provides an awe-inspiringlyonvenient methodology for assessing myosin func-

ion and isolating cells exhibiting enhanced myosinunction compared with their parents.

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303GENETIC TECHNIQUES ENABLING MYOSIN CHARACTERIZATION

Assessing Myosin Function Using Plaque ExpansionRates

The circular nature of a Dictyostelium plaquemakes expansion rate measurements straightfor-ward. We employ plastic sheets used by engineers todraw circles of varying sizes (sheets with sizes in therange 5–30 mm serve nicely). By comparing theplaque to a circle held to the bottom of the plate, onecan quickly determine the largest diameter fullyfilled by the plaque. We have found that increase inplaque diameter is linear for all myosin mutants wehave studied.

Unfortunately, Dictyostelium biology also con-pires against the most rapid achievable plaque ex-ansion. As cells run out of bacteria locally, theyegin to starve, which triggers the developmentalesponse. Part of this response is the cAMP-signaledggregation of neighboring amoebas and initiationf a multicellular stage. This aggregation comes athe expense of expansion of the plaque perimeter, ashese cells are torn between growth and aggrega-ion. Despite this conundrum, wild type plaques doxpand continuously.To enhance the rates of plaque expansion (thus

endering measurements quicker and more accu-ate) we have isolated a mutant Dictyostelium celline that shows expansion rates exceeding those ofild type by a factor of two- to fivefold. This strainas derived by two rounds of mutagenesis of myosinull cells followed by isolation of cells giving rise tohe most rapidly expanding plaques (3). The result-ng cell line is called the SPERA line and is availablerom our laboratory. These cells expand more rap-dly than their wild-type counterparts with or with-ut myosin. They also have acquired the property ofailing to aggregate on the onset of starvation; weave not sought to determine the cause of this de-ect.

Plaque expansion rates are also influenced by thehickness of the bacterial lawn and the percentage ofgar used to make the plates on which the lawns areoured. Using less rich medium to make the platesives rise to thinner lawns and correspondinglyuicker plaque expansion. Similarly, lowering theercentage of agar in the plates enhances plaquexpansion rate.The following is a protocol for generating lawns

or plaque expansion rate assays:

1. Pour 150-mm Petri plates with medium con-

isting of (SM/16) 1 half-normal agar. SM/16 isade by taking the recipe for SM (12) but using

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lucose, peptone, and yeast extract at 1/16th theecommended levels.2. Allow plates to become “optimally dry” by

tanding on a benchtop for 2–6 days. “Optimallyry” is of course operationally defined; if the platesre too dry they develop ridges, whereas if they areoo wet the bacterial lawn becomes runny and de-elops contours and pools.3. Prepare an overnight solution of Klebsiella

erogenes by growing cells in rich bacterial mediumvernight, spinning down cells, and resuspending in.05 vol of HL-5 (without antibiotics!). This solutionan be stored in a refrigerator for at least 1 monthnd used periodically during that time.4. Apply 3–4 ml of K. aerogenes solution to a plate

rom step 1. Do not allow bubbles onto the plateurface. Stored K. aerogenes solution should bearmed prior to applying to plates.5. Ensure coverage of the entire plate surface by

ilting and maneuvering the plate until the liquidas run over the entire surface.6. Aspirate off as much liquid as possible; this

hould include tilting the plate at 15–30°C for 30 s.7. Place the plate in a flat location overnight.

Plaque expansion rates are measured as follows:

1. Take a six-well plate and grow cells in a givenell to near confluence (far fewer cells can be used ifxpedient; larger numbers generally give better re-ults and ensure that all members of a mixed popu-ation are represented).

2. Resuspend cells by gentle trituration.3. Recover cells in a 1.5-ml Eppendorf tube by

entle spinning.4. Aspirate off all but 15–25 ml.5. Place 150-mm Petri plate prepared as above on

a grid with markings for plaques. (For most experi-ments we use a handmade grid with 19 centersmarked. Fewer centers allow data to be taken over alonger period but this is usually unnecessary.)

6. Resuspend cells and carefully drip 7–10 ml ofell suspension onto grid position using a fine-boreipet tip (achieving a round drop is easy but essen-ial).

7. Allow all drops to soak in before moving thelate to growth location.8. Measure plaque sizes daily by comparison to

ircle template.9. Calculate plaque expansion rate by figuring av-

rage growth per day over the period during whichxpansion rates are linear. In our hands, using the

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SPERA strain, we observe expansion rates of 2–3mm/day for myosin null cells and up to 12 mm/dayfor SPERA cells expressing wild-type myosin. Due toinevitable variation between plates, each plateshould contain a control with no myosin or wild-typemyosin and rates should be expressed as percentagegrowth of tested cells compared with this control.We have found expressing growth in this fashionallows reproducibility of 65% when assays are care-ully performed.

ebuilding the Motor: Suppressor Isolation

A minor variation of the plaque expansion ratessay allows for efficient recognition and isolation ofevertants of myosin mutants. Briefly, instead oftarting with a uniform population of cells, a mutantopulation (expressing a myosin known to conferess-than-wild-type rates of plaque expansion) is

utagenized (either before or after application tohe bacterial lawn) and allowed to plaque-expand foreveral days. If the population contains cells ex-ressing a myosin with improved function, theseill manifest as either a “blister” or “petal” of fasterrowing cells emerging from a plaque (in caseshere mutagenesis occurs after plating) or a plaquexpanding more rapidly than the unmutagenizedontrol. Sample protocols are given below both forargeted and untargeted mutagenesis.

Protocol 1: Untargeted Search for Suppressors

One wholly unanticipated but welcome findingfrom our first efforts to isolate suppressors of myosinmutants was that .90% of them proved to beintragenic—occurring within the myosin gene withthe original mutation. Thus, suppressors in generalwill not need to be screened to determine which arenot in the myosin gene. However, more recent re-sults with mutants thought to perturb actin bindinggive as little as 66% in the myosin gene—suggestingthe possibility that these may reside in the actingenes, though that is a leap of faith that we have yetto pursue. Regardless, those seeking intragenic sup-pressors can proceed with good cheer, as they knowin advance that their mutations will generally residein the myosin gene. The basic strategy is to takeadvantage of the power of the plaque expansionprocedure for identifying rapid expanders and the

convenience and safety of ultraviolet light as a mu-tagen. 9

1. Prepare lawns as described above and apply6–18 plaques (depending on expansion rate of mu-tant being reverted: those growing like null can beaccommodated up to 18/150-mm plate; those withmore wild-type rates should be held to 6 candidateplaques/plate).

2. After plaques have cleared and achieved ;2 cmiameters, expose the plates (without lids!) under aV light source (we use as Stratalinker) to 800

mJ/cm2 UV light at 260 nm.3. Return plates to growth conditions. Check

daily for irregular shapes occurring at plaque edges.4. If an aberration occurs that is semicircular and

clearly outgrowing its parent plaque, allow it to ex-pand as far as possible, then harvest cells by scrap-ing the leading edge of the plaque with a steriletoothpick.

5. Release cells into well of six-well dish contain-ing HL-5 and G418 at 5 mg/ml by gently scrapingtoothpick against wall. In some cases, we do notrecover drug resistant cells for some revertants.These are not pursued further.

Protocol 2: Site Specific or Region-Targetedutagenesis

Mutagenesis

For “region-selective” random mutagenesis, weemploy error-prone polymerase chain reaction(PCR) by using Taq polymerase and a small amountof manganese. We usually mutagenize a 300- to700-nt region using the following conditions:

1. For the template, we generally use a 1:100dilution of a miniprep DNA preparation that hasbeen treated with RNase.

2. We employ kinased primers (terminal phos-phates may improve recombination efficiency) withestimated Tm of ;60°C.

3. Each PCR consists of 12.9 ml distilled water,2.5 ml 103 magnesium-free Taq polymerase buffer,.4 ml 25 mM MgCl2, 0.075 ml 25 mM MnCl2, 2 ml of

a 1:100 dilution of miniprep DNA (ribonucleasetreatment of DNA preparations is often importantfor successful PCR in our hands), 1 ml each oligo at20 pmol/ml, 0.7 ml of a solution that is 25 mM foreach dNTP. We generally prepare 10 to 20 times thisamount and then aliquot into 10 or 20 tubes prior toadding 0.5 ml Taq polymerase/tube.

4. PCR conditions are 30 cycles of denature, 45 s,4°C; anneal, 45 s, 58°C; extend, 2.5 min, 72°C.

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305GENETIC TECHNIQUES ENABLING MYOSIN CHARACTERIZATION

5. Clean up reactions using Qiagen Qiaquick Spinkits, but perform the final elution in distilled waterinstead of Buffer EB.

6. Vacuum-dry samples and resuspend in 12 mlTE buffer.

When a specific mutation is to be examined,we introduce the mutation into subcloned DNAusing conventional methods and then amplify anappropriate region of DNA with high fidelity us-ing Pfu polymerase from Stratagene (La Jolla,CA).

Transformation of Dictyostelium

Our current protocol for transformation is as fol-lows:

1. Prepare vector DNA at 1 mg/ml. Ideally, vectorshould be cut at one or more sites, generating a cutor gap that will be fully overlapped by the cotrans-formed DNA. As little as 50 nt homology on eachside of the gap is sufficient.

2. Mix 0.25 ml cut vector with 3 ml PCR fragment(prepared as above or generated from template con-taining mutation(s) of interest) in 0.5 ml Eppendorftube on ice.

3. Grow SPERA (or myosin null) cells overnightor longer on 150-mm Petri plate. Ideally, cellsshould be fully confluent, but with few displacedfloating cells. Harvest cells by gentle triturationwith 12 ml ice-cold Electroporation Buffer (50 mMsucrose, 10 mM KPO4, pH 6.5).

4. Harvest cells by spinning gently for 1 min inabletop centrifuge.

5. Resuspend cells is 1 ml cold Electroporationuffer.6. Mix each DNA aliquot with 50 ml cells and

transfer to ice-cold 0.1-cm gap electroporation cu-vette.

7. Electroporate at 400 V, 1000 ohm parallel re-sistance, 10 mF. Time constants should be in therange 3.5–5 ms.

8. Add 350 ml ice-cold HL-5.9. Gently pipet cells into one well of six-well Petri

plate containing 1.5 ml ice-cold HL-5.10. At 24 h, add G418 to 7.5 mg/ml.11. At 48 h, replace medium with HL-5 contain-

ing 5 mg/ml G418.

12. Replace medium every third day with HL-5

containing 5 mg/ml G418.

Recovery of DNA

We isolate DNA from revertants using a variationof a protocol from the Qiagen Tissue DNA extractionkit. The protocol is as follows

1. Grow cells in six-well Petri dish until “over-crowded” (confluent and many cells floating becausethere is no room to adhere to surface).

2. Resuspend cells by trituration; add 1 ml toEppendorf tube (replace medium with HL-5 andsave until success of preparation is demonstrated).

3. Spin cells gently and aspirate off all medium.4. Resuspend cells in solution ATL.5. Add 20 ml proteinase K solution.6. Incubate 1 h at 55°C.7. Add 40 ml of 10 mg/ml ribonuclease solution;

incubate 10 min at 25°C.8. Add 200 ml solution AL.9. Add 200 ml 100% ethanol; mix thoroughly.10. Heat at 55°C until all particulate matter dis-

solves.11. Apply to Qiagen spin column, spin, and dis-

card liquid.12. Add 500 ml solution AW, spin, discard liquid,

and repeat.13. Release with 125 ml 0.1 mM Tris, pH 8.5; spin

into clean Eppendorf tube to collect.14. Vacuum-dry and resuspend with 15 ml dis-

illed water.15. This material can be transformed directly intoscherichia coli cells by electroporation.

CONCLUDING REMARKS

Dictyostelium is ideally suited for the genetic in-vestigation of the myosin motor cycle. Mutant isola-tion linked with biochemical and structural charac-terization potentially offers insights not readilyaccessible by any other means. By refining and ex-ploiting the biology of Dictyostelium, one can gener-ate conditional mutations and easily generate largenumbers of intragenic suppressors for any mutationthat does not irreparably damage the motor. Thetechniques presented here allow the generationof mutants and identification of the nucleotidechange(s) underlying the relevant phenotype and

should be feasible in any laboratory with access tobasic molecular biology tools. Further details of this

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work can be found at http://research.biology.arizona.edu/myosin.

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3. Patterson, B. (1998) Genetics 149, 1799–1807.

4. Yount, R. G., Lawson, D., and Rayment, I. (1995) Biophys. J.

68, 44s–49s.

5. Manstein, D. J., Ruppel, K. M., and Spudich, J. A. (1989)Science 246, 656–658.

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6. De Lozanne, A., and Spudich, J. A. (1987) Science 236, 1086–1091.

7. Knecht, D. A., and Loomis, W. F. (1987) Science 236, 1081–1086.

8. Pasternak, C., Spudich, J. A., and Elson, E. L. (1989) Nature341, 549–51.

9. Knecht, D. A., and Loomis, W. F. (1988) Dev. Biol. 128,178–184.

0. Knecht, D. A., and Kessin, R. H. (1990) Dev. Genet. 11, 388–390.

1. Patterson, B., and Spudich, J. A. (1995) Genetics 140, 505–515.

2. Sussman, M. (1987) in Dictyostelium discoideum: MolecularApproaches to Cell Biology (Spudich, J. A., Ed.), Vol. 28, pp.9–29, Academic Press: Orlando, FL.

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4. Wu, Y., Nejad, M., and Patterson, B. (1999) Genetics 153,107–116.