Identification and analysis of Dictyostelium actin genes, a family of moderately repeated genes

Cell,

Vol. 15.763-776,

November

1978, Copyright

0 1978 by MIT

Identification and Analysis of Dictyostelium Actin Genes, a Family of Moderately Repeated Genes Karen L. Kindle* and Richard A. Firtelt Department of Biology University of California, San Diego La Jolla, California 92093

Summary Plasmid M6 has been shown to contain sequences complementary to two related abundant mRNA species which differ in length by 100 nucleotides and code for Dictyostelium actin. M6 complementary RNA was isolated by hybridization to immobilized M6 DNA and translated in vitro. The product is identical to major forms of in vivo labeled actin in both mobility on two-dimensional gels and two-dimensional fingerprints of tryptic peptides. Roth plasmid M6 and a second plasmid complementary to the actin mRNA complementary region in M6, pDd actin 2 (McKeown et al., 1976), direct the synthesis in minicells of a number of similar polypeptides that are not seen in minicells containing other recombinant plasmids. Three of these polypeptides are similar in two-dimensional gel mobility to Dtctyostelium actin and bind to DNAase I agarose. The repetition frequency of isolated restriction fragments from actin mRNA complementary plasmid M6 has been examined. The data from two different experfmental approaches (DNA excess hybridizations using plasmid DNA as probe, and hybridization of plasmid probe to DNA blot filters of restriction enzyme-digested Dictyostelium DNA) indicate that the mRNA complementary region is reiterated 15-20 times. When an actin cDNA probe is used in the same experiments, the results suggest that the entire coding region is reiterated. When the two major actin mRNA species are separated and independently translated, each appears to code for one of the two major actin species. The results suggest that there are at least two different functional genes, and possibly more, for Dictyostelium actin. Introduction Actin is a very abundant protein in most eucaryotic ceil types. It is important in many forms of motility including muscle contraction, chromosome movement, cytokinesis, phagocytosis and exocytosis (see Pollard and Weihing, 1974; Goldman, Pollard and Rosenbaum, 1976, for reviews). In mammalian systems, at least three different forms of actin (a, ’ Present address: Department of Chemistry, of Technology, Pasadena, California 91125. t To whom correspondence should be sent.

California

Institute

p and y) have been separated by isoelectric focusing (Gruenstein, Rich and Weihing, 1975; Garrels and Gibson, 1976; Whalen, Butler-Browne and Gros, 1976). The most acidic form, (Y actin, is found only in muscle and is the major form synthesized in fused myoblasts. Amino acid sequence analysis has shown that cardiac and skeletal muscle a actins are not identical and, therefore, must be the products of different genes (Elzinga and Lu, 1976). All tissues, including prefusion myoblasts, contain two other forms of actin, p and y, whose tryptic peptides differ from (Y actin (Gruenstein et al., 1975; Garrels and Gibson, 1976). Hunter and Garrels (1977) have separated three mRNAs that code for the three different forms of actin from early fusion myoblasts, a result which indicates that all three forms of actin are probabiy products of separate genes. In Dictyostelium discoideum, actin is an abundant protein whose relative rate of synthesis changes during the developmental cycle (Woolley, 1972; Spudich, 1974; Tuchman, Alton and Lodish, 1974; Spudich and Cooke, 1975; Alton and Lodish, 1977). Quantitation of the relative rate of actin synthesis during Dictyostelium development shows that it increases between 0 and 3 hr from approximately 6-20% of newly incorporated 35S-methionine. By late in the developmental cycle, the relative rate of actin synthesis decreases to below the level observed in vegetative cells (Tuchman et al., 1974; Alton and Lodish, 1977). Alton and Lodish (1977) have correlated these changes with developmental changes in the relative amount of actin mRNA by assaying the ability to stimulate actin synthesis in a wheat germ in vitro protein synthesizing system. Additional results (A. Jacobson, personal communication) suggest that there is an increase in the relative synthesis of actin mRNA which parallels the increase in actin protein synthesis during early development. In this paper, we describe the isolation and characterization of a recombinant plasmid (M6) carrying a DNA fragment from Dictyostelium which is complementary to actin mRNA. We present evidence that mRNA complementary to M6 DNA is translated in vitro into Dictyostelium actin as defined by mobility in two-dimensional gels and by tryptic peptide analysis, and that M6 plasmid DNA can direct the synthesis of actin in E. coli minicells. In addition, the sequence arrangement of the M6 DNA insert relative to the Dictyostelium genome has been analyzed and the organization of the gene sequences complementary to actin mRNA has been examined. The results show that actin mRNA complementary sequences are reiterated in the genome approximately 15 fold and that flanking sequences are either single-copy or reiterated only a few times.

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We also show that there are at least two forms of actin mRNA which can be translated in vitro into at least two different isoelectric focusing species of actin. For simplicity of presentation, genomic sequences complementary to actin mRNA will be described as actin “genes” although we do not know how many or which of these sequences are transcribed in vivo.

electrofocusing

OH-

Results Isolation and Properties of Plasmid M6 The formation of recombinant plasmids carrying Dictyostelium genomic DNA fragments by the poly(dA)-poly(dT) tailing procedure and the screening for those which are complementary to poly(A)+ RNA are described in Experimental Procedures. Plasmid M6 DNA was isolated from a clone selected as one which is complementary to a large fraction of mRNA. Hybridization of pulse-labeled poly(A)+ RNA to an excess of immobilized plasmid M6 DNA shows that 0.5-l% of newly synthesized RNA from vegetative cells is complemsntary to immobilized M6 DNA and that the fraction of pulse-labeled RNA that hybridizes to M6 DNA increases when RNA is labeled during early development. RNA excess hybridization indicates that the mRNA complementary to M6 is present at a level of -1% of total poly(A)+ RNA in vegetative cells (Kindle, 1978; K. L. Kindle and R. A. Firtel, manuscript in preparation). When 32P-poly(A)+ RNA is size-fractionated on formamide-containing polyacrylamide gels and M6 DNA filters are used to assay complementary RNA in each fraction, a peak of hybridization is obtained indicating that the complementary RNA is approximately 1.3 kb in length (data not shown). The hybridization properties described above for M6 are those which would be expected for a genome sequence complementary to actin mRNA. The following sections present the evidence that M6 does contain DNA sequences complementary to actin mRNA and that the actin gene is repeated in Dictyostelium. Evidence That Clone M6 Hybridizes a Message Which Codes for Actin M6 complementary RNA was purified by two cycles of hybridization and elution from nitrocellulose filters containing M6 DNA. This RNA was then translated in a wheat germ cell-free system, and the products were separated on 12.5% polyacrylamide gels. RNA purified by one cycle of hybridization directs the synthesis of protein products greatly enriched in a protein which co-migrates with Dictyostelium actin. After a second round of hybridization and elution, actin was virtually the only protein synthesized other than those endoge-

Figure 1. Two-Dimensional Display of Translation Products of M6 Complementary RNA RNA purified by two cycles of hybridization to M6 filters was translated in the wheat germ cell-free system and displayed on two-dimensional gels as described by Garrels and Gibson (1976). The first dimension is electrofocusing and the second is SDSpolyacrylamide gel electrophoresis as described in Experimental Procedures. A narrow range of pH 5-7 ampholites were used. The nominal pH range of the first dimension is approximately pH 4.56.2. The gels were fluorographed (Bonner and Laskey, 1974; Laskey and Mills, 1975). (A) SsS-met-labeled translation products of M6 complementary RNA; (B) proteins produced in vitro by total poly(A)+ RNA from Dictyostelium; (C) proteins labeled in vivo from 3-4.5 hr in development. A, and A,, indicate actin proteins; a and b are believed to be premature termination products of actin mRNA translation.

nous to the wheat germ system. Figure 1A shows the analysis of in vitro products on two-dimensional gels. Two spots can be identified which co-

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migrate with two of the major Dictyostelium proteins seen in gels of in vitro translation products programmed by total poly(A)+ RNA (Figure 1 B) and in vivo total pulse-labeled Dictyostelium proteins (Figure IC). These major proteins have been shown to be actin (Alton and Lodish, 1977; C. MacLeod and R. A. Firtel, unpublished observations). There are also two minor spots which can be seen in the in vitro translation products of total poly(A)+ RNA but not in the in vivo labeled protein spots. These lower molecular weight proteins are also seen when gel-fractionated actin mRNA and mRNA purified by hybridization to a recombinant plasmid carrying a cDNA insert complementary to M6 are translated in the wheat germ system, but are present at lower levels with RNA translated in the rabbit reticulocyte MDL (Pelham and Jackson, 1976). For these reasons, we feel that the two lower molecular weight proteins are early termination products of actin. The presence of at least two isoelectric focusing forms of the protein suggest that there are at least two forms of actin in Dictyostelium. To further establish the identity of these proteins as actin, the products of in vitro translation of putative actin mRNA were size-fractionated on a 12.5% polyacrylamide gel and the band that comigrated with Dictyostelium actin was eluted from the gel for tryptic peptide analysis. Figure 2A shows the results of a two-dimensional fingerprint of the in vitro translation product and authentic in vivo labeled Dictyostelium actin. The tryptic peptide maps of the in vitro translated protein are essen-

tially the same as those of the in vivo labeled actin (see Figure 2B). When the in vitro synthesized protein and in vivo labeled actin tryptic peptides are fingerprinted together, the peptides co-migrate, confirming the identity of this protein as Dictyostelium actin (see Figure 2C). Actin mRNA Is Complementary to M6 DNA along Its Entire Length We were interested in determining whether actin mRNA is complementary to M6 DNA along its entire length. 32P in vivo labeled poly(A)+ RNA was hybridized to M6 DNA bound to nitrocellulose filters. The filters were washed and treated with low levels of RNAase to remove the unhybridized, singlestranded RNA tails (Kindle, 1978). The RNAase was inactivated and the filters were washed several times before the RNAase-resistant RNA was eluted. The RNA was then size-fractionated on urea-containing polyacrylamide gels (Spradling, Pardue and Penman, 1977) or methyl mercury-containing agarose gels (data not shown) (Bailey and Davidson, 1976). As shown in Figure 3, two mRNA bands with lengths of 1.25 and 1.35 kb including the poly(A) appear in the control fractions (McKeown et al., 1978). The fractions treated with low levels of RNAase show the same bands, indicating that the entire non-poly(A) length of the RNA is hybridized to M6 DNA. Several lower molecular weight bands can also be observed. At higher RNAase concentrations, these additional smaller bands increase in amount while the fraction of full-length molecules

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is decreased appreciably evidence that the entire tary sequence is present can form heteroduplexes DNA and cloned cDNA mRNA (see Bender et al.,

(data not shown). Further actin mRNA complemenin plasmid M6 is that we 1 .l kb long between M6 complementary to actin 1978).

Synthesis of Actin in Minicells Containing Plasmid M6 To determine whether plasmid M6 contains a potentially functional actin gene sequence, minicell strain ~678-54 (Adler et al., 1967) was transformed with plasmid pMB9, M6, another recombinant plasmid carrying Dictyostelium genomic DNA (KHlO) (K. L. Kindle and R. A. Fir-tel, manuscript in preparation) and a second actin “gene’‘-containing plasmid, pDd actin 2 (McKeown et al., 1978). Minicells from these strains were purified and labeled with Yi-met (Matsumura, Silverman and Simon, 1977), and the proteins were displayed on two-dimensional polyacrylamide gels and localized by fluorography (Bonner and Laskey, 1974; Laskey and Mills, 1975; see Figure 4). Comparisons of the twodimensional array of proteins synthesized by M6 and pDd actin 2 minicells with those of KHlO minicells indicates some proteins in common. These are presumably the products of minicells containing a pMB9 plasmid interrupted at the Eco RI site by a foreign piece of DNA. In addition, both M6 and pDd actin 2-containing minicells produce a large number of polypeptides that are not synthesized in KHlO minicells. When the products from pDd actin 2 minicells are analyzed by two-dimensional electrophoresis with in vivo 35S-met-labeled Dictyostelium proteins included as markers (see Figure 4D), it is clear that a protein produced in both M6 and pDd actin 2 minicells but not in KHlO minicells (labeled A, in Figure 4) has a mobility in the molecular weight dimension that is identical with that of Dictyostelium actin. This protein, however, is slightly more basic than Dictyostelium actin

Figure

3. Ribonuclease

Resistance

of RNA Hybrids

with M6 DNA

52P-poly(A)+ RNA was hybridized to M6 DNA filters in 0.72 M NaCI, 10 mM EDTA (pH 7.2) 55% formamide. 0.2% SDS, 1 mg/ml poly(A) for 2 days at 3PC. After extensive washing in the same buffer lacking poly(A). the filters were separated and digested with RNAase A in 2 x SSC and the RNAase was inactivated by digestion with 0.3 mg/ml proteinase K in 1.2 x SSC, 120 mM TrisHCI, 4 mM EDTA (pH 7.9) 0.6% SDS at 3PC for 1 hr. RNA was eluted in 70% formamide, 4 mM EDTA (pH S) containing 200 fig/ ml tRNA at 60°C for 3 washesof 3 min each. Aflerelution, 1710 vol of 4 M NaAc (pH 4.7) was added, immediately treated with 1% DEP and extracted with a 1 :l mixture of redistilled phenol and chloroform. The RNA was then ethanol-precipitated and sized on 3.5% polyacrylamide gels containing urea (Spradling et al., 1977). RNA eluted from: (a) non-RNAase-treated filter which had previously been washed with 0.045 M NaCI, 55°C; (b) filter treated with 0.03 @g/ml RNAase A.

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electrofocusing

Figure

4. Two-Dimensional

Gel Fractionation

of Proteins

___c_t

Labeled

in Plasmid-Containing

Minicells

Plasmid DNA from pMB9, M6, pDd actin 2 and KHlO were transformed into minicell strain P676-54. Minicells strains were grown in L broth containing 15 pg/ml tetracycline, and minicells were isolated and labeled with 3BS-met as described (Matsumura et al., 1977). Samples were prepared and fractionated on two-dimensional gels as described by Garrels and Gibson (1976). 3-met-labeled proteins from minicells containing plasmid: (A) M6; (6) pDd actin 2; (C) KHlO; (D) pDd actin 2 plus Dictyostelium proteins labeled in vivo with 3-met. The gels were aligned by use of spots common to the gels, some of which are given Arabic numerals. These numbered E. coli-specific spots are only a representation of the spots used for alignment.

(approximately one charge). It is possible that posttranslational modifications of actin which occur in Dictyostelium do not occur readily in minicells, producing an actin protein with an altered pl. There is also a very faint spot among both pDd actin 2 and M6 minicell proteins (labeled 4) which appears to coincide with one of the major Dictyostelium actins (Figure 4A). In addition, a protein labeled B in Figures 4A and 48 is also found in the products of both M6 and pDd actin 2 but not in KHlO. B is slightly more basic than A, and has a slightly higher mobility in the second dimension. There are also some differences between the

proteins produced by the two actin minicell strains: note that protein C is prominent among pDd actin 2 products but missing in M6 minicells. A group of abundant and possibly related polypeptides is present among both M6 and pDd actin 2 minicell proteins, although close examination reveals differences between these sets of proteins. It is interesting to note that most of these proteins have an acidic pl, similar to that of actin. These may be the result of imperfect transcription of the actin gene or translation of the eucaryotic RNA by the procaryotic cellular machinery. Additional evidence that the A,, 4 and B poly-

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peptides are related to actin comes from experiments using DNAase I-agarose as an affinity column for actin (Lazarides and Lindberg, 1974). DNAase I has been shown to have a high affinity for eucaryotic actins (Lazarides and Lindberg, 1974) and has been shown to specifically bind in vivo and in vitro synthesized Dictyostelium actin (C. MacLeod and R. A. Firtel, unpublished observations). pDd actin 2 minicell extract labeled with 35S-met was mixed in batch with DNAase I agarose and the matrix was washed. The bound proteins were eluted and analyzed on two-dimensional polyacrylamide gels. The results are shown in Figure 5. Proteins labeled A,, 4 and B, which migrate similarly to Dictyostelium actin, bind to DNAase I agarose. Note that spot 6 can also be seen. The fact that binding occurs indicates that these spots, which are minor in the total pDd actin P-directed proteins, bind quite specifically and therefore are probably related to actin. It should be noted that the binding efficiency of B, which is slightly more basic than A, and 4 and migrates more rapidly in the SDS dimension, was appreciably less than A, and 4. Spot 6, which binds to DNAase I with low efficiency and is a major E. coli-specific spot, has the mobility expected of E. coli translation factor Tu, which has been shown to bind to DNAase I (Pederson et al., 1976; Beck, Arscott and Jacobson, 1978). The presence of labeled E. coli-specific proteins is due to a slight contamination of the minicell preparation with whole bacteria. OH-

electrofocusing

-

Actin Genes Are Repeated Plasmid M6 has been mapped as described by Bender et al. (1978). The location of multiple restriction endonuclease cleavage sites and the location and transcriptional polarity of the mFlNA complementary region in the Dictyostelium insert have been determined (see Figure 6). Two experi-

pMB9

A

a. Hae III+ HapII b.HaeIlI c.Eco RI + HindlIT d.Eco RI

H+

obcd Figure 6. Restriction Labeled Restriction Blot Filters

Figure 5. DNAase Minicell Extracts Part of the agarose as and eluted resuspended dimensional shows an correspond spot to the

I Agarose-Bound

Proteins

from

pDd

Actin

2

sample shown in Figure 48 was bound to DNAase described (Lazarides and Lindberg, 1974) and washed in 0.2% SDS. The sample was then lyophilized. in isoelectric focusing buffer and analyzed on twogels as described in Experimental Procedures. Insert enlargement of the region of interest. A,, A, and B to the proteins as labeled in Figure 4. The large dark left of the lettered spots does not represent a protein.

abed

o bed

Map of Plasmid Fragments from

M6 and Hybridization M6 to Dictyostelium

of DNA

Restriction map of plasmid M6 was determined as described by Bender et al. (1976). The localization of the mRNA complementary region was determined by hybridization of 52P-poly(A)+ to Southern DNA blot filters of M6 DNA digested with various restriction enzymes and separated on agarose gels. See Bender et al. (1976) for data and detail of methods. A dashed arrow indicates the direction of transcription (5’ + 3’) and the location. of the actin mRNA complementary region. Plasmid M6 was digested to completion with Hap II, and Hap II and Hae Ill, and the fragments were size-fractionated on a 0.6% flat bed agarose gel. The gel was stained and the 1.4 kb Hap II, 2.0 kb Hap II and 1.7 kb Hae Ill-Hap II fragments were isolated and labeled by nick translation as described m Experimental Procedures. The nick-translated fragment DNA was hybridized to Southern DNA blot filters of Dictyostelium DNA which had been digested with various restriction endonucleases and size-fractionated on 0.6% agarose flat bed gels. The filters were autoradiographed after appropriate washings. See Experimental Procedures for details. (A) Hae-Hap 1.7 kb; (B) Hap 2.0 kb; (C) Hap 1.4 kb. An arrow points to the location of the 1.7 kb Hae Ill-Hap II band in the genome.

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mental approaches were used to examine the repetition frequency of the various regions of M6 DNA. In the first, hybridization probes were made from isolated restriction fragments of M6 and hybridized to Southern DNA blot filters (Southern, 1975) carrying size-fractionated Dictyostelium DNA which had been digested to completion with various restriction endonucleases. In the second, restriction fragments labeled by nick translation (Maniatis, Jeffrey and Kleid, 1975) were hybridized to a vast excess of sheared Dictyostelium DNA and the repetition frequency of these sequences in the Dictyostelium genome was determined from the kinetic rate constant. Figure 6 shows the hybridization of nicktranslated restriction fragments to DNA blot filters of genomic DNA. What is most striking is that the 1.7 kb Hae Ill-Hap II fragment, which is complementary to actin mRNA, hybridizes to 15-20 fragments depending upon the enzyme used to digest the Dictyostelium genomic DNA. The 1.3 kb Hae III fragment (data not shown) and the 2.0 kb Hap II fragment, which contain sequences adjacent to those coding for the 5’ end of the mRNA, hybridize strongly to one or two fragments, suggesting that the sequences are present l-2 times per genome. The 1.4 kb Hap II fragment and the 2.5 kb Hae III fragment (data not shown), containing sequences complementary to the 3’ end of those coding for the mRNA, hybridize to 6-10 major bands, suggesting they are repeated several times but less than the actin mRNA complementary fragment. Genomit DNA restriction fragments which hybridize less intensely to sequences flanking the 3’ end of the actin gene may be due to very short repeat sequences or to a longer repeat sequence which is distantly related. The less intense hybridization observed with the 2.0 kb Hap II fragment at the 5’ end of the actin gene is probably due to a short region of homology between the 5’ end of the M6 actin gene and the other genomic actin genes. When plasmid DNA is hybridized to a vast excess of Dictyostelium DNA, the cloned sequences hybridize with kinetics that are characteristic of their repetition frequency in the Dictyostelium genome. Figure 7 shows the hybridization kinetics of the 4.6 kb Eco RI fragment (comprising 80% of the Dictyostelium insert), the 1 .7 kb Hae Ill-Hap II, the 1 .3 kb Hae III and the 2.5 kb Hae III fragments. Approximately three fourths of the 1.7 kb Hae Ill-Hap II fragment carrying the region complementary to actin mRNA renatures with a Cot,,, of about 8 Msec. Since the single-copy component of total Dictyostelium DNA has a CotliZ of 120 Msec under the assay conditions used (Firtel and Bonner, 1972; Firtel et al., 1976), most of the 1.7 kb Hae Ill-Hap II fragment is reiterated lo-20 fold in the genome. The remainder of the fragment renatures with a that it is either single-copy or cot,,*, indicating

Figure 7. Hybridization Isolated M6 Restriction DNA

Kinetics Fragments

of Nick-Translated to a Vast Excess

DNA from of Genomic

M6 was digested with Hae Ill, Hap II, or Hae III and Hap II; the fragments were separated on agarose gels, eluted and labeled by nick-translation as described in the legend to Fig. 6. The labeled fragments were hybridized in solution with a 25-50 fold sequence excess of Dictyostelium DNA. It was estimated that the specific activity of the labeled DNA was 2-4 x lO’cpm/pg. Approximately 3000 cpm of label were used per point. Assays with Sl nuclease were carried out as described in Experimental Procedures. The fraction hybridized was plotted according to Morrow (1974) to correct for cleavage of unhybridized tails by Sl nuclease. (W..m) 4.6 kb Eco RI fragment; (CL-0) 1.7 kb Hae Ill-Hap II fragment; (A-A) 2.5 kb Hae Ill fragment; (O- - -0) 1.3 kb Hae Ill fragment; (A- - -A) 1.3 kb Hae Ill fragment renatured without Dictyostelium driver DNA.

repeated only a few times in the genome. The 1.3 kb Hae III fragment renatures with a CotliZ of about 100, again suggesting that is either single-copy or repeated twice in the Dictyostelium genome. The 2.5 kb Hae III fragment renatures at a lower Cot, suggesting that it is reiterated several times in the genome in agreement with the Southern DNA blot experiments (see Figure 7). The results of these two experimental approaches indicate that the 1.7 kb Hae Ill-Hap II mRNA complementary fragment is composed mainly of a sequence reiterated about 15 times in the genome, and that the cloned sequences flanking this region on the 5’ and 3’ side of the sequences coding for the mRNA are represented only one to two times and several times per genome, respectively.

Evidence That the Entire Actin Gene Sequence Is Repeated It is important to determine whether the entire actin-encoding sequence is repeated. A number of experiments have been carried out to establish this point. Dictyostelium poly(A)+ RNA was partially degraded and end-labeled to a high specific activity using 32P-y-ATP and polynucleotide kinase (Maizels, 1976; Maxam and Gilbert, 1977). M6 complementary RNA was purified by hybridization and elution from M6 DNA attached to nitrocellulose

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filters. When this RNA was hybridized to DNA blot filters of restriction enzyme-digested Dictyostelium DNA, the hybridization pattern was identical to that obtained with the nick-translated 1.7 kb Hae Ill-Hap II fragment (data not shown). This means that at least part of the reiterated sequence is transcribed onto mRNA. Evidence that the entire transcript is repeated comes from the following experiment. A population of recombinant plasmids carrying cDNA made from Dictyostelium poly(A)+ mRNA (W. Rowekamp and R. A. Firtel, manuscript in preparation) was screened for those containing sequences complementary to the 1.7 kb Hae Ill-Hap II fragment of M6. The cDNA was inserted into the Pst I site of pBR322 using poly(dG)-poly(dC) tailing, which allows excision of the insert with Pst I (A. Otsuka, manuscript in preparation; W. Rowekamp and R. A. Firtel, manuscript in preparation). One of the cDNA clones isolated, pcDd actin Bl, contains a 1.0-l .1 kb cDNA fragment approximately 8590% the length of actin mRNA. DNA excess hybridization of the nick-translated cDNA insert to sheared Dictyostelium nuclear DNA showed a single kinetic component with a Cot,, of 8, indicating that (within the limits of the experiment) the entire cloned sequence is repeated (data not shown). Thus at least 8590% of the actin mRNA is complementary to sequences repeated approximately 15 times per genome. Additional evidence that the protein coding portion of actin mRNA is repeated comes from an experiment in which the cDNA insert from pcDd actin Bl was cleaved with Mbo II into two fragments, approximately 600 and 400 nucleotides in length. Both the entire cDNA insert and the two Mbo II fragments were purified, labeled by nick translation and hybridized to Dictyostelium DNA blot filters (see Figure 8). Probes representing both the 5’ end and most of the 3’ end of the mRNA hybridize to the same set of restriction fragments and to the same fragments which hybridize to the 1.7 kb Hae Ill-Hap II fragment from M6. Since pcDd actin Bl contains most of the 5’ and 3’ end of the mRNA sequence (see Bender et al., 1978), we conclude that essentially the entire coding region for actin is repeated. Two Different Forms of Actin mRNA Make Two Different Forms of Actin When Dictyostelium proteins labeled in vivo or synthesized in vitro in response to Dictyostelium RNA are displayed on two-dimensional gels, there are several proteins which migrate with the same mobility in the molecular weight dimension as actin but which differ slightly in isoelectric focusing points. The two forms of mRNA described in Figure 3 must have related sequences since they both

Mbo II-I

Mbolt-2

a b c d Figure 6. Hybridization Fragments of pcDd Filters

ab

c d

of Nick-Translated DNA from Restriction Actin Bl to Dictyostelium DNA Southern

The Dictyostelium insert and the 5’ and 3’ ends of the insert were isolated by digesting pdDd actin Bl with Pst I and Pst I-Mbo II, respectively, and fractionating the fragments on agarose gels. The fragments were labeled by nick translation and hybridized to Dictyostelium DNA blot filters (see legend to Figure 6 for details). The figure shows Dictyostelium DNA Southern filters hybridized to nick-translated probe made from two Mbo II fragments from either end of the Dictyostelium insert. Mbo II-I is the larger fragment: (a) Hap II, (b) Hae III, (c) Eco RI + Hind Ill, (d) Eco RI.

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hybridize almost entirely to a fragment which is too small to encode them both in tandem (M6, 1.7 kb Hae Ill-Hap II fragment), and they both specifically hybridize to pcDd actin 81, the insert of which was presumably copied from a single message (R. A. Firtel, unpublished observations; see McKeown et al., 1978, for additional data). To see whether the two mRNA forms complementary to plasmid M6 DNA direct the synthesis of different forms of actin, the following experiment was performed. 32P-labeled poly(A)+ RNA was size-fractionated on a 3.5% urea polyacrylamide gel (Sprading, Pardue and Penman, 1977), and the RNA was eluted from the gel fractions corresponding to the actin mRNA bands visualized by autoradiography. These RNAs were translated in the rabbit reticulocyte messen-

OH-

Figure

9. Translation

ger-dependent lysate (MDL) (Pelham and Jackson, 1976) and the proteins synthesized were displayed on two-dimensional gels (see Figure 9). The translation products of the lower molecular weight RNA fraction include a protein (labeled A,) (see Figure 9A) which co-migrates with the major and more basic form of actin seen among the total poly(A)+ in vitro translation products shown in Figure 9D. Figure 9B shows that the higher molecular weight actin mRNA band directs the synthesis of the more basic form of actin (A,,), but produces little or no detectable A,. When equal amounts of incorporated counts of the two translation products are mixed, both forms of actin are seen in approximately equal amounts as shown in Figure 9C. Thus it is reasonable to conclude that there are

electrofocusing

Products

of the Two

Different

Actin

mRNA

Species

“P-poly(A)+ RNA was size-fractionated on 3.5% polyacrylamide urea gels (Spradling et al., 1977). The bands corresponding to actin mRNA were identified by autoradiography and cut from the gel. The urea was eluted from the gel pieces, and the RNA was extracted as described by Hunter and Garrels (1977) passed over oligo(dT)-cellulose and precipitated twice with ethanol. The RNA fractions were then translated in the rabbit reticulocyte messenger-dependent lysate using =S-met as label (Pelham and Jackson, 1976). To align the gels, common spots between sets of gels were picked as reference points. Only some of these spots are numbered. The A, and A,, spots in (A) and (8) were aligned relative to the numbered spots in (C) and (D). The positions of mobility of the actin spots A, and A,, relative to the other Dictyostelium protein spots in (D) and (C) have been determined by DNAase I binding studies (C. MacLeod and R. A. Firtel. unpublished observations). Shown are MDL translation products using (A) higher molecular weight actin mRNA; (B) lower molecular weight actin mRNA; (C) equal amounts of 35S-met cpm incorporated from (A) and (B): (D) unfractionated RNA.

Cell 772

at least two different forms of actin which are the products of RNAs that differ in length by about 100 nucleotides are therefore probably products of different actin genes. In Figure 9B there is a second protein which has the same molecular weight as actin A,,, but which focuses at a pl that is somewhat more acidic. Its location on the two-dimensional gels is similar to a minor spot seen in gels of in vivo labeled proteins and may represent a third actin form. Discussion Evidence That M6 DNA Is Complementary to Actin mRNA M6 hybridizes mRNA that codes for Dictyostelium actin. This has been established by detailed char; acterization of the in vitro translation products of mRNA purified by hybridization to and elution from M6 DNA filters. The migration of these proteins is identical to that of Dictyostelium actin on one(data not shown) and two-dimensional gels (see Figure 1), and the tryptic peptides of the protein whose synthesis is directed by M6 complementary RNA co-migrate with those of in vivo labeled Dictyostelium actin. It should be emphasized that the location of actin on two-dimensional gels is characteristic. Using the high resolution two-dimensional gel system of Garrel and Gibson (1976), we have routinely separated at least two major actin protein forms migrating approximately equivalent to a major asymmetrical spot identified as actin by Alton and Lodish (1977) in O’Farrell gels (O’Farrell, 1975). C. Macleod and R. A. Firtel (personal communication) have shown that these actin forms bind to DNAase I. When Dictyostelium mRNA is translated in vitro, actin is the major product (Alton and Lodish, 1977; see Figure 1, this paper). It is important to establish that the reason that M6 complementary RNA appears to direct the synthesis of actin is not due simply to contamination with the abundant actin mRNA. After one cycle of mRNA purification by hybridization and elution from M6 DNA filters, actin becomes a more prominent band among the translation products. After two cycles of hybridization, essentially the only proteins observed (with the exception of the endogenous wheat germ products) are the actin protein spot plus what we believe are early termination products; this is the case even after prolonged autoradiography. When 32P-labeled poly(A)-containing RNA is hybridized to M6 DNA filters and the RNA is eluted and sizefractionated on gels, only two major bands of RNA are observed, both of which appear to code for actin. In addition, when Dictyostelium mRNA is size-fractionated on formamide-containing poly-

acrylamide gels and the RNA is eluted from the gel slices, the peak of hybridization to M6 DNA coincides with the peak of actin translational stimulating activity in the wheat germ in vitro protein synthesizing system (data not shown). These results support the conclusion that M6 is cornpIe: mentary to actin mRNA. M6 probably contains sequences complementary to the entire length of actin mRNA, as suggested by the fact that some of the RNA eluted after a mild RNAase treatment of the M6 DNA:mRNA hybrids is full-length. Furthermore, as shown by Bender et al. (1978), the inserts from actin cDNA-containing recombinant plasmids form heteroduplexes with M6 along their entire length. At higher ribonuclease concentrations, a number of lower molecular weight bands are seen which could be due to cleavage at regions of base mismatch between M6 DNA and actin mRNAs with some heterogeneity in the primary sequence (Kindle, 1978; M. McKeown and R. A. Firtel, unpublished observations; see also McKeown et al., 1978). Further evidence concerning the coding capacity of M6 comes from experiments in which proteins synthesized in minicells in response to plasmid DNAs are analyzed. Both M6 and pDd actin 2 produce an array of proteins that are not seen in pMB9 (data not shown) or in KHlO-containing minicells (see Figure 4). Plasmid pDd actin 2 has been shown to carry two sequences which are complementary to the mRNA complementary region of M6 (see McKeown et al., 1978). In this paper, we show that spot A, seen in both M6 and pDd actin 2specific minicell proteins, migrates at the same molecular weight (see Figures 4B and 4C) as Dictyostelium actin but has a slightly more basic pl. This protein and several others of similar molecular weight and isoelectric point bind specifically to DNAase I agarose. It is probable, therefore, that the pDd actin 2-specific proteins we have seen which bind to DNAase I are related to Dictyostelium actin and may well be the product of transcription and translation of a Dictyostelium actin gene in E. coli minicells. Differences in isoelectric point could be due to incomplete or improper modification of the eucaryotic gene product in a procaryotic cell. Repetition and Organization of Actin Genes The evidence that the entire actin mRNA complementary region (actin “gene”) is repeated approximately 15 fold in the genome is very convincing. The actin mRNA complementary region is localized on the 1.7 kb Hae Ill-Hap II fragment (see Bender et al., 1978). The moderately repeated region representing approximately one fourth of the 4.6 kb Eco RI fragment has been localized to the 1.7 kb mRNA complementary fragment by DNA blot filter hybrid-

Dictyostelium 773

Actin

Genes

izations and DNA excess hybridization kinetic experiments. Moreover, it is estimated that the entire 1050 bp insert from pcDd actin Bl, which is complementary to the 1.7 kb Hae Ill-Hap II fragment (see Bender et al., 1978), is repeated approximately 15 fold on the basis of both renaturation kinetics and independent hybridization of the 5’ and 3’ halves of the insert to DNA blot filters carrying Dictyostelium nuclear DNA. Since the insert from pcDd actin Bl is a copy of approximately 90% the length of actin mRNA, we conclude that the actin mRNA complementary sequences are reiterated. The isolation and analysis of a second recombinant plasmid carrying two actin “genes”, pDd actin 2, (McKeown et al., 1978) makes this conclusive. Whether the multiple actin genes are clustered or dispersed throughout the genome is an extremely interesting and important question but it cannot be definitively answered with the data described in this paper. Our results do indicate, however, that the multiple genes, if clustered in the genome, are not found in the “classical” repeatspacer organization seen in repeated chromosomal 18 and 285 rRNA and 5s rRNA genes of Xenopus (see Brown and Sugimoto, 1973). Analysis of the M6 insert by renaturation kinetics and DNA blot filter hybridizations shows that the sequences adjacent to the actin mRNA complementary region are present in l-2 copies per genome on the 5’ side and several times per genome on the 3’ side, while the fragments carrying the region complementary to actin mRNA or pcDd actin Bl probe hybridize to approximately 15 different restriction fragments on genomic DNA blots. This indicates that all of the actin genes cannot have the same adjacent sequences. A possible explanation for the large number of restriction fragments which hybridize to the 1 .7 kb Hae Ill-Hap II probe on DNA blots is that inserts may be present in some of the genes which contain sites recognized by some of the restriction enzymes utilized in the experiments shown in Figures 6 and 8. This explanation, however, appears to be improbable since the two isolated Mbo II fragments (3’ and 5’ halves) of pcDd actin Bl showed essentially the same hybridization pattern on DNA blot filters. Heterogeneity in Actin Genes The existence of approximately 15 actin genes (mRNA complementary regions) in Dictyostelium immediately leads one to ask whether there is heterogeneity in the coding regions of these genes. Although by definition there are no Hap II or Hae III restriction sites in the 1.7 kb Hae Ill-Hap II fragment of M6, Figures 6 and 8 show that there are DNA fragments significantly smaller than the 1.1-l .2 kb

gene size in the Hae Ill-Hap II digest of total Dictyostelium DNA which hybridize nick-translated DNA from pcDd actin Bl. Assuming that the hybridization is specific and that all the actin genes are complete copies (do not represent partial genes), this can only result if there is heterogeneity within the coding region such that some of the actin mRNA complementary sequences in the genome have at least one additional Hap II and/or Hae III site in the coding region. [Note: although the M6 actin mRNA region overlaps a Hap II site near the 5’ end (for details see Bender et al., 1978), the actin mRNA complementary region on the Hap II 2.0 kb fragment is too short to give a strong hybridization signal to the reiterated fragments shown in Figures 6 and 8.1 Some of the Dictyostelium DNA bands complementary to the Hae Ill-Hap II coding region appear consistently darker than others and presumably hybridize more probe (see Figures 6 and 8). This could be due to the linkage of two genes in a single restriction piece or to the presence of identical duplications of parts of genes, which would also yield greater than molar amounts of DNA complementary to M6 at a specific molecular weight. Another possibility is that the probe is better matched to some repeats than others, resulting in more hybridization and darker bands with the more closely matched sequences. It is also possible that some fragments complementary to M6 cRNA are generated by restriction sites within the genes such that some fragments contain a larger percentage of the coding region than others. This does not appear to be the major cause of variable band intensity and cannot explain the results observed with Hind III and Eco RI (K. L. Kindle, M. McKeown and R. A. Firtel, unpublished observations), since the two Mbo II fragments of the cDNA insert representing the 3’ and 5’ ends of the gene hybridize to most of the same fragments and do so with approximately the same relative intensities. Data presented by McKeown et al. (1978) indeed show that there is some heterogeneity in the genome regions complementary to actin mRNA. This is also clear from heterogeneity in Hap II and/or Hae Ill restriction sites within the actin “genes” (see Figures 6 and 8). At the present time the biological significance of this heterogeneity is not understood, since we do not know how many or which actin mRNA complementary regions are transcribed in vivo. From our analysis of the two actin mRNA forms and the multiple actin proteins, it seems reasonable that at least two of the “genes” are biologically functional. Characterization of Two Actin mRNA Forms When pulse-labeled RNA is hybridized to plasmid

Cell 774

M6, two poly(A)+ RNA species which differ in length by approximately 100 nucleotides are isolated. The two mRNAs must be related in primary sequence since they hybridize along their entire length to the same region of M6. The size of the 1.7 kb Hae lllHap II fragment precludes the possibility that the two mRNAs are complementary to different sequences each 1.2 kb in length. Moreover, additional results indicate that both mRNAs hybridize to pcDd actin Bl carrying a single insert complementary to actin mRNA (M. McKeown and R. A. Firtel, unpublished observations) and hybridize to pDd actin 2 (see McKeown et al., 1976). When the two regions containing the two forms of actin mRNA are excised from a urea-polyacrylamide gel in which total poly(A)+ RNA has been sized by electrophoresis and these fractions are translated in vitro, the higher molecular weight mRNA synthesizes a protein which co-migrates with the more acidic major form of Dictyostelium (labeled A,,) (Figure 9). The lower molecular weight mRNA directs the in vitro synthesis of a protein which co-migrates with the more basic major actin protein (labeled A,). The simplest explanation for these data is that the two mRNAs are transcribed from independent genes and code for two forms of Dictyostelium actin in a manner similar to the different mRNAs for the various forms of mammalian actin (Hunter and Garrels, 1977). It is possible that the two mRNAs could be derived from the same gene by differences in transcription or processing and that the different proteins might somehow reflect these differences. This is improbable, but we have not excluded the possibility because we have not shown primary nucleotide sequence differences in the RNAs. It IS of interest that A, and A,, are not the only prominent spots on these two-dimensional gels; this is somewhat surprising since actin mRNA is rather abundant and these fractions were selected as being enriched in actin message. The two RNA fractions stimulated incorporation of 35S-met in the MDL in vitro system lo-20 fold versus 300 fold for pure poly(A)+ RNA message. This low rate is partially due to the fact that the RNAs were isolated from gels and were not at optimal concentration. Although some of the spots are endogenous to the MDL system, this explanation does not account for all of them. The actin mRNA fractions excised from the gels were identified by alignment with 32P-actin mRNA run in the gel and localized by autoradiography. Any inaccuracy in the alignment might result in fractions which are less enriched in actin mRNA and more enriched in other less abundant messages. Examination of the subcellular localization and relative stability of the two RNA forms did not

reveal any differences. Both forms are present in equal concentrations in pulse-labeled nuclear, cytoplasmic and polysomal fractions and neither was preferentially stable after a 2-3 hr chase period. Neither form was more prominent in a 5 min pulse or a 2 hr pulse. In our experiments, no higher molecular weight nuclear actin mRNA precursor in the poly(A)+ or poly(A)- fractions was detected. It is quite possible that a very transient precursor would not be seen (Kindle, 1978). The results presented here indicate that actin mRNA complementary regions are repeated approximately 15 fold in the genome and that they are not organized in a classic repeat-common spacer arrangement. From the analysis of the actin mRNA, we feel that at least two such genes are active and code for related proteins which show differences in their primary structure. To fully elucidate the structural organization of all the actin “genes” it will be necessary to isolate additional clones carrying genome sequences and additional clones carrying cDNA inserts. At that point, it should be possible to determine which genomic sequences are transcribed by comparing fine structure restriction mapping and sequencing data of the genomic “genes” with actin cDNA inserts. Experimental

Procedures

Strains Dictyostelium discoideum is a wild-type strain NC-4 (Raper. 1935) and Ax-3 an axenic mutant of NC-4 derived by W. F. Loomis, Jr. (Loomis, 1975). The cloning and maintenance of plasmid stocks have been carried out with E. coli C600 (rr-, mr-) derived strains. For minicell experiments, strain P676-54 (Adler et al., 1967) was used. Chemicals Water and all buffers used with RNA were treated with 0.1% diethylpyrocarbonate (DEP, Eastman) and autoclaved before use. Chemicals were reagent grade. Seakem agarose was used for gel electrophoresis; ultra-pure urea was from Schwartz/Mann and sucrose (ribonuclease-free) was from Sigma. Formamide (MCB) was deionized by stirring with mixed bed ion exchange resin shortly before use. Kodak No-screen film or RP X-Omat film and intensifying screens were used for autoradiography. Enzymes All restriction enzymes were prepared from methods obtained from R. Roberts (R. Roberts, 1976; and personal communication). Some commercial preparations from New England Biolabs were also used. All other enzymes were commercial: DNA polymerase I (Boehringer-Mannheim), polynucleotide kinase (PL Biochemicals) ribonuclease A and trypsin (Worthington), proteinase K (E. M. Labs) and pronase (Calbiochem). Radkchemkals Carrier-free s*P-O, in HCI-free water was from New England Nuclear Corporation (NEN). #P-deoxyribonucleotide and aa2Pribonucleotide triphosphates from NEN had a specific activity of 120-250 CVmmole. +*P-ATP was produced by the method of Maxam and Gilbert (1977).

Dictyostelium 775

Isolation

Actin

Genes

and Screening

of Recombinant

Plasmids

To make recombinant plasmids carrying Dictyostelium genomic DNA fragments, nuclear DNA was randomly sheared and inserted into pMB9 using the poly(dA)-poly(dT) tailing procedure (Jackson, Symons and Berg, 1972; Lobban and Kaiser, 1973; Wensink et al., 1974) as described previously (Cockburn, Newkirk and Firtel, 1978). DNA was size-fractionated on sucrose gradients containing 1 .O M NaCI, 10 mM EDTA (pH 8.4) and an 8 kb (average) cut was taken. The DNA was resected with A exonuclease ((PC. 30 min) (Lobban and Kaiser, 1973) as described previously (Cockburn et al., 1978). Plasmid pMB9 DNA was cleaved with a 5 fold excess of Eco Rl sufficient to give a limit digest for 2 hr followed by an additional 5 fold excess of enzyme for an additional 1 hr. After phenol-CHCI, extraction and ethanol precipitation, the plasmid DNA was resected with A exonuclease as described above. Poly(dA) tails were then added to the genomic DNA and poly(dT) tails to the pMB9 DNA using terminal transferase (a gift from W. Salser) according to Wensinket al. (1974). We found that a homopolymer length of -50 nucleotides on both insert and vehicle gave the best transformation efficiency. Plasmid DNA was transformed into E. coli HBlOl as described previously (Cockburn et al., 1978). Bacteria carrying recombinant plasmids were screened for those which were complementary to moderate-high abundance mRNA according to the method of Grunstein and Hogness (1975). 8 X lOa cpm of azP pulse-labeled poly(A)+ RNA from vegetative cells (Fidel and Lodish, 1973) in 1 ml of 55% deionized formamide, 4 x SSC, 0.18 M sodium phosphate, IO mM EDTA (pH 8.8), 0.2% SDS and 1 mg poly(A) were used for each filter. The filters were exhaustively washed in hybridization buffer: poly(A) was not included and technical grade formamide was used. Positive colonies were detected by autoradiography.

Growth

and Development

of Cella

Dictyostelium Ax-3 cells were grown in suspension culture in HL5 medium or in MES buffered HL-5 for labeling with 5*P-0, (Cocucci and Sussman, 1970; Firtel and Lodish, 1973). NC-4 cells were grown on SM agar using a lawn of Klebsiella aerogenes as the food source (Sussman, 1966). For development, cells were washed several times and then resuspended in pad dilution fluid (PDF) (Sussman, 1966; Newell and Sussman. 1989) plus antibiotics (200 fig/ml streptomycin, 100 fig/ml each of chloramphenicol, kanamycin, tetracycline, rifampicin and erythromycin). In experiments which involved labeling cells with 3*P-0,, PDF was buffered with 8 mM MES rather than PO, (Firtel, Baxter and Lodish, 1973). Cells were spread at a density of 4 x lOa-1.5 x 10’ cells per cm* on Whatman 50 filters, which have as an underpad two Whatman 3 filters saturated with PDF. Filters were kept in a humid environment and underpads were changed after about 10 hr of development. Bacteria were grown in L broth or MB casamino acid media (Roberts et al., 1963). For some preparations of plasmid DNA, cells were grown to a Klett of 100 and then 200 pg/ml of chloramphenicol were added and cultures were incubated for an additional 18 hr to allow plasmid amplification (Clewell and Helinski. 1972; Hershfield et al., 1974).

Preparetlon

of DNA

Dictyostelium nuclei were isolated as described (Firtel and Lodish. 1973) and DNA was extracted from nuclei as described by Firtel and Bonner (1972) and modified by Firtel et al. (1976), except that in some cases lysed nuclei were digested with 0.7 mg/ ml predigested pronase [predigested in 0.05 M CaCI,. 0.05 M TrisHCI (pH 7.4) 3PC for 1 hr] in 0.1 M EDTA (pH 8.4). 1% sarcosyl at 50°C for 3 hr. Plasmid DNA was isolated using CsCllethidium bromide equilibrium centrifugation of a high speed supernatant of lysed cells (cleared lysate. Clewell and Helinski, 1972; Katz, Kingsbury and Helinski, 1973).

RNA Purlflcatlon RNA was isolated from whole cells or cell fractions as described previously (Firtel and Lodish. 1973) or by the method of Alton and Lodish. (1977). Poly(U) Sepharose (PL Biochemicals) or oligo(dT)cellulose (T3. Collaborative Research) was used for the purification of poly(A)+ RNA. RNA was passed over the column twice in binding buffer [0.4 M NaCI, 10 mM Tris-HCI (pH 7.4), 1 mM EDTA, 0.2% SDS, 10% formamide], washed in 0.5 x binding buffer and eluted with 70% formamide for poly(U) Sepharose or water for oligo(dT)-cellulose and precipitated with 2.5 vol of ethanol and 108 pg E. coli tRNA (Boehringer-Mannhein) as carrier.

In Vlvo Labeling

of Cells

The RNA of vegetative cells was labeled by harvesting exponentially growing Dictyostelium Ax-3 and cells and resuspsnding them in MES buffered HL-5 media at a concentration of 2 x IO’ per ml, adding 2-3 mCi/ml ‘*P-O,, incubating for 2-3 hr and preparing RNA as described above. Cells were washed in ice cold HL-5 medium before extracting RNA to remove as much free 32P0, as possible. Cells were labeled during development as described previously (Firtel et al., 1973) using 3-5 mCi or ‘?P-0, in 1 ml per 12.5 cm filter. For proteins, cells on 47 mm filters were labeled for 30 min with 20-50 &i of Yt-methionine as described (Firtel et al., 1973). Cell extracts were immediately prepared for one- or two-dimensional gel electrophoresis (Laemmli, 1970; Garrels and Gibson, 1976).

In Vitro Labeling To label RNA to a very high specific activity, RNA was treated with alkali and end-labeled in vitro with y-12P-ATP using polynucleotide kinase (Maizels. 1978; Maxam and Gilbert, 1977). To prepare radioactive RNA complementary to plasmids or plasmid fragments in vitro (cRNA), 0.5-3 pg of DNA were incubated with &*P-ribonucleotide triphosphates and E. coli RNA polymerase under conditions described by Barnes et al. (1975). DNA used as probe for hybridization experiments was labeled by nick translation (Schachat and Hogness, 1973; Maniatis et al., 1975) using u-3zP-deoxyribonucleotide triphosphates and DNA polymerase I (Boehringer-Mannheim). Specific activities in excess of 2-3 x 1O’cpmlpg were achieved using these procedures. cRNA and nick-translated DNA preparations were extracted with 1:l phenol:chloroform and all probes were passed over 20 ml G-50 or G-75 Sephadex to remove unincorporated nucleotide label. They were then ethanol-precipitated using 250 pg E. coli tRNA as carrier.

Rertrktion

Endonuclease

Digestions

Restriction enzyme digestions were generally carried out with a two fold excess of enzyme over that required to digest the DNA, in a small volume (50 ~1) for lo-16 hr at 3PC. Digestions with Mbo II, Hae Ill and Hap II were performed in 8 mM Tris-HCI (pH 7.4), 8 mM NaCI. 8.8 mM Mg acetate, 8 mM 2-mercaptoethanol; Hind II buffer contained 10 mM Tris-HCI (pH 7.9). 60 mM NaCI. 6.6 mM Mg acetate, 6 mM 2-mercaptoethanol; Hind III buffer was 7 mM Tris-HCI (pH 7.4), 80 mM NaCI, 7 mM Mg acetate, 7 mM 2mercaptoethanol. Eco RI digestions were carried out in 100 mM Tris-HCI (pH 7.4), 50 mM NaCI. 7 mM Mg acetate; Pst I buffer was 20 mM Tris-HCI (pH 7.4) 10 mM MgC&, 50 mM (NH&SO, and 6 mM 2-mercaptoethanol. Double digestions were carried out using the enzyme in the lower salt buffer first, after which the buffer was adjusted to conditions for the second enzyme. 10 fig of tRNA were added as carrier and digestions were ethanol-precipitated and resuspended in 0.1 x gel buffer, 0.5% SDS, 20% ficoll and heated at 80°C for 30 set before loading onto agarose gels. Conditions for restriction enzyme digests were obtained from R. J. Roberts (personal communication; Roberts, 1978). Agarose gels, usually 0.6-l .2%, were prepared and run in 4 mm thick slabs (14 x 20 cm) with fourteen slots per gel, using Tris-borate-EDTA as buffer (Helling, Goodman and Boyer. 1974).

Cell 776

To visualize DNA bands, gels were stained with 0.1 *g/ml ethidium bromide and illuminated with long wavelength ultraviolet light. Polyacrylamide gels (4-6%) were crosslinked with bisacrylamide and run in the same buffer. Hind Ill-digested A DNA (Allet and Bukhari. 1975) and Eco RI-digested Dictyostelium nuclear DNA (Firtel et al., 1976) were used as molecular weight markers. Restriction fragments were isolated from preparative gels using a simplified freeze-squeeze method (A. F. Cockburn, W. C. Taylor and R. A. Fidel, manuscript in preparation), passed over SP Sephadex (C25) in 0.2 M Na acetate (pH 4.5) and precipitated with 2.5 vol of ethanol. Preparation of DNA Blot Filters Size-fractionated, restriction enzyme-digested directly onto nitrocellulose filters using the (1975).

DNA was eluted method of Southern

Hybridizations Hybridization experiments with DNA immobilized on filters were carried out in siliconized glassware. Filters to be used for hybridizations with nick-translated DNA were pretreated with W Denhardt’s solution in hybridization buffer (Denhardt, 1966) for at least 8 hr before hybridization, which was carried out in 0.24 M PB (PB is equimolar N&HPO, and NabPO,) and 0.2% SDS at 60°C. Hybridizations were generally performed for 40 hr with gentle agitation. Washing of filters took place in hybridization buffer at the criterion of the hybridization, and washing solution was changed 5-8 times over a number of hours. Autoradiography was performed at room temperature with Kodak No-screen film or with intensifying screens and preflashed RPX-Omat film at -70°C (Bonner and Laskey, 1974; Laskey and Mills, 1975,1977). Hybridizations in solution were performed in sealed microcapillary pipettes as described (Firtel and Bonner, 1972) using NaCI. 10 mM TES buffer, 1 mM EDTA, 0.2% SDS at 20°C below the Tm. NaCl concentrations varied in experiments and all data were corrected for rate differences and plotted as equivalent Cots normalized to 0.18 M NaCl (Britten, Graham and Neufeld, 1974). For DNA excess hybridization experiments, the DNA was prepared by sonicating intermittently at a setting of 6 on a Heat Systems Ultrasonics sonifier in 1 M NaCI, 10 mM EDTA (pH 8.4) on ice. DNA was then ethanol-precipitated, passed over SP Sephadex (C25) in 0.2 M Na acetate (pH 4.5, adjusted to pH 9) extracted with 1 :l phenol:chloroform and precipitated twice with ethanol. Assays by Sl nuclease (for DNA excess hybridization experiments with nick-translated DNA probe) were performed in 0.18 M Na+, 2 mM ZnSO, (pH 4.7) using acetate buffer for 45 min at 42°C. 6 units of Sl nuclease were used for 50 rg of DNA; all samples were precipitated with 10% TCA and 400 rg crude yeast RNA carrier, and filtered through Millipore filters. Data are expressed as percentage of Sl nuclease resistance relative to an undigested sample. To monitor renaturation of the nick-translated probe, controls were run in which tRNA replaced driver DNA, and these were assayed in the same manner. Preparatlon of DNA Filters and Hybridization To immobilize DNA on filters, 50 rg of plasmid DNA was nicked by mild depurination [0.2 M Na acetate (pH 4.2) for 15 min at 55”C]. denatured in alkali at room temperature, placed on ice and neutralized, and then filtered over 47 mm diameter Millipore nitrocellulose filters in 6 x SSC (Gillespie and Spiegelman, 1965). Filters were baked under vacuum for 2-3 hr at 60-65°C and seven 10 mm diameter filters were punched from the larger one. RNA was hybridized in 4 x SSC, 0.12 M phosphate buffer, 10 mM EDTA (pH 7.0), 1 mg/ml poly(A), 55% formamide, 0.2% SDS at 3PC for 36-48 hr. Washing of filters was carried out in hybridization buffer lacking poly(A) at the criterion of the hybridization, and washing solution was changed 5-8 times, once per hour. Isolation of Plasmid Complementary To isolate plasmid complementary

RNA RNA, DNA filters

were

hybrid-

ized with RNA as outlined above and washed extensively with hybridization buffer. The filters were then washed twice in 0.045 M NaCl at 55-60°C to remove nonspecifically-bound RNA, and the remaining hybridized RNA was eluted in 70% formamide, 5 mM EDTA (pH 7.2) at the same temperature. RNA to be used for in vitro translation was ethanol-precipitated twice with 6 pg yeast tRNA carrier (Boehringer-Mannheim); other RNA preparations were ethanol-precipitated with 50-100 fig E. coli tRNA. Analytical Gel Methods Proteins were separated on polyacrylamide-SDS gels using a ratio of 30% polyacrylamide:0.8% bisacrylamide as crosslink (Laemmli. 1970). Two-dimensional gels were run as described by Garrels and Gibson (1976; also personal communication). Electrofocusing was performed using a narrow range ampholite pH 5-7, and the second dimension was electrophoresed on 10% polyacrylamide gels. Gels were infiltrated with PPO, dried and fluorographed with preflashed Kodak RP X-Omat film as described (Bonner and Laskey, 1974; Laskey and Mills. 1975). RNA was analyzed on urea-containing polyacrylamide gels according to Spradling et al. (1977). In Vltro Translation Systems The wheat germ cell-free system was a gift from T. Hunter and G. Ma, who prepared it as described (Hunter et al., 1977). The rabbit reticulocyte messenger-dependent lysate (MDL) was a gift from T. Hunter, who prepared it according to Pelham and Jackson (1976). RNA was translated with Y9-methionine labeled as described in these references, and incorporation was monitored by placing aliquots in 0.1 M KOH, precipitating with TCA in the presence of excess unlabeled methionine, and filtering on Whatman GF/C filters. Tryptk Peptide Maps In viva or in vitro labeled proteins were size-fractionated on 12.5% polyacrylamide, 0.1% bisacrylamide gels (Laemmli, 1970). The gels were then dried and autoradiographed, and the appropriate bands were cut from the gel and fingerprinted as described by Garrels and Gibson (1976) and Gibson (1974). Mlnkell Experiments Plasmids were transformed into minicell strain P678-54 as described (Cohen, Chang and Hsu. 1972), and minicells were isolated and labeled with SsS-methionine using the procedure of Matsumura et al. (1977). Acknowledgments We are extremely grateful to T. Hunter for assistance in running the tryptic peptides and for the gift of rabbit reticulocyte messenger-dependent lysate; to J. Garrels and C. MacLeod for assistance in running the two-dimensional gels and the DNAase I binding experiments: to P. Matsumura for supplying the minicell strain and advice on fractionating minicells and whole cells and to C. Lawrence for DNAase I agarose. The experiment shown in Figure IC was carried out by C. MacLeod; we thank C. MacLeod for allowing us to use this Figure. We thank A. Kimmel and M. McKeown for their critical readings of the manuscript. K.L.K. was the recipient of an NSF predoctoral fellowship and was also supported by an NIH predoctoral training grant. R.A.F. is the recipient of an American Cancer Society Faculty Research Award. This research was supported by grants from the NSF, the American Cancer Society and the NIH to R.A.F. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

April 20, 1978;

revised

July 31, 1978

Dictyostelium 777

Actin

Genes

References

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Adler, H. I., Fisher, W. D.. Cohen, A. and Hardigree, A. A. (1967). Miniature Escherichia co/i cells deficient in DNA. Proc. Nat. Acad. Sci. USA 57, 321-326. Allet. B. and Bukhari, A. I. (1975). and A-mu hybrid DNAs by specific 92, 529-540.

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Alton. T. H. and Lodish, H. F. (1977). Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoidew. Dev. Biol. 60, 160-206. Bailey, J. M. and Davidson, N. (1976). ible denaturing agent for agarose Biochem. 70, 75-65.

Methylmercury as a reversgel electrophoresis. Anal.

Barnes, W. M.. Reznikoff, W. S., Blattner. F. C., Dickson, and Abelson, J. (1975). The isolation of RNA homologous genetic control elements of the lactose operon. J. Biol. 250, 6164-6189.

R. C. to the Chem.

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