Cell,

Vol. 15, 789-800,

November

1975. Copyright

0 1978 by MIT

Multiple, Heterogeneous Dictyostelium Michael McKeown, William C. Taylor,* Karen L. Kindlet and Richard A. Firtel$ Department of Biology University of California, San Diego La Jolla, California 92093 Welcome Benders and Norman Davidson Departments of Biology and Chemistry California Institute of Technology Pasadena, California 91125

Summary We have used an actin gene-containing restriction fragment of plasmid M6 (Kindle and Firtel, 1976) to select a second actin gene-containing plasmid which we have named pDd actin 2. This plasmid has been shown to’contain two actin genes separated by 350 bp of nonactin DNA. When heteroduplexes are formed between any two of the three actin genes present in chimeric plasmids, the region of homology is 1100 + 100 bp. This is close to the minimum length required to code for actin protein. The 1100 bp region of intergene homology corresponds to the 1100 bp homology observed between M6 and the two actin cDNA plasmids pcDd actin Bl and pcDd actin Al (Bender et al., 1976). We have no evidence for additional sequences common to either the 3’ or 5’ ends of the 1100 & 100 bp region of intergene homology. Thermal denaturation experiments show that different pairs of actin genes are diverged from each other by as much as 66%. There are two size classes of mRNA complementary to the three actin genes. These have lengths of 1.25 and 1.35 kb as determined on methyl mercuric hydroxide-containing agarose gels. The possible linkage of these three actin genes to other actin genes is discussed. Introduction The previous papers (Kindle and Firtel, 1978; Bender et al., 1978) have described the isolation and characterization of a recombinant plasmid (M6) containing a 6 kb insert of Dictyostelium DNA which includes a region of approximately 1 .l kb homologous to actin mRNA. This region is repeated approximately 15 times in the genome, as judged by both hybridization kinetics and hybridization to DNA blots of restriction enzyme digests of * Present address: Department of Genetics, University of California, Berkeley, California 94720. t Present address: Department of Chemistry, California Institute of Technology, Pasadena, California 91125. $ To whom all correspondence should be sent. 5 Present address: Department of Biochemistry, Stanford University, Stanford, California 94305.

Actin Genes in

nuclear DNA. Restriction analyses indicated that there was heterogeneity in the primary sequences of these complementary regions. It was also shown that these actin mRNA complementary regions were not tandemly repeated with homogeneous spacer regions, although other linkage patterns could not be excluded. We will refer to the chromosomal regions complementary to actin mRNA as actin genes, although we have not confirmed that any particular one of these regions is transcribed in vivo or gives rise to the transcript subsequently translated into actin. In this paper, we report the isolation of a second actin mRNA complementary plasmid containing two closely linked actin genes. These genes are complementary to the 1 .l kb actin gene region of plasmid M6, but there is considerable sequence heterogeneity between the three gene regions. We also present evidence which suggests that there is an additional actin gene in the genomic region adjacent to the actin gene present in M6. Results Isolation of a Second Recombinant Plasmid Carrying Sequences Complementary to Actin mRNA A population of approximately 3000 chimeric plasmids carrying Dictyostelium nuclear DNA sequences inserted into plasmid vector pMB9 (see Kindle and Firtel, 1978) had previously been screened by the colony filter hybridization technique for those which contain sequence complementary to vegetative poly(A)+ mRNA. Approximately 160 colonies (in addition to those reported previously; Kindle and Firtel, 1978) showed hybridization levels expected for sequences complementary to high and middle abundance mRNAs. Since the actin genes are reiterated approximately 15 fold and since actin mRNA is a highly abundant mRNA, these 160 colonies were screened for those which are complementary to the M6 actin gene region. One such plasmid was found and has been denoted pDd actin 2. A restriction map of this plasmid is shown in Figure 1, along with the map of M6 (Kindle and Firtel, 1978; Bender et al., 1978) for reference. Note that the pMB9 vehicle sequences are in opposite orientations in the two maps. It can be seen that the maps for pDd actin 2 and M6 are not superimposable, indicating that they contain different’ segments of the Dictyostelium genome. pDd Actin 2 Contains Two M6 Complementary Regions Hybridization to DNA blots (Southern, 1975)

indi-

Cell 790

Sub2

I 0

I

,

2

Sub I

3

that both the sub 1 region and the sub 2 region contain an actin gene. Despite the fact that bands in different slots of these filters are not present in equimolar amounts, it is clear that bands hybridize noticeably better to homologous than to heterologous probes. This is especially true for M6 and sub 2, which cross-hybridize very poorly, and implies that considerable heterogeneity exists among the three genes.

I

4

5

I 6

kb

Figure

1. Restriction

Maps

of pDd Actin

2 and M6

The restriction sites shown in pDd actin 2 were mapped by conventional methods involving single and multiple restrictions of plasmid DNA followed by electrophoresis on 1.2% agarose gels. In addition to the enzymes shown, Pst I, Sal I and Hind II were used but did not cleave within the insert region. Hinf I, Mbo I and Alu I cleave the insert many times but the sites have not been mapped. The M6 map is taken from Bender et al. (1978). Bars underneath the restriction maps indicate the regions of homology seen in heteroduplexes between the plasmids when observed in the electron microscope. The small arrows represent the direction of transcription (Bender et al.. 1978). The regions labeled sub 1 and sub 2 above the pDd actin 2 map indicate the regions that have been subcloned into pDd actin P-sub 1 and pDd actin P-sub 2. respectively. Note that the pMB9 vehicle is in an inverted orientation in the two maps.

cated that the M6 actin gene was complementary to the two leftmost Hind III fragments of pDd actin 2. At the 5’ end of the gene in M6, there are a Hind III restriction site and a Hap II restriction site which are separated by approximately 50 bp. Two similar pairs of Hind Ill and Hap II sites are present in pDd actin 2; the separation between the Hind III sites and the Hap II sites is again approximately 50 bp. This similarity in restriction sites suggested to us that pDd actin 2 might contain two actin genes. To test this possibility, the central 1 .4 kb Hind III fragment and the leftward 1.3 kb Hind III fragment were subcloned into the Hind Ill site of the plasmid pBR322 (Bolivar et al., 1977). The resulting plasmids were designated pDd actin 2-sub 1 and pDd actin 2-sub 2, respectively. Figure 2 shows hybridization of the 1.4 and 1.3 kb Hind III fragments from these two plasmids, plus the 1.7 kb Hae Ill-Hap II actin gene piece from M6, to Southern blots made from digests of M6, pDd actin 2, pDd actin 2-sub 1 and -sub 2. The digests of pDd actin 2 have a few visible bands remaining after partial digestion by our Hind III. The hybridization to pBR322 vehicle sequences is probably due to slight contamination of our probes with pBR322 sequences. As can be seen in this figure, both the 1.4 and 1.3 kb Hind III fragments are homologous to the M6 actin gene region; these fragments also contain sequences which cross-hybridize. It is probable, therefore,

Location of M6 Complementary Regions on pDd Actin 2 The locations and lengths of the two actin genes in pDd actin 2 were determined more accurately by using electron microscopy to examine heteroduplexes between pDd actin 2 and M6. When linearized pDd actin 2 and M6 DNAs are hybridized, most of the visible heteroduplexes show hybridization between the pMB9 vehicle sequences without hybridization of the inserted sequences. H forms, when observed, show no hybridization of the vehicle sequences, indicating that the actin genes in the two plasmids have the opposite polarities relative to the pMB9 vehicle. To analyze the actin genes more easily, Barn HI-digested pDd actin 2 was hybridized first to Barn HI-digested pMB9 and then to the central Eco RI fragment of M6 which includes the M6 actin gene. Figure 3 shows such a heteroduplex. There are two regions on pDd actin 2 complementary to the M6 actin gene; both are 1 .l ? 0.1 kb in length. The 5’ to 3’ orientation of the pDd actin 2 actin gene is deduced from the known orientation of the M6 actin gene (Bender et al., 1978); both pDd actin 2 genes are in the same orientation and each has the Hind III and Hap II restriction sites near the 5’ end of the actin gene. The region of M6 homologous to either pDd actin 2 actin gene corresponds exactly to the region homologous to the two actin cDNA plasmids, pcDd actin Al and pcDd actin Bl (Bender et al., 1978). There is no indication of additional common sequences on either side of the cDNA complementary region. The two actin genes of pDd actin 2 did not, however, appear to be identical. Heteroduplexes with only the right (sub 1) actin gene hybridized to an M6 Rl fragment were seen about 5 times as often as heteroduplexes with only the left (sub 2) actin gene hybridized or with both actin genes hybridized (as in Figure 3). In addition, there was a reproducible branch migration intermediate observed in which one M6 actin gene was hybridized to parts of both pDd actin 2 actin genes. As shown in Figure 3, the M6 fragment is hybridized to 0.9 kb of the sub 1 actin gene at the 5’ end and to 0.2 kb of the sub 2 actin gene at its 3’ end. The remaining unhybridized regions of both pDd actin 2 genes

Multiple 791

Actin

P;

Genes

2

3 4

in Dictyostelium

19.2 3

4

f.2

3 4

D. I234

M6

.‘liar-

SUE’ t -Y4ubZ

PROBES Figure 2. Hybridization of Various Actin mids with Different Labeled Actin Genes

Gene-Containing

Plas-

Plasmids pDd actin 2. pDd actin P-sub 1, pDd actin P-sub 2 and M6 were digested with different restriction enzymes. Identical aliquots were layered onto 4 mm 1.2% agarose slab gels such that three groups of four slots were formed, each group containing one aliquot from each of the four digestions. Note that not all bands are present in equimolar amounts. After electrophoresis, Southern blot filters were made from each group and a filter was challenged with either a 32P 1.7 kb Hae Ill-Hap II fragment from M6. or a 32P 1.4 kb sub 1 fragment or 3nP 1.3 kb sub 2 fragment. The order of samples within each group is (1) Hind Ill-pDd actin 2; (2) Hind Ill-pDd actin P-sub 1; (3) Hind Ill pDd actin P-sub 2; (4) Hae Ill plus Hap II M6. The 32P-labeled hybridization probes were (6) 1.4 kb Hind III fragment of sub 1; (C) 1.3 kb Hind III fragment of sub 2; (D) 1.7 kb Hae Ill-Hap II fragment of M6. A photograph (A) of one of the ethidium bromide-stained gels is included. The left-hand channel contains a Hind Ill-Eco RI double digest of phage ,+ DNA.

plus the intervening region are contained in the single-stranded loop. Since this particular configuration is stable enough to be seen reproducibly, the 5’ end of the sub 2 actin gene must be more highly diverged from the M6 actin sequence than the sub 2 actin 3’ end. Heteroduplexes between the two subcloned Hind III fragments of pDd actin 2, sub 1 and sub 2, were made to measure the length of homology between the two actin genes and to determine whether there is any homology between the regions contiguous to the 3’ ends of these genes. In the chimeric plasmids pDd actin 2-sub 1 and pDd actin 2-sub 2, the two fragments proved to be inserted into the plasmid vehicle with opposite polarity. Most of the

heteroduplexes formed were similar to those shown in Figure 4C in which only the plasmid vehicle forms a duplex. Another plasmid, pDd actin 2-sub la, contained the sub 1 fragment inserted into the vehicle with the same polarity as sub 2. Heteroduplexes between sub la and sub 2 resulted in structures similar to that shown in Figure 48. The plasmid vehicle and the actin genes are paired, but the regions contiguous to the 3’ ends of both genes show no homology. The region of homology between sub 1 and sub 2 is 1.1 1 0.1 kb. This measurement was obtained by subtracting the length of the vehicle extending from the Barn HI site to the insertion site (determined from sub 11 sub 2 heteroduplexes) from the length of the shorter duplex region in sub la/sub 2 heteroduplexes. There is again no evidence for any homology other than the intergene homology. We assume that 1 .l kb is the length of the acrin gene since this is the length of the region of homology shared between the three actin genes we have isolated to date. As noted by Bender et al. (1978), it is also the length of the region defined by two cDNA plasmids to actin mRNA. Two repeated gene families in Drosophila melanogaster have been shown to have short direct repeat sequences flanking each gene copy (Finnegan et al., 1977). The actin gene family was examined for terminal repeats, but neither direct nor inverted repeats were found. As shown in the Southern DNA blot hybridization (see Figure 2D, lane 4), a restriction fragment of M6 which includes the 3’ end of the actin gene does not show hybridizaton to the 0.6 kb fragment of M6 which includes the 5’ end. Heteroduplexes between a fragment of M6 containing the 5’ end of the actin gene (from the Hind III site near the 5’ end to the Barn HI site in pMB9) and either pDd actin 2-sub la or pDd actin 2-sub 2 (which both include actin 3’ ends) showed no 5’ end to 3’ end hybridization. Inverted repeats flanking the actin genes were never seen in single-stranded molecules of M6 or pDd actin 2 under conditions where the poly(dA):poly(dT) sequences at the vector-insert junctions were frequently paired. There is no evidence for any inserted, deleted or rearranged segments in any of the three genes relative to the cDNA plasmids or to each other. If any rearrangements exist, they must be smaller than the minimum detectable length in the electron microscope (- 75-100 bp). Length of mRNA Complementary to Actin Genes Dictyostelium actin co-migrates with rabbit 01 actin on one-dimensional SDS-polyacrylamide gels (C. MacLeod and R. A. Firtel, unpublished observation). Rabbit u actin is composed of 374 amino

Cell 792

acids (Elzinga et al., 1973) which require a minimum coding length of 1128 bp. This length is very close to the 1 .l kb intergene homology for our three actin genes. A previous paper (Kindle and Firtel, 1978) has identified two species of actin mRNA in Dictyostelium which differ in length by about 100 bp. For a better determination of mRNA length, M6, pDd actin 2, sub 1 and sub 2 complementary RNAs were electrophoresed on a methyl mercuric hydroxide-containing 1.8% agarose gel (Bailey and Davidson, 1976). Phage T7 early RNA (Studier, 1973; Simon and Studier, 1973) was used as a molecular weight standard. The results are shown in Figure 5. Two bands are present that have lengths of 1.25 and 1.35 kb. These lengths are identical for RNA complementary to all four kinds of DNA. The autoradiograph of the band representing the longer RNA appears to be darker than the band representing the shorter RNA in the four samples. Four faint bands are present above the two actin mRNA bands. The uppermost and lowest of these bands have mobilities equal to those of 26s and 17s rRNA, respectively. The nature of the middle two bands is unknown.

-

EcoRi

I kb

+ I

Heterogeneity of Actin Gene Sequences Hybridization to Southern blots and heteroduplex formation suggest that there is considerable heterogeneity among the three actin genes in our plasmids. To measure the degree of heterogeneity between different actin genes and between cloned and genomic actin sequences, radioactively labeled actin gene sequences were hybridized to an excess of unlabeled actin plasmid DNA or genomic DNA. The hybrids formed were bound to hydroxyapatite and eluted at increasing temperatures. Figure 6 shows the melting profiles for the different plasmids and Figure 7 shows the melting profile of M6 actin DNA hybridized to genomic DNA. Table 1 summarizes the data. The melting profiles are quite broad, especially at low temperatures, even for the melting of homoduplexes. We feel that this result is due at least in part to the use of nick-translated fragments-some of which are probably very short-as tracers. Table 1 shows that for a given pair of actin genes, the melting temperature of the heteroduplexes is essentially independent of which DNA is used as driver and which is used as tracer. The melting profiles of homoduplexes are surprisingly different for the three actin genes. The melting temperatures of the heteroduplexes are considerably depressed. MG-sub 1 hybrids show a melting temperature depression of about 4-6”C, MG-sub 2 hybrids a depression of about 7-9°C and sub l-sub 2 hybrids a depression of about 5-6X. These data are consistent with the idea that the three actin

Eco RI

I

phB9 A

tc +

BamHI \

Figure 3. Heteroduplexes 4.5 kb Eco RI Fragment

Formed of M6

between

pDd Actin

Eco RI

+ 2 and the

pDd actin 2 was first hybridized to Barn HI-digested pMB9 plasmid vehicle and then to the M6 fragment. This page: both actin genes in pDd actin 2 have hybridized to M6 fragments. Facing page: a single M6 fragment has hybridized to 0.9 kb of the 5’ end of the sub 1 actin gene and 0.2 kb of the sub 2 actin gene.

genes are diverged from each other, and implies that the order of relatedness between them is M6sub l-sub 2. The melting temperature depression of MG-genomic hybrids indicates that a large fraction of the genomic actin genes are diverged from the M6 sequence and not merely from the genes in pDd actin 2. Actin Genes May Be Clustered The fact that pDd actin 2 contains two actin genes suggests that other actin genes might also occur in clusters. This is also suggested by Southern DNA blot hybridization of the actin gene probe to various restriction enzyme digests of Dictyostelium

Multiple 793

Actin

Genes

in Dictyostelium

DNA. Hind III, which cleaves at or near the 5’ end of all actin genes examined so far, yields 17 bands when digested Dictyostelium DNA is hybridized with actin probes containing the central region of various actin sequences. Eco RI yields only 14 bands in similar experiments (Kindle and Firtel, 1978, and unpublished observations). To test whether there was another actin gene near one of our cloned actin genes, we identified restriction fragments complementary to the nonactin sequences flanking our cloned actin genes. We then determined whether these fragments contained an actin gene not contained in our clone. Total Dictyostelium DNA was digested with an enzyme known to yield a restriction fragment with one end homologous to our nonactin probe and one end outside the cloned piece. This DNA was electrophoresed in a single very wide (lo-15 cm) slot of an 0.8% 4 mm agarose slab gel, and a single Southern blot filter was made. This was marked with horizontal lines near the top and bottom and cut into ‘/4 inch strips. A set of adjacent strips was challenged with 32P-labeled actin gene DNA or 32Plabeled nonactin restriction fragment or a mixture of both. After the hybridization and washing were complete, the adjacent strips were realigned using the horizontal lines at the top and bottom of the strips. If the nonactin probe we used was part of a genomic restriction fragment which also contained an actin gene, then the band of hybridization for this sequence should have aligned with one of the bands of hybridization to the actin gene. The strip that was hybridized to both the actin gene and the nonactin probe should have shown the same bands as the actin gene alone, with no extra bands. It should be noted that this analysis also yielded

information on the location of restriction sites in the genomic regions adjacent to the segment of the genome included in the chimeric plasmid. We carried out such an analysis for the region to the left of the M6 insert. The leftward 2.5 kb Hae III fragment of M6, used as the nonactin probe, was hybridized to a Southern blot of Hae Ill-digested Dictyostelium DNA. The autoradiogram in Figure 8A shows weak hybridization to a number of bands, but strong hybridization to only a few-notably bands at 7, 5 and 1.6 kb. The 1.6 kb band is too short to have been derived from the same region as our probe, so our probe is probably contained in a Hae III fragment of either 5 or 7 kb in length. The 7 kb fragment can be seen to align with a strong band of actin DNA hybridization while the 5 kb fragment aligns with a weak but nevertheless repeatedly observable band of actin DNA hybridization. These two actin bands can be observed regardless of which actin gene probe is used. In either case, it appears probable that there is another actin gene on the genomic Hae III fragment to the left of the M6 actin gene. The data also suggest that the sequence contained in the 2.5 kb nonactin Hae III fragment of M6 is likely to be found on the same Hae III fragment as at least one more actin gene. Hind III, Hap II and Eco RI digests are very difficult to interpret due to the large number of bands showing weak hybridization and the fact that the region of homology between our probe and the outside restriction fragments is greatly reduced, lowering the intensity of hybridization to these bands. The possibility exists, nevertheless, that there are a Hind III site and a Hap II site about 1.7 kb to the left of the leftward Hind III site of M6 (data not shown). If these sites exist in the region

Cell 794

I

Figure 4. Heteroduplexes actin 2 pDd

Actin2-Subl

,,HindIlI )xFidiii ..__.........___....

~ . ..e

pDd ~

Actin

2-Sub

lo

. ,P

pDd

.. . . . . Actin

. . . .. . . . . . . . . . . . . .

.. .. .

. .

. .

.

.

. .. .. . .. . . . . . . . . . .

.

.

..

Barn HI

Sub’0 /

*

C.

Sub2

. . ..&A%.. .... + Barn HI

SubI

/

the Two

Actin

Genes

of pDd

(A) Schematic of the three hybrid plasmids used. The same Hind III fragment has been inserted with opposite polarity in pDd actin a-sub 1 and -sub la. The dotted line represents the plasmid vehicle, the heavy solid line the actin gene, and the light solid line sequences contiguous to the 3’ ends of the genes. (B) Heteroduplex between pDd actin P-sub la and pDd actin P-sub 2. (C) Heteroduplex between pDd actin 2-sub 1 and pDd actin P-sub 2.

2-Sub2

i.. . b-V

0.

between

. . . . . . . . . . .. .. . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . .

Sub2

i:::~::::::::::::::::::::::;::::::::; Barn HI

adjacent to M6, they might represent the 5’ end of an actin gene with the same polarity as the gene in M6. In analyzing the chromosomal regions around pDd actin 2, we have used both the 0.65 kb Hind Ill-

RI piece to the right of the sub 1 actin gene and the 5.5 kb Hind III-RI piece which contains the rightmost region of pDd actin 2. Using these two probes we have found no evidence for additional actin genes within 7 kb of the right-hand side of the pDd actin 2 insert. We have, however, been able to localize a number of nearby restriction sites. These are shown in Figure 9 along with a summary of the inferred mapping data. Despite the fact that we used a mixture of our three actin genes as the actin probe in these experiments, we observed that not all bands show the same intensity of hybridization. This phenomenon is even more evident when the three actin probes are hybridized separately as in Figure 88. The hybridization patterns for the 1.4 kb Hind III fragment from sub 1 and the 1.7 kb Hae Ill-Hap II fragment from M6 are quite similar. The pattern for

Multiple 795

Actin

Genes

in

Diclyostelium

tion). On the other hand, Kindle and Firtel (1978) have presented evidence that an actin-like protein is produced from plasmids M6 and pDd actin 2 in minicells. This evidence indicates that sequences in both of these plasmids are capable of coding for actin-like proteins. Given a large number of related genes, it is exceedingly difficult to prove that any particular one of the genes is or is not transcribed. If all of the genes have diverged from each other, fine structure restriction mapping and/or partial sequencing of actin genes may indicate at least some of the genes that are transcribed.

284

0.61-

T?

M6

pod A-2

SI

S2

T7

RNA Figure 5. Mobility of Actin Containing Agarose Gel

mRNA

in a Methyl

Mercuric

Hydroxide-

Poly(A)-containing in vivo labeled RNA from vegetative cells was hybridized to nitrocellulose-bound DNA, eluted and electrophoresed on a 1.6% agarose gel containing 5 mM methyl mercuric hydroxide. 32P-labeled T7 early RNA was prepared by the method of Studier (1973). M6, pDd A-2 actin 2, Si and 52 refer to mRNA complementary to M6, pDd actin 2, pDd actin P-sub 1 and pDd actin P-sub 2 DNA.

the 1.3 kb Hind III fragment from sub 2 is noticeably different. It is also interesting that there are certain bands that give weak hybridization to all three probes. Discussion Throughout this paper we have referred to the multiple actin mRNA complementary regions in the Dictyostelium genome as actin genes. At this time, we have no evidence that any of the three actin genes described in this paper are actually transcribed in vivo. Since the actin cDNA plasmid pcDd actin 61 has Hap II and Hae III restriction enzyme cleavage sites not present in any of our three actin genes, it is probable that at least one additional actin gene is functional (our unpublished observa-

Relationship between Moderately Repeated and Low Copy Number Sequences in Actin Clones The nuclear DNA of Dictyostelium is arranged in a short period interspersion pattern with 1200 bp stretches of single-copy DNA alternating with shorter stretches of moderately repeated DNA (Firtel and Kindle, 1975; Firtel, Kindle and Huxley, 1976). This is similar to the pattern observed in Xenopus (Davidson et al., 1973). pDd actin 2 and M6 have been shown to contain repeated DNA interspersed with low copy number sequences. In addition, analysis of heteroduplexes formed between different actin gene regions reveals that the length of homology between different actin genes is only 1 .I kb, approximately the minimum sequence length needed to code for actin. There is no evidence for additional common sequences present at either the 3’ or 5’ ends of the regions complementary to the cDNA plasmids pcDd actin Bl or pcDd actin Al (Bender et al., 1978). Either such sequences do not exist in the case of the three actin genes we have examined, or they do not form stable hybrids under our conditions. mRNA Size Relative to Estimated Gene Size We have estimated the length of intergene homology between actin genes to be 1 .l kb. This length is close to the minimum size necessary to code for actin protein. Two size classes of mRNA, having lengths of 1.25 and 1.35 kb, hybridize to our actin gene-containing plasmids. Kindle (1978) has shown that these two size classes bind equally well to oligo(dT)-celulose and probably have the same length of 3’ poly(A). Since the average length of poly(A) sequences at the 3’ ends of Dictyostelium mRNAs has been shown to be loo-125 nucleotides (Firtel, Kindle and Huxley, 1976), the 1.25 kb mRNA could easily be derived from a 1 .l kb gene. The 1.35 kb mRNA must contain sequences in addition to the 1.1 kb actin coding region and a 3’ poly(A) tail of loo-125 nucleotides. Several investigators have obtained evidence for insertions into the coding regions of genes known to be transcribed, such as the genes for globin

Cell 796

TEMP.‘C Figure

6. Melting

Curves

for Heteroduplexes

TEMP.OC

TEMPoc Formed

between

Various

Actin

Genes

Plasmids M6, pDd actin P-sub 1 and pDd actin P-sub 2 were randomly sheared by mild depurination and hybridized in vast excess (greater than 200 fold) to J*P-nick-translated actin gene containing restriction fragments. Hybridization was at 55°C in 0.12 M PB for at least 20 times the calculated Cot,,, for the unlabeled driver DNA. The samples were then bound to 1 ml hydroxyapatite-CF 11 columns in 0.12 M PB at 55°C and washed with 2 x 1 ml of preheated 0.12 M PB. All columns for a given unlabeled driver DNA were contained in the same variable temperature water bath. Unbound DNA waseluted with 2 x 1 ml of 0.12 M PB after 5 min of equilibration at the temperature of the indicated points. The number of counts not eluted at 55°C but eluted at temperatures up to 97.5-96X inclusive was taken as 100% of the amount bound. This number was at least 30,000 Cerenkov cpm in all experiments. (A) Ualabeled M6 driver: (B) unlabeled pDd actin P-sub 1 as driver; (C) unlabeled pDd actin 2-sub 2 as driver. ( 0..0)32P 1.7 kb Hae Ill-Hap fragment of M6 as tracer. (A-A)32P 1.4 kb Hind III fragment of sub 1 as tracer. (O- - -O)52P 1.3 kb Hind Ill fragment of sub 2 as tracer.

(Jeffreys and Flavell, 1977; Tilghman et al.: 1978), ovalbumin (Breathnach, Mandel and Chambon, 1977) and yeast tRNA (Goodman, Olson and Hall, 1977; Valenzuela et al., 1978). The 5’ ends of the late mRNAs from SV40 are transcribed from regions not immediately adjacent to the coding sequences (Aloni et al., 1977) and the late mRNAs from adenovirus have been found to be derived from regions of the genome that are separated by regions not included in the messages (Klessig, 1977; Berget, Moore and Sharp, 1977; Chow et al., 1977). A significant fraction of the 28s rRNA genes in Drosophila have been found to contain a lengthy insert not present in the 28s RNA (Wellauer and Dawid, 1977; White and Hogness, 1977; Pellegrini, Manning and Davidson, 1977). Electron microscopic examination of heteroduplexes formed between the three actin genes and two actin cDNA plasmids gives no evidence for such insertions in the actin genes we have studied. This does not eliminate the possibility that there are relatively small insertions, such as those found in yeast tRNA, but it does place an upper limit (75-100 bp) on the size of such insertions in these actin genes. The fact that actin-like protein can be synthesized in M6 and pDd actin 2-transformed minicells (Kindle and Firtel, 1978) suggests either that there are no inserts in the actin genes we have described, or that if there are inserts, they must be small or removed by processing in E. coli.

Heterogeneity of Actin Genes Three types of evidence suggest that there is significant heterogeneity among our three isolated actin genes. First, the intensity of hybridization to bands on Southern filters of chimeric plasmid DNA varies greatly depending upon whether a homologous or heterologous probe is used. Second, hybrids are observed five times more often between the sub 1 gene and M6 than between the sub 2 gene and M6 in the electron microscope. Third, heteroduplexes formed between the different actin genes show a significant depression of melting temperature relative to homoduplexes with as much as 7-9°C depression for sub 2:M6 hybrids, indicating a sequence divergence of up to 8% (Wetmur, 1976). In addition, melting profiles between the M6 actin gene and Dictyostelium genomit DNA indicate that there is an average 4°C melting depression between M6 and the other actin. genes. On account of the heterogeneity between the three cloned genes, we assume that this average Tm depression is not due to an anomaly of M6. Since the thermal stability of heteroduplexes was assayed by hydroxyapatite chromatography, our estimates of melting temperature depression are minimal estimates. The fact that branch migration intermediates were seen in heteroduplexes between the M6 actin gene fragment and pDd actin 2 suggests that some regions of actin genes have diverged less than other regions.

Multiple 797

Actin

Genes

in Dictyostelium

A. I234

B. 123

1234

(7.0)

c

6.0)

t

(5.0)

f’~464

(3.0)

Temp. OC Figure 7. Melting Profile of Fragment from M6 Hybridized

the 1.7 kb Hae Ill-Hap to Dictyostelium DNA

II Coding

The 1.7 kb Hae Ill-Hap II fragment was isolated from plasmid M6 and labeled by nick translation (sea Experimental Procedures). One aliquot was allowed to reanneal while a second was hybridized to a vast excess of Dictyostelium DNA under conditions that prevented renaturation of the probe. Each sample was loaded onto hydroxyapatite (HAP) in 0.12 M PB at 51°C. The temperature was raised by 4°C increments, the HAP was washed with 0.12 M PB and the counts removed at each temperature were counted. At gl”C, the column was rinsed with 0.48 M PB to remove any unmelted duplex. (W - -0) Renatured 1.7 kb Hae Ill-Hap II fragment; (C--O) Hae Ill-Hap II fragment hybridized to Dictyostelium DNA.

Table

1. Melting

Temperatures Sub 1

Tracer

Sub2

M8

82

74

76

Sub 2

75

79

72

76

72

60

II

(I.31

Figure

76

Although it is possible for three sequences with this amount of mispairing to code for the same protein, we were surprised at this large difference. It is also possible that, if these sequences actually code for actin, they code for different forms of the protein. Preliminary results from this laboratory (C. MacLeod and R. A. Firtel, unpublished observations; Kindle and Firtel, 1978) indicate that there are indeed multiple forms of Dictyostelium actin differing in isoelectric point but not in molecular weight. It is conceivable that some of the actin mRNA complementary regions are never transcribed.

neighboring

Haem

Dictyostelium

The curves shown in Figures 6 and 7 were used to determine the temperature at which 50% of the cpm bound to HAP at 55 or 5l”C, respectively, had been eluted. All numbers are rounded off to the nearest “C.

Clustering of Actin Get& By hybridizing sequences

(I.61

of Hybrids

Sub 1

M6 Hae Ill-Hap

Sub I + (1.4)

the

actin

8. An Additional

Actin

HapII Gene

Near M6

(A) Total Dictyostelium DNA was digested with Hae Ill and fragments were separated by electrophoresis on a single lo-15 cm wide channel of an 0.6% agarose gel. A single DNA blot filter was made from this gel. The filter was sliced into ‘/d inch strips and each strip was hybridized to a different 3ZP-labeled probe. After thorough washing, the filter strips were realigned and autoradiographed at -70°C. Two different exposures of the same set of filter strips are shown. The hybridization probes were (1 and 4) 32P-labeled actin gene containing fragments from M6, pDd actin P-sub 1 and pDd actin P-sub 2; (2) 32P-labeled 2.5 kb Hae Ill fragment from the left-hand side of the insert of plasmid M6: (3) a mixture of the probes used in (I), (2) and (4). (B) A DNA blot filter was prepared from a Hap II digest of total Dictyostelium DNA and the filter was sliced as in (A). The 3ZP-labeled probes hybridized to each strip were the actin gene containing restriction fragments from (1) pDd actin P-sub I; (2) pDd actin P-sub 2; (3) M6.

gene regions in our plasmids to Southern blots of different restriction enzyme digests of Dictyostelium DNA, we have been able to locate a number of restriction enzyme cleavage sites in the genomic DNA near the regions included in our chimeric plasmids. The mapping of these sites is independent of any conclusions drawn about the location of additional actin genes. While mapping additional restriction sites be-

Cell 798

Figure

9. Summary

of Inferred

Mapping

Data

By hybridizing restriction fragments not containing actin gene sequences to various digests of Dictyostelium DNA, we have inferred the location of a number of restriction enzyme cleavage sites in the genomic regions adjacent to the regions contained in our plasmids. We have also been able to infer the presence of an actin gene to the left of M6. In addition to the sites shown, we believe there is either an Eco RI or a Kpn I restriction site 7.5 kb to the right of the Eco RI site in pDd actin 2. The dashed lines represent the region of the genome near the DNA segments in our plasmids and thicker lines represent the regions actually contained in our plasmids. The solid bars below each line represent actin genes and the arrows represent the direction of transcription.

yond the boundaries of the regions inserted into our plasmids, we also showed that the left-hand end of M6 is homologous to restriction fragments which have the same length as fragments homologous to actin DNA. It is possible that this similarity in fragment size is merely fortuitous. Without having isolated a plasmid containing the outside sequence, this possibility is difficult to disprove. An alternative conclusion is that there is in fact an additional actin gene in the genomic region near M6. For technical reasons, we have successfully examined only one restriction enzyme digest for the presence of additional actin genes near M6, but we feel that the simplest explanation of the data in Figure 8 is that there is another actin gene in the region of the genome to the left of the M6 region. We suspect that the polarity of this gene is the same as the polarity of the M6 actin gene. If our interpretation of the data is correct, an interesting organization is implicated for at least four of the 15-20 actin genes that we believe exist in Dictyostelium. pDd actin 2 is part of a cluster of at least two actin genes and M6 is part of a cluster of at least two actin genes. These genes are diverged from each other by as much as 8-10% of the primary sequence and are separated from each other by single- or low copy sequences of variable length. Furthermore, the sequence between the M6 actin gene and the next actin gene to the left appears to be homologous to a sequence which is near still another actin gene. If all the actin genes in Dictyostelium are involved in a cluster arrangement of some kind, certain predictions can be made about the organization of the DNA within each cluster based upon the evidence presented in this work and in previous pa-

pers. The genes must be separated by heterogeneous DNA sequences since a large number of genomic restriction fragments differing in size hybridize to an actin gene probe. There are 17 bands of actin hybridization after digestion of genomic DNA with Hind Ill (data not shown). The total length of these bands is approximately 130 kb. If fragments of all lengths are included in groups of actin genes, an average spacing of at least 7 kb would be expected between actin genes. Such a large spacing between genes raises the possibility that there are other genes interspersed between the actin genes of a cluster. We are in the process of isolating additional plasmids homologous to actin DNA and to the sequences outside the actin gene regions of pDd actin 2 and M6. Plasmids of this type should allow us to test our inferences about the regions near pDd actin 2 and M6, as well as allowing us to observe higher orders of organization that may have evaded us before. Experimental

Procedures

Materiels Recombinant plasmids were constructed and purified as previously described (Cockburn, Newkirk and Firtel, 1976; Kindle and Firtel, 1976). pDd actin P-sub 1, pDd actin P-sub la and pDd actin P-sub 2 were constructed by restriction with Hind Ill followed by ligation of a mixture of plasmids pDd actin 2 and pBR322 with T4 DNA ligase. After transformation into SF8 (Cameron et al., 1974). ampicillin-resistant, tetracycline-sensitive colonies were screened for the presence of plasmids containing the 1.4 kb Hind Ill fragment of sub 1 or the 1.3 kb Hind III fragment of sub 2. “P-labeled T7 early RNA was prepared by the method of Studier (1973). After lysis and removal of cellular debris, the RNA was ethanol-precipitated and suspended in sample buffer for the methyl mercury gel (25 mM boric acid, 2.5 mM Na,B,O,.lO H,O, 5 mM Na,SO,, 0.5 mM EDTA, 10% glycerol) (Bailey and Davidson, 1976). 3ZP-labeled Dictyostelium mRNA complementary to the three actin genes was prepared by hybridization to filter-bound plasmid DNA as described by Kindle and Firtel (1978). After ethanol precipitation, these DNA samples were suspended in methyl mercury gel sample buffer. The samples were made 5 mM with methyl mercuric hydroxide immediately before loading onto the gel. Methyl Mercury-Contalning Agarose Gels The mobility of actin mRNA relative to T7 early RNA used as a standard was determined in a vertical 1.8% agarose 4 mm slab gel which was 5 mM methyl mercuric hydroxide (Bailey and Davidson, 1976). Electrophoresis was for 14 hr at 1.5 V per cm. The gel was soaked in 0.1 M P-mercaptoethanol, dried under vacuum and autoradiographed using X-ray intensifying screens as described by Laskey and Mills (1977). Hetaroduplex Heteroduplex al. (1978).

Mapping mapping

was performed

as described

by Bender

et

Thermal Denaturation Profiles Driver DNA for the thermal denaturation curves was prepared from whole plasmid DNA by mild depurination. The DNA was brought to 0.1 M sodium acetate at pH 4.2 and incubated at 65°C for 20 min. The sample was then neutralized with 1 M acetic acid,

Multiple 799

Actin

Genes

in Dictyostelium

precipitated with ethanol and resuspended in 0.12 M phosphate buffer (pH 7.0). Nick-translated tracer DNA was added to driver DNA such that the concentration of the driver actin gene sequences was at least 200 times that of the tracer actin gene sequences. The samples were placed in siliconized glass test tubes, covered with mineral oil and immersed in a boiling water bath for 5 min. Hybridizations were then carried out at 55°C. After hybridization, the samples were layered at 55°C onto columns produced by the addition of 1.2 ml of a slurry containing 1 g hydroxyapatite. 1 g CF-11 (Whatmann) and 20 ml 0.12 M phosphate buffer to a glass wool plugged Pasteur pipette. All columns were contained in a single constant temperature water bath. Elution was with 2 x 1 ml of 0.12 M phosphate buffer. Columns were allowed to equilibrate for 5 min at the indicated temperatures before washing. Eluates were collected directly into plastic scintillation vials and counted by Cerenkov radiation. General Procedures All chemicals, enzymes and buffers were as described in Kindle and Firtel (1978). Nick-translated hybridization probes were made as previously described (Kindle and Firtel, 1978). Southern DNA blot filters were made according to the method of Southern (1975). Hybridizations were carried out as described by Kindle and Firtel (1978). Acknowledgments We would like to thank Mary Jane Newkirk for technical assistance and Alan Kimmel, Walter Rowekamp and Carol MacLeod for invaluable discussions. M.M.. K.L.K. and W.B. were NSF predoctoral fellows; W.B. and K.L.K. were further supported as trainees of the NIH. W.C.T. is a postdoctoral fellow of the American Cancer Society. R.A.F. is the recipient of an American Cancer Society Faculty Research Award. This work was funded by grants from the NSF, the American Cancer Society and the NIH to R.A.F., and from the NIH to N.D. 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

Aloni, Y.. Dhar, R.. Laub, O., Horowitz, M. and Khoury, G. (1977). Novel mechanism for RNA maturation: the leader sequences of simian virus 40 mRNA are not transcribed adjacent to the coding sequences. Proc. Nat. Acad. Sci. USA74, 3686-3690. Davidson, N. (1976). Methylmercury agent for agarose gel electrophoresis.

as a Anal.

Bender, W., Davidson, N.. Kindle, K. L., Taylor, W. C.. Silverman, M. and Firtel, R. A. (1978). The structure of M6. a recombinant plasmid containing Dictyostelium DNA homologous to actin messenger RNA. Cell 15. 779-788. Berget, S. M., Moore, C. and Sharp, P. A. (1977). Spliced segments at the 5’ terminus of adenovirus 2 late mRNA. Proc. Nat. Acad. Sci. USA 74, 3171-3175. Bolivar, F., Rodriguez, R. L., Green, P. J., Betlach. M. C., Heyneker, H. L., Boyer, H. W., Crosa. J. H. and Falkow, S. (1977). Construction and characterization of new cloning vehicles. II. A multiple cloning system. Gene2, 95113. Breathnach, J. M., Mandel. min gene is split in chicken

P. and Chambon, DNA. Nature270,

Cameron, J. R.. Panasenko. (1974). In vitro construction

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selection Nat. Acad.

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Chow, L. T.. Gelinas, R. E., Broker, T. R. and Roberts, R. J. (1977). An amazing sequence arrangement at the 5’ ends of adenovirus 2 messenger RNA. Cell 72, l-8 Cockburn, A. F., Newkirk, M. J. and Firtel, R. A. (1976). Organization of the ribosomal RNA genes of Dictyostelium discoideum: mapping of the nontranscribed spacer regions. Cell 9, 805-613. Davidson, E. H., l-tough. B. R.. Amenson. C. S. and Britten, R. J. (1973). General interspersion of repetitive with non-repetitive sequence elements in the DNA of Xenopus. J. Mol. Biol. 77, l-23. Elzinga, M., Collins, J. H., Kuehl. W. M. and Adelstein, R. S. (1973). Complete amino-acid sequence of actin of rabbit skeletal muscle. Proc. Nat. Acad. Sci. USA 70, 2687-2691. Finnegan, D. J., Rubin, G. M., Young, M. W. and Hogness, (1977). Repeated gene families in Drosophila melanogaster. Spring Harbor Symp. Ouant. Biol. 42, 1053-1063.

D. W. Cold

Fidel, R. A. and Kindle, K. (1975). Structural organization of the genome of the cellular slime mold Dictyostelium discoideum: interspersion of repetitive and single-copy DNA sequences. Cell 5, 401-411. Firtel, R. A., Kindle, K. and organization and processing cellular slime mold Dictyostelium 22. Goodman, sequence ing ochre 7453-7457. Jeffreys, contains 1108.

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Kindle, K. L. (1978). Ph.D. Diego, La Jolla, California.

thesis,

University

&globin gene Cell 12, 1097-

of California

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at San

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