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The Dictyostelium discoideum genome contains an estimated 17 to 20 actin genes. We report the identification of a new member of this multigene family, actin 15, and its complete nucleotide sequence and transcription initiation sites. We constructed transformation vectors carrying either the actin 15 promoter fused to the neomycin phosphotransferase gene from transposon Tn903 or the actin 6 promoter fused to the neomycin phosphotransferase gene from Tn5. Cells transformed with the actin 15 vector carried less than five copies of vector DNA, while cells transformed with the actin 6 vector carried more than 200 copies. In both cases, the vector appeared to be integrated into the chromosome as a tandem array. Gene fusion RNAs transcribed from the actin 15 and actin 6 vectors were regulated like endogenous actin genes during D. discoideum development. DNA sequences required for temporal and cell type-specific regulation of these genes were contained within 2.8 kilobases of 5' noncoding DNA for actin 15 and 0.7 kilobases of 5' noncoding DNA for actin 6.

DIRS-1 is a Dictyostelium discoideum transposable element that contains heat shock promoter sequences in the inverted terminal repeats. We showed that transcription of a 4.5-kilobase polyadenylated RNA initiates at a discrete site within the left-terminal repeat of DIRS-1, downstream from heat shock promoter and TATA box sequences. This RNA represents a full-length transcript of DIRS-1. We describe a cDNA clone that contains the 4.1 kilobases of internal sequence of DIRS-1, a cDNA clone that spans the junction between the internal sequences and the right-terminal repeat, and a cDNA clone that appears to have been transcribed from a rearranged genomic copy of DIRS-1. A second DIRS-1 RNA, named E1, is transcribed on the opposite strand of DIRS-1 from the 4.5-kilobase RNA and is under control of the heat shock promoter in the right-terminal repeat. E1 transcription initiates at multiple positions both within and downstream from the right-terminal repeat. The same transcriptional initiation sites are used during normal development and during heat shock, suggesting that in all cases DIRS-1 transcription is regulated by the heat shock promoters contained within the two terminal repeats.

Sequence analysis of genomic clones containing the intact Dictyostelium transposable element DIRS-1 reveals that in five of six cases DIRS-1 has inserted into other DIRS-1 sequences. The nucleotide sequences just beyond the endpoints of the terminal repeats of five different genomic clones can be aligned with different regions of the internal nucleotide sequence of DIRS-1. In the three genomic clones which contain flanking sequences on both sides of the element, both flanking sequences are homologous with DIRS-1. In one of these clones, both extended flanking sequences represent the full 4.1-kilobase EcoRI fragment of DIRS-1, which has been interrupted by the insertion of an intact DIRS-1 element. There is no duplication or deletion (except possibly 1 base) of the DIRS-1 sequence upon insertion of a second DIRS-1 transposon. DIRS-1-into-DIRS-1 insertions can occur in either a colinear or inverted orientation with respect to the target sequence; the target sequence need not be an intact DIRS-1 element. We also describe a cDNA clone which could be derived by transcription of a sequence that resulted from a DIRS-1-into-DIRS-1 insertion and discuss its significance concerning the function of the heat-shock promoters found in the terminal repeats of DIRS-1 and in other DIRS-1-related sequences.

The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1. Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells. We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor. To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells. In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock. Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert. A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.

The gene encoding the prestalk D11 mRNA of Dictyostelium discoideum has been isolated and characterized. Transcriptional mapping and sequence data indicated that the D11 message is unspliced and contains an 846-base open reading frame. The 273 base pairs upstream from the translation initiator codon are 88% A + T, typical of Dictyostelium upstream sequences, and contain no recognizable upstream activator sequence. The deduced D11 protein is exceptionally rich in cysteine residues and consists of a 25-amino acid hydrophobic leader sequence (L) followed by a series of repeats with the structure LA1B1A2B2C1B3C2B4C3B5C4B6, where A, B, and C, are, respectively, amino acid sequences of 39, 18, and 15 residues. The deduced D11 protein shares certain similarities with the Dictyostelium cyclic AMP phosphodiesterase inhibitor protein.

EB4 is one of several cloned cDNAs that is expressed as mRNA only after the aggregation stage of Dictyostelium discoideum differentiation and exclusively in prespore and spore cells (E. Barklis and H. F. Lodish, Cell 32:1139-1148, 1983). We have isolated the unique genome fragment corresponding to the 5' portion of the EB4 message and the EB4 promoter. The EB4 transcript has an unusually long, G + C-rich, 5' noncoding region, but initiates at several start sites within a region of DNA that is 96% A + T. The sequence GTGGTGG, along with slight variations, occurs several times in the promoter. We have used the EB4 promoter to drive the transcription of an EB4/beta-galactosidase fusion transcript in yeast cells. Although the cap sites of the fused transcript in yeast cells are located in the region where multiple EB4 transcripts are initiated in Dictyostelium, the unregulated expression of the fusion transcript in yeast does not mimic the normal regulated pattern of EB4 mRNA expression in D. discoideum.

We tested the effects of inhibitors of protein and RNA synthesis on the disaggregation-mediated destabilization of prespore mRNAs in Dictyostelium discoideum. Incubating disaggregated cells with daunomycin to inhibit RNA synthesis prevented the loss of prespore mRNAs, whereas the inhibitor decreased or did not affect levels of the common mRNAs CZ22 and actin. Protein synthesis inhibitors varied in their effects. Cycloheximide, which inhibited protein synthesis almost completely, prevented the loss of the prespore mRNAs, but puromycin, which inhibited protein synthesis less well, did not. These results indicate that the process of specific mRNA destabilization requires the synthesis of RNA and possibly of protein.

Nuclear processing of mRNA precursors in differentiating multicellular Dictyostelium discoideum aggregates is markedly slower than in growing amoebae. Thus, we have been able to determine the time of nuclear processing of individual mRNA species in postaggregating cells by following the incorporation of 32PO4 into nuclear and cytoplasmic RNA complementary to cloned cDNAs. Precursors of mRNAs synthesized during both growth and differentiation remain in the nucleus for about 25 to 60 min. By contrast, typical mRNAs which are synthesized only by postaggregative cells have nuclear processing times between 50 and 100 min. Depending on the particular mRNA, between 20 and 60% of nuclear transcripts are converted into cytoplasmic mRNA. A third class of mRNAs are transcribed from a set of repetitive DNA segments and are expressed predominantly during differentiation. Nuclear precursors of these mRNAs are extensively degraded within the nucleus or very rapidly after transport to the cytoplasm. Those sequences that are stable in the cytoplasm exit from the nucleus only after a lag of over 2 h. Thus, mRNAs encoded by different genes that are subject to different types of developmental controls display different times of transit to the cytoplasm and different efficiencies of nuclear processing. Differential nuclear processing may contribute to the regulation of the level of individual cytoplasmic mRNAs.

During development of the cellular slime mold Dictyostelium discoideum, approximately 2,000 to 3,000 regulated mRNAs are induced when amoebae enter multicellular aggregates. We used in vitro transcription in isolated nuclei to follow the synthesis of individual mRNA precursors during development; these were quantitated by hybridization to cloned cDNAs or genomic DNAs. Those RNAs that are present at all stages of development--the common RNAs--were transcribed by nuclei from cells at all stages of development. By contrast, those RNAs that are present only after cells begin to aggregate--here called aggregation stage RNAs--were transcribed only by nuclei from cells at the aggregation and postaggregation stages of development. The temporal pattern of in vitro transcription correlated well with the time course of accumulation of different aggregation stage mRNAs. Continued expression of aggregation stage genes normally depends upon cell-to-cell contact or cyclic AMP (cAMP); when cells are disaggregated, the regulated mRNAs are rapidly and specifically degraded. When cAMP is subsequently added to the disaggregated cells, most of the mRNAs reaccumulate. We show here that disaggregation reduced 2- to 10-fold the relative transcription of several aggregation stage RNAs, whereas addition of cAMP to disaggregated cells reinduced the level of regulated gene transcription to values approximating those found in normal postaggregation cells. These results indicate that a representative set of Dictyostelium aggregation stage genes are under transcriptional control; both the transcription and the stability of these mRNAs require either continued cell-to-cell interactions or cAMP.

Synthesis of ribosomes and ribosomal ribonucleic acid (RNA) continued during differentiation of Dictyostelium discoideum concurrently with extensive turnover of ribosomes synthesized during both growth and developmental stages. We show here that the rate of synthesis of 26S and 17S ribosomal RNA during differentiation was less than 15% of that in growing cells, and by the time of sorocarp formation only about 25% of the cellular ribosomes had been synthesized during differentiation. Ribosomes synthesized during growth and differentiation were utilized in messenger RNA translation to the same extent; about 50% of each class were on polyribosomes. Ribosome degradation is apparently an all-or-nothing process, since virtually all 80S monosomes present in developing cells could be incorporated into polysomes when growth conditions were restored. By several criteria, ribosomes synthesized during growth and differentiation were functionally indistinguishable. Our data, together with previously published information on changes in the messenger RNA population during differentiation, indicate that synthesis of new ribosomes is not necessary for translation of developmentally regulated messenger RNA. We also establish that the overall rate of messenger RNA synthesis during differentiation is less than 15% of that in growing cells.