A characterization of the preaggregative period of Dictyostelium discoideum

The preaggregative period of Dictyostelium discoideum has been characterized by measuring the reduction in time for the onset of aggregation under conditions which hinder close cell-cell associations, inhibit protein synthesis, and/or include continuous high concentrations or pulsed low concentrations of exogenous cAMP. The results demonstrate that: the preaggregative period (normally 7 hr for cells from log phase cultures) can be dissected into two distinct components: an initial component which includes the first 4.5 hr, and a second component which includes the last 2.5 hr; the first component will progress at normal rate in the continuous absence of close cell-cell associations (as single amoebae in suspension) or in the continuous absence of de novo protein synthesis; the second component will not progress in the continuous absence of close cell-cell associations or de novo protein synthesis; high concentrations of cAMP continuously present in suspension cultures do not affect progress through the first component, nor do they support progress through the second component; however, if cells are allowed to form close cell-cell associations during progress through the first component, high concentrations of cAMP will support progress through the second component in the absence of close cell-cell associations; these associations, which render cells sensitive to cAMP, will occur in the absence of de novo protein synthesis and before the acquisition of contact sites A; these associations may be completely bypassed if suspended cells are continuously pulsed with low concentrations of cAMP; in this case, pulses of cAMP will support progress through the final component in continuous suspension cultures; and the acquisition of contact sites A will not occur in the absence of progress through the second component; in contrast, the acquisition of cAMP binding sites on the cell's surface will occur. These results are considered in terms of the complexity and regulation of the preaggregative period of Dictyostelium.     

Regulation of protein synthesis during the preaggregative period of Dictyostelium discoideum development: involvement of close cell associations and cAMP

The preaggregative period of Dictyostelium discoideum is composed of two rate-limiting components which exhibit dramatic differences in either their dependency upon, or sensitivity to, close cell-cell associations, inhibitors of protein synthesis, temperature, and pH. The first component comprises the initial 4.5 hr and the second component the last 2.5 hr of the preaggregative period. By pulse-labeling cells with [35S]methionine, separating polypeptides by 2D-PAGE, and semiquantitatively comparing the rates of synthesis of 778 individual polypeptides by fluorography, the following results were obtained: a detailed program of protein synthesis accompanies the preaggregative (0-7 hr) and aggregative (7-10.5 hr) periods of development; this includes significant decreases in the rate of synthesis of 93 polypeptides synthesized during vegetative growth and significant increases in the rate of synthesis of 74 polypeptides either undetectable or synthesized at relatively low rates during vegetative growth; 35 polypeptides are transiently synthesized at different times during the preaggregative and aggregative periods; two peaks of activity are clearly defined for both increases and decreases; these peaks correlate temporally with the first and second rate-limiting components of the preaggregative period; the majority of changes (74%) which occur during the first rate-limiting component will occur in the absence of close cell-cell associations, but the majority (66%) which normally occur during the second rate-limiting component do not occur in the absence of close cell-cell associations; a high concentration of cAMP in the medium of continuous suspension cultures does not stimulate most of the changes which are dependent upon close cell-cell associations; even though cAMP stimulates progress through the second rate-limiting component in suspension cultures first allowed to associate for 4.5 hr ("competent" cells) prior to disaggregation it still does not stimulate most of the changes which are dependent upon close cell-cell associations; and synthesis of only 3 out of 778 polypeptides appears to be stimulated by addition of exogenous cAMP, and only in resuspended cultures of "competent" cells. The prominent role of close cell-cell association and the surprisingly minor effect of cAMP in the regulation of the program of protein synthesis accompanying the preaggregative and aggregative periods of Dictyostelium are discussed, especially as they relate to the effect of cAMP on protein synthesis in suspended cultures of postaggregative cells.     

Cell-type-specific genes expressed late in Dictyostelium development show markedly different responses to 3'' 5'' cyclic AMP

The maintenance of late gene expression by 3'5' cyclic AMP was re-examined using several newly isolated cell-type-specific genes. Expression of all the prespore-enriched genes ceased immediately upon disaggregation of developing cells and pre-existing mRNA was rapidly degraded. For most genes, cAMP had little or no effect either alone or in combination with conditioned medium factors. The expression of the non-cell-type-specific genes 7E and 2C also ceased upon cell disaggregation but cAMP triggered a full re-induction of expression although the timing of the response differed markedly between these two genes. In contrast to earlier interpretations, these data argue that for none of these late prespore genes is cAMP alone sufficient for the maintenance of expression. The responses of the two prestalk mRNAs examined were gene-specific. Prestalk 5D mRNA decayed slowly upon disaggregation and was partially stabilized by cAMP whereas prestalk 5G mRNA increased upon disaggregation and was inhibited by cAMP.     

Cell-cell contact and cAMP regulate the expression of a UDP glucose pyrophosphorylase gene of Dictyostelium discoideum

UDP glucose pyrophosphorylase (UDPGP) (EC.2.7.7.9) is a developmentally regulated enzyme of Dictyostelium discoideum. Two polypeptides of UDPGP are translated from Dictyostelium mRNA. Recently we isolated a cDNA clone which encodes one of the UDPGP polypeptides (B. R. Fishel, J. A. Ragheb, A. Rajkovic, B. Haribabu, C. W. Schweinfest, and R. P. Dottin (1985). Dev. Biol. 110, 369-381). By hybridization with the cDNA and by in vitro translation and immunoprecipitation, we examined the effect of cell-cell contact and cAMP on the regulation of UDPGP expression. Disaggregation of slugs resulted in a rapid loss of UDPGP mRNA. Addition of cAMP to these cells resulted in increased levels of UDPGP mRNA, though not to the same extent as seen during normal development. The two UDPGP polypeptides observed in vitro are coordinately regulated. Unaggregated cells, starved and shaken rapidly in suspension, did not show UDPGP mRNA accumulation. However, addition of cAMP to these cells caused UDPGP induction, suggesting that the requirement for cell-cell contact could be bypassed in part by cAMP addition.     

Dictyostelium discoideum mRNAs developmentally regulated during spore germination have short half-lives.

mRNA decay was studied during spore germination in Dictyoselium discoideum by the use of three previously isolated cDNA clones, pLK109, pLK229, and pRK270, which are specific for mRNAs developmentally regulated during spore germination. The half-life of a constitutive mRNA, pLK125, which is present throughout germination, growth, and development, as also determined. Nogalamycin, a DNA-intercalating compound, was used to inhibit RNA synthesis. Total RNA was isolated at intervals after addition of the drug, and the decay of mRNAs specific for the cDNA clones was determined by both Northern blot and RNA dot hybridization. If nogalamycin was added immediately after activation of dormant spores, neither pLK229 nor pLK109 mRNA decayed, but pLK125 mRNA did decay. Although pLK109 mRNA did not decay under these conditions, the RNA was smaller 1 h after activation than in dormant spores, indicating that it was processed normally. At 1 h after activation, pLK229-, pLK125-specific mRNAs decayed exponentially, with half-lives of 24, 39, and 165 min, respectively. Under the same conditions, decay of pLK109-specific mRNA was biphasic. Thirty-eight percent of the mRNA decayed with a half-life of 5.5 min, and the remainder decayed with a half-life of 115 min. It seems likely that nogalamycin inhibits the synthesis of an unstable component of the mRNA degradative pathway which is needed continuously for the decay of pLK109 mRNA. By extrapolating the curve representing the rapidly decaying component, a half-life of 18 min was calculated for pLK109-specific mRNA. The mRNAs developmentally regulated during spore germination have half-lives shorter than that of the constitutive messenger and shorter than the average half-life of 3 to 4 h previously determined for total Dicyostelium polyadenylated mRNA.     

Changes during differentiation in requirements for cAMP for expression of cell-type-specific mRNAs in the cellular slime mold, Dictyostelium discoideum

A number of genes encoding developmentally regulated mRNAs in the cellular slime mold, Dictyostelium discoideum, have been described. Many of these are regulated by cAMP. Analysis of the earliest time at which elevated levels of cAMP can induce the expression of these mRNAs reveals a more complex pattern of regulation in which genes change in their ability to be induced in response to cAMP with developmental stage. A prestalk mRNA (C1/D11) previously thought not be regulated by elevated levels of cAMP is inducible by cAMP between aggregation and loose mound stage; later in development its expression becomes independent of elevated cAMP. The early prespore genes (prespore class I) also show two modes of regulation; early in development they are induced independently of continuous elevated levels of cAMP, while later in development their expression is dependent upon elevated cAMP. The period during development when the prestalk genes are cAMP inducible precedes by 2 hr the first time at which either the early prespore class I or late prespore class II mRNAs are inducible by continuous elevated levels of cAMP. Previous analysis of these mRNAs has been carried out using Dictyostelium cells grown axenically. In this report we have studied the developmental expression of these mRNAs in cells grown on bacteria. A substantial shutoff of the class I prestalk and early prespore (class I) mRNAs not seen in axenically grown cells is observed when bacterially grown cells are plated for development. Less than 10% of the maximal level of these mRNAs remains in the cells at the time of mature spore and stalk differentiation. Additionally, in the bacterially grown cells two distinct patterns of developmental regulation are observed for mRNAs which in axenically growing cells appear to be constitutively expressed throughout growth and development.     

A cytosolic cyclic AMP-dependent protein kinase in Dictyostelium discoideum. II. Developmental regulation

The cAMP-dependent protein kinase of the cellular slime mold, Dictyostelium discoideum, is developmentally regulated; there is an approximately 4-fold increase in activity during development. The incorporation of [3H]leucine into the enzyme demonstrates that there is de novo synthesis of the cAMP-dependent protein kinase. The activities of the catalytic and regulatory subunits increase in parallel. The maximal rate of increase of cAMP-dependent protein kinase activity precedes "tip" formation, a stage of development characterized by a sharp increase in mRNA complexity. The high level of cAMP-dependent protein kinase activity, attained at this stage of development, persists when aggregates are dispersed and the amoebae are kept in suspension without added cAMP. The synthesis of the developmentally regulated mRNAs under these conditions is dependent on exogenous cAMP. The increase in cAMP-dependent protein kinase activity during development does not require sustained cell-cell contact insofar as it occurs in single cell suspensions of amoebae. Furthermore, the increase does not require exogenous cAMP, although added cAMP stimulates the synthesis of the enzyme to a level higher than that found, when cAMP is not added. These observations support the hypothesis that in D. discoideum cAMP-dependent protein kinase mediates the effects of cAMP on development.     

Disaggregation-induced changes of developmentally regulated proteins in Dictyostelium discoideum

By means of two-dimensional gel electrophoresis, we analyzed proteins present in a slug-shaped tissue mass of D. discoideum and examined the changes in their amounts after disaggregation of the slugs. Of approximately one hundred polypeptides, six were found to decrease in amount after disaggregation. The decreases of four polypeptides were inhibited by the presence of 1 mM cAMP or 250 micrograms/ml cycloheximide. The decreases of the two other proteins were not suppressed by cAMP or cycloheximide. The patterns of proteins present in vegetative and aggregative cells were also examined. None of the six proteins which showed a decrease after slug disaggregation was found in vegetative or preaggregative cells. These results indicate that both synthesis and degradation of these proteins are controlled by cell-cell contact.     

Metallothionein mRNA stability in chicken and mouse cells

Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.     

Regulation of discoidin I gene expression in dictyostelium discoideum by cell-cell contact and cAMP

We have previously presented evidence that cell-cell contact is the normal developmental signal to deactivate discoidin I gene expression in D discoideum [Berger EA, Clark JM: Proc Natl Acad Sci USA 80:4983, 1983]. Here we provide genetic evidence to support this hypothesis by examining gene expression in a cohesion-defective mutant, strain EB-21, which enters the developmental program but is blocked at the loose mound stage. When this strain was developed in suspension, the cells remained almost entirely as single amoebae, unlike the wild type, which formed large multicellular aggregates. In both strains, discoidin I mRNA levels were low in vegetative cells but rose sharply during the first few hours of development. However, the peak level reached at 8 hr in EB-21 exceeded that observed in wild type, and while the level declined markedly over the next few hours in wild type, it remained highly elevated in the mutant. Thus, there was a correlation between the inability of EB-21 to form normal cell-cell contacts and its deficiency in inactivating discoidin I gene expression. Previous studies from several laboratories, including this one, have demonstrated that exogenously added cAMP can block or reverse the changes in gene expression normally seen upon cell disaggregation. This has led us to propose that cAMP serves as a second messenger regulating the expression of contact-regulated genes. Here we provide additional support for this hypothesis. Intracellular cAMP levels rapidly dropped several-fold when wild type tight cell aggregates were disaggregated and remained low as the cells were cultured in the disaggregated state. Furthermore, overexpression of discoidin I mRNA late in development in EB-21 was corrected by addition of high concentrations of cAMP. These results are consistent with a second messenger function for cAMP in the contact-mediated regulatory response, and they indicate that the cAMP response machinery for discoidin I gene expression is capable of functioning in the cohesion-defective EB-21 strain.     

Proteins and messenger RNAs of the transforming region of wild-type and mutant adenoviruses

We have studied the proteins encoded by the transforming region of the closely related human adenovirus serotypes 2 and 5. Messenger RNAs complementary to the two parts of this region, E1A and E1B, were prepared separately by hybridization to cloned DNA fragments encompassing 0.8 to 4.5 map units (for E1A) and 9.8 to 11.1 map units (for E1B). These RNAs were further fractionated by electrophoresis through agarose gels containing methylmercuric hydroxide, and then translated in vitro to identify the proteins encoded by each RNA species. E1A and E1B RNAs isolated at early and at late times after infection were compared. Three size classes of E1A mRNA direct the synthesis of at least five proteins: a28K³ protein encoded by a 0.6 kb mRNA, 42K and 54K proteins encoded by a 0.9 kb mRNA(s), and 48K and 58K proteins encoded by a 1.1 kb mRNA(s). The mRNA for the 28K protein accumulates preferentially at late times. Three size classes of early E1B mRNA direct the synthesis of three proteins: a 15K protein encoded by a 0.9 kb mRNA, an 18K protein encoded by a 1.2 kb mRNA, and a 57K protein encoded by a 2.6 kb mRNA. The mRNA for the 15K protein continues to accumulate at late times, and an additional 22K protein is made, while the 18K and 57K proteins are synthesized poorly, if at all, with late RNA.Substantially different E1A and E1B proteins are encoded by RNA from cells infected with the adenovirus type 5 mutants dl311, dl312, dl313, dl314 and hr1, which are all defective for replication on human cells and, except for dl311, for transformation. dl312, dl314 and hr1 are also defective for early viral gene expression. No viral mRNA could be detected in either dl312 or dl314-infected cells. hr1-infected cells contain a 0.9 kb mRNA encoding E1A 54K and 42K, but instead of 58K and 48K, the 1.1 kb hr1-E1A mRNA is translated into a 26K protein. The E1B mRNAs are present in substantially decreased amounts in hr1-infected cells. dl311-infected cells contain E1A mRNAs of 1.1 and 0.9 kb, encoding 38K and 34K proteins, respectively, and normal E1B mRNAs. The dl313 mRNAs of 1.1 and 0.9 kb contained fused E1A and E1B sequences and were translated into 40K and 36K proteins, respectively. These results are related to the mRNA structures and the biological activity of regions of the individual proteins.     

Accumulation of the early histone messenger RNAs during the development of Strongylocentrotus purpuratus

The developmental profile of amounts of the mRNAs for the early histones was determined. Sea urchin embryos (Strongylocentrotus purpuratus) were labeled with ³H nucleoside, and the RNA was extracted and fractionated by electrophoresis on polyacrylamide gels. Radioactivity in the resolved mRNA bands was determined and converted to molar quantities by knowledge of the precursor pool-specific activity. Between the 16- and the 200-cell stages 7–10 × 10⁶ molecules of each core histone mRNA, and 2.5 × 10⁵ molecules of H1 mRNA accumulate. During the subsequent few hours of cleavage the accumulated mRNAs disappear with a half-life of 1.5–2 hr. It is argued that mRNA half-life may be regulated during cleavage.