Gene-specific expression of the actin multigene family of Dictyostelium discoideum

We have investigated the expression of 14 cloned genes of the 20-member actin multigene family of Dictyostelium discoideum using gene-specific mRNA complementary probes and an RNase protection assay. Actin gene expression was studied in vegetative cells and in cells at a number of developmental stages chosen to represent the known major shifts in actin mRNA and protein synthesis. At least 13 of these genes are expressed. A few genes are expressed very abundantly at 10% or more of total actin mRNA; however, the majority are maximally expressed at 1 to 5% of actin message. Although all of the genes are transcribed in vegetative cells, most genes appear to be independently regulated. Actin 8 appears to be transcribed at constant, high levels throughout growth and development. Actin 12 mRNA is maximally expressed in vegetative cells but the level is reduced appreciably by the earliest stage of development examined, while Actin 7 mRNA is specifically induced approximately sevenfold at this time. The rest of the genes appear to be induced 1.5 to 2-fold early in development, coincident with the increase in total actin mRNA. Since 12 of the genes code for extremely homologous proteins, it is possible that the large number of actin genes in Dictyostelium is utilized for precise regulation of the amount of actin produced at any stage of development, even though individual gene expression appears in some cases to be very stage-specific. In addition to these 13 actin genes, at least two and possibly four more genes are known to be expressed, because they are represented by complementary DNA clones, and an additional one or two expressed genes are indicated by primer extension experiments. Only one known gene, Actin 2-sub 2, is almost certainly a pseudogene. Thus the vast majority of Dictyostelium actin genes are expressed.     

Properties of a 5'-AMP specific nucleotidase which accumulates in one cell type during development of Dictyostelium discoideum

5′-AMP nucleotidase activity accumulates during the culmination stage of development in a thin layer of cells at the prestalk-prespore interface of Dictyostelium discoideum. In this report we characterize a highly purified preparation of this enzyme in an attempt to determine the physiological significance of the accumulation and localization of the activity during cellular differentiation. A pH optimum of 9.5 was determined using nine different buffer systems tested over a range of pH from 3 to 13.5. The Michaelis constants for p-nitrophenylphosphate (NPP) and 5′-AMP were 1.8 and 1.2 mm, respectively. Substrate concentrations of 5′-AMP in excess of 2.5 mm were found to inhibit the activity. Little or no effect on the activity of the enzyme was observed in the presence of EDTA, Mg²⁺, Mn²⁺, Ca²⁺, Fe²⁺, or Zn²⁺ ions. However, the enzyme appears to be a zinc metalloprotein as evidenced by its inhibition with 1,10-phenanthroline and recovery of activity in the presence of zinc. Other inhibitors of enzymatic activity include dithiothreitol and imidazole. The enzyme was bound by calcium phosphate, but could not be immobilized on matricies containing other substrate or product analogs, including 5′-AMP, cyclic AMP, ATP, phenylalanine, blue dextran, and Procion Red HE3B. The hydrophobicity of 5′-AMP nucleotidase was demonstrated by its strong affinity for immobilized alkyl and ω-amino alkyl ligands, as well as phenyl Sepharose. Isoelectric focusing of the enzyme in granulated gel required both the presence of detergent to prevent aggregate formation and precipitation of the enzyme, and the addition of zinc after focusing to reverse Ampholine inhibition. Apparently, Ampholine chelates zinc away from the enzyme much like 1,10-phenanthroline. Using this method, the isoelectric point of 5′-AMP nucleotidase was found to be 4.5–4.9, with a 30% recovery of the applied activity.     

Site-specific phasing in the chromatin of the rDNA in Dictyostelium discoideum

The rDNA in Dictyostelium discoideum is organized in linear, extrachromosomal, palindromic dimers of approximately 88 X 10(3) bases in length. The dimers are repeated about 90 times per haploid genome. Using indirect end-labeling, we have mapped micrococcal nuclease and DNAase I-sensitive sites in the chromatin near the rDNA telomeres. This region is 3' to the 36 S rRNA coding region and contains a single 5 S rRNA cistron but is primarily non-coding. We have observed somewhat irregularly spaced but specific phasing of nuclease-sensitive sites relative to the underlying DNA sequence. Comparison of the sites in chromatin with those in naked DNA reveals an unusual and striking pattern: the sites in naked DNA that are attacked most readily by both nucleases, presumably because of the specificity of the nucleases for certain sequences or physical characteristics of the DNA, appear to be the same sites that are most protected in chromatin. This pattern extends over most of a 10(4) base region, from the sequence immediately distal to the 36 S rRNA coding region and extending to the terminus. Although much of the sequence-specific phasing is irregularly spaced, salt extraction data are consistent with the presence of nucleosomes. In addition, phasing in the terminal region may be directed partially by proteins that do not bind DNA as tightly as do core histones. We present a model for phasing in spacer regions in which the sequence preferences of nucleases such as micrococcal nuclease and DNAase I may be useful tools in predicting nucleosome placement.     

Cyclic AMP inhibits dedifferentiation in the cellular slime mold Dictyostelium discoideum

When aggregating amoebas of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis is reinitiated, the amoebas will reaggregate in less than $\text{1}\text{10}\text{th}$ the original time. When aggregating amoebas are disaggregated and resuspended either in full nutrient medium or in buffered salts solution containing dextrose, they retain this developmentally acquired capacity to rapidly reaggregate for approximately 1 hr and then lose it completely in a synchronous and discrete step which we have referred to as the “erasure event.” In this report, it is demonstrated that micromolar concentrations of cAMP completely block this transition from the developmental to vegetative state, and that other cyclic nucleotides also inhibit it, but they do so at 20-fold higher concentrations. Neither the hydrolysis products of cAMP nor the vegetative chemoattractant folic acid inhibit dedifferentiation at concentrations as high as 10^?3M, demonstrating a specificity for cyclic nucleotides and cAMP in particular. The addition of cAMP at any time during the lag period preceding the erasure event inhibits it and addition immediately after the erasure event reverses it. Since cAMP may inhibit the transition from the developmental to vegetative state intracellularly or extracellularly, we have also examined the intracellular concentration of cAMP and the levels of cAMP binding sites on the cell surface during the erasure process. Evidence is presented that the majority of cAMP binding sites on the cell surface are not necessary for the inhibition of erasure by cAMP. The results of these latter studies are discussed in terms of alternative models for the involvement of cAMP in the transition from the developing to vegetative state.     

Morphogenesis in the slime mold Dictyostelium discoideum. 1. The accumulation and erasure of "morphogenetic information".

When developing cultures of the cellular slime mold Dictyostelium discoideum are disaggregated and morphogenesis immediately reinitiated, they recapitulate the morphogenetic scheme, but at an increased rate. Employing this feature of the system, we have identified time periods when “information” accumulates for specific morphogenetic events. In this case, we have defined “morphogenetic information” as the reduction in time for the appearance of a particular morphology during morphogenetic recapitulation. Accumulated information can be erased by disaggregating developing cultures and reinoculating them into liquid growth medium. Erasure occurs as a discrete event and can be blocked by inhibiting protein synthesis.By comparing morphogenesis in log-phase and stationary-phase cultures, at least two parallel timers have been distinguished, one specific for aggregation and one or more specific for morphogenetic events following aggregation. Approaches to the molecular identity of morphogenetic information and morphogenetic timers are discussed.     

An investigation of the structure of Alfalfa mosaic virus by small-angle neutron scattering

Small-angle neutron scattering experiments have been performed on the tubular bottom component of Alfalfa mosaic virus (AMV) and the “30 S” particle (a quasispherical reassembled AMV coat protein particle) with the aim of determining the internal structure of the virus. Scattering curves were obtained out to a resolution of $\text{1}\text{50}\text{A}\text{?}{}^{\mathrm{?1}}$ at a number of H₂O/²H₂O ratios and were analysed using a model fitting technique. This involves calculating the scattering intensity due to a parameterised distribution of scattering density representing the particle and comparing this to the experimental data after taking into account the effect of instrumental smearing. The use of the contrast variation method enables the internal consistency of the model to be well tested.Three models are used in an attempt to explain the scattering curve of the 30 S particle. A single homogeneous shell is shown to be inadequate and two other models introducing the presumed T = 1 icosahedral symmetry of the particle are presented and discussed. The most satisfactory of these consists of 60 spherical monomers of radius 19 Å symmetrically placed in pairs about the 2-fold icosahedral positions.The analysis of the bottom component data has yielded a low resolution model for the virus, which is shown to be consistent with its composition as given by earlier physico-chemical measurements. In the model the RNA is uniformly packed throughout the interior of the capsid (which is cylindrical with hemispherical ends) out to a radius of about 65 Å and with a packing fraction of 20%. Within the limitations of an homogeneous shell model, the protein capsid has an outer radius of 94 Å and thickness of 23 Å, but arguments are presented based on the marked lattice structure of the cylindrical capsid and the analysis of the scattering data of the 30 S particle, that this model underestimates the thickness of the protein shell and that it in fact makes contact with the RNA at about 65 Å.     

Conserved structural features are found upstream from the three co-ordinately regulated discoidin I genes of Dictyostelium discoideum

The discoidin I genes of Dictyostelium form a small, co-ordinately regulated multigene family. We have sequenced and compared the upstream regions of the DiscI-alpha, -beta and -gamma genes. For the most part the upstream regions of the three genes are non-homologous. The upstream sequences of the beta and gamma genes are exceedingly A + T-rich, while those of the alpha gene are less so. All three genes have a relatively G + C-rich region 20 to 40 base-pairs in length, found approximately 200 base-pairs 5' to the messenger RNA start site. This G + C-rich region 5' to the beta and gamma genes is flanked by short inverted repeats. Within this region, there is an 11 base-pair exact homology between the alpha and gamma genes, and a less perfect homology between these genes and the beta gene. The homology is flanked at a short distance by interspersed G and T residues. The gamma gene is greater than 90% A + T for greater than 800 base-pairs upstream. Further upstream there is a G + C-rich region that is also found inverted approximately 3.5 X 10(3) base-pairs away. The gamma and beta genes are tandemly linked, and the entire approximately 500 base-pair intergene region between the 3' end of the gamma gene and the 5' end of the beta gene is A + T-rich (approximately 90%) with the exception of the homology region 5' to the gamma gene. We demonstrate also the presence of a discoidin I pseudogene fragment having only 139 base-pairs of discoidin homology with greater than 8% mismatch. It is flanked upstream by five 39 base-pair G + C-rich repeats, and downstream by sequences that are extremely A + T-rich. We discuss the possible significance of the conserved G + C-rich structures on discoidin I gene expression.     

Direct visualization of collagens and procollagens in polyacrylamide gels.

This report describes a procedure that results in the rapid visual identification of collagens and procollagens in polyacrylamide gels. The technique results in a pink stain for collagenous proteins and a blue stain for all other proteins. The color difference has been evaluated spectrophotometrically. The absorbance maxima for collagen-Coomassie blue R250 complexes in gels is 520–535 nm, and the maxima for all other protein-Coomassie blue R250 complexes that we tested is 550–560 nm. This technique will facilitate the identification of collagenous proteins in complex mixtures of proteins derived from cell membranes, whole cell extracts, conditioned media, and extracellular matrices. We use the technique to detect procollagens in human diploid fibroblast conditioned media. The technique is simple, relatively rapid, has utility for proteins extracted by a variety of methods, and is applicable to all polyacrylamide gel systems in general use.     

Evidence for sub-families of actin genes in Dictyostelium as determined by comparisons of 3' end sequences

We have sequenced the 3′ end of five actin genomic clones and three actin complementary DNA clones from Dictyostelium. Comparison of the sequences shows that the protein coding regions are highly conserved, while the region corresponding to the 3′ untranslated regions are divergent. Additional analysis indicates regions of homology in the 3′ untranslated region between sets of actin genes. Southern DNA blot hybridization studies using labeled 3′ ends suggest that there are sub-families of actin genes that are related within the 3′ untranslated regions. No homology is found in the sequences outside the messenger RNA encoding regions. Analysis of the sequence data has shown that the difference in length between the ~1.25 × 10³ and ~1.35 × 10³ base actin messenger RNAs is in the lengths of the 3′ untranslated region.     

The phosphorylation of membranal proteins in Dictyostelium discoideum during development

The pattern of membranal phosphoproteins in Dictyostelium discoideum changes during development (D. S. Coffman, B. H. Leichtling, and H. V. Rickenberg, 1981, J. Supramol. Struct. Cell. Biochem. 15, 369–385). Phosphorylation of six membranal proteins occurred concomitantly with their synthesis. Cyclic AMP stimulated the precocious synthesis of a phosphoprotein, of molecular weight 80,000, which corresponds to contact sites A. Phosphoserine was the only phosphorylated amino acid found in the five phosphoproteins examined. In at least two phosphoproteins, that corresponding to contact sites A and a phosphoprotein of molecular weight 64,000, the phosphate moiety did not turn over.     

Phosphodiesterase induction in Dictyostelium discoideum by inhibition of extracellular phosphodiesterase activity

Adenosine 3′,5′-monophosphate (cAMP) is a chemoattractant in Dictyostelium discoideum; it also induces phosphodiesterase activity. Recently it was shown (M. H. Juliani, J. Brusca, and C. Klein, (1981)Develop. Biol.83, 114–121) that N⁶-(aminohexyl)adenosine 3′,5′-monophosphate (hexyl-cAMP) effectively induced phosphodiesterase activity, while this compound was chemotactically inactive and did not effectively bind to the cell surface receptor for cAMP. It was suggested that hexyl-cAMP and cAMP induce phosphodiesterase activity via a chemoreceptor-independent mechanism. In another recent report (P. J. M. Van Haastert, R. C. Van der Meer, and T. M. Konijn (1981)J. Bacteriol.147, 170–175) investigation of induction of phosphodiesterase by several cAMP derivatives revealed that phosphodiesterase induction and chemotaxis had similar cyclic nucleotide specificity. Based on this result it was suggested that cAMP induces phosphodiesterase activity via activation of the chemotactic receptor. In this report we show that hexyl-cAMP transiently inhibits extracellular and cell surface phosphodiesterase. This transient inhibition of the inactivating enzyme and the permanent release of small amounts of cAMP by the cells leads to a transient increase of extracellular cAMP levels. Hexyl-cAMP does not inhibit beef heart phosphodiesterase, and is not degraded by this enzyme. Addition of hexyl-cAMP to a cell suspension containing beef heart phosphodiesterase does not result in an accumulation of extracellular cAMP, and phosphodiesterase induction is absent. We conclude that hexyl-cAMP inhibits phosphodiesterase activity which leads to the accumulation of cAMP; consequently cAMP binds to the chemotactic cAMP receptor resulting in the induction of phosphodiesterase activity.     

Sequence and expression of the discoidin I gene family in Dictyostelium discoideum

We have isolated recombinant phage and plasmids containing the four developmentally regulated discoidin I genes of Dictyostelium discoideum. Two of the genes are linked within 0.5 × 10³ bases with the same polarity. S¹ nuclease mapping shows that at least three members of the gene family are expressed and that the 5′ ends of the mRNAs start at equivalent sites. The genes have homologous 5′ untranslated regions and extremely divergent 3′ untranslated regions. In addition, some of the genes are flanked by homologous repeat sequences. The genes encode three different isoelectric forms of the protein. Examination of nucleotide sequences in the protein coding region shows that most nucleotide changes in the 5′ half of the gene result in amino acid substitutions while most base substitutions in the 3′ half are neutral.     

Inhibition of cell differentiation and cell cohesion by tunicamycin in Dictyostelium discoideum

The effects of tunicamycin on protein glycosylation and cell differentiation were examined during early development of Dictyostelium discoideum. Tunicamycin inhibited cell growth reversibly in liquid medium. At a concentration of 3 μg/ml, tunicamycin completely inhibited morphogenesis and cell differentiation in developing cells. These cells remained as a smooth lawn and failed to undergo chemotactic migration. The expression of EDTA-resistant contact sites was also inhibited. The inhibition by tunicamycin was reversible if cells were washed free of the drug within the first 10 hr of incubation. After 12 hr of development, cells were protected from the drug by the sheath. When cells were treated with tunicamycin during the first 10 hr of development, incorporation of [³H]mannose and [³H] fucose was inhibited by approximately 75% within 45 min while no significant inhibition of [³H]leucine incorporation was observed during the initial 3 hr of drug treatment. The inhibition of protein glycosylation was further evidenced by the reduction in number of glycoproteins “stained” with ¹²⁵I-labelled con A. A number of developmentally regulated high-molecular-weight glycoproteins, including the contact site A glycoprotein (gp80), were undetectable when cells were labelled with [³H]fucose in the presence of tunicamycin. It is therefore evident that glycoproteins with N-glycosidically linked carbohydrate moieties may play a crucial role in intercellular cohesiveness and early development of D. discoideum.     

Quantitative analysis of cyclic AMP waves mediating aggregation in Dictyostelium discoideum

We have previously reported the detection of cAMP waves within monolayers of aggregating Dictyostelium discoideum cells (K. J. Tomchik and P.N. Devreotes, 1981, Science 212, 443-446). The computer-assisted analysis presented here of the fluorographic images of the cAMP waves reveals (1) all the waves have a consistent width and height; (2) cAMP concentrations within centers of concentric aggregation territories oscillate periodically while at spiral centers the concentration builds up to a plateau value within 2 mm; (3) cells within the region of intersection of two oppositely directed cAMP waves are stimulated to produce more cAMP than those responding to a single wave; (4) cells start to move when the cAMP level begins to increase and cease movement when the peak cAMP concentration reaches the cell.     

Purification of human skin tyrosinase and its protein inhibitor: properties of the enzyme and the mechanism of inhibition by protein

Tyrosinase from normal human skin was purified to high specific activity; 228 nmol of dopa formed/min/mg protein. The properties of the purified enzyme differ from those of the same enzyme in crude homogenates. The activity of the purified enzyme is not affected by dopa. It is not inhibited by excess tyrosine and exhibits no lag in its rate at 4 mm concentration of ascorbic acid. This preparation is free of peroxidase and yet will catalyze both hydroxylation of tyrosine to dopa and its further oxidation to dopa quinone with fourfold more activity with dopa as substrate suggesting that mammalian tyrosinase catalyzes both reactions rather than dopa oxidation alone as suggested by M. Okun, L. Edelstein, R. Patel, and B. Donnellan (1973, Yale J. Biol. Med.46, 535–540). A protein present in the cytosol and melanosomes that constitutes 30% of soluble epidermal proteins was purified and found to inhibit tyrosinase competitively with tyrosine. Its molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 66,000.     

Modulation of the cAMP relay in Dictyostelium discoideum by ammonia and other metabolites: possible morphogenetic consequences

Using a perfusion technique (P.N. Devreotes, P.L. Derstine, and T.L. Steck, 1979, J. Cell Biol. 80, 291-299), it has been shown that cAMP secretion by aggregation-competent cells in response to an exogenous cAMP signal is significantly reduced by exposure to NH4Cl or any of a set of carboxylic acids that includes propionate, succinate, pyruvate, and acetate. The effects of NH4Cl and any of the carboxylic acids are additive and the combinations restrict cAMP secretion to barely detectable or insignificant levels. The inhibitions are rapidly expressed, and are reversible. The activity of NH4Cl is marked at pH 7.2 and undetectable at pH 6.2. Hence, NH3 is presumably the active molecular species. Propionate activity is significantly greater at pH 6.2 than 7.2, indicating that the un-ionized acid is the active species. The data presented herein indicate that these effects are exerted via two separate and independent routes. During exposure of cAMP-stimulated cells to NH4Cl, the decrease in intracellular cAMP accumulation was even greater than the decrease in extracellular accumulation. Hence, NH3 appears to act as a cAMP accumulation inhibitor (CAI). In contrast, exposure to carboxylic acid concentrations that drastically reduce extracellular cAMP accumulation can actually enhance or, at worst, only slightly reduce intracellular accumulation. Hence, the carboxylic acids appear to act as cAMP release inhibitors (CRI). Stationary phase cells incubated on solid substratum in the presence of NH4Cl plus succinate (or propionate) for 18 hr failed to exhibit even the earliest signs of aggregation. If then harvested and redeposited in the absence of the metabolites, they proceeded through the morphogenetic sequence with approximately normal kinetics, suggesting that no significant morphogenetic competence had been achieved during their previous tenure. The morphogenetic implications of cAMP relay modulation are discussed.