Search

In the development of the cellular slime mold Dictyostelium discoideum there is a stage in which the aggregated amoebae form a migrating slug that moves forward in a polar fashion, showing sensitive orientation to environmental cues, as well as early signs of differentiation into anterior prestalk and posterior prespore cells. Heretofore it has been difficult to follow the movement of the individual cells within the slug, but a new method is described in which small, flat (one cell thick) slugs are produced in a glass-mineral oil interface where one can follow the movement of all the cells. Observations of time-lapse videos reveal the following facts about slug migration: (i) While the posterior cells move straight forward, the anterior cells swirl about rapidly in a chaotic fashion. (ii) Turning involves shifting the high point of these hyperactive cells. (iii) Both the anterior and the posterior cells move forward on their own power as the slug moves forward. (iv) There are no visible regular oscillations within the slug. (v) The number of prestalk and prespore cells is proportional for a range of sizes of these mini-slugs. All of these observations on thin slugs are consistent with what one finds in normal, three-dimensional slugs.

When Dictyostelium discoideum cells are drawn into a fine glass capillary, they rapidly begin the first steps toward the formation of prestalk and prespore zones. Some of the events occur within a minute or two, whereas others follow later. The cells in the front segment are actively motile and those in the hind segment are passive. The volumes of the segments are proportional for different-sized cell masses, and those proportions are the same as those found in normal slugs. When the cells are stained with the vital dye neutral red, the anterior zone becomes darker simultaneously with the formation of the division line. Green fluorescent protein expressed from a stalk-specific promoter is synthesized mostly in the anterior end. Later, this capillary prestalk zone shows a sharp increase in alkaline phosphatase activity, which is known to be characteristic of prestalk cells.

Rat-liver chromatin has bee fractionated into transcriptionally active and inactive regions [Gottesfeld et al. (1974) Proc. Nat. Acad. Sci. USA 71, 2193-2197] and the distribution of nuclease-resistant complexes in these fractions has been investigated. About half of the DNA of both fractions is resistant to attack by tne endonuclease DNase II. The nuclease-resistant structures of inactive chromatin are DNA-histone complexes (v-bodies) which sediment at 11-13 S. Template-active chromatin yields two peaks of nuclease-resistant nucleoprotein. These complexes sediment at 14 and 19 S, and contain DNA, RNA, histone, and nonhistone chromosomal proteins. Polyacrylamide gel electrophoresis reveals a complex pattern of chromatin proteins, suggesting that the complexes are heterogeneous in composition.

The nucleotide sequences of the recombinant DNA inserts of three bacterial plasmid clones containing nearly all of the rat serum albumin mRNA have been determined. A statistical analysis of the nucleotide sequence reveals a pattern of repeated internal homology that confirms the "intragenic triplication" model of albumin evolution.

Chromatin from Friend leukemia cells labeled with [14C]thymidine for 24 hr followed by [3H]thymidine for 10 min is converted into nucleosomes by staphylococcal nuclease at only half the rate that total chromatin is converted. Polyacrylamide gel electrophoresis of nucleosomal DNA from cells labeled for 24 hr with [14C]thymidine followed by 10 min with [3H]thymidine demonstrates that the internucleosomal spacer of newly replicated chromatin is approximately 20 base pairs shorter than that of total chromatin. The implications of this difference for models of chromatin structure are discussed.

The physical properties of rat liver chromatin and nucleosomes acetylated with acetic anhydride were examined in order to clarify the mechanism by which chemical acetylation of histones increases template activity in vitro [Marushige, K. (1976) Proc. Natl. Acad. Sci. USA 73, 3937-3941]. Acetylation was found to have dramatic effects on the magnesium solubility, nuclease sensitivity, thermal denaturation, and sedimentation of chromatin and nucleosomes. The significance of the results to models of gene activation and chromatin replication is considered.

The properties of chromatin containing newly synthesized DNA and protein were investigated. A fraction of chromatin enriched in newly replicated DNA was isolated by means of its increased density in metrizamide relative to bulk chromatin. The DNA of this fraction appeared to be packaged into nucleosomes but at a reduced nucleosomal spacing. Although pulse-labeled DNA was present in this dense fraction, nucleosomes labeled with short pulses of arginine or acetate were of normal density. The data presented are consistent with the conclusion that newly replicated DNA is associated with preexisting histones in a short-lived, compact structure whereas newly synthesized histones are deposited at normal spacing some distance from the replication fork.

Evidence is presented to support the hypothesis that, in addition to its possible role in mediating chemo- and phototaxis, ammonia (NH(3)) is also the key substance responsible for directing thermotaxis of the migrating slugs of Dictyostelium discoideum. NH(3) is produced by the cells of the slug and we show that high and low concentrations of NH(3) decrease the speed of the amoebae while intermediate concentrations increase their speed. NH(3) production by amoebae is affected by temperature: the greater the temperature, the more NH(3) is produced. From these facts we speculate that both the positive and the negative thermotaxis found in slugs can be explained by temperature gradients stimulating regional differences in NH(3) production, and depending upon the temperature, the amount of NH(3) will either be in the range that stimulates or inhibits the rate of movement. If this explanation is correct, then minute localized differences in the production of NH(3) and their differential effect on cell speed could account for all the directed movements of the cell masses of these slime molds.

We have isolated cDNA clones for the maize leaf enzymes phosphoenolpyruvate (P-ePrv) carboxylase [orthophosphate:oxaloacetate carboxy-lyase (phosphorylating) EC 4.1.1.31] and pyruvate,orthophosphate (Prv,P(i)) dikinase (ATP:pyruvate,orthophosphate phosphotransferase, EC 2.7.9.1) by exploiting the light-inducibility and large size of the mRNAs (3.5 kilobases) that encode the two enzymes. The clones were identified by hybrid-selection and immunoprecipitation assays. From a maize genomic library, two different types of genomic clones were screened with both the P-ePrv carboxylase and the Prv,P(i) dikinase cDNA clones. Information from these genomic clones and genome blots indicates that the P-ePrv carboxylase gene family has at least three members and the Prv,P(i) dikinase family at least two. Transcripts for both enzymes were detected in green leaves, etiolated leaves, and roots. The results show that the P-ePrv carboxylase mRNAs in green leaves and roots are encoded by different genes. Whereas the P-ePrv carboxylase mRNAs in all three tissues appear to be the same size, the Prv,P(i) dikinase mRNA in green leaves is about 0.5 kilobases longer than the Prv,P(i) dikinase mRNAs in etiolated leaves and roots. It is possible that all these Prv,P(i) dikinase transcripts are encoded by one gene, and the size differences may correspond to the presence or absence of a sequence encoding a chloroplast transit peptide.

A fraction of rat-liver chromatin that is transcriptionally active in vivo has been purified 6- to 7-fold over whole chromatin. This was accomplished by selectively shearing chromatin with DNase II followed by fractionating the released portion on the basis of its solubility properties in 2 mM MgCl(2). The resulting soluble material comprises 11% of the total chromatin DNA and is impoverished in histone and enriched in nonhistone protein. Compared with unsheared chromatin, this minor fraction exhibits marked differences in chromosomal protein species. DNA renaturation studies indicate that this fraction is composed of a specific subset of whole genomal DNA sequences. Furthermore, DNA.RNA hybridization experiments suggest that almost 60% of the nonrepetitious DNA sequences of this minor fraction could code for cellular RNA.

Nuclear RNA of high molecular weight, approximately 15 to 30 x 10(3) nucleotides long, isolated from rat ascites cells, has been shown previously to contain both middle repetitive and single-copy sequences. However, it was not known whether these sequences were located on separate molecules or whether they were interspersed within the same molecule. The present communication distinguishes between these possibilities and shows that a large proportion of nuclear RNA of high molecular weight consists of interspersed middle repetitive and single-copy sequences.

Fractionation of heat-shocked Drosophila melanogaster Kc cells reveals that both the small heat shock proteins (hsp28, -26, -23, and -22) and vimentin-like intermediate filament proteins (IFPs) are abundantly represented in the nuclear fraction. Cofractionation of the IFPs with nuclei is due to the collapse of the IFP network against the nucleus upon heat shock, raising the possibility that cofractionation of the small hsps is by a similar mechanism. Indirect immunofluorescence supports this possibility. In salivary glands, both the hsps and the IFPs are cytoplasmic after mild-to-moderate heat shocks and only enter the nucleus upon severe--indeed, lethal--shocks. Double-label experiments with Schneider line 2 cells show that the IFPs and small hsps colocalize to the same perinuclear aggregates in 70% of the cells examined. Thus, the small hsps are associated with the cytoskeleton rather than with nuclear structures.

We have determined the sequences of the recombinant DNA inserts of three bacterial plasmid cDNA clones containing most of the rat alpha a-fetoprotein mRNA. The resultant nucleotide sequence of alpha-fetoprotein was exhaustively compared to the nucleotide sequence of the mRNA encoding rat serum albumin. These two mRNAs have extensive homology (50%) throughout and the same intron locations. The amino acid sequence of rat alpha-fetoprotein has been deduced from the nucleotide sequence, and its comparison to rat serum albumin's amino acid sequence reveals a 34% homology. The regularly spaced positions of the cysteines found in serum albumin are conserved in rat alpha-fetoprotein, indicating that these two proteins may have a similar secondary folding structure. These homologies indicate that alpha-fetoprotein and serum albumin were derived by duplication of a common ancestral gene and constitute a gene family.

Some species of cellular slime mold do not respond to cyclic AMP as an acrasin, or chemoattractant. In one such species, Polysphondylium violaceum, we have isolated and purified its acrasin and determined some of its chemical properties, which lead us to believe it is a small molecule of less than 1500 daltons. One possibility is that it might be a peptide. The acrasin specifically attracts the amoebae of P. violaceum and P. pallidum and fails to do so for six species of Dictyostelium tested. We also have evidence for a specific acrasinase that inactivates the Polysphondylium acrasin.

With immunofluorescent techniques it has been possible to show that bound cyclic AMP is uniformly distributed in the nucleus and cytoplasm of a number of species of cellular slime molds. One species (which does not respond to cyclic AMP as an acrasin) is an exception and has its cyclic AMP concentrated in the nucleus during the feeding and aggregation stage. In cell masses of Dictyostelium discoideum that show early signs of differentiation the anterior, prestalk cells contain more cyclic AMP than the posterior, prespore cells.

Two lines of evidence derived from fusion gene constructs indicate that sequences residing in the 5'-nontranslated region of a cell cycle-dependent human H3 histone mRNA are involved in the selective destabilization that occurs when DNA synthesis is terminated. The experimental approach was to construct chimeric genes in which fragments of the mRNA coding regions of the H3 histone gene were fused with fragments of genes not expressed in a cell cycle-dependent manner. After transfection in HeLa S3 cells with the recombinant plasmids, levels of fusion mRNAs were determined by S1 nuclease analysis prior to and following DNA synthesis inhibition. When the first 20 nucleotides of an H3 histone mRNA leader were replaced with 89 nucleotides of the leader from a Drosophila heat-shock (hsp70) mRNA, the fusion transcript remained stable during inhibition of DNA synthesis, in contrast to the rapid destabilization of the endogenous histone mRNA in these cells. In a reciprocal experiment, a histone-globin fusion gene was constructed that produced a transcript with the initial 20 nucleotides of the H3 histone mRNA substituted for the human beta-globin mRNA leader. In HeLa cells treated with inhibitors of DNA synthesis and/or protein synthesis, cellular levels of this histone-globin fusion mRNA appeared to be regulated in a manner similar to endogenous histone mRNA levels. These results suggest that the first 20 nucleotides of the leader are sufficient to couple histone mRNA stability with DNA replication.

Protein degradation appears to be essential for normal differentiation in the cellular slime mold Dictyostelium discoideum. Several protease inhibitors block normal differentiation, and in most cases this inhibition can be reversed by addition of amino acids. For example, chloroquine, which inhibits slime mold cathepsin B activity, interferred with development by blocking sorocarp formation, and this inhibition was reversed by the addition of amino acids. Tosyllysyl chloromethyl ketone also blocked development, and this inhibition was reversed by simultaneous additions of amino acids and glutathione. Moreover, the addition of antipain and leupeptin delayed sorocarp formation. These results, together with the finding reported earlier that cathepsin B activity is differentially localized in the prestalk-prespore zones of the migrating slugs, suggest that proteolysis might play a regulatory role in cellular slime mold differentiation.

The structure of Dictyostelium discoideum chromatin has been studied by the following techniques: electron microscopy, staphylococcal nuclease digestion, acrylamide gel electrophoresis, sucrose gradient centrifugation, and melting. The basic unit of chromatin is the nucleosome, which is a particle 98.6 A in diameter. Approximately 50% of the chromatin is protected from nuclease digestion, but this decreases when protease activity is not inhibited. The nucleosome contains 187 base pairs of DNA, including a 137-base-pair core and a 50-base-pair linker. The monomer nucleosome has an s20,w value of 11.5 S on isokinetic sucrose gradients. When the chromatin is melted, four transitions are observed, at 54.5 degrees, 66.7 degress, 74.9 degrees, and 79.7 degrees. The structure of Dictyostelium chromatin is very similar to that seen in higher eukaryotes.

Long and short repetitive sequences of rat DNA can be isolated and characterized. Long [greater than 1.5 kilobases (kb)] sequences can be separated from short (0.2-0.4 kb) sequences by exclusion chromatography after renaturation of 4-kb DNA fragments to a repetitive Cot and digestion with the single-strand-specific S1 nuclease. (Cot is the initial concentration of DNA in mol of nucleotides/liter multiplied by time in sec.) Long repetitive DNA can be driven by an excess of whole rat DNA can also be used to drive tracer quantities of either long (self-renaturation) or short repetitive DNA. Both the extent and the rate of the renaturations are found to be similar, suggesting that long and short DNA fragments share sequences. When long repetitive DNA is used to drive whole DNA tracers of various lengths, a 3.2-kb interspersion period is found. These data are consistent with the concept that short repetitive sequences are present within long repetitive DNA sequences in the rat genome.

Hitherto it has not been possible to obtain spore and stalk cell differentiation of the cellular slime molds in submerged cultures. It is shown here that cells, when placed in roller tubes under an atmosphere of oxygen, will form clumps and differentiate in 48-72 hr into mature spores and stalk cells. Although differentiation occurs without the normal morphogenetic movements, there is the appearance of an anteroposterior polarity of the cells in the clump. In addition to oxygen we examined a number of other factors that affect differentiation.

The aggregation chemoattractant (or acrasin) of Polysphondylium violaceum, a species of cellular slime mold that does not respond chemotactically to cAMP, has been identified. It was extracted and purified from aggregating amoebae, then analyzed for amino acid composition and by IR and mass spectrometry. The active molecule is N-propionyl-gamma-L-glutamyl-L-ornithine-delta-lactam ethyl ester (Mr, 327), which we have named glorin. The compound has been synthesized and shows normal chemotactic activity with the amoebae of P. violaceum.

Previously we showed that the rising cell masses of cellular slime molds orient away from high concentrations of ammonia gas, presumably by speeding up the cells on one side. Here we show that in the same way NH(3) could also be involved in the highly sensitive phototaxis found in the migrating slugs of Dictyostelium discoideum. We have evidence that light increases their speed of migration and their production of NH(3). Since unilateral light is concentrated on the distal side of a cell mass by the "lens effect," this leads to the obvious hypothesis that the light stimulates the local production of NH(3), which, in turn, stimulates the cells in the illuminated region to move faster.

Full-length radiolabeled albumin and alpha-fetoprotein (AFP) cDNAs were synthesized from pure albumin and AMP mRNA preparations by using avian myeloblastosis virus reverse transcriptase (RNA-dependent DNA polymerase). The cDNAs have been used to quantitate the number of albumin and AFP genes in different rat tissues by two independent methods, both of which yielded similar results. First, the kinetics of the association of these cDNAs with nuclear DNA from rat liver, rat kidney, and Morris hepatoma 7777 under conditions of vast DNA excess indicated that the albumin and AFP mRNA's are transcribed from "nonrepetitive DNA." Second, saturation hybridization experiments in which a constant amount of rat liver DNA or Morris hepatoma 7777 was hybridized with increasing amounts of cDNA to albumin mRNA have shown the presence of 1--2 albumin genes per rat haploid genome. The number of AFP genes obtained in similar titration experiments was approximately 2--3. This was true whether rat liver DNA or hepatoma 7777 DNA was used in the reassociation experiments. When high molecular weight DNA preparations from both these tissues were digested with the restriction endonuclease EcoRI and the fragments were transferred to a nitrocellulose filter, the albumin and AFP [32P]cDNA probes hybridized to different sets of DNA fragments. However, each probe gave the same hybridization pattern whether Buffalo rat liver DNA or hepatoma 7777 DNA was utilized.

The rat serum albumin gene has been isolated from a recombinant library containing the entire rat genome cloned in the lambda phage Charon 4A. Preliminary R-loop and restriction analysis has revealed that this gene is split into at least 14 fragments (exons) by 13 intervening sequences (introns), and that it occupies a minimum of 14.5 kilobases of genomic DNA.

Two major nonhistone polypeptides from rat liver chromatin have been identified as myosin and actin. Preliminary observations indicate that three other chromatin polypeptides of molecular weights 50,000, 34,000, and 32,000 are tubulin and heavy and light tropomyosin, respectively. A sixth component of molecular weight 65,000 which has been purified and electrophoreses as a single band on sodium dodecyl sulfate-polyacrylamide gels may be composed in part of protease-digested myosin. These six polypeptides together account for as much as 38% of the nonhistone protein mass of chromatin in this tissue.

Rat liver chromatin has been fractionated into two fractions on the basis of precipitability in standard saline after mild treatment with DNase II. The major portion of liver chromatin contains little nonhistone protein and is enriched in histones, while a minor portion of such chromatin, with which RNA polymerase is associated, is highly enriched in proteins other than histone and impoverished in histones. Although the relative concentrations of the several histone species present in two chromatin fractions are identical, binding of histones to DNA in the minor fractions appears to be modified, presumably by the presence of proteins other than histones.

The hypothesis that insulin may serve as a derepressor of genetic information in the liver of diabetic rats has been tested by comparing the template activity for RNA synthesis of chromatin from liver of insulin-treated diabetic rats to that of chromatin from liver of insulin-deficient diabetic rats. The template activity of the chromatins of insulin-treated diabetic rats is found to be 28 per cent greater than that of chromatin from liver of diabetic rats not treated with insulin. Time course studies show that the template activity of liver chromatin of rats injected with a single dose of insulin reaches a peak at two hours, which is some hours before the appearance of a typical insulin-induced liver enzyme, glucokinase. We conclude that insulin derepresses genetic material of the diabetic liver genome that is repressed in the absence of insulin.

Chromosomal RNA, which is a component of the chromosomes of higher organisms, and whose participation is required for sequence-specific interaction of chromosomal proteins with chromosomal DNA, occurs in chromosomes bound to DNA. We have found that the DNA sequences to which chromosomal RNA binds are repetitive ones.

Chromosomal RNA was isolated from several rat tissues by a new technique. It is shown by RNA-DNA hybridization that each tissue contains a distinct population of chromosomal RNA sequences. This finding is compatible with the proposal that chromosomal RNA is involved in the process of gene activation.