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.     

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.     

Isolation of a multigene family containing human alpha-tubulin sequences

The boundaries of the origin of polyoma DNA replication have been analyzed using a set of deletion mutants. The majority of these had small deletions, 5 to 30 base-pairs in size, which together removed most of the non-translated sequences of the genome. The phenotype of the mutants was characterized by analysis of infectivity, transforming ability and DNA synthesis. All mutants with reduced or abolished infectivity had corresponding defects of viral DNA synthesis. The effect of the deletion was cis-acting, since the replication of the mutants was not stimulated by the presence of wild-type DNA. Deletions causing a reduction of DNA synthesis were found at two sites. The first at the 32 base-pair inverted repeat sequence and the neighbouring A · T tract previously implicated in the initiation of DNA synthesis, and the second close to the late genes. The two sites were separated by at least 60 base-pairs of non-essential DNA. Only one mutant with a deletion at the second site was unable to express early gene functions.The mutants were constructed by linearization, shortening and recircularization of polyoma DNA inserted into the plasmid pBR322. The mutagenesis was directed at restriction endonuclease BglI or PvuII cleavage sites. The BglI-directed mutagenesis was focussed to polyoma DNA by using as a vector a derivative of pBR322 resistant to cleavage by BglI.     

The absence of a growth inhibitor during the log phase of growth of Dictyostelium discoideum

When amebae of the cellular slime mold Dictyostelium discoideum Ax-3 reach stationary phase in liquid nutrient medium, an extracellular factor appears which will inhibit cell multiplication when added to a logarithmically multiplying culture (Yarger et al., 1974). Recently, Hanish (1975) presented evidence that an inhibitor also accumulated during log phase and concluded that the cessation of cell multiplication and the final cell concentration were controlled by the accumulation of this inhibitor to an effective level. We have reexamined this extremely interesting observation, but have found no evidence to support Hanish's conclusions.     

Multiple,heterogeneous actin genes in Dictyostelium

We have used an actin gene-containing restriction fragment of plasmid M6 (Kindle and Firtel, 1978) 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 B1 and pcDd actin A1 (Bender et al., 1978). 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 6–8%. 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.     

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.     

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.     

Identification and analysis of Dictyostelium actin genes,a family of moderately repeated genes

Plasmid M6 has been shown to contain sequences complementary to two related abundant mRNA species which differ in length by 100 nucleotides and code for Dictyostellum actin. M6 complementary RNA was isolated by hybridization to immobilized M6 DNA and translated in vitro. The product is identical to major forms of in vivo labeled actin in both mobility on two-dimensional gels and two-dimensional fingerprints of tryptic peptides. Both plasmid M6 and a second plasmid complementary to the actin mRNA complementary region in M6, pDd actin 2 (McKeown et al., 1978), direct the synthesis in minicells of a number of similar polypeptides that are not seen in minicells containing other recombinant plasmids. Three of these polypeptides are similar in two-dimensional gel mobility to Dictyostelium actin and bind to DNAase I agarose.The repetition frequency of isolated restriction fragments from actin mRNA complementary plasmid M6 has been examined. The data from two different experimental approaches (DNA excess hybridizations using plasmid DNA as probe, and hybridization of plasmid probe to DNA blot filters of restriction enzyme-digested Dictyostelium DNA) indicate that the mRNA complementary region is reiterated 15–20 times. When an actin cDNA probe is used in the same experiments, the results suggest that the entire coding region is reiterated.When the two major actin mRNA species are separated and independently translated, each appears to code for one of the two major actin species. The results suggest that there are at least two different functional genes, and possibly more, for Dictyostelium actin.     

In situ inactivation of E. coli uridylylation cycle is independent of the state of covalent modification of the components of the glutamine synthetase cascade

Permeabilization of nitrogen-starved cells of Escherichia coli W with Lubrol WX leads to a selective inactivation of the uridylyl-removing uridylyltransferase (UR/ UTase) enzyme of the glutamine synthetase (GS) cascade system; whereas similar treatment does not affect activity of UR/UTase in cells grown under conditions of nitrogen excess (10 mm glutamine) (Mura, U., and Stadtman, E. R. (1981) J. Biol. Chem.256, 13014–13021). The possibility that susceptibility to Lubrol inactivation is related to differences in the state of adenylylation of GS and/or in the state of uridylylation of the P_II protein was investigated. Permeabilized cells from nitrogen sufficient as well as from nitrogen-limited growth medium were exposed to Lubrol after prior incubation under conditions that lead to high or low states of GS adenylylation and high or low P_IID/P_IIA ratios. Integrity of UR/UTase was monitored by measuring the capacity of UTP to stimulate the deadenylylation of GS in situ. The results showed that the inactivation of UR/UTase by Lubrol is not affected by the states of GS adenylylation or P_II uridylylation.     

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.     

Studies on the mode of action of calciferol: Comparison of the biochemical properties of an antiserum and the chick intestinal receptor both specific for 1,25-dihydroxyvitamin D3

The structural requirements for the interaction of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] with an anti-1,25(OH)₂D₃ antiserum and with the natural cytosolic receptor for 1,25(OH)₂D₃ isolated from chick intestine have been evaluated quantitatively. The antiserum was raised in a rabbit against a 1,25(OH)₂D₃-hemisuccinate derivative which was linked to bovine serum albumin at the C-3 position of the steroid. For these cross-reaction studies structural analogs of 1,25(OH)₂D₃ were used in competitive protein binding assays; their ability to interact with the binding proteins was expressed as relative competitive index (RCI) values where the RCI of 1,25(OH)₂D₃ is defined to be 100. The results indicate that the 25-hydroxyl group is the most important hydroxyl for the interaction of 1,25(OH)₂D₃ with this antiserum. The absence of this hydroxyl group decreases the RCI value to 0.2. Lack of the hydroxyl at carbon-3 or carbon-1 decreases the RCI value to 33 or 25, respectively, indicating that the specificity of this antiserum for the A ring is much lower than for the side chain. The high specificity for the side chain is underlined by the fact that insertion of an additional hydroxyl group at C-24 or C-26 of 1,25(OH)₂D₃ decreases the binding affinity to the antiserum markedly. The chick intestinal mucosal receptor shows a comparable high specificity for the side chain of 1,25(OH)₂D₃, but an even higher specificity for the A ring in comparison to the antiserum. With the intestinal receptor, the 3-hydroxyl is only 1/ 10th as important as the 1-hydroxyl group and the 25-hydroxyl group for the binding process. Scatchard analysis showed a K_D value of 1.7 × 10^?10m for the antiserum and 2.3 × 10^?10m for the chick intestinal mucosal receptor for the equilibrium binding of 1,25(OH)₂D₃ at 2 °C. The association rate constant at 2 °C was determined to be 5.8 × 10⁷ M^?1 min^?1 for the antiserum and 0.55 × 10⁷ M^?1 min^?1 for the receptor, indicating a 10-fold more rapid association of 1,25(OH)₂D₃ to the antiserum in comparison to the receptor. Furthermore, the dissociation process was found to be slower for the chick intestinal receptor (dissociation rate constant 3.6 × 10^?5 min^?1 versus 21.0 × 10^?5 min^?1).     

Comparison of the applicability of several allosteric models to the pH and 2,3-bis(phospho)glycerate dependence of oxygen binding by human blood

Oxygen saturation curves of blood were measured by the mixing method at several concentrations of protons and bis(phospho)glycerate. Various theoretical models for the co-operativity of oxygen binding by haemoglobin were then tested for their ability to fit the experimental curves. The effects of pH on oxygen binding could be described by both the Monod, Wyman & Changeux, and the Herzfeld & Stanley models, with most success when protons were assumed to affect oxygen affinity directly rather than through effects on the quaternary-state equilibrium. When the combined effects of pH and bis(phospho)glycerate were considered, however, all the versions of the Monod model that were used, including the three-state version, were unsuccessful. The best fit to the saturation curves was obtained with the Herzfeld & Stanley model, with protons acting as a direct effector of oxygen affinity, and bis(phospho)glycerate acting to lower oxygen affinity as well as influencing the quaternary-state equilibrium.     

X-ray crystallographic structure of the light-harvesting biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus and its resemblance to globin structures

The structure of the biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 3 A resolution by X-ray diffraction methods. Phases have been obtained by the multiple isomorphous replacement method. The electron density map could be improved by solvent flattening and has been interpreted in terms of the amino acid sequence. The protein consists of three identical (alpha-beta)-units which are arranged around a threefold symmetry axis to form a disc of approximate dimensions 110 A X 30 A with a central channel of 35 A in diameter. This aggregation form is supposed to be the same as that found in the rods of native phycobilisomes. Both subunits, alpha and beta, exhibit a similar structure and are related by a local twofold rotational axis. Each subunit is folded into eight helices and irregular loops. Six helices are arranged to form a globular part, whereas two helices stick out and mediate extensive contact between the subunits. The arrangement of the helices of the globular part resembles the globin fold: 59 equivalent C alpha-atoms have a root-mean-square deviation of 2 X 9 A. The chromophores attached to cystein 84 of the alpha- and beta-subunits are topologically equivalent to the haem. All three chromophores of C-phycocyanin, open-chain tetrapyrroles, are in an extended conformation. alpha 84 and beta 84 are attached to helix E (globin nomenclature), beta 155 is linked to the G--H loop. The shortest centre-to-centre distance between chromophores in trimer is 22 A.     

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.     

Control of the mitochondrial inner membrane permeability by sulfhydryl groups

The effect of some thiol alkylating agents (N-substituted maleimide derivatives) on the permeability of the mitochondrial inner membrane was investigated. Several experimental approaches were used to study the modifications of the permeability properties. Alkylation of sulfhydryl groups led to an increase in the nonspecific permeability as judged by (i) the augmentation of the rate of osmotic shrinkage of mitochondria induced by polyethylene glycol, (ii) the sensitization of succinate dehydrogenase toward oxaloacetate, (iii) the enhancement of the oxidation rate of exogenous NADH, and (iv) the increase of the sucrose permeable space. The sulfhydryl groups involved in the maintenance of the selective permeability were shown to be located in the hydrophobic core of the membrane. Energization of mitochondria provoked an unmasking of these sulfhydryl groups. When magnesium ions were present in the incubation medium, N-substituted maleimide derivatives promoted gross modifications of the intramitochondrial ionic contents. Effluxes of endogenous calcium ions, inorganic phosphate, adenine nucleotides, and NAD(P)H were established. It was concluded that sulfhydryl groups probably play a crucial role in the maintenance of the membrane integrity and thus control the mitochondrial inner membrane permeability.     

The procaryotic nature of the fatty acid synthetase of developing Carthamus tinctorius L. (safflower) seeds

All component activities involved in the synthesis of fatty acid were detected in crude extracts of developing safflower seeds. The crude extracts were fractionated into three portions by polyethylene glycol (0–5, 5–15, and 15% supernatant). Acetyl-CoA:acyl carrier protein (ACP) transacylase was precipitated about 66% by 5% polyethylene glycol. β-Ketoacyl-ACP reductase and enoyl-ACP reductase I were completely recovered in the 5–15% fraction. β-Ketoacyl-ACP synthetase and enoyl-ACP reductase II were in the 15% supernatant fraction. Malonyl-CoA:ACP transacylase and β-hydroxyacyl-ACP dehydrase were distributed into both fractions of 5–15 and 15% supernatant. When the 5–15% fraction was gel-filtrated on Sephadex G-200 column, β-hydroxyacyl-ACP dehydrase and malonyl-CoA:ACP transacylase were clearly separated from other enzymes, but β-Ketoacyl-ACP reductase and enoyl-ACP reductase I overlapped. However, by hydroxyapatite chromatography, these two reductases were clearly separated. Properties of each enzyme were examined with the samples fractionated by polyethylene glycol. β-Ketoacyl-ACP reductase preferably utilized NADPH (K_m = 16 μM) as hydrogen donor. The K_m for acetoacetyl-ACP was 9 μm. β-Hydroxyacyl-ACP dehydrase had a K_m of 12 μm for crotonyl-ACP. Enoyl-ACP reductase had two forms, I and II, and these two reductases differed from each other as follows: (a) separation by polyethylene glycol (15%) fractionation; (b) the optimum pH; (c) the hydrogen donor specificity; (d) the substrate specificity. From these results, it is concluded that the FAS system of developing safflower seeds was nonassociated and similar to the procaryotic type of Escherichia coli.     

The structure of a platinum(II) complex of cytidine-3'-monophosphate.

The structure of the cis-[Pt(NH₃)₂(3′-CMP)₂]^2? ion, isolated in a partially protonated form as its cesium salt, has been analyzed by single-crystal x-ray diffraction methods. The 3′-CMP ligands bind in a monodentate fashion through their N(3) atoms: in contrast to the structure of [Pt(en)(5′-CMP)]₂, no covalent platinum-phosphate bonding is found. This compound represents the first example of a 1:2 cis-metal/cytosine complex structurally characterized.     

Zn(II)-induced dimerization of human carbonmonoxy hemoglobin

Binding of Zn(II) to the carbon monoxide complex of human hemoglobin was shown by equilibrium sedimentation and sedimentation velocity experiments at pH 7.0 to induce the dissociation of liganded tetramers to dimers but not to monomers. These results provide direct confirmation of previous kinetic and gel filtration experiments (R. D. Gray, (1980) J. Biol. Chem.255, 1812–1818) that Zn(II) binding to liganded hemoglobin produces a change in aggregation state of liganded hemoglobin.     

N-acetyl conjugates of basic amino acids newly identified in rat urine

Ninhydrin-negative conjugates of basic amino acids were isolated from rat urine and were characterized. The following conjugates of basic amino acids are the compounds newly identified in animal urine specimens, N^α-acetyl-N^π-methylhistidine, N^α-(N-acetyl-β-alanyl)histidine (N-acetylcarnosine), N^α-acetyl-N^G,N^′G-dimethylarginine, N^α-acetyl-N^G,N^G-dimethylarginine, and N^α-acetyl-N^?,N^?,N^?-trimethyllysine.