Isolation and characterization of megaplasmid DNA from lithoautotrophic bacteria

A method is described for the preparative isolation of megaplasmids ranging in size from 340 to 700 kb. These plamids were isolated from chemolithoautotrophic bacteria including the species Alcaligenes, Pseudomonas, and Paracoccus. The procedure was based on alkaline sodium dodecyl sulfate lysis of the cells, followed by heat treatment, salt precipitation, several phenol extractions, dialysis steps, and proteinase and RNase treatment. The various parameters were evaluated and controlled. Hydrogen-oxidizing-ability (Hox) encoding plasmids were compared by EcoRI restriction enzyme analysis. pHG plasmids from Alcaligenes eutrophus wild-type strains appeared to be closely related; plasmids derived from the type strain TF93 and from A. hydrogenophilus exhibited major differences in restriction sites. Two cryptic plasmids harbored by Pseudomonas facilis and Paracoccus denitrificans showed scarcely detectable similarity to the plasmid species of Alcaligenes.     

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.     

Isolation and characterization of a tryptic fragment containing the thioesterase segment of fatty acid synthetase from the uropygial gland of goose.

Trypsin treatment of purified fatty acid synthetase from the uropygial gland of goose released a 33,000 molecular weight peptide from the 270,000 molecular weight synthease. A combination of ammonium sulfate precipitation, Sephadex G-100 gel filtration, anion-exchange chromatography with QAE-Sephadex, and cation-exchange chromatography with cellulose phosphate gave rise to the first homogeneous preparation of the 33,000 molecular weight fragment containing fatty acyl-CoA thioesterase activity. Amino acid composition of this peptide was quite similar to that of the intact fatty acid synthetase except for a lower valine content; a partial specific volume of 0.734 was calculated for the thioesterase fragment. The pH optimum for the thioesterase was near 7.5 and the enzyme showed a high degree of preference for CoA esters of fatty acids with 16 or more carbon atoms. Palmitoyl-CoA inhibited the enzyme and therefore the rate of hydrolysis was not proportional to the amount of protein at low concentrations. Inclusion of bovine serum albumin in the reaction mixture prevented this inhibition. Disregarding the substrate inhibition, an apparent K_m of 5 × 10^?5m and a V of 340 nmol/min/mg were calculated. The thioesterase was inhibited by active serine-directed reagents such as phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate as well as by SH-directed reagents as p-chloromercuribenzoate and N-ethylmaleimide. The isolated thioesterase fragment generated antibodies in rabbits and the antithioesterase inhibited the enzymatic activity of fatty acid synthetase. The antithioesterase showed immunoprecipitant lines with fatty acid synthetase from the uropygial gland and the synthetase from the liver of goose. Anti-fatty acid synthetase prepared against the enzyme from the gland cross-reacted with the thioesterase segment. Even though the synthetase from the uropygial gland synthesizes multimethyl-branched fatty acids in vivo, the thioesterase segment of this synthetase appears to be quite similar to that isolated from the rat.     

The production of oxyluciferin during the firefly luciferase light reaction.

The luciferase-product complex (E · P) was isolated from the reaction mixture after light emission had occurred. The spectral properties of the product in the E · P complex are similar to those of oxyluciferin, with a broad absorption at 385 nm. The enzyme from the complex regains full activity upon the addition of substrates. The product is not covalently bound to the enzyme and readily dissociates in the presence of 6 m urea. The isolated E · P complex was found to have 1 mol of oxyluciferin per 100,000 daltons of luciferase. No AMP could be detected in the E·P complex unless inorganic pyrophosphatase was present during the reaction. In that case 1 mol of AMP per 100,000 daltons was found.Stopped flow studies showed that an increase in 385 nm absorption occurred concomitant with light emission. Measurement of the initial rate of product formation and the rate of photon emission showed they were identical, suggesting that oxyluciferin is indeed the light-emitting product. In the initial burst of the reaction two oxyluciferin moles per 100,000 daltons of luciferase are formed. A plot of the log of the initial rate of product formation was biphasic, indicating that the first mole of product is formed at a faster rate than the second. These results are consistent with previous experiments. However, they do not resolve the question of the molecular weight of the catalytically active species.     

Isolation and characterization of an acyl-coenzyme A carboxylase from an erythromycin-producing Streptomyces erythreus

Streptomyces erythreus produces erythromycin presumably from methylmalonyl-coenzyme A, (CoA) which might be generated by carboxylation of propionyl-CoA. A biotin-containing enzyme which carboxylates acetyl-CoA, propionyl-CoA, and butyryl-CoA was purified to near homogeneity from S. erythreus using DEAE-cellulose, affinity chromatography on monomeric avidin-Sepharose, and blue Sepharose. The enzyme carboxylates propionyl-CoA (100%) with a K_m of 0.09 mm and V of 0.86μmol/mg/min, acetyl-CoA (16%) with a K_m of 0.17 mm and V of 0.08μmol/mg/min, and butyryl-CoA (7.7%) with a K_m of 0.67 mm and V of 0.044 μmol/mg/min. The native enzyme has a molecular weight of 537,000 and consists of two types of subunits with molecular weights of 67,000 and 61,000, respectively, indicating an octameric α₄β₄ type of structure. Biotin is associated with the large subunit (α). The enzyme has a pH optimum between 7.5 and 7.8. It is stimulated (three- to fourfold) by K⁺, Rb⁺ and Cs⁺ but not by Na⁺ or Li⁺ and is inhibited by high concentrations of NH₄⁺ and C1^?. Neither citrate nor free CoA stimulated the enzyme. The enzyme was shown to be stereospecific and generated onlyS-methylmalonyl-CoA from the carboxylation of propionyl-CoA. The present case appears to be the first enzyme possibly involved in erythromycin production to be isolated in homogeneous form.     

Purification and partial characterization of the catalytic subunit of cAMP-dependent protein kinases from reticulocytes.

The catalytic subunit of cyclic AMP-dependent protein kinases from rabbit reticulocytes has been purified to near homogeneity. It has a molecular weight of 43,000 as judged from gel filtration and by polyacrylamide gel electrophoresis in the presence of sodium dodecyi sulfate and appears to be similar in physical properties and substrate specificity to the comparable enzyme isolated from muscle or liver. The enzyme phosphorylates histones, a protein of 40 S ribosomal subunits from reticulocytes and from Artemia salina, and the low molecular weight heat-stable phosphatase inhibitor (G. A. Nimmo and P. Cohen, 1978, Eur. J, Biochem.87, 341–351). No evidence has been obtained for a direct or indirect role of this enzyme in the regulation of protein synthesis.     

Flash spectroscopic characterization of photosynthetic electron transport in isolated heterocysts

Electron transport was studied in heterocysts of the filamentous cyanobacterium Anabaena 7120 using spectral and kinetic analysis of absorbance transients elicited by single turnover flashes. Consistent photosynthetic turnovers were observed only in the presence of an exogenous source of reductant; therefore measurements were routinely made under a gas phase containing H2. Prominent absorbance changes corresponding to the oxidation of cytochrome c (554 nm) and the reduction of cytochrome b563 (563 nm) were observed. Under the most reducing conditions (99% H2/1% O2) cytochrome b563 was partially reduced between flashes in a slow, dark reaction. At 10-15% O2, the slow, dark reduction of cytochrome b563 was eliminated. Cytochrome turnover ceased entirely at high O2 concentrations (30%) but was restored by the addition of 25 microM KCN, demonstrating an interaction between the photosynthetic and respiratory electron transfer chains. Strobilurin A slowed the re-reduction of cytochrome c and eliminated the appearance of reduced cytochrome b563 by blocking electron transfer between reduced plastoquinone and the cytochrome b/f complex. Inhibition at a second site was apparent with 2-(n-heptyl)-4-hydroxyquinoline N-oxide, which blocked the reoxidation of cytochrome b563 but had little effect on cytochrome c relaxation. In uncoupled heterocysts, the rates of cytochrome c re-reduction and cytochrome b563 reduction were equal. Additional unassigned absorbance changes at 475 nm, 515 nm, and 572 nm were partially characterized. No absorbance change corresponding to an electrochromic shift was observed.     

Proteolytic fragmentation of Helix pomatia alpha-hemocyanin: isolation of a functionally active chemically pure domain and evidence for subunit heterogeneity.

α-Hemocyanin of the vineyard snail, Helix pomatia, is a large oligomer composed of 20 subunits with a molecular weight of 360,000 ± 30,000. Limited proteolysis showed these subunits to be composed of about seven structural domains, each having one oxygen binding site (1). This paper describes the production of these structural domains by tryptic digestion of $\text{1}\text{10}$ hemocyanin molecules. The digestion pattern was followed as a function of time by examining the proteolytically obtained fragments electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels. Based on the molar ratio of each fragment present during digestion the first part of the reaction sequence for trypsinolysis could be deduced. This reaction scheme was simulated by means of an analog computer. Pseudo-first-order reaction rate constants of the various proteolytic cleavages were estimated from the computer generated time course of digestion. On the basis of these results it is postulated that Helix pomatia α-hemocyanin possesses at least two kinds of subunits which differ in their proteolytic susceptibility. These subunits occur in equimolar amounts. A functionally active domain with a molecular weight of about 50,000 has been isolated from a tryptic digest of hemocyanin subunits. This domain seems to be chemically pure, as suggested by the unique sequence of its first six amino acids, viz: Lys-Val-His-Leu-Asn-Lys.     

Isolation and characterization of the hydrophobic COOH-terminal domains of the Sindbis virion glycoproteins

Digestion of intact Sindbis virions with α-chymotrypsin produced a single membrane-associated peptide derived from each of the two virion glycoproteins (referred to as RE1 and RE2, or roots derived from E1 and E2, respectively). Amino acid composition data and NH₂-terminal sequence analysis established their location at the extreme COOH-terminal end of each glycoprotein. RE1 and RE2 are rich in hydrophobia amino acids and insoluble in aqueous solutions in the absence of detergents, and show differential solubility in organic solvent systems designed for the extraction of lipids. Essentially all of the covalently attached palmitic acid associated with E1 and E2 was found to be clustered in their hydrophobic, membrane-associated roots. Beginning six to seven residues from their NH₂ termini, RE1 and RE2 contain uninterrupted sequences of hydrophobic amino acids similar in terms of amino acid composition and length to the transmembrane anchors found in other bitopic integral membrane proteins. By comparing the sequence and composition data obtained here with the sequences of E1 and E2 deduced from complementary DNA sequence analysis (Rice & Strauss, 1981) we can make several observations. First, following their uncharged, putative intramembrane segments (33 and 26 amino acids, respectively), E1 and E2 contain clusters of predominantly basic amino acids. By structural analogy to known transmembrane proteins, E1 probably spans the bilayer but contains only a few residues exposed on the inner face of the virion envelope. In contrast, E2 and PE2 (the precursor to E2), which have been shown to span the bilayer completely, contain an additional 33 COOH-terminal residues, which could be either exposed on the cytoplasmic face of the lipid bilayer or which could loop back into the membrane. This region at the extreme COOH-terminal end of E2, which is protected by the virion envelope from digestion by a-chymotrypsin, contains a second uncharged domain (23 amino acids in length) whose orientation is unknown, but which may be involved in the highly specific interaction of the transmembrane glycoproteins in the plasma membrane with the cytoplasmic nucleocapsid during budding.     

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.     

Protocatechuate 3,4-dioxygenase. Resonance Raman studies of the oxygenated intermediate.

Resonance Raman spectra of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa have been investigated during the reaction of the enzyme with substrate and oxygen. It is found that the spectrum of the turned-over enzyme is indistinguishable from that of the resting enzyme in the absence of substrate, and is characterized by resonance-enhanced tyrosinate ring vibrational modes at 1263 and 1174 cm^?1. In the ternary ESO₂ complex, however, the tyrosinate vibrational modes are shifted to 1252 and 1165 cm^?1, respectively. There is no evidence for any dioxygen vibrations in the spectra of ESO₂ complexes prepared with ¹⁶O₂, ¹⁸O₂, and ¹⁶O¹⁸O in the region between 1300 and 200 cm^?1. The results of this resonance Raman study are interpreted to indicate that molecular oxygen is attached only to the substrate (but not iron) in the stable intermediate, and that the concomitant rearrangement at C4 of the substrate induces a substantial change in geometry of the tyrosine residues associated with the iron complex. Furthermore, the optical spectrum of the ESO₂ complex (λ_max = 520 nm) is dominated by tyrosinate → Fe(III) charge transfer and contains little or no peroxide → Fe(III) charge transfer. These results invalidate the previously advanced analogy in spectral properties between this enzyme and the respiratory protein, oxyhemerythrin.     

Nitrogen and ammonia assimilation in the cyanobacteria: regulation of glutamine synthetase.

Glutamine synthetase, the first enzyme of the ammonia assimilatory pathway, has been purified from Anabaena sp. CA by use of established procedures and by affinity chromatography as a final step. No adenylylation system controlling glutamine synthetase activity was found. The enzyme shows a marked specificity for Mg²⁺ in the biosynthetic assay and Mn²⁺ in the transferase assay. Under physiological conditions, Co²⁺ produces a large stimulatory effect on the Mg²⁺-dependent biosynthetic activity. The enzyme is inhibited by the feedback modifiers l-alanine, glycine, l-serine, l-aspartate, and 5′-AMP. Inhibition by l-serine and l-aspartate is linear, noncompetitive with respect to l-glutamate with apparent K_i values of 3 and 13 mm, respectively. Cumulative inhibition is seen with mixtures of l-serine, l-aspartate, and 5′-AMP. The results indicate that, in vivo, divalent cation availability and the presence of feedback inhibitors may play the dominant role in regulating glutamine synthetase activity and hence ammonia assimilation in nitrogen-fixing cyanobacteria.     

Preliminary characterization of chelation-sensitive nucleoprotein particles

Nucleoprotein particles (B2), isolated following digestion of calf thymus chromatin with micrococcal nuclease, are resolved on a non-chelating Bio-Gel A-5m column. B2 protein electrophoresis showed the presence of several H1 species and several nonhistone proteins but was depleted in core histones. DNA electrophoresis demonstrated that native B2 DNA has a length of about 46 base pairs. On DNA sequencing gels, the length distribution of denatured B2 DNA ranged from 12 to 35 bases with a weighted average chain length of about 26 bases. Depletion of a 20 base band in B2 DNA suggested specific protection of internucleosomal DNA sites during the nuclease digestion.     

The viscoelastic properties of actin solutions

The viscoelastic properties in actin solutions were investigated by measuring their elastic modulus and viscous modulus using a rheometer. The polymerization/gelation process of actin solutions was accompanied by an increase of both parameters, indicating the formation of a protein network. High shear rotational motion destroyed this network which, however, would reanneal if left undisturbed. At 25 °C under low ionic strength conditions, the viscoelastic moduli of a Spudich-Watt globular (G) actin preparation increased with time, while G-actin, purified by gel filtration maintained low viscoelastic moduli. The rigidity of the filamentous (F) actin network in a solution of Spudich-Watt actin, measured by the elastic modulus, was somewhat lower than that of gel-filtration-purified actin at the same protein concentration. The crosslink density of these F-actin networks was estimated, using models from rubber elasticity theory. The calculated density was 1 crosslink/50 actin monomers for the purified actin and 1 crosslink/120 actin monomers for Spudich-Watt actin. The results are consistent with the idea that a small amount of regulatory factor(s), which could be removed by the gel filtration step, modulates the structure of an actin network.     

Scanning electron microscopy of Drosophila melanogaster embryogenesis: III. Formation of the head and caudal segments

The formation of both the anterior most and posterior most segments in higher dipteran embryos involves complex movements of primordia which can be best visualized with the scanning electron microscope. During head formation, the gnathocephalic segments partially involute through the stomodeum. The labial segment forms the floor of the mouth, and the fused maxillary and mandibular segments form the lateral sides of the mouth. The involuted clypeolabrum forms the roof of the mouth. Invaginations of cells for segmentally derived sense organs can be found prior to involution on all the gnathocephalic and thoracic segments as well as on the labrum. The antennal sense organ derives from the lateral surface of the procephalic lobe. Following involution of the mouth parts, the dorsal ridge, which arises just anterior to the first thoracic segment, is drawn over the dorsal procephalic lobe producing the deep dorsal sac. The optic lobes of the brain invaginate anterior to the dorsal ridge just prior to the covering over of the head. The formation of the anal segment is similarly complex. Two rudimentary segments are found posterior to the eighth abdominal segment. During shortening of the germ band, the posterior most segment is drawn around the posterior tip of the embryo to lie ventrally. Two large anal pads form lateral to the anus from this segment. The next segment, following dorsal closure, produces a pair of anal sense organs and a central tuft of setae. Finally, the eighth abdominal segment gives rise to the posterior spiracles. Following dorsal closure these three segments fuse to produce the terminal (anal) segment of the larva.     

Restricted fragmentation of poliovirus type 1, 2 and 3 RNAs by ribonuclease III.

Cleavage of the genome RNAs of poliovirus type 1, 2 and 3 with the ribonuclease III of Escherichia coli has been investigated with the following results: (1) at or above physiological salt concentration, the RNAs are completely resistant to the action of the enzyme, an observation suggesting that the RNAs lack “primary cleavage sites”; (2) lowering the salt concentration to 0.1 m or below allows RNase III to cleave the RNAs at “secondary sites”. Both large and small fragments can be obtained in a reproducible manner depending on salt conditions chosen for cleavage. Fingerprints of three large fragments of poliovirus type 2 RNA show that they originate from unique segments and represent most if not all sequences of the genome. Based upon binding to poly(U) filters of poly(A)- linked fragments, a physical map of the large fragments of poliovirus type 2 RNA was constructed. The data suggest that RNase III cleavage of single-stranded RNA provides a useful method to fragment the RNA for further studies.     

The in vivo effect of benzamide and phenobarbital on liver enzymes: poly(ADP-ribose) polymerase, cytochrome P-450, styrene oxide hydrolase, cholesterol oxide hydrolase, glutathione S-transferase and UDP-glucuronyl transferase

Rats fed a synthetic diet containing 0.25% benzamide, 0.1% phenobarbital, separately or in combination, for two weeks showed a significant augmentation in the activity of nuclear poly(ADP-ribose) polymerase as well as changes in various nuclear, microsomal and cytosolic liver enzymes involved in the metabolism of xenobiotics. A selective depression of microsomal styrene oxide hydrolase activity by benzamide feeding, and a contrasting augmentation by phenobarbital, were confirmed by immunological titration of the enzyme-protein content suggesting actual enzyme repression and induction. The NAD content of these livers is not altered significantly as a result of benzamide and phenobarbital feeding, indicating that the changes in enzymes are not a result of non-specific toxic effects.     

Isolation and partial characterization of a rat hepatoma heparan sulfate proteoglycan

A proteoglycan was isolated from a Morris rat hepatoma by sequential precipitations with ammonium sulfate and cetyl pyridinium chloride followed by chromatography on Sepharose CL-4B and DEAE-cellulose. The proteoglycan has a molecular weight of about 1.5 × 10⁵ with 40,000 molecular weight glycosaminoglycan side chains, identified as heparan sulfate based on resistance to chondroitinase and susceptibility to nitrous acid treatment. Immunological studies showed that the protein core of this proteoglycan is immunologically distinct from a rat yolk sac tumor chondroitin sulfate proteoglycan (Å. Oldberg, E. G. Hayman, and E. Ruoslahti, 1981,J. Biol. Chem.256, 10847–10852), but resembles a heparan sulfate proteoglycan isolated from a basement membrane-producing mouse tumor (J. R. Hassell, P.M. Robey, H.-J. Barrach, J. Wilczek, S. R. Rennard, and G. R. Martin, 1980, Proc. Nat. Acad. Sci. USA77, 4494–4498).     

The interferon-induced enzyme oligo-isoadenylate synthetase: rapid determination of its in vitro products

A procedure for rapid isolation of the products of the interferon-induced enzyme, oligoisoadenylate synthetase, is described. After incubation of [α-³²P]ATP with the poly(rI): (rC)-adsorbed fraction of cellular proteins, the products are treated with phosphatase. Aliquots of the nucleotides are then applied on small columns which contain 300 μl of alumina powder and eluted with 3 ml of 1 m glycine-HCl buffer, pH 2.0. The labeled free phosphate, released by the phosphatase treatment, is efficiently adsorbed by the alumina, while the phosphatase-resistant cores of the oligo-isoadenylates, up to the length of pentamer, are eluted. Larger oligomers are only partially recovered. We successfully applied this method for determination of the level of the enzyme in multiple samples of cell homogenates.     

Enrichment for auxotrophic and heat-sensitive mutants of Neurospora crassa by tritium suicide.

Tritium suicide is shown to be an effective technique for mutant enrichment in Neurospora crassa. When mutagenized conidia were labelled to a high specific radioactivity either with a tritiated amino acid mixture or with [5-3H]uridine at a non-permissive temperature and stored at 4 degrees C to accumulate decays, there was a 13-15 fold enrichment for temperature-sensitive mutants relative to the original mutagenized cultures. For a wild type culture of Neurospora crassa labelled with [5-3H]uridine at 35 degrees C the probability of cell killing per tritium decay was calculated to be 3.64 X 10(-5).