Extracellular proteins of Vibrio cholerae: molecular cloning, nucleotide sequence and characterization of the deoxyribonuclease (DNase) together with its periplasmic localization in Escherichia coli K-12

The gene encoding the extracellular DNase of Vibrio cholerae was cloned into Escherichia coli K-12. A maximal coding region of 1.2 kb and a minimal region of 0.6 kb were determined by transposon mutagenesis and deletion analysis. The nucleotide sequence of this region contained a single open reading frame of 690 bp corresponding to a protein of Mr 26,389 with a typical N-terminal signal sequence of 18 aa which, when removed, would give a mature protein of Mr 24,163. This is in good agreement with the size of 24 kDa, calculated directly by Coomassie blue staining following sodium dodecyl sulphate-polyacrylamide gel electrophoresis and indirectly via a DNA-hydrolysis assay. The protein is located in the periplasmic space of E. coli K-12 unlike in V. cholerae where it is excreted into the extracellular medium. The introduction of the DNase gene into a periplasmic (tolA) leaky mutant of E. coli K-12 facilitates the release of the protein, further confirming the periplasmic location.     

Nucleotide sequence of the cDNA and the derived amino acid sequence for the major antigenic protein of foot and mouth disease virus, type Asia 1 63/72.

A 0.9 kb cDNA for the foot and mouth disease virus (FMDV) type Asia 1 63/72, cloned in the plasmid pUR222 by dC/dG tailing method, was expressed into a protein which was immunogenic in guinea pigs and cattle. The protein purified to homogeneity was found to be basic and of 38 kDa. A sequence of 879 nucleotides of the inserted cDNA was obtained. The nucleotide sequence was 65% GC-rich and was homologous to the gene for VPI of FMDV types A5, OIK and C3 to the extent of 35-40%. From the nucleotide sequence, a sequence of 293 amino acids was derived which contained 43 arginine, 4 lysine, 7 glutamic acid and 18 aspartic acid residues making the protein highly basic. The molecular weight was calculated to be 31.6 kDa. The 38 kDa protein produced by the cloned cDNA is a fused protein composed of the 293 amino acids; 5 and 55 amino acids of the alpha-complementation protein of the beta-galactosidase at the N and C terminal, respectively, and 5 amino acid coded by the dG/dC tails used for cloning the cDNA.     

The isolation of duplex DNA fragments containing (dG.dC) clusters by chromatography on poly(rC)-Sephadex.

Duplex DNA containing oligo(dG.dC)-rich clusters can be isolated by specific binding to poly(rC)-Sephadex. This binding, probably mediated by the formation of an oligo(dG.dC)rC+ triple helix, is optimal at pH 5 in 50% formamide, 2 M LiCl; the bound DNA is recovered by elution at pH 7.5. Using this method we find that the viral DNAs PM2, lambda and SV40 contain at least 1, 1 and 2 sites for binding to poly(rC)-Sephadex, respectively. These binding sites have been mapped in the case of SV40; the binding sites can in turn be used for physical mapping studies of DNAs containing (dG.dC) clusters. Inspection of the sequence of the bound fragments of SV40 DNA shows that a (dG.dC)6-7 tract is required for the binding of duplex DNA to poly(rC)-Sephadex. Although about 60% of rabbit DNA cleaved with restriction endonuclease KpnI binds to poly(rC)-Sephadex, no binding is observed for the 5.1 kb DNA fragment generated by KpnI digestion, which contains the rabbit beta-globin gene. This indicates that oligo(dG.dC) clusters are not found close to the rabbit beta-globin gene.     

Complete nucleotide sequence and genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens

The complete nucleotide sequence of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens has been determined. The plasmid genome comprises 10,207 bp and has a dA + dT content of 75%. Functions have been tentatively assigned to 6 of the 10 open reading frames and an origin-like region of repeated sequence identified. The codon usage of this extremely dA + dT rich plasmid is highly unusual and displays a pronounced preference for codons with the lowest dG + dC content. Only one of the genes from pIP404 was expressed at a significant level in Escherichia coli, suggesting that the atypical codon usage could represent a major obstacle to heterologous gene expression.     

Detection of triple-helix related structures adopted by poly(dG)-poly(dC) sequences in supercoiled plasmid DNA.

Negative superhelical strain induces the poly(dG)-poly(dC) sequence to adopt two totally different types of triple-helices, either a dG.dG.dC triplex in the presence of Mg(+)+ at both neutral and acidic pHs or a protonated dC+.dG.dC triplex in the absence of Mg(+)+ ions at acidic pH (1). To examine whether there are still other types of non-B DNA structures formed by the same sequence, we constructed supercoiled plasmid DNAs harboring varying lengths of the poly(dG) tract, and the structures adopted by each supercoiled plasmid DNA were studied with a chemical probe, chloroacetaldehyde. The potential of a poly(dG)-poly(dC) sequence to adopt non-B DNA structures depends critically on the length of the tract. Furthermore, in the presence of Mg(+)+ and at a mildly acidic pH, in addition to the expected dG.dG.dC triplex detected for the poly(dG) tracts of 14 to 30 base pairs (bp), new structures were also detected for the tracts longer than 35 bp. The structure formed by a poly(dG) tract of 45 bp revealed chemical reaction patterns consistent with a dG.dG.dC triplex and protonated dC+.dG.dC triple-helices fused together. This structure lacks single-stranded stretches typical of intramolecular triplexes.     

Synthesis of part of a mouse immunoglobulin light chain in a bacterial clone

We have cloned double stranded cDNA sequences encoding a mouse immunoglobulin light chain (L-321) into the PstI site of the beta-lactamase gene of plasmid pBR322 by the oligo (dG)-oligo (dC) tailing procedure. Escherichia coli X1776 transformed by the recombinant plasmids were screened for the expression of L-321 antigenic determinants by a newly developed in situ radio-immunoassay. One out of seven transformants screened was found to synthesize an L-chain like protein. Each bacterial cell produces about 550 molecules of the L-chain sequence. Preferential segregation of the L-chain sequence to the periplasmic space suggest covalent attachment of the L-chain sequence to the N-terminal portion of beta-lactamase. Restriction mapping of the plasmid DNA isolated from the positive clone indicated the presence of a DNA sequence coding for the entire constant region and extending into the variable region for a length corresponding to about 40 amino acid residues. The orientation of the cloned cDNA with respect to the plasmid DNA is compatible with the formation of a fused beta-lactamase-L-321 peptide.     

In vitro synthesis of uniform poly(dG)-poly(dC) by Klenow exo- fragment of polymerase I

In this paper, we describe a production procedure of the one-to-one double helical complex of poly(dG)–poly(dC), characterized by a well-defined length (up to 10 kb) and narrow size distribution of molecules. Direct evidence of strands slippage during poly(dG)–poly(dC) synthesis by Klenow exo⁻ fragment of polymerase I is obtained by fluorescence resonance energy transfer (FRET). We show that the polymer extension results in an increase in the separation distance between fluorescent dyes attached to 5′ ends of the strands in time and, as a result, losing communication between the dyes via FRET. Analysis of the products of the early steps of the synthesis by high-performance liquid chromatography and mass spectroscopy suggest that only one nucleotide is added to each of the strand composing poly(dG)–poly(dC) in the elementary step of the polymer extension. We show that proper pairing of a base at the 3′ end of the primer strand with a base in sequence of the template strand is required for initiation of the synthesis. If the 3′ end nucleotide in either poly(dG) or poly(dC) strand is substituted for A, the polymer does not grow. Introduction of the T-nucleotide into the complementary strand to permit pairing with A-nucleotide results in the restoration of the synthesis. The data reported here correspond with a slippage model of replication, which includes the formation of loops on the 3′ ends of both strands composing poly(dG)–poly(dC) and their migration over long-molecular distances (μm) to 5′ ends of the strands.     

The binding of the chromosomal protein HMG-2a to DNA regions of reduced stabilities

The interaction of immunopurified high mobility group 2a protein (HMG-2a) with DNA was examined by the nitrocellulose filter binding assay. The relative binding activity of HMG-2a for synthetic polynucleotides was: (dI).(dC) greater than (dA-dT).(dA-dT) greater than (dA).(dT) much greater than (dG).(dC) greater than (dG-dC).(dG-dC). The protein also exhibited a marked preference for (A + T)-rich restriction fragments derived from rat and Drosophila satellites, yeast centromeres, phage lambda, and the ovalbumin gene and its 5' flanking sequences. These preferential DNA interactions occurred at ionic strengths and temperatures within the physiological range which argue for an in vivo role of DNA stability in dictating the genomic distribution of the large Mr HMG proteins.     

RNautophagy/DNautophagy possesses selectivity for RNA/DNA substrates

Lysosomes can degrade various biological macromolecules, including nucleic acids, proteins and lipids. Recently, we identified novel nucleic acid-degradation systems termed RNautophagy/DNautophagy (abbreviated as RDA), in which RNA and DNA are directly taken up by lysosomes in an ATP-dependent manner and degraded. We also found that a lysosomal membrane protein, LAMP2C, the cytoplasmic region of which binds to RNA and DNA, functions, at least in part, as an RNA/DNA receptor in the process of RDA. However, it has been unclear whether RDA possesses selectivity for RNA/DNA substrates and the RNA/DNA sequences that are recognized by LAMP2C have not been determined. In the present study, we found that the cytosolic region of LAMP2C binds to poly-G/dG, but not to poly-A/dA, poly-C/dC, poly-dT or poly-U. Consistent with this binding activity, poly-G/dG was transported into isolated lysosomes via RDA, while poly-A/dA, poly-C/dC, poly-dT and poly-U were not. GGGGGG or d(GGGG) sequences are essential for the interaction between poly-G/dG and LAMP2C. In addition to poly-G/dG, G/dG-rich sequences, such as a repeated GGGGCC sequence, interacted with the cytosolic region of LAMP2C. Our findings indicate that RDA does possess selectivity for RNA/DNA substrates and that at least some consecutive G/dG sequence(s) can mediate RDA.     

Cationic metal-specific structures adopted by the poly(dG) region and the direct repeats in the chicken adult beta A globin gene promoter.

Naturally occurring contiguous deoxyguanine residues and their surrounding sequences in the chicken adult beta A globin gene promoter were analyzed for their inherent potential to adopt non-B DNA structures in supercoiled plasmid DNA. In particular, cationic effects on structure were studied by treating the supercoiled plasmid DNA harboring the chicken adult beta A globin 5' flanking sequence with an unpaired DNA base-specific probe, chloroacetaldehyde in the presence of either Mg++, Cu++, Zn++, Ca++ or Co++ ions. The chloroacetaldehyde-reactive bases were mapped at a single base resolution by a chemical cleavage method that specifically cleaves DNA at the chloroacetaldehyde modified sites. These experiments revealed that while Mg++ and Ca++ ions induce a dG.dG.dC triple helix structure at the contiguous dG residues, Zn++, Cu++ and Co++ ions induce yet another structure at the direct repeats immediately 5' of the dG residues. When Mg++ and Zn++ ions are both present, Zn++ inhibits the dG.dG.dC triplex at the contiguous dG residues and induces a particular non-B DNA structure at the adjacent direct repeats. The specific induction of non-B DNA structures by metal ions at the two adjacent sequences within the promoter region may be of biological significance.     

Nucleotide sequence of bovine prolactin messenger RNA. Evidence for sequence polymorphism

Hybrid molecules containing DNA sequences complementary to bovine pituitary mRNA were constructed in the Pst I site of pBR322 by the dC . dG tailing technique. Recombinant plasmids containing bovine prolactin (bPRL) sequences were amplified in bacteria and identified by hybridization to purified [32P]bPRL cDNA sequences. Nucleotide sequence analysis was performed on the inserts from two of the positive clones. One clone, pBPRL72, contained a 982-base pair insert that included 67 nucleotides of the 5'-untranslated region, the complete coding region of the preprolactin protein (690 nucleotides), and the entire 3'-untranslated region (150 nucleotides) of bPRL mRNA. The nucleotide sequence analysis of clone pBPRL72 predicted the sequence of a 30-amino acid signal peptide and confirmed the published amino acid sequence of the protein with one exception. A comparison of the pBPRL72 cDNA sequence with a second bPRL clone, pBPRL4, revealed four silent nucleotide differences. Three of the base changes occurred in the third position of amino acid codons, and one occurred in the 3'-noncoding region. The sequence polymorphism suggests the existence of alleles or multiple loci for bPRL that do not alter the protein structure.     

Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.     

Molecular cloning of DNA complementary to bovine growth hormone mRNA

We have cloned DNA complementary to mRNA coding for bovine growth hormone (bGH). Double-stranded DNA complementary to bovine pituitary mRNA was inserted into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E. coli x 1776. A recombinant plasmid containing bGH cDNA ws identified by hybridization to cloned rat growth hormone cDNA. It contains the entire coding and 3'-untranslated regions and 31 bases in the 5'-untranslated region. Nucleotide sequence analysis determined the sequence of the 26-amino acid signal peptide and confirmed the published amino acid sequence of the secreted hormone at all but 2 residues. Codon usage is nonrandom, with 81.7% of the codons ending in G or C. The nucleotide sequence of bGH mRNA is 83.9% homologous with rat GH mRNA and 76.5% homologous with human GH mRNA, while the respective amino acid sequence homologies are 83.5% and 66.8%.     

Magnesium-dependent supercoiling-induced transition in (dG)n.(dC)n stretches and formation of a new G-structure by (dG)n strand.

Plasmids containing (dG)27.(dC)27 inserts (pPG27), (dG)37.(dC)37 inserts (pPG37), and (dG)24C(dG)21.(dC)24G(dC)21 inserts (pPG46C) were constructed for the study of structural transitions within (dG)n.(dC)n stretches. Two-dimensional gel electrophoresis has shown that a Mg2+-dependent supercoiling-induced structural transition takes place at pH 8 in plasmid pPG46C. The transition occurs at -0=0.06 and involves a supercoiling release corresponding to 5 superhelical turns. After denaturation of the restriction fragments containing (dG)n.(dC)n inserts, the strands do not renature completely and (dG)n-containing strand migrates in PAGE much faster than the (dC)n-containing one. Chemical modification experiments with the (dG)n-strand have revealed the periodic nature of the protection of guanines against dimethyl sulfate methylation. The (dG)n strand in the presence of Mg2+ forms complexes with the complementary (dC)n strand, which differ from the native duplex in mobility. We believe these effects to be due to the formation of an intrastrand structure within the (dG)n strand stabilized by G.G interactions (we called it G-structure), which in the presence of Mg2+ forms an interstrand complex. with the (dC)n strand.     

The mechanism of action of virginiamycin M on the binding of aminoacyl-tRNA to ribosomes directed by elongation factor Tu

Mitochondrial DNA from Ustilago cynodontis has been investigated in several of its properties. Its dG + dC content is equal to 33.5%; its buoyant density (1.698 g/cm3) is higher, by 5 mg/cm3, and its melting temperature (82.5 degrees C) is lower than expected for a bacterial DNA having the same base composition; the first derivative of its melting curve indicates a large compositional heterogeneity, its molarity of elution from hydroxyapatite is high, 0.28 M phosphate, and allows its partial separation from nuclear DNA. Degradation by micrococcal nuclease indicates that about 25% of the DNA is formed by stretches having no more than 15% dG + dC. Finally, the unit size of mitochondrial genome is about 50 X 10(6). In most of its properties, the mitochondrial genome of U. cynodontis presents strong analogies with that of Saccharomyces cerevisiae. A parallel investigation on mitochondrial DNA from Acanthamoeba castellanii which has as genome unit size of only 27 X 10(6), has shown that this shares with the former the dG + dC content (32.9%), the melting temperature (82.5 degrees C), a large compositional heterogeneity and a very similar pattern of micrococcal nuclease degradation; its buoyant density (1.692 g/cm3) and its molarity of elution from hydroxyapatite (0.25 M phosphate) are, however, normal, probably because of a different short-sequence pattern and the fact that its dA + dT-rich stretches are shorter, on the average.     

Poly(dG)-poly(dC) sequences, under torsional stress, induce an altered DNA conformation upon neighboring DNA sequences

Supercoiled plasmid DNAs (at bacterial superhelical density) harboring the homopurine-homopyrimidine sequence, poly(dG)-poly(dC), were reacted with bromoacetaldehyde (BAA), a reagent that reacts with unpaired DNA bases. Not only did the poly(dG)-poly(dC) sequence react with BAA but, surprisingly, neighboring sequences located 3' to the contiguous G sequences also reacted. The altered conformation in the poly(dG)-poly(dC) sequence and in the neighboring sequence occurred in the same supercoiled plasmid DNA molecule. Furthermore, the occurrence of an "unpaired" conformation in the neighboring sequence is strictly due to a positional effect, since it is observed when the poly(dG)-poly(dC) segment is adjacent to a variety of neighboring sequences.     

A bacterial cell that synthesizes a protein containing the antigenic determinants of rat prolactin.

Bacterial minicells containing three different recombinant plasmids with rat prolactin cDNA sequences inserted at the Pst I site of pBR322 via the poly(dG):poly(dC) joining technique were examined for the expression of rat prolactin antigenic determinants. The three prolactin coding sequences were in the same orientation as the coding sequence of the ampicillin-resistance gene of pBR322. The presence of each of the three recombinant plasmids induced some prolactin synthesis by the bacteria as measured by immunoprecipitation with anti-prolactin antisera. About 10% of the protein synthesized from one of the plasmids, prl 3, precipitated with the antisera. These prolactin antigenic determinants were part of a larger fused protein.     

Insertion of a rabbit beta-globin gene sequence into an E. coli plasmid.

Double stranded DNA has been synthesized in vitro from rabbit globin messenger RNA and elongated with homopolymeric dG tails. An E. coli plasmid was cleaved by EcoRI. The cohesive ends were repaired and dC tails added, to permit reconstitution of the EcoRI sites upon annealing with the dG elongated globin DNA. Transformation of E. coli with the globin-plasmid DNA hybrid has yielded a clone which harbours a recombinant plasmid (pCR1-betaG1), as demonstrated by hybridization experiments with radioactive globin cDNA. The sequence carried by the recombinant plasmid corresponds to part of the gene sequence coding for the beta chain of rabbit globin. Circular DNA of the purified recombinant plasmid exhibits sensitivity to EcoRI.     

Solution structure of the N-(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dA in a DNA duplex.

Solution structural studies have been undertaken on the aminopyrene-C(8)-dG ([AP]dG) adduct in the d(C5-[AP]G6-C7). d(G16-A17-G18) sequence context in an 11-mer duplex with dA opposite [AP]dG, using proton-proton distance and intensity restraints derived from NMR data in combination with distance-restrained molecular mechanics and intensity-restrained relaxation matrix refinement calculations. The exchangeable and nonexchangeable protons of the aminopyrene and the nucleic acid were assigned following analysis of two-dimensional NMR data sets on the [AP]dG.dA 11-mer duplex in H2O and D2O solution. The broadening of several resonances within the d(G16-A17-G18) segment positioned opposite the [AP]dG6 lesion site resulted in weaker NOEs, involving these protons in the adduct duplex. Both proton and carbon NMR data are consistent with a syn glycosidic torsion angle for the [AP]dG6 residue in the adduct duplex. The aminopyrene ring of [AP]dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs and is in contact with dC5, dC7, dG16, dA17, and dG18 residues that form a hydrophobic pocket around it. The intercalated AP ring of [AP]dG6 stacks over the purine ring of dG16 and, to a lesser extent dG18, while the looped out deoxyguanosine ring of [AP]dG6 stacks over dC5 in the solution structure of the adduct duplex. The dA17 base opposite the adduct site is not looped out of the helix but rather participates in an in-plane platform with adjacent dG18 in some of the refined structures of the adduct duplex. The solution structures are quite different for the [AP]dG.dA 11-mer duplex containing the larger aminopyrene ring (reported in this study) relative to the previously published [AF]dG.dA 11-mer duplex containing the smaller aminofluorene ring (Norman et al., Biochemistry 28, 7462-7476, 1989) in the same sequence context. Both the modified syn guanine and the dA positioned opposite it are stacked into the helix with the aminofluorene chromophore displaced into the minor groove in the latter adduct duplex. By contrast, the aminopyrenyl ring participates in an intercalated base-displaced structure in the present study of the [AP]dG.dA 11-mer duplex and in a previously published study of the [AP]dG.dC 11-mer duplex (Mao et al., Biochemistry 35, 12659-12670, 1996). Such intercalated base-displaced structures without hydrogen bonding between the [AP]dG adduct and dC or mismatched dA residues positioned opposite it, if present at a replication fork, may cause polymerase stalling and formation of a slipped intermediate that could produce frameshift mutations, the most dominant mutagenic consequence of the [AP]dG lesion.