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PubMed1994

Nature and distribution of chromosomal intertwinings in Saccharomyces cerevisiae.

To elucidate yeast chromosome structure and behavior, we examined the breakage of entangled chromosomes in DNA topoisomerase II mutants by hybridization to chromosomal DNA resolved by pulsed-field gel electrophoresis. Our study reveals that large and small chromosomes differ in the nature and distribution of their intertwinings. Probes to large chromosomes (450 kb or larger) detect chromosome breakage, but probes to small chromosomes (380 kb or smaller) reveal no breakage products. Examination of chromosomes with one small arm and one large arm suggests that the two arms behave independently. The acrocentric chromosome XIV breaks only on the long arm, and its preferred region of breakage is approximately 200 kb from the centromere. When the centromere of chromosome XIV is relocated, the preferred region of breakage shifts accordingly. These results suggest that large chromosomes break because they have long arms and small chromosomes do not break because they have small arms. Indeed, a small metacentric chromosome can be made to break if it is rearranged to form a telocentric chromosome with one long arm or a ring with an "infinitely" long arm. These results suggest a model of chromosomal intertwining in which the length of the chromosome arm prevents intertwinings from passively resolving off the end of the arm during chromosome segregation.

Centromere↗
PubMed1986

Characterization of the stability functions and inverted repeat structure X3 of the IncFI plasmid ColV2-K94.

A region of the IncFI plasmid ColV2-K94 which showed homology to the sop partitioning genes of F was cloned and characterized in an attempt to study the stability functions of this element. The sop region contained the incD incompatibility determinant common to many IncFI plasmids, but could not confer on ColV2-K94 miniplasmids the same stable inheritance found in the intact ColV2-K94; thus, other functions appear to be required for efficient plasmid maintenance. Adjacent to the area of sop homology was the X3 region, which was found to contain three inverted IS1-like sequences. The X3 region of ColV2-K94 was similar in organization to the aerobactin iron uptake region of ColV3-K30, but ColV2-K94 lacked the ability to synthesize either the aerobactin siderophore or its outer membrane receptor.

Bacteriocin Plasmids↗
PubMed1993

Structural organization of a cryptic plasmid, pMA1, from Microcystis aeruginosa f. aeruginosa Kützing.

The 2287-bp cryptic plasmid, pMA1, from Microcystis aeruginosa f. aeruginosa Kützing, a unicellular cyanobacterium originally derived from Kasumigaura lake, was completely sequenced and analyzed. The predicted amino acid sequence (253 residues) of an open reading frame had identities of 47%, 48%, and 53% with replication-associated proteins of Bacillus amyloliquefaciens's plasmid, pFTB14, B. subtilis BAA1's pBAA1, and B. coagulans's pBC1, respectively, when conservative amino acid substitutions were included. Such high-level identities were also shown with rep proteins and ori regions in a group of Gram-positive bacterial plasmids such as Lactococci and Staphylococci that are known to replicate via single-stranded intermediates. The pMA1 does hybridize with a plasmid, pUS1-3, derived from another unicellular cyanobacterium, Synechocystis sp. PCC 6803. Novel features of pMA1 are discussed.

Amino Acid Sequence↗
PubMed1995

Structure and replication of cryptic plasmids, pMA1 and pMA2, from a unicellular cyanobacterium, Microcystis aeruginosa.

The 4993-bp cryptic plasmid, pMA2, from a cyanobacterium, Microcystis aeruginosa f. aeruginosa Kützing, originally derived from Kasumigaura lake, was completely sequenced and analyzed. Plasmid pMA2 had a unique sequence motif CTTGATT, which was proposed to be a nicking site on the smaller plasmid pMA1 (2287-bp) by the presence of single-stranded DNA susceptible to S1 nuclease. We had detected the occurrence of the single-stranded pMA1 in the living M. aeruginosa cells. This was also the case of pMA2. Thus, we suggest that both plasmids, pMA1 and pMA2, replicate through the rolling circle mechanism. By computer analysis of the pMA2 sequence, two open reading frames were found: one had a predicted molecular size of 30,440 Da and the other on a complementary one had a predicted molecular size of 10,852 Da. No rep protein was detected. Replication mechanisms of the plasmids are discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Sequence specific modulation of DNA restriction enzyme cleavage by minor groove binders.

The inhibition of restriction endonuclease cleavage by a series of bisquaternary ammonium derivatives (BQA-derivatives) which bind to the minor groove of DNA has been studied. The derivatives considered included six sequence-selective binders (SN 6570, SN 6999, SN 6050, SN 6132, SN 6131 and SN 18071) and four non-specific binders (SN 6113, SN 5754, SN 6324 and SN 4094) and can be distinguished by their activity on restriction endonucleases. Digestion experiments with pUC19 DNA were monitored electrophoretically using the transition of the covalently closed circular (ccc) DNA into the linear double stranded (lds) one. Only the sequence-specific binders inhibit the cleavage activity of restriction endonucleases EcoRI, SspI and DraI with four and six dAdT-base pairs within their restriction sites, while the activity of SalI and BamHI with less than four dAdT-sequences was unaffected. In contrast, the non-specific binding ligands were incapable of suppressing enzyme digestion. The inhibition of the restriction endonuclease PvuII indicates that ligand binding in close vicinity to the cleavage sites is also involved in the enzyme inhibition. The dAdT-content in proximity to the palindromic sequences of three DraI cutting sites in pUC19 DNA explains why the derivative SN 6053 protects these sequences in different manners. Gel shift experiments indicated that BQA-derivatives inhibit the DNA-enzyme complex formation if the ligand was added to the DNA before the enzyme. In contrast, complex formation between DNA and enzyme remained unchanged when the enzyme was added first.

Deoxyribonucleases, Type II Site-Specific↗
PubMed1991

Xylose (glucose) isomerase gene from the thermophile Clostridium thermohydrosulfuricum; cloning, sequencing, and expression in Escherichia coli.

The xylose isomerase gene from the thermophile Clostridium thermohydrosulfuricum has been cloned, using a fragment of the Bacillus subtilis gene as a probe. The complete nucleotide sequence of the gene was analyzed. C. thermohydrosulfuricum is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. Comparison with amino acid sequences from other xylose isomerases showed that amino acids involved in substrate binding and isomerization are well conserved. Purification of the enzyme produced in E. coli was done by heating a cell-free extract at 85 degrees C for 10 min, giving a 20-fold purified enzyme. The native enzyme is a homomeric tetramer with a molecular weight of 200,000.

Aldose-Ketose Isomerases↗
PubMed1991

[Cloning of the Arthrobacter globiformis fcbA gene for dehalogenase and construction of a hybrid pathway of 4-chlorobenzoic acid degradation in Pseudomonas putida].

The Artrobacter globiformis KZT1 fcbA gene responsible for dehalogenase (4-chlorobenzoate-4-hydroxylase) activity was cloned in Escherichia coli and Pseudomonas putida cells. The character of the fcbA gene expression was studied. Notwithstanding amplification of the gene dose and control of the inducible Plac promoter, the level of substrate dehalogenation by recombinant E. coli strains was lower, as compared with that in the original KZT1 strain. Cloning of the fcbA gene in P. putida KZ6R cells utilizing 4HBA resulted in a recombinant pathway of 4CBA degradation, which proved more effective for substrate consumption, in comparison with the original KZT1 strain.

Arthrobacter↗
PubMed1991

Putative structure of the FGF6 gene product and role of the signal peptide.

The human FGF6 gene is an oncogene related by sequence similarities to the fibroblast growth factor (FGF) gene family, which encodes mitogenic peptides implicated in various physiological processes including angiogenesis, morphogenesis, tissue regeneration and survival and oncogenesis. Nucleotide sequence analysis of the FGF6 gene and of cDNA clones revealed an open reading frame able to code for a protein of 208 residues. The FGF6 protein shares 32-70% residues with the other members of the family within the C-terminal two-thirds of the molecule. In vitro, three in-frame ATG codons are able to initiate the translation of three peptides of 175, 198 and 208 residues. These three peptides differ at their amino termini with respect to the relative position of a hydrophobic leader peptide, which extends from residues 16 to 40, and is therefore absent from the shorter (175 amino acids) form. In-vitro analysis indicates that this signal peptide is able to drive the FGF6 protein through the endoplasmic reticulum, where it becomes glycosylated. The presence of this signal peptide sequence appears essential for the in vivo transforming capacity of the FGF6 gene.

Amino Acid Sequence↗
PubMed1990

[Expression of delta-endotoxin gene from Bacillus thuringiensis var. berliner 1715 in Escherichia coli and Bacillus megaterium cells].

Effects of the structure of plasmids carrying the cloned delta-endotoxin gene (tox) ot Bacillus thuringiensis and of the culture media on the expression of the gene have been studied. The DNA region located upstream from the crystal protein gene promoter inhibited the expression of the tox gene in Escherichia coli cells, but enhanced the expression in Bacillus megaterium cells grown in LB medium. The upstream DNA region did not affect the tox gene expression when Bacillus megaterium cells were grown in SSM medium.

Bacillus megaterium↗
PubMedDate not supplied

[Structure-functional characteristics of pBS195 plasmids from a broad range of gram-positive and gram-negative hosts].

The ability of plasmid pBS195 to be inherited by a broad host range including both gram-positive and gram-negative bacteria attracts special attention to its molecular genetic organization. Physical map of the plasmid has been constructed and the gene for kanamycin resistance and oriV homologous to the one of pUC19 have been localized. The determinants for kanamycin-resistance have been shown to share no homology with the genes for the I and II type phosphotransferases and type II nucleotide-transferase. The minicells of Escherichia coli x925 harboring pBS195 produce two plasmid-encoded proteins with mol. masses 26 and 34 kD found in SDS-PAGE. The plasmid determines an oxygenase activity and shares homology with a nahC gene in the EcoRI-BamHI region (0 bp-1.6 kbp). The enzyme was identified as a methylpyrocatehase possessing high activity in Escherichia coli cells and substrate specificity uncharacteristic of this type enzymes.

Catechol 1,2-Dioxygenase↗
PubMed1996

Structure of a Gelasinospora linear plasmid closely related to the kalilo plasmid of Neurospora intermedia.

We have determined the complete nucleotide sequence of a linear mitochondrial plasmid from a natural isolate of a homothallic species of Gelasinospora. The plasmid genome is 8231 bp long. It carries terminal inverted repeats of 1137 bp. Extending inwards from the terminal repeats are two long open reading frames coding for putative proteins with similarity to DNA and RNA polymerases. These are separated by a short intergenic region. The plasmid sequence shows remarkable similarity to that of the Neurospora intermedia senescence-plasmid kalilo. Overall the two plasmids have a similar genetic organization and are clearly homologous at the sequence level. The main differences are in the intergenic region and in the terminal repeats.

Amino Acid Sequence↗
PubMed1999

A single immunization with a plasmid encoding hepatitis C virus (HCV) structural proteins under the elongation factor 1-alpha promoter elicits HCV-specific cytotoxic T-lymphocytes (CTL).

Recent studies have raised the possibility that DNA-based vaccination may prove useful for generating virus-specific cytotoxic T-lymphocytes (CTL) responses. Recently, a plasmid containing the human elongation factor 1alpha(EF1-alpha) promoter, pEF321, was reported to be a versatile expression vector for gene expression in mammalian cells in vitro. In the present study, we assessed the capability of a novel plasmid, pEFCE1E2, encoding hepatitis C virus (HCV) structural proteins (core, E1 and E2) under the EF1-alpha promoter to generate CTL against HCV in vivo. BALB/c mice were immunized with the pEFCE1E2 but not with a plasmid possessing the same cDNA under the cytomegalovirus developed HCV-specific effector cells by a single immunization. These effector cells elicited by pEFCE1E2 immunization were CD8(+) and major histocompatibility complex class I restricted. These studies provided evidence for the potential utility of the EF1-alpha promoter for development of DNA vaccines against HCV infections.

Amino Acid Sequence↗
PubMed2004

Genetic vaccination of mice with plasmids encoding the NS1 non-structural protein from tick-borne encephalitis virus and dengue 2 virus.

Although there is a safe, inexpensive and efficacious vaccine against yellow fever, vaccination against other flavivirus diseases is less successful. There is no licensed vaccine against dengue fever and current vaccines against tick-borne encephalitis (TBE) and Japanese encephalitis are expensive and require several injections. Furthermore novel vaccines containing only virus envelope proteins may raise fears over antibody mediated enhancement (ADE) of disease. Here we report the successful use of genetic vaccination against TBE in an experimental animal model using a plasmid containing the coding sequence of a non-structural protein (NS1). Such vaccines would provide inexpensive protection against disease, without raising concerns over inducing ADE on subsequent exposure to heterotypic infectious virus. Attempts to generate chaemeric plasmids to protect against both TBE and dengue fever were less successful. Although these chaemeric plasmids directed the synthesis and secretion of the virus NS1 protein normally, no protection was observed against either TBE or dengue fever.

Amino Acid Sequence↗
PubMed2004

The activity of a single-stranded promoter of plasmid ColIb-P9 depends on its secondary structure.

The leading region of the conjugal bacterial plasmid ColIb-P9 contains three dispersed repeats of a 328 bp sequence homologous to Frpo, a sequence from plasmid F that acts as a promoter in single-stranded DNA. One of these sequences, ssi3, inactive in the double-stranded form, promoted in vitro transcription exclusively from the single strand that is transferred during conjugation. Promoter activity was dependent on the presence of RNA polymerase holoenzyme containing sigma 70. Transcription initiated from the position predicted from folding the single-stranded DNA to form a pseudo double-stranded hairpin structure containing recognizable -35 and -10 promoter elements. Footprinting of RNA polymerase holoenzyme on single-stranded ssi3 DNA was consistent with this suggestion. Mutagenesis of the putative -35 region inactivated the promoter, but random mutations in the -10 region had little effect. The putative -10 region is a poor match to the consensus sequence and contains mismatched bases. Elimination of these mismatches invariably destroyed single-strand promoter activity. These observations reveal the crucial contribution of the unpaired bases in the -10 region in potentiating the formation of the productive open complex with RNA polymerase.

Bacteriocin Plasmids↗