Identification of the origin of replication of the eukaryote Dictyostelium discoideum nuclear plasmid Ddp2

Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryote Dictyostelium discoideum. We have identified two functional domains, a large open reading frame (Rep gene) and a 626-bp fragment containing an origin of replication (ori). The ori, when cloned into a shuttle vector, confers stable extrachromosomal replication in D. discoideum, provided that the Rep gene, which acts in trans, is integrated into the host genome. Ddp2 carries a 501-bp imperfect inverted repeat, and part of the ori overlaps with one of these repeats. The ori sequence contains two direct repeats of 49 bp comprising two 10-bp "TGTCATGACA" palindromes separated by a poly(T.A) sequence. Deletion of either 49-bp repeat abolished extrachromosomal replication.     

Location of the origin of replication for the 7.5-kbChlamydia trachomatis plasmid

The hypothetical origin of replication for the 7.5-kb plasmid common toChlamydia trachomatis is believed to be in a region of the plasmid that contains four 22-bp tandem repeats preceded by an A-T-rich region. To test this hypothesis, replication of plasmid DNA in metabolically active reticulate bodies of the Lymphogranuloma venereum biovar ofC. trachomatis was examined by electron microscopy. The results presented show that the origin of replication appears to be near the tandem repeats of pCHL2. In addition, replication of the 7.5-kb plasmid is unidirectional, and the copy number during replication is 7–10. The evidence presented suggests thatC. trachomatis has a homologue to theEscherichia coli dnaA gene and that this homologue might be involved in replication of theC. trachomatis 7.5-kb plasmid.     

The sequence and organization of Ddp2, a high-copy-number nuclear plasmid of Dictyostelium discoideum

The complete nucleotide sequence of the plasmid Ddp2 found in the nucleus of the simple eukaryote Dictyostelium discoideum is reported. This 5852-bp plasmid contains a 2661-bp open reading frame (ORF), named the "Rep gene," and 501-bp imperfect inverted repeats. A 1762-bp section of Ddp2, which includes one of the 501-bp repeat sequences, could be deleted without abolishing extrachromosomal replication. Deletion of the second 501-bp repeat, or interruption of the Rep gene, removed the ability to replicate extrachromosomally. We suggest that Ddp2 encodes a protein, "REP," that positively regulates replication initiation, a regulatory mechanism different from that of the yeast 2 mu plasmid which also possesses inverted repeat sequences. Ddp2 has a structure similar to that of plasmid pDG1, found in an unidentified isolate of Dictyostelium, with a similar sized ORF and inverted repeats. A common evolutionary origin is suggested by considerable sequence homology between the ORFs of pDG1 and Ddp2.     

Identification of the minimal essential region for the replication origin of miniF plasmid

Summary The minimal replication origin of miniF plasmid was found to lie within a region of 217 bp in length. This region contains an AT cluster and the four 19 bp direct repeats responsible for incompatibility, termed incB. Its location coincides with that of ori2 of plasmid F, previously inferred to be the replication starting point by electron microscopic analysis (Eichenlaub et al. 1981).Abbreviations kb kilobase(s) - bp base pairs - Ap ampicillin - Tc tetracycline     

Plasmid maintenance functions encoded on Dictyostelium discoideum nuclear plasmid Ddp1.

All of the plasmid-carried genes expressed during vegetative growth are essential for long-term maintenance of plasmid Ddp1 in the nucleus of Dictyostelium discoideum. Deletion of Ddp1 genes expressed only during development had no detectable effect on plasmid maintenance. Deletion of vegetatively expressed genes, either singly or in pairs, resulted in (i) a rapid loss of plasmid from cells grown in the absence of selection for plasmid retention, (ii) variation in the proportion of monomer to multimer forms of the plasmid molecules, and/or (iii) abnormalities in plasmid copy number. At least two plasmid-encoded gene products influence patterns of expression of plasmid genes.     

Anatomy of the replication origin of plasmid ColE2-P9

The plasmid ColE2-P9 origin is a 32-bp region which is specifically recognized by the plasmid-specified Rep protein to initiate DNA replication. We analyzed the structural and functional organization of the ColE2 origin by using various derivatives carrying deletions and single-base-pair substitutions. The origin may be divided into three subregions: subregion I, which is important for stable binding of the Rep protein; subregion II, which is important for binding of the Rep protein and for initiation of DNA replication; and subregion III, which is important for DNA replication but apparently not for binding of the Rep protein. The Rep protein might recognize three specific DNA elements in subregions I and II. The relative transformation frequency of the autonomously replicating plasmids carrying deletions in subregion I is lower, and nevertheless the copy numbers of these plasmids in host bacteria are higher than those of the wild-type plasmid. Efficient and stable binding of the Rep protein to the origin might be important for the replication efficiency to be at the normal (low) level. Subregion II might be essential for interaction with the catalytic domain of the Rep protein for primer RNA synthesis. The 8-bp sequence across the border of subregions II and III, including the primer sequence, is conserved in the (putative) origins of many plasmids, the putative Rep proteins of which are related to the ColE2-P9 Rep protein. Subregion III might be required for a step that is necessary after Rep protein binding has taken place.     

The in vivo replication origin of the yeast 2 microns plasmid

We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.     

Recombination between an Flac and a mini-FKm plasmid deleted for an origin of replication

The mini-F plasmid specifying resistance to kanamycin (Km), pML31, contains an origin of replication at kilobase coordinate 42.6 in the F DNA sequences. In previous research we found that this origin could be deleted by recombinant DNA techniques without the loss of plasmid maintenance functions. In this report we show that the deleted plasmid, designated pMF21, has normal incompatibility properties and a recA⁺-dependent ability to form cointegrates with an Flac plasmid. By comparison, pML31 does not form cointegrates with the Flac plasmid at a detectable frequency. The frequency for spontaneous loss of the Lac⁺ phenotype in strains containing pMF21:Flac cointegrates resembles that of the Flac plasmid; however, in some Lac⁻ variants the Km^r phenotype is retained. Examination of the plasmid DNA in four of these Lac⁻Km^r clones revealed two with normal pMF21 plasmids and two with plasmids intermediate in size between pMF21 and the Flac.     

Functional organization of the plasmid pT181 replication origin

Replication of the staphylococcal plasmid pT181 is initiated at the origin (ori) with the introduction of a site-specific nick by the plasmid-encoded initiator protein RepC. Deletion analysis showed that a sequence of about 70 base-pairs is required for full ori function, including the ability to compete with a co-resident wild-type origin for the trans-acting RepC protein. A shorter sequence of 43 base-pairs is sufficient for origin function in the absence of competition. Single and double point mutations within these 43 base-pairs were used to determine the sequence requirement for replication within the minimal origin. Deletion mutants and point mutants were tested in replication and competition assays in vivo and in vitro, and in a RepC-mediated nicking assay.     

湖北双蝴蝶的组织培养研究

以湖北双蝴蝶带芽茎段、不带芽茎段及叶片为外植体,以MS为基本培养基,通过添加不同的植物生长物质种类和浓度配比,建立湖北双蝴蝶组培快繁体系。结果如下:在所有实验方案中,带芽茎段的出愈率最高,是理想的离体快繁材料。较适宜的初代培养基为MS+BA2.0mg/L+蔗糖3.0%,增殖培养基为MS+BA2.0mg/L+NAA0.1mg/L+蔗糖3.0%,而根的诱导则在1/2MS+NAA0.5mg/L+蔗糖1.5%的培养基上进行较为适宜。同时对组织培养过程中湖北双蝴蝶植株再生的方式进行了讨论。     

Cytochrome b of the Dictyostelium discoideum plasma membrane

Cytochrome b of the plasma membrane of Dictyostelium discoideum was investigated in purified plasma membranes and in solubilized form. The membrane-bound cytochrome b can be reduced by NADH. This reduction is inhibited by p-hydroxymercuribenzoate. The reduced cytochrome b does not react with carbon monoxide. Its apparent molecular weight lies between 13000 and 16000. Tryptic digestion yields a large, heme-containing peptide with an apparent molecular weight between 12000 and 15000. After solubilization with cholate, cytochrome b can be enriched by reversed-phase HPLC, indicating that it contains also a hydrophobic component. With these properties, cytochrome b of the D. discoideum plasma membrane resembles microsomal cytochrome b₅.     

The large endogenous plasmid of Anacystis nidulans: Mapping, cloning and localization of the origin of replication

Summary Anacystis nidulans contains two cryptic plasmids of 8.0 (pANS) and 48.5 (pANL) kilobasepairs (kbp). A clone bank of the large plasmid pANL consisting of 7 Bam HI fragments has been established. The cloned fragments were used as radioactive probes to Bam HI, Sal I, Hind III and Eco R1 digests of pANL in blot hybridization experiments to verify the clones and map the restriction fragments. Further size characterization of the physical map was done by restriction analysis of the cloned fragments. The origin of replication has been located in the largest Bam HI fragment of the large plasmid.     

In vivo definition of the functional origin of replication (ori(+ )) of the promiscuous plasmid pLS1

The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein. The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins. Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline. The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex. Sequence analysis of the NocR binding site indicated the presence immediately downstream of the –10 sequence of the nocR promoter of a 12 by putative operator overlapping a consensus gyrase recognition sequence and an 18 by long alternating purine-pyrimidine sequence. These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.     

Localization of the replication origin of plasmid pE194.

The pE194 replication origin was localized to a 265-base-pair interval by analyzing the ability of purified pE194 restriction fragments to direct replication of heterologous plasmids. Replication was dependent upon RepF protein supplied in trans. The origin region contained a GC-rich dyad symmetry which may serve as the RepF target.     

The Rep protein binding elements of the plasmid ColE2-P9 replication origin

The plasmid ColE2-P9 (ColE2) origin (32bp) is specifically recognized by the plasmid-specified Rep protein that initiates DNA replication. The ColE2 origin is divided into at least three functional subregions (I, II, and III), and three sites (a, b, and c) found in subregions I and II play important roles in Rep protein binding. We performed SELEX experiments of plasmid ColE2 to determine the optimal sequences for specific binding of the Rep protein. From these experiments, we obtained a common 16-bp sequence (5'-TGAGACCANATAAGCC-3'), which corresponds to about one half of the minimal ColE2 origin and contains sites a and b. Gel mobility shift assays using single-point mutant origins and the Rep protein further indicated that high affinity sequence-specific recognition by the Rep protein requires sites a, b, and c, but that mutations in site c were less disruptive to this recognition than those in sites a and b.