Construction of an extrachromosomally replicating transformation vector for Dictyostelium discoideum

An extrachromosomally replicating transformation vector for Dictyostelium discoideum has been constructed using sequences of the endogenous Dictyostelium plasmid Ddp2. This transformation vector pnDeI (9.6 kb) replicates as a high copy number plasmid in Dictyostelium and is located in the nucleus. It has been constructed as shuttle vector containing the Escherichia coli vector pUC19 for replication and selection in E. coli and a part of the Tn903 transposon which confers resistance to G418 for selection in Dictyostelium. In order to show that the vector can be used for cloning and stable propagation of Dictyostelium DNA, a fragment of the Dictyostelium alpha-actinin gene that was marked with a synthetic oligonucleotide was cloned into pnDeI and found to be stably maintained in the extrachromosomal vector without undergoing noticeable recombination with the endogenous gene.     

Identification of an endogenous plasmid in Dictyostelium discoideum

A plasmid has been discovered in a strain of the eukaryote, Dictyostelium discoideum, which has an unstable, non-chromosomal, cobalt resistance phenotype. The plasmid, termed Ddp1, is ˜13.5 kbp in size and is found in the nucleus. It has an A-T content typical of Dictyostelium DNA as judged by its restriction enzyme digestion pattern, and it is not related to either mitochondrial or ribosomal DNA. Similar or identical plasmids have been found in two original, cobalt-sensitive, isolates, NC4 and V12, but no plasmid was detected in three other isolates (WS472, WS526, WS584). The plasmid codes for non-essential functions since it is absent from the latter isolates, and it is lost from mutant strains which are capable of axenic growth.     

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.     

The structure of M6, a recombinant plasmid containing Dictyostelium DNA homologous to actin messenger RNA

The recombinant plasmid M6 contains a DNA sequence from the cellular slime mold Dictyostelium discoideum which hybridizes to actin messenger RNA. The plasmid contains 6 kilobase pairs (kb) of Dictyostelium DNA inserted into a pMB9 vector. Ten cleavage sites for four different restriction enzymes have been mapped. Other work has shown that a central restriction fragment, 1.7 kb in length, contains sequences repeated about fifteen times in the genome, and that this fragment hybridizes to actin mRNA. Heteroduplexes between M6 and pDd actin 2, a chromosomal plasmid which contains two copies of the actin repeated sequence, were used to define the position of this repeat in M6. Two plasmids with inserts of cDNA made from actin mRNA were heteroduplexed to M6 to define the position and orientation of the message complementary region. This orientation was confirmed by inserting the fragment into phage λ and determining which of the separated λ strands was complementary to actin mRNA. An electron microscope technique has been developed for identifying poly(dA) sequences by hybridizing to them dBrU polymers attached to suitable markers. The mapping of the (dA) tracts that occur in the Dictyostelium insert of M6 is described here. The positions of the A:T tracts do not correlate in any simple way with the position of the actin gene sequence.     

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.     

Dictyostelium 17S, 25S, and 5S rDNAs lie within a 38,000 base pair repeated unit.

Mapping with the restriction enzymes Sal 1 and R1 has generated a picture of the organization of Dictyostelium ribosomal DNA. The DNA which codes for 17S and 25S ribosomal RNAs is located within a stretch of repetitive DNA at least 38,000 base pairs long. This repeated unit includes 5S DNA, linked to 25S DNA. Two techniques were especially useful in the mapping: "cloning" 14S + 25S DNA on the plasmid pMB9 to amplify individual R1 fragments, and digesting DNA with R1 in the presence of the antibiotic distamycin A to produce specific partial digestion products.     

A Dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant

Dictyostelium discoideum DNA fragments have been inserted into the chimeric bacterium-yeast plasmid YEp13. Recombinant plasmids were used to transform yeast using a strain of Saccharomyces cerevisiae deficient in OMP decarboxylase activity. Several clones were selected for growth in uracil-free medium. One clone was further analysed and contains a plasmid with a segment of D. discoideum DNA which complements a yeast ura3 mutation.     

Characterization of a new repetitive sequence in the Dictyostelium discoideum genome

A repetitive DNA sequence was isolated from a Dictyostelium discoideum genomic plasmid library of BglII-digested DNA ligated to the BamHI site in pBR322. This clone, called pBS582, hybridized to a large number of phage lambda Dictyostelium genomic clones. Southern blot analysis indicated that pBS582 DNA hybridized to many differently sized genomic DNA fragments generated by digestion with Eco RI, AvaI, or HindIII. Restriction maps of pBS582 and five genomic clones showed that the flanking regions of each of the genomic clones were different. These findings indicate that the sequence specific to pBS582 is scattered throughout the Dictyostelium genome and is reiterated approximately 100 times in the haploid genome. Northern blot analysis revealed that RNA which hybridized to pBS582 DNA was present during all stages of growth and development and did not seem to be developmentally regulated. Southern blot analysis of DNAs from other slime molds (D. giganteum, D. purpureum, and Polysphondylium violaceum) were performed to determine whether the pBS582 sequence was present in other species of slime molds. Hybridization of pBS582 was observed to DNA from the two Dictyostelium species but not to Polysphondylium. It may thus be possible to use hybridization of specific sequences as a biochemical tool to study the relatedness of different slime mold species and their molecular taxonomy.     

An inverse PCR technique to rapidly isolate the flanking DNA of Dictyostelium insertion mutants

Restriction enzyme mediated integration is a widely used and effective method for insertional mutagenesis in Dictyostelium discoideum. In this method, plasmid rescue is used to clone the genomic deoxyribonucleic acid (DNA) sequences that flank the insertion site. For this to be effective, it is necessary to first find a convenient restriction enzyme site within the genomic DNA. This is a time-consuming process that requires Southern blot analysis of the mutant DNA. In addition, plasmid rescue requires transformation into highly competent Escherichia coli. Problems can arise owing to unstable genomic sequences, damage to the plasmid DNA and exogenous plasmid contamination. We have established a simple and rapid polymerase chain reaction-based technique that works for all mutants and circumvents the need for Southern blot analysis and plasmid rescue.     

利用盘基网柄菌表达可溶性人Fas配体

用PCR扩增从激活的人中性粒细胞中得到的编码可溶性Fas配体胞外区中第141个到第281个氨基酸的cDNA ,将其与hCG-β信号肽片段融合到质粒MB12neo中,随后导入到盘基网柄菌AX3细胞中,得到分泌性表达hFasL的重组菌AX3-H3。为提高shFasL的表达量,对质粒pMB12neo作了改造,得到衍生质粒pMB74。利用质粒pMB74克隆表达shFasL ,得到高通量表达shFasL的重组菌AX3_pLu8。在复杂培养基HL_5C中,重组菌的细胞密度可达(1.5～2 )×10⁷ mL ,AX3-H3及AX3_pLu8分泌的shFasL浓度分别为23.5 μg/L及206μg/L。利用合成培养基SIH培养重组菌AX3-H3及AX3-pLu8,细胞密度均达到(4～5)×10⁷m/L ,shFasL浓度则分别达到111μg/L和420μg/L。     

The 3'-noncoding region of the chick myosin light-chain gene hybridizes to a family of repetitive sequences in the slime mold Dictyostelium discoideum

During studies aimed at isolating myosin-specific genomic clones in Dictyostelium, we probed a lambda genomic library with a chicken myosin light-chain sequence (pML10). Many lambda recombinant Dictyostelium clones hybridized to the pML10 cDNA insert, indicating that this sequence was reiterated in the Dictyostelium genome. It was found that the 3'-noncoding region (pML10-NC) alone was responsible for these results. Dictyostelium DNA contained approximately 65 copies of a sequence(s) similar but not identical to that of pML10-NC. Southern blot analysis showed that pML10-NC hybridized to many Dictyostelium genomic DNA fragments of varying sizes generated by digestion with EcoRI, HindIII, or AluI. In addition, each of the Dictyostelium clones was different in its size, restriction map, and flanking sequences. It seems likely, therefore, that the sequences which hybridized to pML10-NC are scattered throughout the Dictyostelium genome and similar but not identical to each other or to pML10-NC. Thus, probing with pML10-NC has allowed us to select a family of closely related but not identical sequences. These D. discoideum sequences are not found in other slime mold species. No RNA complementary to pML10-NC was found in vegetative cells, 18 h culmination stage, spores, or 1- and 2-h germinating spores. pML10-NC-related sequences were present in two other Dictyostelium species but were absent in the related genus Polysphondylium.     

Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum.

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.     

A linear shuttle vector for yeast and the hypotrichous ciliate Stylonychia

A linear plasmid was constructed in vitro using the telomeres of the rDNA of Tetrahymena pyriformis. These telomeres were added to a yeast circular vector containing an ARS sequence from Dictyostelium, the LEU2 gene of yeast and the neo gene from Escherichia coli Tn5 fused with a eukaryotic promoter. The resulting plasmid was used to transform yeast. During the replication of the linear plasmid in yeast it was spontaneously modified at the extremity by the addition of 300 bp of yeast telomeric sequence for each end. Total DNA prepared from yeast transformants was used to transform the hypotrichous ciliate Stylonychia lemnae. The same plasmid isolated from Stylonychia can again be replicated in yeast.     

Quantification of DNA uptake by Dictyostelium discoideum amoebae and the stability of the DNA during growth and development

We have developed a simple and accurate method to determine the amount of intact plasmid DNA taken up and retained by Dictyostelium discoideum amoebae during various transformation protocols. We have used this method to compare the efficiency of three different methods for introducing foreign DNA into D. discoideum amoebae. Both a calcium phosphate and a spheroplast fusion procedure gave good uptake, but no intracellular plasmid DNA was detectable after calcium chloride treatment. The exogenous DNA was rapidly lost after transformation but was 20-fold more stable during starvation rather than growth conditions, suggesting a possible approach to improving transformation efficiency. No transient expression of neomycin phosphotransferase activity of any of the heterologous animal or plant promoters used could be detected using a sensitive gel assay procedure.     

Cloning and analysis of a genomic fragment of Dictyostelium discoideum hybridizing to an RNA specifically accumulated in spore cells

A marker for Dictyostelium discoideum spore RNA differentiation has been isolated from a genomic DNA library by differential screening and a recombinant plasmid containing a genomic sequence complementary to spore specific RNA has been characterized. Only a small portion (~1 kb) of the 4.7-kb genomic insert is transcribed. The genomic organization of this spore specific gene shows a unique sequence flanked by reiterated sequences.