Structural organization of the genome of Dictyostelium discoideum: Analysis by EcoR1 restriction endonuclease

Firtel, Richard A.; Cockburn, Andrew; Frankel, Gary; Hershfield, Vickers

doi:10.1016/0022-2836(76)90294-1

Cited by 67 publications

(24 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The relatively high copy number of the A6NPTII vector is consistent with the observation of a vector-sized band in ethidium bromide-stained gels of EcoRI-digested DNA from A6NPTII transformants (solid arrowhead, top right panel). The intensity of the vector band was comparable to the intensity of the flanking EcoRI fragments of rDNA, which are present at several hundred copies per haploid genome (18) with other clonal isolates and with populations of transformants, indicating that the vector copy number in transformed cells is a vector-specific property.…”

Section: Resultsmentioning

confidence: 67%

Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors.

Knecht¹,

Cohen²,

Loomis³

et al. 1986

Mol. Cell. Biol.

196

136

View full text Add to dashboard Cite

(19,25,32,33,46). With the exception of two quite divergent actinrelated proteins, all of these genes appear to encode substantially the same actin polypeptide. Differences in the length of the 3' untranslated region of these RNAs account for the two mRNA size classes (33). The different actin genes can be grouped into families based on homologies in their 3' untranslated sequences (33,45 (19,25,32,45,55). S1 nuclease and RNase mapping (25, 32, 47) of the 5' ends of the actin mRNAs and primer extension analysis (55) indicate that individual actin genes are differentially transcribed during development.We report the identification and complete nucleotide sequence of a genomic clone that encodes the actin mRNA previously identified by the cDNA clone cActin III-12/Al (33) and by primer extension as actin III (55). Following the system of nomenclature used by

show abstract

Section: Resultsmentioning

confidence: 67%

Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors.

Knecht¹,

Cohen²,

Loomis³

et al. 1986

Mol. Cell. Biol.

196

136

View full text Add to dashboard Cite

show abstract

“…Late-logarithmic phase T. brucei genomic DNA was extracted as described (23) and analyzed by the method of Southern (24). The DNA was resolved by electrophoresis on 0.6% agarose gels, transferred onto a GeneScreenPlus membrane, and hybridized to DNA probes radiolabeled by random priming (25).…”

Section: Methodsmentioning

confidence: 99%

Stable integrative transformation of Trypanosoma brucei that occurs exclusively by homologous recombination.

Eid

Sollner‐Webb

1991

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

A calmodulin-neomycin-resistance fusion gene was introduced into Trypanosoma brucei by electroporation, and stably transformed cell lines were obtained. In all of the transformants, the fusion gene had integrated into the host genome at the cognate locus, evidently by homologous recombination within flanking calmodulin DNA. This unusual observation distinguishes trypanosomes as the only eukaryote other than yeast known to undergo gene targeting in essentially 100% of the stable transformants. It should now be possible to systematically manipulate the trypanosome genome, directing predetermined mutations to virtually any chromosomal locus.Trypanosoma brucei are parasitic protozoa and the causative agent of sleeping sickness, a major disease in Africa. This organism exhibits many fascinating biological phenomena including antigenic variation (1, 2), polycistronic transcription (1, 3), trans-splicing of RNA (4, 5), RNA editing (6, 7), and stage-dependent mitochondrial activation (8). To better study these and other features of T. brucei, a stable transformation system involving homologous gene targeting was sought, for this would allow directed mutation of the genome as well as long-term expression of introduced DNA. Such homologous gene targeting has proven extremely valuable in numerous species to selectively alter genes of interest in the host chromosome. In yeast, virtually 100% of transfected DNA that integrates into the host genome does so by homologous recombination (9,10); other species show homologous gene targeting as well, but with lower efficiency ("'0.01%-1% of the integrants in mammalian cells; refs. 11-13). Although stable transformation of another trypanosomatid, Leishmania, has been reported (14,15), the introduced DNA remained extrachromosomal, precluding the possibility of mutating the host genome.In this paper, we present evidence for stable integrative transformation of T. brucei by a fusion gene construct, CNeoC. This construct, which contains the neomycinresistance (neo) coding region flanked by extensive 5' and 3' segments of the T. brucei calmodulin locus, was designed to favor homologous targeting (16) into the chromosomal calmodulin locus. After electroporation of procyclic cultures of T. brucei with the excised fusion gene, G418-resistant cells were obtained. DNA analysis from seven of the resultant stable cell lines (detailed in this report) and preliminary evidence from 20 additional similarly derived lines showed that in all cases the neor fusion gene was integrated into the calmodulin locus of the host genome, evidently by homologous recombination. MATERIALS AND METHODSThe CNeoC Plasmid. The CNeoC plasmid (see Fig. 1) contains the translation initiation codons ofboth the T. brucei calmodulin gene (17) and the neomycin phosphotransferase (neo') gene (18) in frame and 36 base pairs apart, the translation termination regions of both genes, and the calmodulin poly(A) addition region (19). It also includes extensive flanking calmodulin sequences, extending upstream to the EcoRI...

show abstract

“…The majority of the labeled and unlabeled fragments were purified by agarose gel electrophoreses. Restriction endonuclease reactions were stopped with gel loading buffer and loaded directly on a flat-bed agarose gel containing 1 ,ug of ethidium bromide per ml and run as described (13). The mobility of the fragment was visualized during the run with short wavelength UV light.…”

Section: Observations)mentioning

confidence: 99%

Unusual nucleotide sequences at the 5' end of actin genes in Dictyostelium discoideum.

Firtel¹,

Timm²,

Kimmel³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The nucleotide sequences at the 5' end of one actin cDNA and six actin genomic clones from Dictyostelium have been determined. The amino acid sequences derived from the nucleotide sequences show strong conservation for six of the seven genes relative to the NH2-terminal regibn of Physarum actin. The region 5' to the AUG initiating codon is >90% A+T residues in all of the genes.Actin, a ubiquitous protein in eukaryotic cells, is associated with a large number of cellular functions including contraction and motility (1,2 the untranslated region adjacent to the 51 end of the initiating AUG codon of all of the actin genes analyzed is extremely A+T rich. Except for the low G+C content there is little additional sequence homology in this region among the various genes.MATERIALS AND METHODS Labeling of DNA Fragments. Plasmid DNA was digested with appropriate restriction endonucleases and was then heated at 650C for 5 min. The reaction was diluted 1:2 with 15 mM Tris (pH 8.0) and the 5'-terminal phosphate residue was removed by digestion with calf intestine alkaline phosphatase for 45 min at 37'C. The phosphatase was removed either by three phenol/chloroform extractions (in 0.2% sodium dodecyl sulfate/0.8 M LiCl/50 mM Tris, pH 9.5) followed by two ethanol precipitations or by isolation of DNA fragments by electrophoresis on agarose or acrylamide gels. Gel-purified fragments were free of phosphatase activity.Nondenatured DNA fragments were labeled in vitro by using ['y-32P]ATP according to the method of Maxam and Gilbert (10). [,y-32P]ATP (estimated specific activity > 8000 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) was synthesized according to the method of Johnson and Walseth (11). In some cases, DNA restriction fragments were end-labeled by using DNA polymerase I (Klenow fragment) and [a-32P]dXTPs (12). After labeling, the material was precipitated with ethanol, resuspended in restriction buffer, and digested with the appropriate restriction endonuclease. The majority of the labeled and unlabeled fragments were purified by agarose gel electrophoreses. Restriction endonuclease reactions were stopped with gel loading buffer and loaded directly on a flat-bed agarose gel containing 1 ,ug of ethidium bromide per ml and run as described (13). The mobility of the fragment was visualized during the run with short wavelength UV light. After appropriate separation of the fragment, a small trough (extending across the distance of the slot and approximately 0.5-1 cm wide) was cut in the gel immediately to the anode side of the fragment. The agarose was removed and the trough was filled with running buffer. The DNA fragment was isolated by electrophoreses into the trough at 350 V. The movement of the DNA into the trough was visualized by UV light. DNA in the trough was removed periodically (at approximately 1-min intervals) to avoid electrophoresis of the fragment into the agarose at the opposite edge of the trough. Eluted DNA was then extracted once with phenol/chloroform as described above and precipitated twice with ethanol...

show abstract

Structural organization of the genome of Dictyostelium discoideum: Analysis by EcoR1 restriction endonuclease

Cited by 67 publications

References 21 publications

Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors.

Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors.

Stable integrative transformation of Trypanosoma brucei that occurs exclusively by homologous recombination.

Unusual nucleotide sequences at the 5' end of actin genes in Dictyostelium discoideum.

Contact Info

Product

Resources

About