Structure of two tRNA genes from Dictyostelium discoideum

Dingermann, Theo; Bertling, Wolf; Brechner, Thomas; Nerke, Käthe; Peffley, Dennis M.; Sogin, Mitchell L.

doi:10.1093/nar/14.2.1127

Cited by 10 publications

(7 citation statements)

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“…Many tRNA genes were physically mapped with tRNA gene family-specific probes. Thus, twa loci were identified with a tRNATrP gene fragment (35), tva loci were identified with a tRNAVal(GUU) gene fragment (36), tvb loci were identified with a tRNAVal(GUA) gene fragment (37), and tea loci were identified with a tRNAGlU gene fragment (37). The tva and tvb loci were previously mapped genetically (38).…”

Section: Methodsmentioning

confidence: 99%

Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome.

Kuspa

Loomis

1996

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

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High resolution gene maps of the six chromosomes ofDictyostelium discoideum have been generated by a combination of physical mapping techniques. A set of yeast artificial chromosome clones has been ordered into overlapping arrays that cover >98% of the 34-megabase pair genome. Clones were grouped and ordered according to the genes they carried, as determined by hybridization analyses with DNA fragments from several hundred genes. Congruence of the gene order within each arrangement of clones with the gene order determined from whole genome restriction site mapping indicates that a high degree of confidence can be placed on the clone map. This clone-based description of the Dictyostelium chromosomes should be useful for the physical mapping and subcloning of new genes and should facilitate more detailed analyses of this genome.Few multicellular organisms have the advantages found in Dictyostelium discoideum of haploid growth and development, convenient molecular genetics, and a small genome (1, 2). The relative ease of isolating mutations that affect endocytosis, cellular motility, signal transduction, and morphogenesis has attracted attention from a number of fields (for recent reviews, see ref.3). As a consequence of having a genome of only 34 megabase pairs (Mb) in size, genes of interest can be readily cloned following screens of cell populations mutated by plasmid insertion facilitated by restriction enzyme-mediated integration (4). Homologous recombination functions efficiently in Dictyostelium such that endogenous genes can be routinely replaced by modified versions. The consequences of genetic alterations in various functions can then be readily determined since the organism grows as single cells that can be induced to develop synchronously into fruiting bodies over a period of 24 h.Segregation patterns from heterozygous diploids have established linkage groups that can be assigned to one or another of the six chromosomes that carry the genes of Dictyostelium (5). Recently, over 200 genes have been mapped to one or another of the six chromosomes by a combination of linkage to parasexually assigned loci and long range restriction mapping using restriction fragment length polymorphisms (RFLPs) generated from random plasmid integrations along the chromosomes (6, 7). To increase the resolution of the resulting physical maps and to provide whole genome coverage in cloned fragments, we have generated a 5-fold redundant library of yeast artificial chromosome (YAC) clones carrying large inserts (200 kb average) of Dictyostelium genomic DNA (8). We have now ordered the YAC clones by hybridizing sets of clones with DNA from previously mapped and sequenced genes and anonymous genomic and cDNA fragments. By using probes that recognize over 400 loci, we have been able to construct long contiguous arrays of overlapping YACs (contigs) that span the chromosomes and together account for over 98% of the genome.

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Section: Methodsmentioning

confidence: 99%

Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome.

Kuspa

Loomis

1996

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

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“…We recently described the sequence of a tRNAGlu(GAA) gene from D. discoideum strain AX2 (Dingermann et al, 1986). This gene resides on a 2.9-kb genomic Eco RI fragment and is a typical eukaryotic tRNA gene, i.e., the CCA 3'-terminal end is not encoded, invariant or semi-invariant nucleotides found in all eukaryotic tRNA genes (Sprinzl et al, 1987) are present at corresponding positions, the putative 5' ICR and 3' ICR correlate with established consensus sequences (Sharp et al, 1985), and an oligo(dT) stretch is located on the noncoding strand within the proximal 3'-flanking region of the tRNA gene.…”

Section: Resultsmentioning

confidence: 99%

“…Nuclear DNA from D. discoideum strain AX2 was digested with Eco RI and size-fractionated on an 0.7% agarose gel. Fragments were transferred to nitrocellulose filters and hybridized with a nick-translated 390-bp Sau 3A genomic DNA fragment containing a tRNAGlu(GAA) gene from D. discoideum (Dingermann et al, 1986). At least 18 different-sized fragments were observed in many independent experiments (see also Fig.…”

Section: Resultsmentioning

confidence: 99%

“…DNA fragments were transferred and fixed to GeneScreen membranes and incubated with a nick-translated 390-bp 5a« 3A genomic DNA fragment from the tRNAGlu(GAA)l gene from D. discoideum (Dingermann et al, 1986). As observed earlier (Dingermann et al, 1987a), restriction fragments containing Haploid segregant strains, designated HU (or HUD) followed by a number, were derived from diploids following thiabendazoleinduced nondisjunction and chromosome loss (Welker and Williams, 1980).…”

Section: Resultsmentioning

confidence: 99%

“…Libraries were screened with a nick-translated 390-bp Sau 3A genomic DNA fragment containing a tRNAGlu(GAA) gene from D. discoideum (Dingermann et al, 1986). Genomic Eco RI fragments containing tRNAGlu(GAA) genes were recloned into plasmid yRMPl (Dingermann et al, 1987b), and tRNA genes were sequenced as double-stranded plasmids following the dideoxy chain-termination method (Sanger et al, 1977).…”

Section: Enzymes and Chemicalsmentioning

confidence: 99%

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tRNA^Glu(GAA) Genes from the Cellular Slime MoldDictyostelium discoideum

Dingermann

Brechner²,

Marschalek³

et al. 1989

DNA

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The haploid genome of the cellular slime mold Dictyostelium discoideum contains at least 18 gene copies coding for a tRNAGlu(GAA). Using a combination of parasexual genetic analysis and molecular biology techniques, 14 of the 18 individual members of this gene family could be assigned to particular linkage groups. According ot this analysis four tRNAGlu genes are located on group I (C, H, I, K), two genes on group II (D,J), seven genes on either group III or VI (A, B, E, F, L, M, N), and one gene on group VII (G). Eight of the tRNAGlu(GAA) genes have been cloned and characterized. All genes are identical in that part of the gene which corresponds to the mature tRNA, thus representing true nonallelic members of this gene family. Different members of this gene family can be distinguished from each other because they reside on restriction fragments of different lengths and because each gene contains unique 5'- and 3'-flanking regions. Nevertheless, a certain degree of sequence conservation within these flanking regions is apparent for members of this gene family. According to in vivo expression analyses of individual genes in Saccharomyces cerevisiae, all isolated tRNAGlu(GAA) copies represent functional transcription units.

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Nonsense suppression in Dictyostelium discoideum

et al. 1990

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We describe the generation of Dictyostelium discoideum cell lines that carry different suppressor tRNA genes. These genes were constructed by primer-directed mutagenesis changing a tRNA(Trp)(CCA) gene from D. discoideum to a tRNA(Trp)(amber) gene and changing a tRNA(Glu)(UUC) gene from D. discoideum to a tRNA(Glu)(ochre) as well as a tRNA(Glu)(amber) gene. These genes were stably integrated into the D. discoideum genome together with a reporter gene. An actin 6::lacZ gene fusion carrying corresponding translational stop signals served as a reported. Active beta-galactosidase is expressed only in D. discoideum strains that contain, in addition to the reporter, a functional suppressor tRNA. Both amber suppressors are active in D. discoideum without interfering significantly with cell growth and development. We failed, however, to establish cell lines containing a functional tRNA(Glu)(ochre) suppressor. This may be due to the fact that nearly every message from D. discoideum known so far terminates with UAA. Therefore a tRNA capable of reading this termination codon may not be compatible with cell growth.

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Structure of two tRNA genes from Dictyostelium discoideum

Cited by 10 publications

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Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome.

Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome.

tRNA^Glu(GAA) Genes from the Cellular Slime MoldDictyostelium discoideum

Nonsense suppression in Dictyostelium discoideum

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Structure of two tRNA genes from Dictyostelium discoideum

Cited by 10 publications

References 0 publications

Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome.

Ordered yeast artificial chromosome clones representing the Dictyostelium discoideum genome.

tRNAGlu(GAA) Genes from the Cellular Slime MoldDictyostelium discoideum

Nonsense suppression in Dictyostelium discoideum

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tRNA^Glu(GAA) Genes from the Cellular Slime MoldDictyostelium discoideum