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T Dingermann

Publications and source records attributed to T Dingermann.

At least 91 records · Page 5Linked to original sources
PubMed1988

Structural requirements for the synthesis of tRNATrp from Dictyostelium discoideum in yeast.

Dictyostelium tRNA genes can generally be expressed in vivo in yeast. Among tested Dictyostelium tRNA genes a tRNATrp gene containing a 13 bp intron is transcribed with particularly poor apparent efficiency and the intron is not removed. Elimination of the intron from the gene increases the amount of transcription products significantly. Splicing can only occur if minimal base-pairing of the anticodon with intron sequences is possible. Accumulation of tRNA gene transcripts decreases with the inability of intron splicing. Products of neither amber (UAG) nor opal (UGA) suppressor variants of the tRNATrp gene from Dictyostelium are able to suppress corresponding non-sense mutations in defined structural yeast genes. This also holds true for suppressor tRNA gene variants with precisely deleted intron regions.

Dictyostelium↗
PubMed1987

Influence of different 5'-flanking sequences of tRNA genes on their in vivo transcription efficiencies in Saccharomyces cerevisiae.

We have investigated the influence of 5'-flanking sequences on the in vivo transcription activities in yeast. Since eukaryotic tRNA genes belong to multi-copy gene families monitoring of the activity of a particular tRNA gene is not possible. We therefore used two different tRNA genes from the cellular slime mould Dictyostelium discoideum which are efficiently transcribed and processed in vivo in yeast. The original 5'-flanking sequences of the two tRNA genes were replaced by random plasmid sequences. The modified tRNA genes were introduced into Saccharomyces cerevisiae and bulk tRNAs from the transformants were analyzed for the presence and the relative number of Dictyostelium tRNA gene transcripts. Substantial differences of steady-state levels of RNA transcribed were detected dependent on the 5'-flanking sequence of the tRNA gene. Minute structural changes, such as inserting two additional nucleotides in front of a tRNA gene, can lead to drastic activity changes. The efficiency of tRNA gene transcription can be conferred by sequences located more than 40 nucleotides upstream from the 5' end of the mature tRNA coding region.

Base Sequence↗
PubMed1987

Primer extension analysis of tRNA gene transcripts synthesized in vitro and in vivo.

The primer elongation method has been adapted to analyze tRNA gene transcripts. The primer used to direct cDNA synthesis from a corresponding tRNA template, in the presence of AMV reverse transcriptase, was a restriction fragment, or a synthetic oligonucleotide, containing exclusively coding nucleotides of a tRNA gene. This method not only allows one to identify the exact 5'-end of mature tRNA, but also 5'-ends of primary transcripts are readily determined. Further, analysis of tRNAs synthesized in vitro, as well as tRNAs produced in vivo in homologous and heterologous organisms can be studied. Purification of the tRNAs questioned, from bulk tRNA, is not necessary.

Animals↗
PubMed1987

Chromosomal mapping of tRNA genes from Dictyostelium discoideum.

Different wild-type isolates of Dictyostelium discoideum exhibit extensive polymorphism in the length of restriction fragments carrying tRNA genes. These size differences were used to study the organisation of two tRNA gene families which encode a tRNA Val(GUU) and a tRNA Val(GUA) gene. The method used involved a combination of classical D. discoideum parasexual genetics and molecular genetics. The tRNA genes were mapped to specific linkage groups (chromosomes) by correlating the presence of polymorphic DNA bands that hybridized with the tRNA gene probes with the presence of genetic markers for those linkage groups. These analyses established that both of the tRNA gene families are dispersed among sites on several of the chromosomes. Information of nine tRNA Val(GUU) genes from the wild-type isolate NC4 was obtained: three map to linkage group I (C, E, F), two map to linkage group II (D, I), one maps to linkage group IV (G), one, which corresponds to the cloned gene, maps to either linkage group III or VI (B), and two map to one of linkage groups III, VI or VII (A, H). Six tRNA Val(GUA) genes from the NC4 isolate were mapped: one to linkage group I (D), two to linkage group III, VI or VII (B, C) and three to linkage group VII or III (A, E, F).

Chromosome Mapping↗
PubMed1985

Characterisation of a Dictyostelium discoideum DNA fragment coding for a putative tRNAValGUU gene. Evidence for a single transcription unit consisting of two overlapping class III genes.

A genomic DNA fragment from Dictyostelium discoideum was characterized. This DNA, although 74% d(A + T)-rich, codes for a putative tRNAValGUU. The tRNAVal gene overlaps at its 5' half with another RNA polymerase III transcription unit. This RNA polymerase III transcription unit can be folded into a tRNA-like shape and is comprised of significant amounts of invariant and semi-invariant nucleotides present in all eukaryotic tRNAs. This unit contains the two promoter blocks defined for RNA polymerase III, which are homologous to recently defined promoter elements to the extent of 76-88% (A block) and 86-93% (B block) respectively [Sharp et al. (1981) Proc. Natl Acad. Sci. USA 78, 6657-6661]. Both of the overlapping class III genes are transcribed in germinal vesicle extracts prepared from Xenopus laevis oocytes as a single transcription unit, resulting in an unusually large product compared to primary transcripts of other tRNA genes. The unit is not transcribed in HeLa extracts but it competes very strongly for transcription factor(s) under the conditions of stable transcription complex formation. Although the whole unit is transcribed, it is believed that only one functional product is formed. Therefore we define the tRNA-like structure, coded for on this class III transcription unit, as a putative tRNA 'pseudogene' meaning that, although it is transcribed by RNA polymerase III, it is not likely to mature to a functional tRNA.

Base Sequence↗
PubMed1985

Processing of precursor tRNAs in Drosophila. Processing of the 3' end involves an endonucleolytic cleavage and occurs after 5' end maturation.

Transfer RNA biosynthesis is a complex process which includes size trimming and nucleotide modification of an initial tRNA precursor. We have examined the temporal order and the nature of tRNA processing events in a Drosophila in vitro transcription/processing system using Drosophila tRNA genes as templates. RNA sequence analysis of processing products indicates that processing at both 5' and 3' ends occurs by endonucleolytic cleavage. The time course of processing of an initial tRNA precursor to mature tRNA reveals that trimming at the 5' end precedes 3' end maturation.

Animals↗
PubMed1985

Escherichia coli supH suppressor: temperature-sensitive missense suppression caused by an anticodon change in tRNASer2.

We describe the cloning and the DNA sequence of the Escherichia coli supH missense suppressor and of the supD60(Am) suppressor genes. supH is a mutant form of serU which codes for tRNASer2. The supH coding sequence differs from the wild-type sequence by a single nucleotide change which corresponds to the middle position of the anticodon. The CGA anticodon of wild-type tRNA and CUA anticodon of supD tRNA is changed to CAA in supH tRNA, which is expected to recognize the UUG leucine codon. We propose that the supH suppressor causes the insertion of serine in response to this codon. The temperature sensitivity caused by supH may be due to a conformation of the CAA anticodon in the supH tRNASer that is slightly different than that in the corresponding tRNALeu species.

Anticodon↗
PubMed1985

Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP(Am) suppressor gene.

We describe the cloning and the DNA sequence of an amber suppressor allele of the Escherichia coli leuX (supP) gene. The suppressor allele codes for a tRNA with anticodon CUA, presumably derived by a single base change from a CAA anticodon. The mature coding sequence of the leuX gene is preceded by a putative Pribnow box sequence (TATAAT) and followed by a termination signal. The sequence of the leuX-coded tRNA is compared with the sequences of the four remaining tRNALeu isoacceptors of E. coli and with two tRNALeu species from bacteriophage T4 and T5. The conserved nucleotides in these seven tRNAs recognized by E. coli leucyl-tRNA synthetase are located mainly in the aminoacyl stem and in the D-stem/loop region.

Anticodon↗
PubMed1984

The extent of a eukaryotic tRNA gene. 5'- and 3'-flanking sequence dependence for transcription and stable complex formation.

We have examined the 5'-and 3'-flanking sequence requirements for the "wild type transcription properties" of a Drosophila tRNA Arg gene through the use of transcription assays in cell-free extracts. Thirty-three base pairs of the 5' flank immediately adjacent to the sequence encoding the mature tRNA are necessary for efficient transcription in Drosophila Kc cell extract. Sequences affecting factor binding to form stable transcription complexes extend more than 60 base pairs into the 5' flank, and approximately 35 base pairs into the 3' flank. HeLa cell extract exhibits dependence, albeit reduced, on the same 5'-flanking sequence; it also has 3'-flanking sequence requirements for maximal stable complex formation. This requirement of in vitro transcription for flanking sequence is not dependent on the use of a homologous system, but is dependent on the cellular source of the extract.

Animals↗
PubMed1983

Each element of the Drosophila tRNAArg gene split promoter directs transcription in Xenopus oocytes.

The intragenic control regions of a eukaryotic tRNA gene have been examined by transcribing mutant forms of a Drosophila tRNAArg gene either by injection into the nucleus of Xenopus oocytes or in extracts prepared from isolated oocyte nuclei. These experiments demonstrate that the selection of the transcription initiation site is a complex mechanism that involves the T-control region, the D-control region, and sequences 5' adjacent to the D-control region. In this study either "half" of the Drosophila tRNAArg gene promoted transcription in Xenopus oocytes. This finding supports a recent model for eukaryotic tRNA gene transcription (Dingermann et al., 1983, J. Biol. Chem. 258, 10395-10402) that proposes transcription initiation is dependent on the ability of specific DNA sequences to sequester two RNA polymerase III transcription factors.

Animals↗
PubMed1983

Stable transcription complex formation of eukaryotic tRNA genes is dependent on a limited separation of the two intragenic control regions.

We have examined the transcriptional role of the DNA region which lies between the two intragenic control sequences (D-control and T-control) of tRNA genes. Deletion templates (3' and 5') of a Drosophila tRNAArg gene, which contain either the D- or T-control region, were joined together through XhoI linkers such that the mutant tRNA genes formed now contain an internal cloning site. DNA fragments of different lengths were inserted into the newly formed cloning site to create a series of mutant tRNAArg genes which have an increased separation between the two intragenic control regions of 12 to 1530 nucleotides. Increased separation of the two intragenic control regions did not alter the transcription initiation or termination sites from those of the wild type tRNAArg gene. Transcription, while most efficient in the wild type gene, is still efficient when the two regions are further separated by a distance of 12-77 nucleotides. However, any further increase in length of the sequence separating the control regions resulted in a decreased transcription efficiency and in a decreased ability to compete in the binding of transcription factors in Drosophila Kc cell extracts. The reduction in transcription efficiency is directly related to the decreased ability of the insertion mutant tDNAs to form stable transcription complexes. The tRNAArg gene forms detectable stable complexes up to a separation of the two intragenic control regions by approximately 200 to 400 base pairs. These results suggest a model for tRNA gene transcription that involves factor recognition of sequences within each control region and that the control regions interact only via these factors.

Base Sequence↗
PubMed1983

Transcription of eukaryotic tRNA genes in vitro. I. Analysis of control regions using a competition assay.

The regions of a Drosophila tRNAArg gene responsible for the "wild type" in vitro transcription level were determined by a transcription-competition assay. Cell-free transcription extracts programmed with 5' and 3' deletion mutants of the tRNAArg gene were used to quantitate the efficiency of transcription and to measure the ability of these DNAs to compete for transcription factors compared to the wild type tRNAArg gene. The results show that those portions of the gene which code for the D-stem/D-loop and T-stem/T-loop of the tRNA product are the regions responsible for competitive ability. These regions were previously shown to contain the intragenic control sequences for eukaryotic tRNA gene transcription and are respectively referred to as the D- and T-control regions. The presence of both the D- and T-control regions is essential for maximum competitive strength. The 5'-flanking and 5' stem regions adjacent to the D-control region have a function in the competitive ability of the D-control region while the 3'-flanking and the 3' stem regions adjacent to the T-control region have a function in the competitive ability of the T-control region. These results are consistent with a model for promotion of tRNA gene transcription that involves recognition by transcription factors of the two control regions. Optimal binding of the transcription factors is dependent upon sequences adjacent to and flanking the intragenic control regions.

Animals↗
PubMed1983

Transcription of eukaryotic tRNA genes in vitro. II. Formation of stable complexes.

Drosophila tRNA genes form stable transcription complexes in vitro, as we have demonstrated by kinetic analyses of transcription experiments in Drosophila Kc cell extracts. tRNA genes added to transcriptionally active cell-free extracts rapidly and stably sequester a transcription factor, inhibiting transcription of a tRNA gene added later. We describe a simplified competition assay dependent on the ability of tRNA genes to form stable complexes. Through the use of this assay with deletion mutations of a Drosophila tRNAArg gene, we demonstrate that stable transcription complex formation is dependent on the DNA region extending from the 5' end of the sequence encoding the T-stem of the tRNA to more than 10 base pairs downstream from the transcription termination sequence. Stable transcription complex formation involves an initial, rapid factor binding followed by rearrangement of the gene-factor complex to a transcriptionally active state. Factor binding to form the stable transcription complex is kinetically dependent on the sequence 5' to the gene region encoding the D-stem, and thermodynamically dependent on the gene region encoding the D-stem and -loop.

Animals↗
PubMed1982

The 5- flanking sequences of Drosophila tRNAArg genes control their in vitro transcription in a Drosophila cell extract.

The transcription efficiencies of four Drosophila tRNAArg genes located in a tRNA gene cluster at region 42A on chromosome 2, and containing identical coding sequences, were studied in Drosophila Kc cell extracts. Transcription is modulated by the 5' flanking sequences; efficient transcription is dependent on the presence of an optimal 5' flanking sequence. One of the genes, p17D Arg, is not transcribed in the homologous extract but does compete with the other genes for transcription factors. Deletion of a specific sequence from the 5' flank of the gene of p17D Arg leads to an increase in transcription efficiency. All tRNAArg genes are efficiently transcribed in extracts from HeLa cells. However, introduction of small amounts of Drosophila extract reduces the efficiency of transcription in HeLa extracts. This is due to incompatibility between transcriptional components of the two extracts.

Animals↗
PubMed1982

The minimum intragenic sequences required for promotion of eukaryotic tRNA gene transcription.

Transcription of eukaryotic tRNA genes is controlled by two intragenic regions, the D-control region (which in the tRNA codes for the D-stem and -loop) and the T-control region (which in the tRNA codes for the T psi C loop). To determine whether these sequences alone are sufficient to promote tRNA gene transcription in vitro, the two control regions of a Drosophila tRNAArg gene were cloned separately from the context of the parental DNA (these constructions are called tRNA minigenes). The tRNA minigene that contains both intragenic control regions supports in vitro RNA synthesis in Xenopus laevis oocyte and HeLa cell transcription systems. The mutant which has deletions to nucleotide 7 within the mature tRNA coding region, pArg5.7, and minigenes derived from it do not support RNA synthesis in a Drosophila Kc cell transcription system. Xenopus and Hela extracts transcribe pArg5.7 albeit at reduced levels compared to the wild-type gene. The tRNA minigene that contained only the D-control region was not able to support RNA synthesis in any of these three transcription systems. A mutant tRNA gene comprising the 3' half of the tRNAArg gene similarly was not able to support RNA synthesis. These experiments show that the DNA sequence from nucleotides 7-58, which contains both intragenic control regions of the tRNA gene, possesses sufficient information to initiate specific transcription by RNA polymerase III in Xenopus and HeLa systems. The transcription efficiency of this tRNA minigene however is reduced to about 20% the transcription level of the wild type tRNA gene. This lowered level of transcriptional efficiency results from deleting the ends of the native tRNA gene and its adjacent flanking sequences. The affects of deleting 5' sequences are most pronounced in the Drosophila transcription system.

Animals↗
PubMed1982

Eukaryotic tRNA gene transcription is controlled by signals within and outside the mature coding sequence.

We have identified the region within a eukaryotic tRNA gene required for initiation of transcription. These results were obtained by systematically constructing deletions extending from the 5'- or the 3'-flanking regions into a cloned Drosophila tRNAArg gene using nuclease BAL-31. Two control regions within the coding sequence were identified. The first was essential for transcription and was contained between nucleotides 8 to 25 of the mature tRNA sequence. Genes devoid of the second control region, which was contained between nucleotides 50 to 58 of the mature tRNA sequence, could be transcribed but with reduced efficiency. Thus, the promoter regions within a tRNA gene encode the tRNA sequences of the D-stem and D-loop, the invariant U at position 8, and the semi-invariant GTpsi C sequence. While transcription of Drosophila tRNA genes is controlled by signals within the mature tRNA coding region deletion analysis has revealed an oligonucleotide sequence in the 5'-flanking region of a Drosophila tRNA2Lys gene to be responsible for the poor transcriptional activity of this and of other tRNA genes. The oligonucleotide responsible for transcriptional repression is GGCAGTTTTTG and is located 13 nucleotides upstream from the mature tRNA coding sequence. Since the sequence of the undecanucleotide is well conserved within the 5'-flanking region of Drosophila tRNA2Lys genes an investigation of why the transcription of all these genes is not similarly repressed revealed that the position of this oligonucleotide, relative to the mature coding sequence, influences the extent of transcriptional repression.

Animals↗