Biomedical subjects

R Marschalek

Publications and source records attributed to R Marschalek.

49 records · Page 3Linked to original sources

PubMed1992

Structure of DRE, a retrotransposable element which integrates with position specificity upstream of Dictyostelium discoideum tRNA genes.

Different Dictyostelium discoideum strains contain between 2 and 200 copies of a retrotransposable element termed DRE (Dictyostelium repetitive element). From the analysis of more than 50 elements, it can be concluded that DRE elements always occur 50 +/- 3 nucleotides upstream of tRNA genes. All analyzed clones contain DRE in a constant orientation relative to the tRNA gene, implying orientation specificity as well as position specificity. DRE contains two open reading frames which are flanked by nonidentical terminal repeats. Long terminal repeats (LTRs) are composed of three distinct modules, called A, B, and C. The tRNA gene-proximal LTR is characterized by one or multiple A modules followed by a single B module (AnB). With respect to the distal LTR, two different subforms of DRE have been isolated. The majority of isolated clones contains a distal LTR composed of a B module followed by a C module (BC), whereas the distal LTR of the other subform contains a consecutive array of a B module, a C module, a slightly altered A module, another B module, and another C module (BC.ABC). Full-length as well as smaller transcripts from DRE elements have been detected, but in comparison with the high copy number in D. discoideum strains derived from the wild-type strain NC4, transcription is rather poor.

Amino Acid Sequence↗

PubMed1992

Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.

Animals↗

PubMed1991

Transfer RNA genes from Dictyostelium discoideum are frequently associated with repetitive elements and contain consensus boxes in their 5' and 3'-flanking regions.

A total of 68 different tRNA genes from the cellular slime mold Dictyostelium discoideum have been isolated and characterized. Although these tRNA genes show features common to typical nuclear tRNA genes from other organisms, several unique characteristics are apparent: (1) the 5'-proximal flanking region is very similar for most of the tRNA genes; (2) more than 80% of the tRNA genes contain an "ex-B motif" within their 3'-flanking region, which strongly resembles characteristics of the consensus sequence of a T-stem/T-loop region (B-box) of a tRNA gene; (3) probably more than 50% of the tRNA genes in certain D. discoideum strains are associated with a retrotransposon, termed DRE (Dictyostelium repetitive element), or with a transposon, termed Tdd-3 (Transposon Dictyostelium discoideum). DRE always occurs 50 (+/- 3) nucleotides upstream and Tdd-3 always occurs 100 (+/- 20) nucleotides downstream from the tRNA gene. D. discoideum tRNA genes are organized in multicopy gene families consisting of 5 to 20 individual genes. Members of a particular gene family are identical within the mature tRNA coding region while flanking sequences are idiosyncratic.

Animals↗

PubMed1991

Glycosylation sites identified by detection of glycosylated amino acids released from Edman degradation: the identification of Xaa-Pro-Xaa-Xaa as a motif for Thr-O-glycosylation.

Here we report the use of automated Edman degradation of covalently linked glycopeptides to identify positively the sites of O- and N-glycosylation. The O-glycosidic linkage of carbohydrate to the hydroxy amino acids Ser and Thr is a major form of post-translational modification. However, unlike Asn-linked glycosylation, which is identified by the consensus sequence Asn-Xaa-Thr/Ser, no simple motif conferring O-linkage to Thr and Ser has been described. After sequencing glycopeptides derived from two cell surface glycoproteins, a Thr-O-glycosylation motif of Xaa-Pro-Xaa-Xaa, where at least one Xaa = Thr(Sac), has been defined. This motif predicts the site(s) of Pro- associated Thr-O-glycosylation in O-glycosylated proteins, although it is clear that there are also other forms of Thr-O-glycosylation not associated with Pro.

Amino Acid Sequence↗

PubMed1991

Nuclear factors which bind to Dictyostelium discoideum transfer RNA genes.

RNA Polymerase III transcription factors from the cellular slime mold Dictyostelium discoideum were characterized, based on their stable binding to isolated tRNA genes. Different protein complexes are sequestered on DNA fragments containing tRNA genes depending on the conditions by which the nuclei were extracted. Binding specificity was determined through competition assays using competitor tRNA genes from the same gene family, from different gene families and from truncated tRNA genes. The complex with the highest multiformity of interdependent proteins is able to assemble with low affinity on a B-block-free tDNA template, whereas most lower molecular weight complexes require the presence of an intact B-block promoter element in order to assemble.

Animals↗

PubMed1990

Genomic organization of the transposable element Tdd-3 from Dictyostelium discoideum.

The transposable element Tdd-3 from D. discoideum has been described originally in 1984 (Poole and Firtel, 1984). Additional copies of this element were discovered in the course of a recent study on tRNA gene organization in D. discoideum. Five out of 24 independently isolated tRNA genes proved to be associated with Tdd-3 elements. The surprising observation that all the elements always occurred within the 3'-flanking sequences of the Dictyostelium tRNA genes suggested the possibility of a general position specific integration of Tdd-3 elements upon transposition. Therefore we isolated additional Tdd-3 elements from various genomic D. discoideum libraries in order to test this hypothesis. Several new Tdd-3 elements were found associated with various tRNA genes. Additionally we identified Tdd-3 elements organized in tandem array or in association with RED (Repetitive Element of Dictyostelium), another repetitive element recently identified by our laboratory. In all cases a B-box equivalent of the eukaryotic gene-internal RNA polymerase III promoter was identified upstream of all Tdd-3 elements.

Base Sequence↗

PubMed1990

Temperature sensitive synthesis of transfer RNAs in vivo in Saccharomyces cerevisiae.

Dictyostelium discoideum tRNA genes can be expressed efficiently in vivo in yeast, and transcription products are processed to mature tRNAs. However, primary transcripts of a variant tRNA(Val)(UAC) gene are processing deficient under standard growth conditions (30 degrees C), due to a slightly altered 5' flanking region. A stable extended amino acid acceptor stem, which seems to be required to compensate a G5-G68 mismatch, cannot form. This mismatch destabilizes secondary and probably tertiary structures to such an extent that recognition of processing enzyme(s) under normal conditions (30 degrees C) is impaired. Growing yeast cells at reduced temperature (22 degrees C) can phenotypically complement the processing defect. This observation provides a new concept for the temperature dependent expression of protein coding genes which carry a nonsense codon. Translation of corresponding messages can be controlled by products of a temperature sensitive su-tRNA gene. We successfully tested this concept with two amber suppressors derived from a tRNA(Glu)(UUC) gene from D. discoideum. One of the variant tRNA genes codes for a product with a destabilized amino acid acceptor stem. Primary transcripts of this particular su-tRNA(Glu)(CUA) gene are processed only at reduced growth temperatures and consequently function as temperature sensitive suppressors only under these conditions.

Base Sequence↗

PubMed1989

Transfer RNA genes: landmarks for integration of mobile genetic elements in Dictyostelium discoideum.

In prokaryotes and eukaryotes mobile genetic elements frequently disrupt the highly conservative structures of chromosomes, which are responsible for storage of genetic information. The factors determining the site for integration of such elements are still unknown. Transfer RNA (tRNA) genes are associated in a highly significant manner with different putative mobile genetic elements in the cellular slime mold Dictyostelium discoideum. These results suggest that tRNA genes in D. discoideum, and probably tRNA genes generally in lower eukaryotes, may function as genomic landmarks for the integration of different transposable elements in a strictly position-specific manner.

Base Sequence↗

PubMed1989

CMER, an RNA encoded by human cytomegalovirus is most likely transcribed by RNA polymerase III.

Through computer analysis of a human cytomegalovirus (HCMV) genomic region, previously identified to be homologous to human genomic DNA, an element showing significant similarity to the 3'-internal control region (3'-ICR or B-block) of a eukaryotic RNA polymerase III promoter could be detected. This region-located on the EcoRI b fragment within the UL segment of the viral genome of HCMV strain AD 169-cannot be transcribed in vitro in an RNA polymerase III specific transcription system. However, this part of the viral genome is able to compete for components of the RNA polymerase III transcription complex as shown in template exclusion experiments and by gel retardation assays. Two different synthetic oligonucleotides complementary to the 3'-ICR and to nucleotides located immediately downstream of this promoter element can anneal specifically to a HCMV-encoded ribonucleic acid (termed CMER) synthesized in human foreskin fibroblasts (HFF) late in virus replication. As a consequence of identifying the transcription initiation point by primer extension analyses the position of the 5'-internal control region (5'-ICR or A-block) of the CMER gene could be uncovered. Both identified control regions (the A-block as well as the B-block) of the transcription unit exhibit significant similarities to corresponding regulatory elements of other class III genes, including virus encoded class III genes. Initiation of in vivo transcription occurs 15 nucleotides upstream of the 5'-border of the 5'-ICR and the two non-contiguous gene internal promoter elements are separated by 79 nucleotides.

Base Sequence↗

PubMed1989

tRNAGlu(GAA) genes from the cellular slime mold Dictyostelium discoideum.

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.

Base Sequence↗

PubMed1988

A family of non-allelic tRNA(ValGUU) genes from the cellular slime mold Dictyostelium discoideum.

A haploid genome of the cellular slime mold Dictyostelium discoideum contains at least 14 non-allelic gene copies coding for a tRNA(ValGUU). The structure, genomic organization, and expression of these genes have been analyzed in relation to stages of the developmental cycle. So far, 13 tRNA(ValGUU) genes have been isolated and characterized. All genes contain identical mature tRNA-coding regions, and consequently identical gene internal promoter elements. However, different genes differ with respect to their 5'- and 3'-flanking regions, although a certain degree of sequence conservation seems apparent. Different members of this tRNA gene family appear to be randomly dispersed along the seven D. discoideum chromosomes, and not clustered at any one genomic location. In vivo expression of individual genes was studied in yeast. All but one tRNA(ValGUU) gene are actively transcribed, though with different efficiencies. There is also evidence that not all of these tRNA genes are constitutively transcribed in Dictyostelium throughout the developmental cycle. One characteristic primary transcript can only be detected in cells of the late preaggregation phase, whereas growing cells, cells in the stationary phase or cells harvested 4 h after the onset of development do not seem to carry this transcript. This product seems to be transcribed from a gene of an unusual structure. Although this particular gene has not yet been isolated, it can be predicted from the sequence of the cDNA synthesized from primary transcription products of this putative gene, that it is composed of nt 1-54 of a 3'-truncated tRNA(ValGUU) gene linked to a bona fide tRNA(ValGUU) gene.

Alleles↗

PubMed1988

Identification of a protein factor binding to the 5'-flanking region of a tRNA gene and being involved in modulation of tRNA gene transcription in vivo in Saccharomyces cerevisiae.

Control mechanisms of tRNA gene transcription were studied in vivo in Saccharomyces cerevisiae. In order to be able to monitor in vivo transcription products of an individual tRNA gene, a 'tester gene' was used which is readily transcribed in vivo in yeast but does not cross-hybridize with any cellular yeast tRNA. A series of insertion mutants were constructed, modifying thereby the immediate and further distant 5'-flanking region of the 'tester tRNA gene'. Small linker molecules of different length and different sequence were inserted at positions -3 and -56 on the non-coding strand. Resulting tRNA gene variants were transformed into yeast cells and in vivo synthesized products were monitored by primer extension analysis. From the experimental data we suggest that a few essential nucleotides within the flanking region are able to determine the in vivo transcription activity of the 'tester tRNA gene'. Our results are rationalized on a biochemical level by protein binding assays: At least one protein binds to the 5'-flanking region of the 'tester tRNA gene' and different protein complexes are sequestered on active or less active tRNA gene variants.

Base Sequence↗

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↗