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T Seebeck

Publications and source records attributed to T Seebeck.

67 records · Page 4Linked to original sources
PubMed1983

Tubulin genes of Trypanosoma brucei: a tightly clustered family of alternating genes.

African trypanosomes are the causative agents of many medically and economically important diseases of man and domestic animals. The cell body of these blood-dwelling protozoa is enveloped with a dense layer of pellicular microtubules, which confer both motility and mechanical stability on these cells; microtubules are also important components of the flagellum. The major structural components of the microtubuli are two related proteins, alpha- and beta-tubulin. We have analyzed the genomic organization of alpha- and beta-tubulin genes in Trypanosoma brucei. In this organism, the majority of these genes are arranged in a single, tightly packed cluster of alternating alpha- and beta-tubulin genes with a basic repeat length together of 3.6 kilobase pairs. A genomic library of T. brucei was constructed by using the phage vector A 1059, and recombinant phages carrying tubulin genes were isolated by screening the library with heterologous chicken tubulin cDNA probes. The results of restriction endonuclease and hybridization analysis of DNA isolated from recombinant phages, and subcloned fragments thereof, were compatible with the restriction maps derived from digestion and Southern blot hybridization of genomic DNA.

Animals↗
PubMed1982

Sequence-specific cleavage of chromatin by staphylococcal nuclease can generate an atypical nucleosome pattern.

We have investigated the nucleosomal organization of ribosomal genes in the acellular slime mold Physarum polycephalum. When probed with staphylococcal nuclease, the ribosomal genes appear to be uniformly packed in nucleosomes, in an arrangement which is indistinguishable from the pattern obtained with bulk chromatin. During this study, an unusual pattern of digestion was obtained from a DNA region immediately upstream of the initiation site of rRNA transcription, in addition to the nucleosomal profile, a second regular ladder of fragments with a repeat length of 30-40 basepairs was generated from this region. We established that this pattern of degradation reflects the strong preference of staphylococcal nuclease for certain nucleotide arrangements on the DNA, rather than a particular chromatin configuration. These observations clearly show that great caution needs to be exerted whenever data from staphylococcal nuclease digestions are interpreted in terms of chromatin structure.

Base Sequence↗
PubMed1980

Processing of ribosomal precursor RNAs in Physarum polycephalum.

The processing of intermediates of Physarum rRNA has been investigated using two cloned rDNA restriction fragments as sequence specific probes. The size of the largest stable pre-rRNA detected is 11.8 Kb, as determined by denaturing gel electrophoresis. R-loop analysis revealed that this pre-rRNA fraction mainly consists of transcripts which do not contain the two insertion sequences present in the 26 S gene. However, a small number of molecules are found which still contain one or the other, or both, of these introns. These observations suggest that the two introns are transcribed but spliced out in a random order before RNA synthesis has reached the 3-end of the primary transcript. Transcription starts at about 17.7 Kb and stops at 4.4 Kb as measured from the ends of the linear palindromic rDNA molecule. Besides the 11.8 Kb transcript several further ribosomal RNA precursors have been identified, ranging in size from 2.5 to 8.8 Kb. In addition, the genes for 5.8 S RNA have been located by R-loop mapping.

Cloning, Molecular↗
PubMed1979

Accessibility of the ribosomal genes to micrococcal nuclease in Physarum polycephalum.

In Physarum polycephalum most genes coding for ribosomal RNA are not integrated in chromosomes, but are located in many copies in the nucleolus as plasmid-like palindromic DNA molecules. To find out whether coding sequences of rDNA are organized in a chromatin-like structure similar to that of bulk chromatin, nuclei were treated with micrococcal nuclease and DNA fragments were isolated. From bulk chromatin multimers of a basic unit of 170-180 base pairs were obtained. Nuclease fragmented DNA hybridized with labelled 19-S + 26-S rRNA was found to give the same saturation value as did unfragmented control DNA. No preferential degradation of ribosomal genes to acid soluble products was observed. A more detailed analysis of the nuclease degradation products was carried out with fragments separated by preparative gel electrophoresis. DNA eluted from the gels was hybridized in solution with labelled 19-S + 26-S rRNA. The coding sequences of rRNA were found to be degraded to approximately nucleosome size slightly more quickly than was the DNA of bulk chromatin. However, the distribution of the rDNA fragments on the gels did not coincide with the distribution of the fragments derived from bulk chromatin nucleosomes and their oligomers. The amount of rDNA in the interband regions was about intermediate between that found in the two adjacent bands. These results lead to the conclusion that the ribosomal genes, most of which are presumably active during rapid growth, are protected by proteins, probably histones. However, the ribosomal genes are present in a structure differing in some way from that of bulk chromatin.

DNA↗
PubMed1979

Isolation of a minichromosome containing the ribosomal genes from Physarum polycephalum.

The ribosomal genes of Physarum polycephalum are located on linear DNA molecules (rDNA) of 38 x 10(6) daltons present in each nucleolus in 100--200 copies. The rDNA is not part of the large chromosomal DNA. These molecules have now been solubilized in the form of transcriptionally active deoxyribonucleoprotein complexes from nucleoli purified through Percoll gradients. The ribosomal chromatin (rDNP) sediments in sucrose gradients as a homogeneous fraction with an approximate sedimentation coefficient of 100 S. It contains alpha-amanitin-resistant RNA polymerase I activity and rDNA as its major (or sole) DNA component. Buoyant density analysis indicates that ribosomal chromatin contains, besides RNA polymerase I, significant amounts of additional protein. RNA synthesized in vitro by the endogenous RNA polymerase is complementary to rDNA and is transcribed mainly from the sequences coding for 19S and 26S ribosomal RNA.

Amanitins↗
PubMed1979

Polyamine-mediated phosphorylation of a nucleolar protein from Physarum polycephalum that stimulates rRNA synthesis.

An acidic nucleolar phosphoprotein with a subunit M(r) of 70,000 was purified as an apparent dimer of 139,000 from isolated nuclei of the slime mold Physarum polycephalum. The protein was purified without the aid of strong dissociating agents after its selective phosphorylation in isolated nuclei by a polyamine-mediated reaction. Its amino acid composition resembled that of a nucleolar phosphoprotein from Novikoff hepatoma ascites cells. The phosphoprotein stimulated rRNA synthesis 5-fold by RNA polymerase I within a nucleolar, ribosomal deoxyribonucleoprotein complex isolated from nucleoli of P. polycephalum. It was also identified as a component of the complex. It bound with high affinity and specificity to the palindromic ribosomal DNA of 38 x 10(6)M(r) from P. polycephalum, which contained two coding sequences for 5.8S, 19S, and 26S rRNA. It also bound to three fragments of ribosomal DNA of M(r) 21.2 x 10(6), 17.1 x 10(6), and 8.1 x 10(6), prepared by cleavage with restriction endonucleases HindIII, PstI, and BamHI, respectively. All of these fragments included the symmetry axis of the palindromic ribosomal DNA. The phosphoprotein that had been treated with alkaline phosphataseagarose to hydrolyze the phosphate groups did not stimulate transcription and did not bind to ribosomal DNA or to the restriction fragments indicated. We have thus isolated a specific phosphoprotein with the capacity to stimulate transcription of a specific set of genes in a eukaryote. These findings suggest that this phosphoprotein may specifically regulate functions of ribosomal DNA in a manner dependent on its degree of phosphorylation.

Amino Acids↗
PubMed1978

Degradation of the ribosomal genes by DNAse I in Physarum polycephalum.

Treatment of nuclei from Physarum polycephalum with DNAse I leads to DNA fragments with a regular pattern of multiples of 10 nucleotides, when analyzed on gels under denaturing conditions as has been shown for other eukaryotes. Reports from Weintraub and Axel lead to the conclusion, that active genes are preferentially digested by DNAse I. When Physarum chromatin is degraded by DNAse I, the ribosomal genes are no longer available for hybridization with 19-S and 26-S rRNA and are thus preferentially destroyed. Degradation of chromatin from nuclei in mitosis, where no rRNA is synthesized and from nuclei in late G2 phase, where rRNA synthesis is maximal, leads to the same proportion of the ribosomal sequences being lost for hybridization. Therefore the preferential degradation of the ribosomal genes in Physarum by DNAse I probably does not reflect the actual momentary activity of these genes. This suggests that DNAse I treatment may distinguish between active chromatin and very strongly repressed chromatin.

Cell Nucleus↗
PubMed1977

Synthesis of Epstein-Barr virus DNA in vitro: effects of phosphonoacetic acid, N-ethylmaleimide, and ATP.

Nuclei from superinfected Raji cells synthesized Epstein-Barr Virus (EBV) DNA in vitro in the absence of cell DNA synthesis. The synthesis of EBV DNA in vitro was inhibited by phosphonoacetic acid and N-ethylmaleimide, and maximum synthesis was achieved in the absence of an ATP-regenerating system. Nuclei from mock-infected cells required an ATP-regenerating system for maximum DNA synthesis.

Acetates↗
PubMed1974

Polyoma viral DNA replicated as a nucleoprotein complex in close association with the host cell chromatin.

Polyoma viral DNA is shown to be replicated in close association with the mouse cell chromatin. Two virus-specific nucleoprotein complexes, designated complex A and B, can be dissociated from the isolated chromatin by gentle homogenization in 0.5 M NaCl. Complex A contains only replicating polyoma (Py) DNA whereas complex B contains only mature Py DNA I. The results show, furthermore, that complex A, containing viral DNA in different stages of replication, and complex B are both nucleoproteins with the same buoyant density. The data presently available suggest that newly synthesized stretches of Py DNA are immediately complexed with mouse cell histones and that complex B becomes the "core" of progeny Py virions. These results suggested that Py-induced replication of the mouse cell chromatin may be necessary to provide replicating Py DNA with histones.

Animals↗
PubMedDate not supplied

The repetitive microtubule-associated proteins MARP-1 and MARP-2 of Trypanosoma brucei.

The microtubular membrane skeleton of Trypanosoma brucei contains two closely related, repetitive, high-molecular-weight microtubule-associated proteins, MARP-1 and MARP-2 (MARP for Microtubule-Associated Repetitive Proteins). Their structure is unusual in that they consist of tandemly arranged, strongly conserved 38-amino-acid repeat units over almost their entire length of about 320 kDa. Their nonrepetitive N and C ends are comparatively short. The predicted amino acid sequences reveal a gradient of similarity between MARP-1 and MARP-2 which increases from the N-terminus (no significant similarity) through the repeat domain (50% similarity) to the C-terminus (94.5% similarity). Transfection of mammalian cell lines with recombinant fragments of MARP-2 demonstrate that the nonrepetitive C-terminus of MARP-2 binds specifically to microtubules. This C-terminus does not show sequence similarity with any other microtubule-associated proteins and thus appears to represent a novel type of microtubule-binding domain.

Amino Acid Sequence↗