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Biomedical subjects

A Kuhn

Publications and source records attributed to A Kuhn.

At least 19 recordsLinked to original sources
PubMed1992

Dual role of the nucleolar transcription factor UBF: trans-activator and antirepressor.

In a reconstituted system consisting of partially purified RNA polymerase I (pol I) and the initiation factors TIF-IA, TIF-IB, and TIF-IC, the nucleolar factor UBF (upstream binding factor) stimulates transcription from the rRNA-encoding DNA (rDNA) promoter at least 50-fold. This activation is not observed at high template concentrations or in the presence of highly purified pol I. Template commitment experiments suggest that UBF activates transcription by relieving inhibition exerted by a negative-acting factor(s) in the polymerase fraction that competes for TIF-IB binding to the rDNA promoter and prevents the formation of preinitiation complexes. Using purified histone H1 bound to DNA as a model for the repressed state of the rDNA promoter, we show that UBF counteracts H1-mediated repression of pol I transcription. The implications of these findings are discussed with respect to the protein-protein and protein-DNA interactions at the rDNA promoter and the possible involvement of UBF in control of ribosomal gene transcription.

Animals↗
PubMed1992

Distinct domains of an oligotopic membrane protein are Sec-dependent and Sec-independent for membrane insertion.

Leader peptidase of Escherichia coli spans the plasma membrane twice with its amino terminus on the periplasmic surface of the membrane and its large carboxyl-terminal domain protruding into the periplasm. To monitor the transfer of the amino terminus of leader peptidase to the periplasm, we have constructed a fusion protein between the 18-residue amino-terminal periplasmic domain of Pf3 bacteriophage coat protein and the beginning of leader peptidase. We find that neither the SecA or SecY proteins nor a transmembrane electrochemical potential is required for insertion of the amino terminus, while the transfer of the carboxyl-terminal domain of leader peptidase has these requirements. The first 35 residues of leader peptidase, which include the first hydrophobic domain and the carboxyl-terminal positively charged cluster, are sufficient to insert the amino terminus. When positively charged residues are introduced before the first transmembrane segment, translocation of the amino terminus is abolished. These studies in protein membrane topogenesis, showing that there are different requirements for amino and carboxyl termini insertion, indicate that multiple mechanisms exist even within the same protein.

Adenosine Triphosphatases↗
PubMed1992

The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation.

UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C-terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.

Amino Acid Sequence↗
PubMed1992

Bacteriophage T4 gene 21 encodes two proteins essential for phage maturation.

The T4 prohead protease (T4 PPase) is the key enzyme in the morphopoietic pathway of the T4 phage head. It is responsible for the proteolytic processing of all head proteins allowing protein rearrangement and head expansion. To study its biochemistry and gene regulation, T4 gene 21 was cloned into an expression vector under the control of the inducible tac promoter. Two proteins of apparent molecular weights of 21.5 and 27.5 kDa were detected after induction. These proteins are synthesized using two different start codons in the same reading frame. Destruction of either start codon resulted in the loss of the respective protein. Complementation experiments with bacteriophage T4 21(-)-infected cells showed that both proteins are functional in vivo and essential for T4 phage assembly.

Base Sequence↗
PubMed1992

[Chronic lichenoid keratosis].

We report on a 41-year-old woman with keratosis lichenoides chronica, a disorder first described by Kaposi in 1886 as "lichen moniliformis", who later also developed chronic lymphatic leukaemia. Since Kaposi's original report, 38 additional cases have been reported. Occurrence of keratosis lichenoides chronica associated with malignant disorders has not previously been described.

Adult↗
PubMed1991

Use of site-directed mutagenesis to define the limits of sequence variation tolerated for processing of the M13 procoat protein by the Escherichia coli leader peptidase.

Leader peptidase cleaves the leader sequence from the amino terminus of newly made membrane and secreted proteins after they have translocated across the membrane. Analysis of a large number of leader sequences has shown that there is a characteristic pattern of small apolar residues at -1 and -3 (with respect to the cleavage site) and a helix-breaking residue adjacent to the central apolar core in the region -4 to -6. The conserved sequence pattern of small amino acids at -1 and -3 around the cleavage site most likely represents the substrate specificity of leader peptidase. We have tested this by generating 60 different mutations in the +1 to -6 domain of the M13 procoat protein. These mutants were analyzed for in vivo and in vitro processing, as well as for protein insertion into the cytoplasmic membrane. We find that in vivo leader peptidase was able to process procoat with an alanine, a serine, a glycine, or a proline residue at -1 and with a serine, a glycine, a threonine, a valine, or a leucine residue at -3. All other alterations at these sites were not processed, in accordance with predictions based on the conserved features of leader peptides. Except for proline and threonine at +1, all other residues at this position were processed by leader peptidase. None of the mutations at -2, -4, or -5 of procoat (apart from proline at -4) completely abolished leader peptidase cleavage in vivo although there were large effects on the kinetics of processing.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Palmoplantar keratoderma with tonotubular keratin.

A 61-year-old man with palmoplantar keratoderma with an unusual tonotubular keratin is reported. The histologic findings, genetic transmission, and clinical course were similar to epidermolytic palmoplantar keratoderma (Voerner type), but keratinocytes ultrastructurally displayed a tonotubular cytoskeleton, which has not been previously described, instead of a tonofilamentous one. Electrophoretically, we found no difference in the keratin pattern of normal plantar skin, skin in palmoplantar keratoderma of Voerner, and that of our patient's skin. Therefore the tubular keratin most likely formed as a result of a posttranslational change of keratin polymerization.

Humans↗
PubMed1990

The function of a leader peptide in translocating charged amino acyl residues across a membrane.

Insertion of bacteriophage coat proteins into the membrane of infected bacterial cells can be studied as a model system of protein translocation across membranes. The coat protein of the filamentous bacteriophage Pf3--which infects Pseudomonas aeruginosa--is 44 amino acids in length and has the same basic structure as the coat protein of bacteriophage M13, which infects Escherichia coli. However, unlike the Pf3 coat protein, the M13 coat protein is synthesized as a precursor (procoat) with a typical leader (signal) sequence, which is cleaved after membrane insertion. Nevertheless, when the gene encoding the Pf3 coat protein is expressed in E. coli, the protein is translocated across the membrane. Hybrid M13 and Pf3 coat proteins were constructed in an attempt to understand how the Pf3 coat protein is translocated without a leader sequence. These studies demonstrated that the extracellular regions of the proteins determined their cellular location. When three charged residues in this region were neutralized, the leader-free M13 coat protein was also inserted into the membrane. Differences in the water shell surrounding these residues may account for efficient membrane insertion of the protein without a leader sequence.

Amino Acid Sequence↗
PubMed1990

Specific interaction of the murine transcription termination factor TTF I with class-I RNA polymerases.

The 18-base-pair sequence element AGGTCGACCAGTACTCCG (the Sal box) signals termination of mouse ribosomal gene transcription. This sequence is recognized by a sequence-specific DNA-binding protein, TTF I, which mediates the termination of transcription by RNA polymerase I (pol I). Subsequently, the ends of the primary transcripts are trimmed by 10 nucleotides in a sequence-dependent 3'-terminal processing reaction. We have now investigated whether TTF I bound to its target sequence will block elongation by any RNA polymerase by steric hindrance, or whether it is specific for elongation by pol I. The results demonstrate that TTF I directs transcription termination with RNA polymerase I from species as divergent as mouse and yeast, but fails to affect elongation by heterologous polymerases (eukaryotic RNA polymerases II and III, Escherichia coli or bacteriophage T3 RNA polymerase). By contrast, purified lac repressor bound to its operator sequence stops elongation by both RNA polymerase I and II.

Animals↗
PubMed1990

Efficient translocation of positively charged residues of M13 procoat protein across the membrane excludes electrophoresis as the primary force for membrane insertion.

The coat protein of bacteriophage M13 is inserted into the Escherichia coli plasma membrane as a precursor protein, termed procoat, with a typical leader peptide of 23 amino acid residues. Its membrane insertion requires the electrochemical potential but not the cellular components SecA and SecY. Since the electrochemical gradients result in the periplasmic side of the membrane being positively charged, the membrane potential could contribute to the transfer of the negatively charged central region of procoat across the membrane. Here we demonstrate that the central domain following the leader peptide can be translocated across the membrane even when the net charge of the region is changed from -3 to +3. This rules out an electrophoresis-like insertion mechanism for procoat. We also show that the sec independence of procoat insertion is linked to the presence of the second apolar domain. The deletion of most of the second apolar domain from a procoat fusion protein results in sec dependent membrane insertion of the hybrid protein. Moreover, like other proteins that require the sec genes, translocation of this sec dependent procoat protein is inhibited when positively charged residues are introduced after the leader peptide. Loop models involving one or two hydrophobic regions are presented that account for the differences in tolerance of positively charged residues.

Amino Acid Sequence↗
PubMed1990

Initial steps in protein membrane insertion. Bacteriophage M13 procoat protein binds to the membrane surface by electrostatic interaction.

Bacteriophage M13 procoat protein is synthesized on free polysomes prior to its assembly into the inner membrane of Escherichia coli. As an initial step of the membrane insertion pathway, the precursor protein interacts with the cytoplasmic face of the inner membrane. We have used oligonucleotide-directed mutagenesis to study the regions of the procoat protein involved in membrane binding. We find that there is an absolute requirement for positively charged amino acids at both ends of the protein. Replacing these with negatively charged residues resulted in an accumulation of the precursor in the cytoplasm. We propose that the positively charged amino acids are directly involved in membrane binding, possibly directly to the negatively charged phospholipid head groups. This was tested in vitro with artificial liposomes. Whereas wild-type procoat interacted with these liposomes, we found that procoat mutants with negatively charged amino acids at both ends did not bind. Therefore, we conclude that newly synthesized M13 procoat protein binds electrostatically to the negatively charged inner membrane of E. coli.

Amino Acid Sequence↗
PubMed1990

A 140-base-pair repetitive sequence element in the mouse rRNA gene spacer enhances transcription by RNA polymerase I in a cell-free system.

We show that the repetitive 140-base-pair (bp) elements present in the spacer of mouse rRNA genes function as enhancers for RNA polymerase I. Attachment of these elements to the rDNA promoter stimulates rRNA synthesis both in vivo and in vitro. The cis-activating effect of the spacer repeats is orientation-independent and increases with increasing numbers of the 140-bp elements. Competition experiments demonstrate that the spacer repeats bind one or more of the transcription factors interaction with the rDNA promoter. Both the 140-bp elements and the core promoter act cooperatively and thus are functionally linked. The 60/81-bp enhancer repeats from Xenopus laevis rDNA compete for a murine transcription factor(s) and stimulate transcription often fused to the mouse rDNA promoter. The results indicate that despite the marked species specificity of rDNA transcription initiation, common factors may interact with both the rDNA promoter and the enhancer.

Animals↗
PubMed1989

[Lymphomatoid papulosis resembling ecthyma. A case report].

A 54-year-old female patient recurrently developed disseminated papules, red-brown in color and partly ulcerous, which spontaneously disappeared after 3-6 weeks. Histological examination revealed dense infiltration of atypical lymphocytes with epidermotropism. Immunohistological analysis showed predominantly helper T-cells (CD 4). Accordingly, we established the diagnosis of lymphomatoid papulosis. There was no evidence of malignant lymphoma.

Biopsy↗
PubMed1989

Molecular basis for the regulation of cell fate by the lethal (2) giant larvae tumour suppressor gene of Drosophila melanogaster.

Tumour suppressor genes act as recessive determinants of cancer. Their function is required for normal cell growth and differentiation during development. When both alleles of these developmental genes are inactivated, cell growth becomes unrestricted. In Drosophila, a series of genes have been identified which when mutated produce tissue-specific tumours. Of these the lethal(2)giant larvae (l(2)gl) gene is the best studied. Homozygous l(2)gl mutations cause the development of malignant tumours in the brain and the imaginal discs. Genomic DNA from the l(2)gl locus has been cloned, introduced back into l(2)gl mutant animals by P-element-mediated transformation and shown to restore normal development. The nucleotide sequence of the l(2)gl gene (13.1 kb) has been determined, as well as the sequences of the two classes of transcripts. These transcripts encode two polypeptides of 127 kDa and 78 kDa, respectively. Both proteins have been immunologically identified. Analyses of the spatial distribution of both l(2)gl transcripts and proteins revealed that during early embryogenesis the l(2)gl gene is uniformly expressed in all cells and tissues. In late embryos, the l(2)gl expression becomes gradually restricted to tissues presenting no morphological or neoplastic alteration in the mutant animals. Further mosaic experiments pointed out that the critical period for the establishment of tumorigenesis is limited to early embryogenesis at a time when the l(2)gl expression is most intense in all cells.

Animals↗
PubMed1989

3'-end formation of mouse pre-rRNA involves both transcription termination and a specific processing reaction.

We have studied the sequence requirements for 3'-end formation of rDNA transcripts in a cell-free system and show that the generation of correct ends of mouse pre-rRNA is brought about by a two-step process that involves a bona fide termination reaction, followed by a specific trimming of the primary transcript by 10 nucleotides. We show that termination of mouse ribosomal gene transcription by RNA polymerase I (pol I) takes place in front of an 18-bp DNA sequence element (the 'Sal box'), which was previously shown to function as termination signal. Termination of pol I transcription occurs at a fixed distance (11 bp) upstream of the Sal box, independent of the sequence of adjacent gene regions. The processing reaction, however, is strongly influenced by sequences flanking the termination signal at the 5' site. Substitution of a cluster of T residues by guanines within the region of 3'-end formation abolishes the 3'-terminal trimming of the primary transcript. Interestingly, this 3'-terminal processing event, which can be uncoupled from the termination reaction, requires both a correct 3' end and specific sequences in the 3'-terminal region of the primary transcript. Read-through transcripts generated in the extract system or by SP6 RNA polymerase are no substrate for the processing nuclease(s). Because the termination and processing activity can be separated chromatographically, the nucleolytic activity does not reside in TTF-I, the factor that binds to the Sal box and directs transcription termination.

Animals↗
PubMed1989

Immunostaining of keratin and vimentin in epidermis: comparison of different post-embedding immunogold techniques for electron microscopy.

The present study compares different post-embedding staining methods, including conventional and low-temperature embedding techniques, for demonstration of the keratin and vimentin cytoskeleton of epidermal cells, applying commercially available polyclonal and monoclonal antibodies. Immunogold staining (5-nm particles) was performed on the following material: (a) osmium-fixed and Durcupan-embedded material, etched with various solutions; (b) aldehyde-fixed material embedded in Lowicryl K4M at 4 degrees C and -35 degrees C; (c) aldehyde-fixed material embedded in Lowicryl K11M at -60 degrees C with and without cryoprotection with glycerol. In conventionally embedded material we failed to demonstrate intermediate filaments, whereas they were stained after low-temperature embedding with Lowicryl, i.e., K4M 4 degrees C, K4M -35 degrees C, and K11M -60 degrees C. The keratin and vimentin cytoskeleton reacted exclusively with polyclonal antibodies. The best results for antigenicity as well as structural preservation were achieved by post-embedding staining of K4M -35 degrees C-embedded material. Antibodies to keratin stained the cytoskeleton in keratinocytes of all epidermal layers. Filaments were decorated in a linear alignment. Antibodies to vimentin stained the cytoskeleton of Langerhans cells and melanocytes. In these cells a linear distribution pattern of the reaction product along the filaments and an extrafilamentous cluster formation were observed, indicating staining of vimentin and a vimentin-associated protein.

Antibodies, Monoclonal↗