Characterisation of pericentrometric and sticky intercalary heterochromatin in Ornithogalum longibracteatum (Hyacinthaceae).

The hexaploid liliaceous plant Ornithogalum longibracteatum (2n=6x=54) has a heterochromatin-rich bimodal karyotype with large (L) and small (S) chromosomes. The composition and subgenomic distribution of heterochromatin was studied using molecular and cytological methods. The major component of centromeric heterochromatin in all chromosomes is Satl, an abundant satellite DNA with a basic repeat unit of 155 bp and an average A+T content (54%). The major component of the large blocks of intercalary heterochromatin in L chromosomes is Sat2, an abundant satellite DNA with a basic repeat unit of 115 bp and a high A+T content (76%). Additionally, traces of Sat2 can be detected at the centromeric regions of S chromosomes, while minor amounts of Satl are discernible in intercalary heterochromatin of L chromosomes. The chromosomal localisation pattern of Sat2 is consistent with the fluorescent staining pattern obtained with the A+T-specific DNA ligand 4'-6-diamidino-2-phenylindole (DAPI). A+T-rich intercalary heterochromatin is sticky and tends to associate ectopically during mitosis. Sister chromatid exchange clustering was found at the junctions between euchromatin and heterochromatin and at the centromeres. The pattern of mitosis-specific phosphorylation of histone H3 was not uniform along the length of the chromosomes. In all L and S chromosomes, from early prophase to ana-/telophase, there is hyperphosphorylation of histone H3 in the pericentromeric chromatin and a slightly elevated phosphorylated histone H3 level at the intercalary heterochromatin of L chromosomes. Consequently, the overall phosphorylated histone H3 metaphase labelling resembles the distribution of Satl in the karyotype of O. longibracteatum.

The human but not the Xenopus RNA-editing enzyme ADAR1 has an atypical nuclear localization signal and displays the characteristics of a shuttling protein.

The RNA-editing enzyme ADAR1 (adenosine deaminase that acts on RNA) is a bona fide nuclear enzyme that has been cloned from several vertebrate species. Putative nuclear localization signals (NLSs) have been identified in the aminoterminal regions of both human and Xenopus ADAR1. Here we show that neither of these predicted NLSs is biologically active. Instead, we could identify a short basic region located upstream of the RNA-binding domains of Xenopus ADAR1 to be necessary and sufficient for nuclear import. In contrast, the homologous region in human ADAR1 does not display NLS activity. Instead, we could map an NLS in human ADAR1 that overlaps with its third double-stranded RNA-binding domain. Interestingly, the NLS activity displayed by this double-stranded RNA-binding domain does not depend on RNA binding, therefore showing a dual function for this domain. Furthermore, nuclear accumulation of human (hs) ADAR1 is transcription dependent and can be stimulated by LMB, an inhibitor of Crm1-dependent nuclear export, indicating that hsADAR1 can move between the nucleus and cytoplasm. Regulated nuclear import and export of hsADAR1 can provide an excellent mechanism to control nuclear concentration of this editing enzyme thereby preventing hyperediting of structured nuclear RNAs.

A Caenorhabditis elegans cohesion protein with functions in meiotic chromosome pairing and disjunction.

We have studied four Caenorhabditis elegans homologs of the Rad21/Scc1/Rec8 sister-chromatid cohesion protein family. Based on the RNAi phenotype and protein localization, it is concluded that one of them, W02A2.6p, is the likely worm ortholog of yeast Rec8p. The depletion of C. elegans W02A2.6p (called REC-8) by RNAi, induced univalent formation and splitting of chromosomes into sister chromatids at diakinesis. Chromosome synapsis at pachytene was defective, but primary homology recognition seemed unaffected, as a closer-than-random association of homologous fluorescence in situ hybridization (FISH) signals at leptotene/zygotene was observed. Depletion of REC-8 also induced chromosome fragmentation at diakinesis. We interpret these fragments as products of unrepaired meiotic double-stranded DNA breaks (DSBs), because fragmentation was suppressed in a spo-11 background. Thus, REC-8 seems to be required for successful repair of DSBs. The occurrence of DSBs in REC-8-depleted meiocytes suggests that DSB formation does not depend on homologous synapsis. Anti-REC-8 immunostaining decorated synaptonemal complexes (SCs) at pachytene and chromosomal axes in bivalents and univalents at diakinesis. Between metaphase I and metaphase II, REC-8 is partially lost from the chromosomes. The partial loss of REC-8 from chromosomes between metaphase I and metaphase II suggests that worm REC-8 might function similarly to yeast Rec8p. The loss of yeast Rec8p from chromosome arms at meiosis I and centromeres at meiosis II coordinates the disjunction of homologs and sister chromatids at the two meiotic divisions.

Brix from xenopus laevis and brx1p from yeast define a new family of proteins involved in the biogenesis of large ribosomal subunits.

A clone was isolated from a cDNA library from early embryos of Xenopus laevis that codes for a highly charged protein containing 339 amino acids. Two putative nuclear localization signals could be identified in its sequence, but no other known motifs or domains. Closely related ORFs are present in the genomes of man, C. elegans, yeast and Arabidopsis. A fusion protein with GFP expressed in HeLa cells or Xenopus oocytes was found to be localized in the nucleolus and coiled (Cajal) bodies. Moreover, immunoprecipitation experiments demonstrated that the new Xenopus protein interacts with 5S, 5.8S and 28S RNAs of large ribosomal subunits. The name Brix (biogenesis of ribosomes in Xenopus) is proposed for this protein and the corresponding gene. In Saccharomyces cerevisiae, the essential gene YOL077c, now named BRX1, codes for the Brix homolog, which is also localized in the nucleolus. Depletion of Brx1 p in a conditional yeast mutant leads to defects in rRNA processing, and a block in the assembly of large ribosomal subunits.

Chromatin-association of the Polycomb group protein BMI1 is cell cycle-regulated and correlates with its phosphorylation status.

The human proto-oncogene Bmi1 is a member of the mammalian Polycomb Group (Pc-G) genes. The subnuclear distribution of the BMI1 protein was studied in several primary human and tumor-derived cell lines using immunohistochemical and biochemical methods. In primary and tumor cells, nuclear BMI1 shows a fine-grain distribution over chromatin, usually dense in interphase nuclei and significantly weaker along mitotic chromosomes. In addition, BMI1 preferentially associates with several distinct heterochromatic domains in tumor cell lines. In both primary and tumor cell lines a marked cell cycle-regulation of Pc-G-chromatin interaction is observed: nuclear BMI1-staining dissipates in late S phase and is re-established early in G(1)-phase. Chromatin-association of BMI1 inversely correlates with its phosphorylation status in a cell cycle-dependent fashion: at G(1)/S, hypophosphorylated BMI1 is specifically retained in the chromatin-associated nuclear protein fraction, whereas during G(2)/M, phosphorylated BMI1 is not chromatin-bound. Our findings indicate a strict cell cycle-controlled regulation of Pc-G complex-chromatin association and provide molecular tools for improving our understanding of Pc-G complex regulation and function in mammalian cells.

The RNA-editing enzyme ADAR1 is localized to the nascent ribonucleoprotein matrix on Xenopus lampbrush chromosomes but specifically associates with an atypical loop.

Double-stranded RNA adenosine deaminase (ADAR1, dsRAD, DRADA) converts adenosines to inosines in double-stranded RNAs. Few candidate substrates for ADAR1 editing are known at this point and it is not known how substrate recognition is achieved. In some cases editing sites are defined by basepaired regions formed between intronic and exonic sequences, suggesting that the enzyme might function cotranscriptionally. We have isolated two variants of Xenopus laevis ADAR1 for which no editing substrates are currently known. We demonstrate that both variants of the enzyme are associated with transcriptionally active chromosome loops suggesting that the enzyme acts cotranscriptionally. The widespread distribution of the protein along the entire chromosome indicates that ADAR1 associates with the RNP matrix in a substrate-independent manner. Inhibition of splicing, another cotranscriptional process, does not affect the chromosomal localization of ADAR1. Furthermore, we can show that the enzyme is dramatically enriched on a special RNA-containing loop that seems transcriptionally silent. Detailed analysis of this loop suggests that it might represent a site of ADAR1 storage or a site where active RNA editing is taking place. Finally, mutational analysis of ADAR1 demonstrates that a putative Z-DNA binding domain present in ADAR1 is not required for chromosomal targeting of the protein.

Meiotic pairing and segregation of translocation quadrivalents in yeast.

Meiotic pairing and segregation were studied in three different heterozygous reciprocal translocation strains of the baker's yeast, Saccharomyces cerevisiae. Pachytene translocation quadrivalents were identified by a combination of immunofluorescence and fluorescence in situ hybridization and the karyotypes of meiotic products were determined by pulsed-field gel electrophoresis. The translocations differed with respect to the relative sizes of the chromosomes involved and the positions of translocation breakpoints, and produced translocation quadrivalents of widely different shapes. This allowed us to study the influence of the morphology of quadrivalents on their segregation behaviour. In all cases alternate predominated over adjacent segregation. 3:1 disjunction of chromosomes was more frequent when translocation breakpoints were close to the centromeres. If a translocation breakpoint was distant from the centromere, the occurrence of an intervening chiasma influenced the pattern of segregation. In general, quadrivalent formation and segregation resembled the behaviour of translocation heterozygotes in most higher eukaryotes. We therefore conclude that, although chromosome condensation does not occur in yeast metaphase, centromere orientation and chromosome disjunction are governed in a way similar to that of higher eukaryotes.

The double-stranded RNA-binding domains of Xenopus laevis ADAR1 exhibit different RNA-binding behaviors.

We have cloned cDNAs encoding two versions of Xenopus double-stranded RNA adenosine deaminase (ADAR1). Like ADAR1 proteins from other species Xenopus ADAR1 contains three double-stranded RNA-binding domains (dsRBDs) which are most likely required for substrate binding and recognition of this RNA-editing enzyme. Analysis of mammalian ADAR1 identified the third dsRBD in this enzyme as most important for RNA binding. Here we analyzed the three dsRBDs of Xenopus ADAR1 for their in vitro RNA-binding behavior using two different assays. Northwestern assays identified the second dsRBD in the Xenopus protein as most important for RNA binding while in-solution assays demonstrated the importance of the third dsRBD for RNA binding. The differences between these two assays are discussed and we suggest that both the second and third dsRBD of Xenopus ADAR1 are important for RNA binding in vivo. We show further that all three dsRBDs can contribute to a cooperative binding effect.

The double-stranded RNA-binding protein X1rbpa promotes RNA strand annealing.

RNA-annealing activity is a common feature of several RNA-binding proteins. The Xenopus RNA-binding protein X1rbpa is composed of three tandemly arranged double-stranded RNA-binding domains (dsRBDs) but lacks any other catalytic or functional domains, therefore making the assessment of biological functions of this protein rather difficult. Here we show that full-length X1rbpa but also isolated dsRBDs from this protein can facilitate RNA strand annealing. RNA annealing can be efficiently inhibited by heparin. However, dsRBDs with a neutral pI still promote strand annealing, suggesting that charged residues within the dsRBD are important for strand annealing. Additionally, mutant versions of the dsRBD, unable to bind dsRNA in northwestern assays, were tested. Of these, some show RNA-annealing activity while others fail to do so, indicating that RNA annealing and dsRNA binding are separable functions. Our data, together with the previously reported association of the protein with most cellular RNAs, suggests an RNA chaperone-like function of X1rbpa.

PARP is important for genomic stability but dispensable in apoptosis.

Mice lacking the gene encoding poly(ADP-ribosyl) transferase (PARP or ADPRT) display no phenotypic abnormalities, although aged mice are susceptible to epidermal hyperplasia and obesity in a mixed genetic background. Whereas embryonic fibroblasts lacking PARP exhibit normal DNA excision repair, they grow more slowly in vitro. Here we investigated the putative roles of PARP in cell proliferation, cell death, radiosensitivity, and DNA recombination, as well as chromosomal stability. We show that the proliferation deficiency in vitro and in vivo is most likely caused by a hypersensitive response to environmental stress. Although PARP is specifically cleaved during apoptosis, cells lacking this molecule apoptosed normally in response to treatment with anti-Fas, tumor neurosis factor alpha, gamma-irradiation, and dexamethasone, indicating that PARP is dispensable in apoptosis and that PARP-/- thymocytes are not hypersensitive to ionizing radiation. Furthermore, the capacity of mutant cells to carry out immunoglobulin class switching and V(D)J recombination is normal. Finally, primary PARP mutant fibroblasts and splenocytes exhibited an elevated frequency of spontaneous sister chromatid exchanges and elevated micronuclei formation after treatment with genotoxic agents, establishing an important role for PARP in the maintenance of genomic integrity.

Xlrbpa, a double-stranded RNA-binding protein associated with ribosomes and heterogeneous nuclear RNPs.

We have cloned and characterized Xlrbpa, a double-stranded RNA-binding protein from Xenopus laevis. Xlrbpa is a protein of 33 kD and contains three tandemly arranged, double-stranded RNA-binding domains (dsRBDs) that bind exclusively to double-stranded RNA in vitro, but fail to bind either single-stranded RNA or DNA. Sequence data and the overall organization of the protein suggest that Xlrbpa is the Xenopus homologue of human TAR-RNA binding protein (TRBP), a protein isolated by its ability to bind to human immunodeficiency virus (HIV) TAR-RNA. In transfection assays, TRBP has also been shown to inhibit the interferon-induced protein kinase PKR possibly by direct physical interaction. To determine the function of Xlrbpa and its human homologue we studied the expression and intracellular distribution of the two proteins. Xlrbpa is ubiquitously expressed with marked quantitative differences amongst all tissues. Xlrbpa and human TRBP can be detected in the cytoplasm and nucleus by immunofluorescence staining and Western blotting. Sedimentation gradient analyses and immunoprecipitation experiments suggest an association of cytoplasmic Xlrbpa with ribosomes. In contrast, a control construct containing two dsRBDs fails to associate with ribosomes in microinjected Xenopus oocytes. Nuclear staining of Xenopus lampbrush chromosome preparations showed the association of the protein with nucleoli, again indicating an association of the protein with ribosomal RNAs. Additionally, Xlrbpa could be located on lampbrush chromosomes and in snurposomes. Immunoprecipitations of nuclear extracts demonstrated the presence of the protein in heterogeneous nuclear (hn) RNP particles, but not in small nuclear RNPs, explaining the chromosomal localization of the protein. It thus appears that Xlrbpa is a general double-stranded RNA-binding protein which is associated with the majority of cellular RNAs, ribosomal RNAs, and hnRNAs either alone or as part of an hnRNP complex.

Comparative mutational analysis of the double-stranded RNA binding domains of Xenopus laevis RNA-binding protein A.

Xenopus laevis RNA-binding protein A is a ubiquitously expressed, double-stranded RNA-binding protein that is associated with the majority of cellular RNAs, ribosomal RNAs, and hnRNAs. X. laevis RNA-binding protein A contains three copies of the double-stranded RNA-binding domain (dsRBD) in tandem arrangement. Two of them, xl1 and xl2, belong to the type A group of dsRBDs that show strong homologies to the entire length of a defined consensus sequence. The xl3 domain, in contrast, is a type B dsRBD which only matches the basic C-terminal end of the dsRBD consensus sequence. Here we show that only xl2 but neither xl1 nor xl3 are able to bind double-stranded RNA substrates in vitro, suggesting that different dsRBD copies have varying RNA binding activities. By fine mapping mutagenesis of the isolated xl2 domain, we identified at least two central aromatic amino acids and a C-terminal alpha-helix that are indispensable for dsRNA binding. Furthermore, we show that different charge distributions within the C-terminal alpha-helices of xl1 and xl2 seem responsible for the different RNA binding behaviors of these two dsRBDs. Analyses of the RNA binding properties of constructs containing various combinations of different dsRBDs reveal that type A dsRBDs exhibit a cooperative binding effect, whereas type B dsRBDs show a rather low binding activity, thus contributing only to a minor extent to a stable RNA-protein interaction.

Mechanisms of distamycin A/DAPI chromosome staining. I. Competition binding effects of nonintercalative DNA groove-binding agents in situ and in vitro.

The molecular mechanism underlying distamycin A-induced differential DAPI fluorescent staining of metaphase chromosomes was studied in Sus scrofa domestica both cytologically, using, besides DAPI, two isomeric derivatives of DAPI (D288.45 and D288.48), and molecularly, by in vitro competitive-binding studies using S. scrofa satellite DNA and synthetic DNA polymers. Significant differences in heterochromatin staining were observed between D288.45 and D288.48. Distinct distamycin A/DAPI bands were obtained with DAPI and D288.45 but not with D288.48. Circular dichroism measurements were performed to characterize the displacement of DAPI from its DNA binding sites by distamycin A and also netropsin. Distamycin A was most effective in displacing DAPI when DAPI was bound to contiguous clusters of AT base pairs and much less effective in displacing DAPI bound to GC or mixed AT/GC base-pair sequences. The results of these competitive-binding studies provide the basis of a molecular explanation of the quenching phenomenon of distamycin A counterstaining on chromosomal DAPI fluorescence.

Assembly and localization of the U1-specific snRNP C protein in the amphibian oocyte.

To study the intranuclear localization of the U1-specific snRNP C protein and its assembly into U1 snRNPs, we injected transcripts encoding a myc-tagged C protein into amphibian oocytes. The distribution of protein translated from the injected RNA was essentially the same in continuous and pulse-label experiments. In both cases the C protein localized within the germinal vesicle in those structures known to contain U1 snRNPs, namely the lampbrush chromosome loops and hundreds of extrachromosomal granules called snurposomes. Oocytes were also injected with an antisense oligodeoxynucleotide that caused truncation of U1 snRNA at the 5' end. In these oocytes, myc-tagged C protein localized normally in the germinal vesicle and could be immunoprecipitated together with truncated U1 snRNA. These experiments suggest that the C protein can enter the germinal vesicle on its own and there associate with previously assembled U1 snRNPs. In transfected tissue culture cells, the myc-tagged C protein localized within the nucleus in a speckled pattern similar to that of endogenous U1 snRNPs.

A conserved double-stranded RNA-binding domain.

We have identified a double-stranded (ds)RNA-binding domain in each of two proteins: the product of the Drosophila gene staufen, which is required for the localization of maternal mRNAs, and a protein of unknown function, Xlrbpa, from Xenopus. The amino acid sequences of the binding domains are similar to each other and to additional domains in each protein. Database searches identified similar domains in several other proteins known or thought to bind dsRNA, including human dsRNA-activated inhibitor (DAI), human trans-activating region (TAR)-binding protein, and Escherichia coli RNase III. By analyzing in detail one domain in staufen and one in Xlrbpa, we delimited the minimal region that binds dsRNA. On the basis of the binding studies and computer analysis, we have derived a consensus sequence that defines a 65- to 68-amino acid dsRNA-binding domain.

Transcription on lampbrush chromosome loops in the absence of U2 snRNA.

The five small nuclear RNAs (snRNAs) involved in splicing occur on the loops of amphibian lampbrush chromosomes and in hundreds to thousands of extrachromosomal granules called B snurposomes. To assess the role of these snRNAs during transcription and to explore possible relationships between the loops and B snurposomes, we injected single-stranded antisense oligodeoxynucleotides (oligos) against U1 and U2 snRNA into toad and newt oocytes. As shown before, antisense U1 and U2 oligos caused truncation of U1 and complete destruction of U2 snRNAs, respectively. However, injection of any oligo, regardless of sequence, brought on dramatic cytological changes, including shortening of the chromosomes and retraction of the lateral loops, with concomitant shutdown of polymerase II transcription, as well as disappearance of some or all of the B snurposomes. When injected oocytes were incubated for 12 h or longer in physiological saline, these changes were reversible; that is, the chromosomes lengthened, transcription (detected by 3H-UTP incorporation) resumed on newly extended lateral loops, and B snurposomes reappeared. In situ hybridization showed that loops and B snurposomes had negligible amounts of U2 snRNA after recovery from injection of the anti-U2 oligo, whereas these structures had normal levels of U2 snRNA after recovery from a control oligo. Thus, the morphological integrity of B snurposomes and lampbrush chromosome loops is not dependent on the presence of U2 snRNA. Because transcription occurs in the absence of U2 snRNA, we conclude that splicing is not required for transcription on lampbrush chromosome loops.

Meiotic chromosome behaviour reflects levels of sequence divergence in Sus scrofa domestica satellite DNA.

We present a general model for the evolution of chromosome-specific satellite DNA subfamilies. Sus scrofa domestica has a bimodal karyotype with two autosomal subsets of 12 meta-/submetacentric (Mc) and 6 acrocentric (Ac) chromosome types (Mc and Ac "subgenomes"). We show that the centromeric heterochromatin is characterised by two distinct satellite DNA families designed Mc1 and Ac2. Mc1 is a diverse satellite family of the Mc subgenome of which certain members with a 100 bp repeat unit are found to occur at the pericentromeric regions of each Mc autosome, while others are chromosome-specific, e.g. clone Mc pAv1.5, a higher order repeat variant, which hybridises specifically to chromosome 1. Ac2 is a homogeneous satellite occurring at the subterminal pericentromeric regions of all Ac autosomes. DNA sequence analyses showed that all clones investigated are built up from a 14 bp repeat unit which is highly conserved. In situ hybridisation to meiotic pachytene nuclei revealed a distinct spatial arrangement of the Ac2 centromeric satellite.

[The history of roentgen diagnosis in urology]. / Zur Geschichte der Röntgendiagnostik in der Urologie.

W. C. Röntgen first published the discovery of x-rays on December 28th, 1895. The extraordinary significance for diagnostics was immediately recognized all over the world. Already as early as January 16th, 1896, pictures produced in Vienna were reported in the "Wiener klinische Wochenschrift". The urological x-ray diagnostics attempted at first the visualization of kidney- and bladderstones. First successful results were demonstrated already in the summer of 1896 by D'Arsonval in Paris, by McIntyre in Glasgow and by A. v. Frisch in Vienna in 1897. The endeavors to demonstrate the collecting system by filling with a contrast medium began in 1905, the intravenous pyelography in 1923. However, it took a long time for finding contrast media of acceptable toxic levels. The arteriography as envisaged in the beginning could be realized in 1929 but first gained its practical application during the middle of this century. Air as contrast medium--as for instance in the pneumoperitoneum--as well as lymphography lost their usefulness with the emergence of the computertomography since 1973.