 RNDr. Karel Koberna, CSc. E-mail: koberna@biomed.cas.cz tel: +420241062697 Ph.D. Students:Mgr. Anna Ligasová Technical Assistant:Lucie Anderlová Laboratory of Cell Biology is dealing with two basic subjects: Organization of ribosomal genes and replication of chromatin. Organization of ribosomal genesApproximately 400 copies of ribosomal genes are present in the human diploid genome. They are organized in the form of head-to-tail tandem repeats. One such repeat consists of the external non-transcribed spacer and the transcribed sequence of about 13 kbp giving rise to 18S, 5.8S and 28S rRNA. The tandem repeats are found at well-described positions within short arms of five pairs of acrocentric chromosomes: 13, 14, 15, 21 and 22. Only certain portion of ribosomal genes is transcriptionally active. These active genes are transcribed in the specific nuclear compartment named nucleolus. This structure accommodate also processes of rRNA maturation and assembly of pre-ribosomal particles. Several problems are addressed with respect to ribosomal genes.
Our electron microscopy data specified dense fibrilar components (DFC) as nucleolar subcompartments in which rRNA synthesis takes place (Fig. 1). Stereological analysis of clustered transcription signal further suggested that these Christmas trees may be contorted in the space and exhibit a DNA compaction ratio on the order of 4-5,5. Next, we have shown that replicating rDNA foci are "switched off" for transcription, but can be "switched on" for it following the replication. Replication of chromatinReplication of the two template strands at DNA replication forks is a highly coordinated process that ensures accurate and efficient duplication of eukaryotic genome. It comprises functioning of many factors. DNA replication begins at multiple sites, termed origins of replication. After recognition of these origins, two replication forks moving in opposite directions until adjacent replication units (replicons) meet are established. In mammalian cells, replicons are heterogeneous in size with the most frequent dimension in the range of 75-150 kbp. The timing of replication of individual replicons is apparently regulated process. In this respect, active genes are replicated early in S phase, whereas inactive genes and repetitive sequences replicate later. Furthermore, it seems that replicons are frequently arranged in groups and all of the replicons in each group are firing at the similar time in S phase. Five basic tasks are followed.
Our results showed that nuclear chromatin of HeLa cells is nuclei is replicated via domains with maximum size around 210 nm. According to these results, described domains (Fig. 2) probably represent basic replication arrangement of clustered replicons replicated at the same time and therefore, we called them replication units (RU). We suggest that chromatin is organized into boxes of several synchronously replicated replicons and these boxes exhibit similar size. This finding is consistent with two basic models of interphase chromatin arrangements: model of chromonema fiber and model of condensed radial loops. Fig. 1: Electron microscopy mapping of transcription signal in a thin sectioned HeLa cell.
Fig. 2: Mapping of replication sites in HeLa cells.
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