[Transcriptional regulation macronuclear protein-coding genes in stichotrichous ciliates].

The concept of transcriptional regulation and base promoter structure of genes in eukaryotic organisms rests on two approaches--the experimental (using different methods of directional mutagenesis) and the comparative molecular (comparison of nucleotide sequences in promoter regions of different genes). Investigation of ciliates has led researchers to the conclusion that the protein-coding genes of these organisms lack the classical eukaryotic regulatory elements in the promoter region. This conclusion is based mainly on the usage of the comparative approach, while experimental investigations of such genes are practically absent so far. In the present paper, the comparative molecular analysis of the promoter regions of genes and analysis of the functional role of tubulin, cathepsin and Hsp 70 5'-uncoding areas was performed using new experimental data obtained during investigation of Stylonychia lemnae alpha-tubulin gene. We suggest a new classification of mechanism of transcriptional regulation of protein-coding genes in stichotrichous ciliates.

[Stylonychia lemnae as a model organism for studies of macronuclear minichromosomes of the ciliates].

The unique structural and functional organization of macronuclear (somatic nucleus) genome of the spirotrichous ciliates, exemplified by Stylonychia lemnae, has been reviewed. Data on the architecture of S. lemnae nuclear apparatus at interphase and during vegetative cell division, conjugation or autogamy are summarized. Special attention being paid to the structural and functional peculiarities of short macronuclear minichromosomes known to contain protein-coding regions, 5'- and 3'-flanking nontranslated regions, and telomeres. A hypothesis, previously put forward, according to which in the spirotrichous ciliates the telomeres themselves may serve as starting points of replication in minichromosomes, has now received its further substantiation. The recent experimental data, which confirm that 5'-nontranscribed DNA leader sequence of alpha1- and alpha2-tubulin-encoding minichromosomes display at least several regulatory elements typical for eukaryote promoter (TATA-box, CAAT-box, transcriptional initiator), are discussed. Up to now, there is no confirmation with regard to a possible existence in the spirotrichous minichromosomes of specific regulatory sequences capable of controlling both replication and transcription processes.

Identification and dynamics of Cajal bodies in relation to karyosphere formation in scorpionfly oocytes.

In oocyte nuclei of the scorpionfly, Panorpa communis, we have recently defined a population of nuclear bodies (NBs) that contain some components of Cajal bodies (CBs). In the present study, we used several criteria [presence of coilin, U7 snRNA, RNA polymerase II (pol II) and specific ultrastructure] to identify these NBs as CBs. The essential evidence for CB identification came from experiments with microinjection of fluorescein-tagged U7 snRNA. Consistent with the U7 data, we found pol II and pre-mRNA splicing factor, SC35, in Panorpa oocyte CBs. We show here that the dynamics of CBs differs from that in somatic cells and correlates with the level of oocyte chromosome condensation. We also found that the significant increase of CB size is accompanied by condensation of the chromosomes in the karyosphere, which is indicative of a decline in transcription. Using immunogold microscopy we determined that pol II and coilin are shared by CBs and the granular material associated with condensed chromosomes in the Panorpa karyosphere. The colocalization of pol II, U7 snRNA and splicing factors with CBs at the inactive stage of late oogenesis suggests that the latter may serve as storage domains for components that were earlier engaged in RNA transcription and processing.

Initial steps of infection of the ciliate Paramecium with bacteria Holospora sp.

New light and electron microscope data on the initial steps of endocytobiosis establishment between the ciliate Paramecium and specific intranuclear bacteria Holospora are provided. At the cytoplasmic step of infection bacteria of all Holospora species are found in a vesicle originating from the membrane of the host cell phagosome. The association between host cell microfilaments and the bacterium bearing vesicle may suggest a possible involvement of the ciliate cytoskeleton in the transportation of bacteria to the host cell nucleus. The authors subdivide the process of infection into 6 steps. Some strains of P. caudatum never take up infectious Holospora bacteria in the course of phagocytosis.

Minichromosomal DNA replication in the macronucleus of the hypotrichous ciliate Stylonychia lemnae is independent of chromosome-internal sequences.

The origins of DNA replication in prokaryotes and eukaryotes are typically defined by cis-acting sequences. However, in ciliates, evidence suggests that the replication of short macronuclear minichromosomes may not require such determinants. In hypotrichous ciliates, macronuclei contain millions of gene-sized minichromosomes, which generally have a single protein-coding region, two short noncoding flanks and, on each end, a short telomere consisting of a double-stranded repeat region and a single-stranded 3' overhang. Electron microscopic studies that showed that replication of minichromosomes initiates at or near telomeres and the discovery of a primase activity synthesizing RNA primers over the whole 3' telomeric overhang in vitro suggested that minichromosome replication starts directly at telomeres. Conversely, many minichromosomes contain an AT-rich, semi-conserved, palindromic sequence motif in their subtelomeric regions and it has been proposed that this motif is involved in regulating minichromosomal replication. To analyze what sequences or structures of the minichromosomes are essential for DNA replication, we stably transfected genetically modified alpha1-tubulin-encoding minichromosomes into the hypotrichous ciliate Stylonychia lemnae. Cotransfection of mutated and control minichromosomes revealed that noncoding regions can be deleted or replaced with unrelated sequences without affecting minichromosome replication efficiency in vegetatively growing cells. Similarly, replacement of the coding region resulted in a minichromosome that was stably maintained in transfected cells at the same high copy number for many months. In contrast, alpha1-tubulin-encoding minichromosomes without telomeres were rapidly lost after transfection. Hence, DNA replication of the alpha1-tubulin-encoding minichromosome does not depend on chromosome-internal sequences but may depend on telomeres.

Bacterial protease ECP32 specifically hydrolyzing actin and its effect on cytoskeleton in vivo.

A procedure for isolation of bacterial protease ECP32 yielding 100 microg of the enzyme from 10 liters of the Escherichia coli strain A2 liquid culture has been developed. The procedure includes chromatography, ultrafiltration, and PAGE under non-denaturing conditions. The purified preparation contained about 80% ECP32 and did not exhibit ATPase activity. Polyclonal ECP32-specific antibodies have been produced, and a two-stage procedure for the isolation of protease ECP32 involving affinity chromatography has been elaborated. Microinjection of the purified ECP32 into Amoeba proteus cells caused reversible distortions in amoeba locomotion. The effect was not observed upon inhibition of the protease activity by the ECP32-specific antibodies. The results indicate that bacterial protease ECP32 may be used for the analysis of actin functions in vivo.

[The characteristics of the transport into the nucleus of conjugates with normal and mutant nucleophilic sequences]. / Kharakteristika transporta v iadro kon''iugatov s normal'noi i mutantnoi nukleofil'nymi posledovatel'nostiami.

A study was made of the transport of conjugates of a fluorescently labeled protein and a synthetic peptide, corresponding to the nuclear localization sequence of the SV 40 large T-antigen, into the nuclei of cultured human (HeLa and A431) and murine (HER14) cells. A possibility for such conjugates to be transported into the nuclei of digitonin-permeabilized cells, without addition of exogenous cytosol, was demonstrated. A quantitative comparison of the transport levels of constructions with normal or altered (K128T) peptide sequence was performed, and a low selectivity of nuclear transport with this alteration in the digitonin-permeabilized cell system was revealed, whereas constructions with the mutant sequence, when injected into live cells, remained in the cytoplasm. ATP-dependent transport of constructions with the mutant sequence into permeabilized cell nuclei was demonstrated, with a considerable part of this transport being NEM-insensitive. A suggestion is put forward that there are several variants of the nucleophilic sequence containing protein transport in the digitonin-permeabilized cell system.

[A device for immobilizing small biological objects under light optical study]. / Prisposoblenie dlia obezdvizheniia melkikh biologicheskikh ob''ektov pri ikh svetoopticheskom izuchenii.

A device constructed on the base of a slide and a coverslip is proposed for immobilisation of small biological objects. The device permits performance of gradual and reversible squeezing of live micro-objects. Using the above device it is possible to watch one and the same living object, (for example, a ciliate) repeatedly within a prolonged period of time.