[Functional Characterization of Septin Complexes].

Septins belong to a family of conserved GTP-binding proteins found in majority of eukaryotic species except for higher plants. Septins form nonpolar complexes that further polymerize into filaments and associate with cell membranes, thus comprising newly acknowledged cytoskeletal system. Septins participate in a variety of cell processes and contribute to various pathophysiological states, including tumorigenesis and neurodegeneration. Here, we review the structural and functional properties of septins and the regulation of their dynamics with special emphasis on the role of septin filaments as a cytoskeletal system and its interaction with actin and microtubule cytoskeletons. We also discuss how septins compartmentalize the cell by forming local protein-anchoring scaffolds and by providing barriers for the lateral diffusion of the membrane proteins.

The Role of Pnut and its Functional Domains in Drosophila Spermatogenesis.

The Drosophila Pnut protein belongs to the family of septins, which are conserved GTPases participating in cytokinesis and many more other fundamental cellular processes. Because of their filamentous appearance, membrane association, and functions, septins are considered as the fourth component of the cytoskeleton, along with actin, microtubules, and intermediate filaments. However, septins are much less studied than the other cytoskeleton elements. We had previously demonstrated that the deletion of the pnut gene leads to mitotic abnormalities in somatic cells. The goal of this work was to study the role of the pnut in Drosophila spermatogenesis. We designed a construct for pnut RNA interference allowing pnut expression to be suppressed ectopically. We analyzed the effect of pnut RNA interference on Drosophila spermatogenesis. Germline cells at the earliest stages of spermatogenesis were the most sensitive to Pnut depletion: the suppression of the pnut expression at these stages leads to male sterility as a result of immotile sperm. The testes of these sterile males did not show any significant meiotic defects; the axonemes and mitochondria were normal. We also analyzed the effect of mutations in the Pnut's conservative domains on Drosophila spermatogenesis. Mutations in the GTPase domain resulted in cyst elongation defects. Deletions of the C-terminal domain led to abnormal testis morphology. Both the GTPase domain and C-terminal domain mutant males were sterile and produced immotile sperm. To summarize, we showed that Pnut participates in spermiogenesis, that is, the late stages of spermatogenesis, when major morphological changes in spermatocytes occur.

[Analysis of Phenotypic Manifestation of peanut Gene Expression Suppression by RNAi in Drosophila Oogenesis].

The peanut gene functions in Drosophila melanogaster oogenesis were studied. It was demonstrated that the suppression of peanut expression by RNA interference in the ovary follicular cells results in the violation of oocyte polarization, anomalous cytokinesis in the chorion cells, and violation of the chromatin condensation in follicular cells. No oogenesis violations were observed in females with decreased peanut gene expression or an absence of the Pnut protein in the ovary generative cells. However, embryos produced by such females had a decreased survival rate caused by two death peaks.

Functional analysis of mutant and wild-type Drosophila origin recognition complex.

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants. Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA(+) nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.

Assembly of functionally active Drosophila origin recognition complex from recombinant proteins.

In eukaryotes the sites for the initiation of chromosomal DNA replication are believed to be determined in part by the binding of a heteromeric origin recognition complex (ORC) to DNA. We have cloned the genes encoding the subunits of the Drosophila ORC. Each of the genes is unique and can be mapped to discrete chromosomal locations implying that the pattern and developmental regulation of origin usage in Drosophila is not regulated solely by a large family of different ORC proteins. The six-subunit ORC can be reconstituted with recombinant proteins into a complex that restores DNA replication in ORC-depleted Drosophila or Xenopus egg extracts.

Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes.

The origin recognition complex (ORC) is required to initiate eukaryotic DNA replication and also engages in transcriptional silencing in S. cerevisiae. We observed a striking preferential but not exclusive association of Drosophila ORC2 with heterochromatin on interphase and mitotic chromosomes. HP1, a heterochromatin-localized protein required for position effect variegation (PEV), colocalized with DmORC2 at these sites. Consistent with this localization, intact DmORC and HP1 were found in physical complex. The association was shown biochemically to require the chromodomain and shadow domains of HP1. The amino terminus of DmORC1 contained a strong HP1-binding site, mirroring an interaction found independently in Xenopus by a yeast two-hybrid screen. Finally, heterozygous DmORC2 recessive lethal mutations resulted in a suppression of PEV. These results indicate that ORC may play a widespread role in packaging chromosomal domains through interactions with heterochromatin-organizing factors.

[A new class of small RNP (alpha-RNP) containing antisense RNA in K-562 cells. III. The DNA-binding activity of nuclear alpha-RNP. The Signal Transmission from Growth Factors Group of the Medical Faculty, F. Schiller University, Jena, Germany]. / Novyi klass malykh RNP (al'fa-RNP), soderzhashchikh antismyslovuiu RNK, v kletkakh linii K-562. III. DNK-sviazyvaiushchaia aktivnost' iadernykh al'fa-RNP. Gruppa "Peredacha signala s rostovykh faktorov" pri Meditsinskom fakul'tete Universiteta im. F. Shillera, Iena, Germaniia.

DNA-binding activity of small nuclear alpha-RNP identified in acid-soluble fraction of chromatin of human proerythroleukemic cell line K-562 was studied using the technique of gel retardation. We found that nuclear alpha-RNP isolated from K-562 cells through treatment with dimethylsulfoxide, an agent inducing differentiation, acquire a capacity to specific interaction with Alu repeats of DNA leading to the formation of alpha-RNP-Alu-DNA complexes; nuclear alpha-RNP from cells that were not treated with dimethylsulfoxide do not show such capacity, although they are tightly bound with chromatin in the cell. Thus, the capacity of nuclear alpha-RNP to direct interaction with DNA Alu repeats appearing after the induction of K-562 cells to differentiation along erythroid pathway is an inducible property. We discuss hypothesis about the involvement of nuclear alpha-RNP in the control of expression of inducible genes at the level of chromatin and interaction with DNA.

p53 inhibits RNA polymerase III-directed transcription in a promoter-dependent manner.

Wild-type p53 represses Alu template activity in vitro and in vivo. However, upstream activating sequence elements from both the 7SL RNA gene and an Alu source gene relieve p53-mediated repression. p53 also represses the template activity of the U6 RNA gene both in vitro and in vivo but has no effect on in vitro transcription of genes encoding 5S RNA, 7SL RNA, adenovirus VAI RNA, and tRNA. The N-terminal activation domain of p53, which binds TATA-binding protein (TBP), is sufficient for repressing Alu transcription in vitro, and mutation of positions 22 and 23 in this region impairs p53-mediated repression of an Alu template both in vitro and in vivo. p53's N-terminal domain binds TFIIIB, presumably through its known interaction with TBP, and mutation of positions 22 and 23 interferes with TFIIIB binding. These results extend p53's transcriptional role to RNA polymerase III-directed templates and identify an additional level of Alu transcriptional regulation.

Flanking sequences of an Alu source stimulate transcription in vitro by interacting with sequence-specific transcription factors.

An Alu source gene, called the EPL Alu, was previously isolated by a phylogenetic strategy. Sequences flanking the EPL Alu family member stimulate its RNA polymerase III (Pol III) template activity in vitro. One cis-acting element maps within a 40-nucleotide region immediately upstream to the EPL Alu. This same region contains an Ap1 site which, when mutated, abolishes the transcriptional stimulation provided by this region. The flanking sequence, as assayed by gel mobility shift, forms sequence-specific complexes with several nuclear factors including Ap1. These results demonstrate that an an ancestral Alu source sequence fortuitously acquired positive transcriptional control elements by insertion into the EPL locus, thereby providing biochemical evidence for a model which explains the selective amplification of Alu subfamilies.

[The DNA-binding activity of the membrane protein annexin II and its interaction with antibodies to the chromatin nuclear ribonucleoprotein (alpha-RNP)]. / DNK-sviazyvaiushchaia aktivnost' primembrannogo belka anneksina II i ego vzaimodeistvie s antitelami k iadernomu ribonukleoteinu khromatina (al'fa-RNP).

By screening with labeled Alu DNA, a clone was isolated from cDNA expression library, which appeared identical in sequence to the well-known Ca-phospholipid-binding protein annexin II. To evidence the DNA-binding activity of recombinant annexin II and its presence in the cell nucleus, we have expressed full-length mouse annexin II cDNA in bacteria with pGEMEX vector. The expressed protein was studied with electrophoretic mobility shift assay and for its reaction with polyclonal antibody to chromatin-associated ribonucleoprotein (alpha-RNP), which is one of the major acid-dissolvent components of the nucleus. The obtained results confirm the DNA-binding activity of recombinant annexin II. Annexin II reacts with polyclonal antibody to rat alpha-RNP. So, annexin II is a major nuclear DNA-binding protein in mammalian cells.

Specific Alu binding protein from human sperm chromatin prevents DNA methylation.

A protein from human sperm nuclei that specifically binds to Alu DNA repeats has been purified. The specific DNA binding site of this protein within the Alu sequence has been mapped by methylation interference and electrophoretic mobility shift assays. This sperm Alu binding protein selectively protects Alu elements from methylation in vitro and may be responsible for the unmethylated state of Alu sequences in the male germ line resulting in a parent-specific differential inheritance of Alu methylation.

Phylogenetic isolation of a human Alu founder gene: drift to new subfamily identity [corrected].

A severe bottleneck in the size of the PV Alu subfamily in the common ancestor of human and gorilla has been used to isolate an Alu source gene. The human PV Alu subfamily consists of about one thousand members which are absent in gorilla and chimpanzee DNA. Exhaustive library screening shows that there are as few as two PV Alus in the gorilla genome. One is gorilla-specific, i.e., absent in the orthologous loci in both human and chimpanzee, suggesting the independent retrotranspositional activity of the PV subfamily in the gorilla lineage. The second of these two gorilla PV Alus is present in both human and chimpanzee DNAs and is the single PV Alu known to precede the radiation of these three species. The orthologous Alu in gibbon DNA resembles the next older Alu subfamily. Thus, this Alu locus is originally templated by a non-PV source gene and acquired characteristic PV sequence variants by mutational drift in situ, consequently becoming the first member and presumptive founder of this PV subfamily.

Mobility of short interspersed repeats within the chimpanzee lineage.

The PV subfamily of Alu repeats in human DNA is largely composed of recently inserted members. Here we document additional members of the PV subfamily that are found in chimpanzee but not in the orthologous loci of human and gorilla, confirming the relatively recent and independent expansion of this Alu subfamily in the chimpanzee lineage. As further evidence for the youth of this Alu subfamily, one PV Alu repeat is specific to Pan troglodytes, whereas others are present in Pan paniscus as well. The A-rich tails of these Alu repeats have different lengths in Pan paniscus and Pan troglodytes. The dimorphisms caused by the presence and absence of PV Alu repeats and the length polymorphisms attributed to their A-rich tails should provide valuable genetic markers for molecular-based studies of chimpanzee relationships. The existence of lineage-specific Alu repeats is a major sequence difference between human and chimpanzee DNAs.

Binding specificity of human nuclear protein interacting with the Alu-family DNA repeats.

We have demonstrated earlier that human cells contain nuclear protein interacting with conserved GC-rich sequence motifs of human Alu-family DNA repeats. One of these sequences is located in the region between elements A and B of bipartite RNA polymerase III promoter of Alu (AB-region). In this study we have used a DNase I footprinting assay with an Alu restriction subfragment covering AB-region, as well as a gel mobility shift assay with appropriate synthetic oligonucleotides to analyse in more detail the interaction of the protein with AB-region. We have also used antibodies raised against a zinc-finger peptide to examine the presence of a zinc-finger in the Alu-binding protein. The results indicate that AGG triplets may be important for high-affinity binding of the protein to DNA, and that the Alu-binding protein is a zinc-finger protein.

[Various properties of cathepsin G and elastase from swine peripheral blood neutrophils]. / Nekotorye svoistva katepsina G i élastazy iz neitrofilov perifericheskoi krovi svin'i.

Using gel filtration through Sephadex G-100 and bioaffinity chromatography on contrical-Sepharose, cathepsin G and elastase were isolated from pig peripheral blood neutrophil granules and purified to homogeneity. Both enzymes hydrolyzed the total histone from calf thymus as well as synthetic substrates--tert-butoxy-L-alanine p-nitrophenyl ester (elastase) and benzoyltyrosine ethyl ester (cathepsin G). The use of natural and synthetic protease inhibitors showed that both enzymes were related to the group of serine proteases. The molecular mass of the cathepsin G subunit as determined by SDS polyacrylamide gel electrophoresis is 28-29 kD, that of elastase--30-31 kD. The pH optima for the hydrolysis of proteinaceous and synthetic substrates for cathepsin G and elastase are 8.0-8.5 and 7.0-7.5, respectively. The isoelectric points for elastase and cathepsin G are 9.7-10.0 and greater than 10, respectively; the temperature optima--30-40 degrees C and 50-60 degrees C, respectively. The amino acid composition of the two enzymes from pig granulocytes revealed a high content of arginine and was similar to that of human granulocytes.