[Transcriptional activity of nuclear factor kappa B (NFkappaB) in posttraumatic sensory neurons (histochemical study)].

Nuclear factor kappa B (NFkappaB) is a ubiquitous nuclear transcription factor that regulates the expression of a number of genes involved in cell survival, immune and inflammatory processes. It has been hypothesized that after nerve injury, the release of specific cytokines may provide a stimulus for activation of the transcription factor NF-kappaB in adult dorsal root ganglia (DRG) neurons exerting the protective effect on the sensory neurons. However, the complexity of this transcription factor has led to some misleading conclusions about NF-kappaB signalling in injured DRG neurons. The goal of the present study is to find out whether NF-kappaB is involved in the transcriptional regulation of genes in adult primary sensory neurons after peripheral nerve transection. In this series of experiments, we used a transgenic line of NF-kappaB reporter mice in which activation of NF-kappaB drives the expression of the lac-z gene. We show that the expression of beta-galactasidase (beta-gal) is not detected in injured DRG neurons and contralateral neurons. However, a strong beta-gal expression was detected in the muscle at the injury site. It may reflect the repressive influence of additional signalling cascades on NF-kappaB activity in sensory neurons.

Posttraumatic activity of signal pathways of nuclear factor κB in mature sensory neurons.

The aim of this study was to clear out whether injury to the peripheral nerve leads to activation of nuclear factor κB in mature spinal ganglia. Analysis of matrix RNA of nuclear factor κB-dependent genes (monocyte chemoattractant protein MCP-1 and inhibitor of nuclear factor κB IκBα) showed different levels of expression of these genes in the spinal ganglia in vivo after axotomy and in vitro after TNF-α stimulation. On the other hand, DNA-binding activity of nuclear factor κB increased in the spinal ganglia 6 h after axotomy and after 10-min incubation of sensory neuron culture with TNF-α. These data attest to possible involvement of nuclear factor κB in the posttraumatic regulation of gene transcription in spinal ganglion cells.

Development and characterization of Thy*IκBα-SI transgenic mice.

We developed and characterized a new transgenic model where NF-κB activity is inhibited only in mature neurons. Transgenic mouse strain Thy*IκBα-SI was created using trans-dominant super inhibitor NF-κB (IκBα-SI), which is a multimutant form of IκBα inhibitory protein cloned into specific neutral Thy-1.2 cassette. Detailed molecular analysis showed that the transgene and its product (IκBα-SI protein) are expressed in the nervous system of transgenic mice. In situ hybridization showed that Thy*IκBα-SI in the nervous system is expressed exclusively in neurons. The developed model provides wide opportunities for studying functional role of NF-κB in mature neurons in the central and peripheral nervous system in vivo.

[The levels of key nucleolar proteins in the lymphoid cells of healthy individuals and patients with lymphoproliferative diseases].

Immunocytochemical staining with specific antibodies was used to study the expression of three nucleolar proteins (fibrillarin, B23/nucleofozmin, and SURF6), which were involved in pRNA maturation, in the lymphoid cells of healthy individuals and patients with lymphoproliferative diseases and to compare it with the expression of the known proliferation marker Ki-67 protein. The results indicated that fibrillarin was detectable at the comparable level in the lymphoid cells of the patients and in the peripheral blood lymphocytes of the healthy individuals. In one fourth of the patients, the proportion of cells containing B23/nucleofozmin was noticeably higher than that in the lymphocytes of donors; however, there was no great difference in patients with different types of the disease. The number of SURF6-positive cells was directly correlated with that of Ki-67-positive cells. The maximum level (47-67%) of SUR6-positive lymphoid cells was found in splenic lymphosarcomas and mantle cell lymphoma. The findings suggest that SURG6 protein may be of additional diagnostic and prognostic value.

[Properties and functions of a new nucleolar protein, Surf-6, in 3T3 mouse cells].

The localization of the specific protein Surf-6 from nucleoli of eukaryotic cells in mitosis and its sensitivity to the treatment of cells with RNase A and DNase I in situ were studied. It was shown that, in interphase nucleoli of 3T3 mouse cells, Surf-6 is probably associated with RNA and practically is not associated with DNA. In mitosis, Surf-6 appears in forming nucleoli after the known RNA-binding proteins fibrillarin and B23/nucleofozmin, which are involved in the early and late stages of the assembly of ribosomal particles, respectively. These observations and the regularities of migration of early and late proteins of ribosome assembly to nucleoli in the telophase of mitosis led us to the presumption that Surf-6 is involved in the terminal stages of the assembly of ribosomal particles in murine cells. An immunoblot analysis of the Surf-6 content in synchronized 3T3 cells showed for the first time that Surf-6 is present at all stages of the cell cycle but its content markedly decreases when cells enter the G0 period. Conversely, the activation of cells for proliferation is accompanied by an increase in the Surf-6 content. These observations allow one to regard Surf-6 as a marker of the cell proliferative state and suggest its implication in the regulation of the cell cycle. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

Growth hormone-releasing hormone (GHRH) neurons in GHRH-enhanced green fluorescent protein transgenic mice: a ventral hypothalamic network.

The hypothalamic GHRH neurons secrete pulses of GHRH to generate episodic GH secretion, but little is known about the mechanisms involved. We have made transgenic mice expressing enhanced green fluorescent protein (eGFP) specifically targeted to the secretory vesicles in GHRH neurons. GHRH cells transported eGFP from cell bodies in the arcuate nucleus to extensively arborized varicose fiber terminals in the median eminence. Patch clamp recordings from visually identified GHRH cells in mature animals showed spontaneous action potentials, often firing in short bursts up to 10 Hz. GHRH neurons received frequent synaptic inputs, as demonstrated by the recording of abundant inward postsynaptic currents, but spikes were followed by large after-hyperpolarizations, which limited their firing rate. Because many GHRH neurons lie close to the ventral hypothalamic surface, this was examined by wide-field binocular epifluorescence stereomicroscopy. This approach revealed an extensive horizontal network of GHRH cells at low power and individual fiber projections at higher power in the intact brain. It also showed the dense terminal projections of the GHRH cell population in the intact median eminence. This model will enable us to characterize the properties of individual GHRH neurons and their structural and functional connections with other neurons and to study directly the role of the GHRH neuronal network in generating episodic secretion of GH.

A secreted fluorescent reporter targeted to pituitary growth hormone cells in transgenic mice.

In stable transfection experiments in the GH-producing GC cell line, a construct containing the entire signal peptide and the first 22 residues of human GH linked in frame with enhanced green fluorescent protein (eGFP), produced brightly fluorescent cells with a granular distribution of eGFP. This eGFP reporter was then inserted into a 40-kb cosmid transgene containing the locus control region for the hGH gene and used to generate transgenic mice. Anterior pituitaries from these GH-eGFP transgenic mice showed numerous clusters of strongly fluorescent cells, which were also immunopositive for GH, and which could be isolated and enriched by fluorescence-activated cell sorting. Confocal scanning microscopy of pituitary GH cells from GH-eGFP transgenic mice showed a markedly granular appearance of fluorescence. Immunogold electron microscopy and RIA confirmed that the eGFP product was packaged in the dense cored secretory vesicles of somatotrophs and was secreted in parallel with GH in response to stimulation by GRF. Using eGFP fluorescence, it was possible to identify clusters of GH cells in acute pituitary slices and to observe spontaneous transient rises in their intracellular Ca2+ concentrations after loading with Ca2+ sensitive dyes. This transgenic approach opens the way to direct visualization of spontaneous and secretagogue-induced secretory mechanisms in identified GH cells.

Differentially expressed genes during in vitro differentiation of murine embryonic stem cells transduced with a human erythropoietin receptor cDNA.

Our previous study demonstrated that transduction of murine embryonic stem (ES) cells with a human erythropoietin (Epo) receptor (R) cDNA resulted in enhanced erythropoiesis in developing embryonic bodies (EBs). To address possible mechanisms of gene regulation, we compared gene expression between hEpoR cDNA-transduced ES (ES-hEpoR) cells and parental ES cells during in vitro differentiation induced by withdrawal of leukemia inhibitory factor (LIF) and cultured in the absence of Epo using differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR). A total of 48 differentially expressed cDNA fragments were found; 12 were sequenced and five were confirmed by Northern blot analysis to be up- or down-regulated in ES-hEpoR cells during differentiation compared to parental ES cells. In a GenBank search of the five putatively regulated cDNA fragments, two fragments shared high sequence homology to two known genes: the Surf-6 gene and the gene for calcyclin binding protein. Northern blot analysis demonstrated that 2.5-kb and 0.3-kb transcripts of the Surf-6 gene were expressed in undifferentiated ES-hEpoR and parental ES cells at a low level, but this expression was enhanced from day 2 to 14 of differentiation after withdrawal of LIF and culture in the presence of Epo. Furthermore, the enhanced expression of these two transcripts was also noticed in EML-C1 cells, a murine multipotential hematopoietic cell line that has erythroid differentiation potential in response to Epo. In summary, our results demonstrate that Surf-6 gene expression is regulated during differentiation of hematopoietic stem/progenitor cells in response to Epo, suggesting a possible role for Surf-6 gene in erythropoiesis.

Isolation and genomic analysis of the human surf-6 gene: a member of the Surfeit locus.

The human Surfeit locus contains at least six tightly clustered genes (Surf-1 to Surf-6) of which five (Surf-1 to Surf-5) have been characterised and found not to share any sequence homology. The organisation and juxtaposition of the Surfeit genes are conserved between human and mouse. The Surf-6 gene that encodes a novel nucleolar-matrix protein with nucleic-acid binding properties has been characterised in mouse. In this work, we have isolated and analysed the human Surf-6 homologue and determined its genomic organisation in the Surfeit locus. The human Surf-6 gene has five exons spread over a distance of 4.3kb and has features of a housekeeping gene being ubiquitously expressed, having its 5' end located within a CpG rich island and lacking a canonical TATA box. The intragenic region between the 3' end of the Surf-5 gene and the 5' end of the Surf-6 gene is 3.2kb and contains a pseudogene of the ribosomal protein gene rpL21. The putative human Surf-6 protein is 361 amino acids long and includes motifs found in both the mouse and fish Surf-6 homologues, which may underlie the functions of Surf-6. Three amino acid polymorphisms have been detected at codons 163, 175 and 311 by SSCP analysis.

Concerted evolution within a trypsin gene cluster in Drosophila.

A cluster of four trypsin genes has previously been localized to cytological position 47D-F of the Drosophila melanogaster genome. One of these genes had been sequenced, and the presence of the other three genes was identified by cross-hybridization. Here, we present the DNA sequence of the entire genomic region encoding these four trypsin genes. In addition to the four previously inferred genes, we have identified a fifth trypsin-coding sequence located within this gene cluster. This new gene shows a high degree of sequence divergence (more than 30%) from the other four genes, although it retains all of the functional motifs that are characteristic of trypsin-coding sequences. In order to trace the molecular evolution of this gene cluster, we isolated and sequenced the homologous 7-kb region from the closely related species Drosophila erecta. A comparison of the DNA sequences between the two species provides strong evidence for the concerted evolution of some members of this gene family. Two genes within the cluster are evolving in concert, while a third gene appears to be evolving independently. The remaining two genes show an intermediate pattern of evolution. We propose a simple model, involving chromosome looping and gene conversion, to explain the relatively complex patterns of molecular evolution within this gene cluster.

The SURF-6 protein is a component of the nucleolar matrix and has a high binding capacity for nucleic acids in vitro.

The recently identified novel protein SURF-6 is shown to be a component of the nucleolar matrix. Immunofluorescence analysis demonstrated that SURF-6 was localized in residual nucleoli of in situ nuclear matrix preparations of mouse fibroblast cells (NIH 3T3), which were depleted of soluble and chromatin related proteins. Immunoblot analysis of biochemical nucleolar subfractions confirmed that SURF-6 was present in the nucleolar matrix fraction, and was absent from the fractions of soluble proteins released by DNase or RNase. The capacity of SURF-6 to bind nucleic acids was investigated in vitro. Both endogenous SURF-6 from nuclear extracts and recombinant SURF-6 exhibited a strong binding capacity for nucleic acids. It was shown that SURF-6 bound to both DNA and RNA, however, it showed stronger binding to RNA. The presence and nuclear distribution of SURF-6 during the cell cycle was explored by immunofluorescence analysis. It was shown that SURF-6 was always found in the nucleolus regardless of the phase of the cell cycle suggesting that it is a structural protein constitutively present in nucleolar substructures. The colocalization of SURF-6 with the major nucleolar proteins B23 and fibrillarin, which are known to be involved in the processing of ribosomal RNA (rRNA), was examined both in interphase and mitosis by double immunolabeling of cells. SURF-6 was found to be largely coincident with both proteins in interphase and it was distributed in the same cellular locations, namely the perichromosomal layer, the cytoplasm and prenucleolar bodies, in mitosis. However, colocalization of SURF-6 with fibrillarin and B23 was only partial in interphase, and the dynamics of its localization was not completely the same as those of either fibrillarin or B23 during mitosis. Taken together, these results indicate that SURF-6 is a novel nucleolar matrix component and imply that SURF-6 might support nucleolar matrix structure and function(s) via its association with nucleic acids. We propose that SURF-6 may be involved in processing of rRNA, based on its cytological characteristics, but at stages in ribosomal biogenesis which are different from those for fibrillarin and B23.

The Surf-6 gene of the mouse surfeit locus encodes a novel nucleolar protein.

The Surfeit locus contains the tightest cluster of mammalian genes so far described. The five Surfeit genes (Surf-1 to -5) that have been previously isolated and characterized do not share any DNA or amino acid sequence homology. These Surfeit genes appear to be housekeeping genes, with the Surf-3 gene encoding the 1.7a ribosomal protein and the Surf-4 gene encoding an integral membrane protein most likely associated with the endoplasmic reticulum. In this work, we have isolated the Surf-6 gene, a sixth member of the Surfeit locus. The Surf-6 gene contains four exons spanning a genomic region of 14 kb and specifies a mRNA of 2,571 bases. Surf-6 has features common to housekeeping genes because its transcript is present in every tissue tested, its 5' end is associated with a CpG-rich island, and its promoter does not contain a canonical TATA box. The Surf-6 long open reading frame encodes a novel highly basic polypeptide of 355 amino acids (28% Arg and Lys). By immunofluorescence and immunoblot analyses, the Surf-6 protein has been found to be located in the nucleolus and by immunocytochemical microscopy to be localized predominantly in the nucleolar granular component, a structure that is involved in ribosome maturation. These results indicate that the novel Surf-6 gene is involved in a nucleolar function.

Isolation and characterization of a full-length trypsin-encoding cDNA clone from the Lepidopteran insect, Choristoneura fumiferana.

The protease-encoding genes of Lepidopteran insects are of interest because they are adapted to functioning at very high pH optima, in the range of pH 10-12. Here, we report the isolation and sequence characterization of a trypsin-encoding cDNA clone from the spruce budworm, Choristoneura fumiferana.

A short 5'-flanking region mediates glucose repression of amylase gene expression in Drosophila melanogaster.

Expression of the alpha-amylase gene is highly repressed by dietary glucose in Drosophila melanogaster larvae. Here, we show that glucose repression is controlled by DNA sequences that are located upstream of the transcribed region. Recombinant gene constructions, in which the amylase promoter sequences were fused with the transcribed region of the Adh gene, were expressed in transgenic Drosophila larvae. The expression of ADH from the recombinant gene was shown to be subject to glucose repression. The function of potential regulatory cis-acting elements within the glucose responsive upstream region was examined by deletion analysis and by site-directed mutagenesis, coupled with expression assays in transformed larvae. The upstream deletion analysis showed that essential elements, both for overall activity and for glucose repression of the amylase gene, are located within a 109-bp region upstream of the transcription start site. Site-directed mutagenesis of these upstream sequences showed that the TATA motif, at position -31, and a novel 36-bp element, at position -109, were necessary for full activity of the amylase promoter. None of the introduced mutations resulted in loss of glucose responsiveness. These results indicate that glucose repression, in Drosophila, is mediated by transcriptional mechanisms that involve multiple, functionally redundant DNA elements.

Functional conservation of a glucose-repressible amylase gene promoter from Drosophila virilis in Drosophila melanogaster.

Previous studies have demonstrated that the expression of the alpha-amylase gene is repressed by dietary glucose in Drosophila melanogaster. Here, we show that the alpha-amylase gene of a distantly related species, D. virilis, is also subject to glucose repression. Moreover, the cloned amylase gene of D. virilis is shown to be glucose repressible when it is transiently expressed in D. melanogaster larvae. This cross-species, functional conservation is mediated by a 330-bp promoter region of the D. virilis amylase gene. These results indicate that the promoter elements required for glucose repression are conserved between distantly related Drosophila species. A sequence comparison between the amylase genes of D. virilis and D. melanogaster shows that the promoter sequences diverge to a much greater degree than the coding sequences. The amylase promoters of the two species do, however, share small clusters of sequence similarity, suggesting that these conserved cis-acting elements are sufficient to control the glucose-regulated expression of the amylase gene in the genus Drosophila.

Amylase cis-acting sequences mediate the alleviation of glucose repression by cAMP in Drosophila.

alpha-Amylase gene expression is highly repressed by dietary glucose in Drosophila melanogaster. This glucose effect can be alleviated by exogenous adenosine 3':5'-cyclic monophosphate (cAMP). Here, we show that the relief of glucose repression by cAMP occurs at the level of amylase mRNA abundance. Furthermore, exogenous cAMP was shown to alleviate glucose repression of the transient expression of an amylase gene construct in transformed Amynull larvae. This construct contains only 109 base pairs of the promoter region; this is the minimal length of upstream sequence which is necessary for wild-type levels of amylase gene expression. These results indicate that cis-acting promoter elements located close to the transcriptional start site of the Drosophila amylase gene mediate both glucose repression and the cAMP-derepression effects.

Isolation of three novel Drosophila melanogaster genes containing repetitive sequences rich in GCN triplets.

Several cDNA and genomic clones were isolated from Drosophila melanogaster gene libraries by hybridization with a region of a mammalian gene that contains a simple repetitive sequence of six GCN repeats. One of the cDNA clones, E6, was completely sequenced and it was shown that it contains a region of 16 GCN repeats; these repeats encode a polyalanine stretch within a long open reading frame. The sequencing of three different genomic clones (A, B, and D) revealed that all the isolated Drosophila clones are similar to one another in a short region containing variable numbers of the GCN repeat. The genomic clone B was found to be the genomic counterpart of the cDNA clone E6. The other genomic clones, A and D, also hybridize with Drosophila cDNA clones at high stringency. These results indicate that the short GCN repetitive sequences, which we have named ala, are found within transcribed regions of the Drosophila genome. These Drosophila genes containing the ala repeat do not show significant sequence similarity to any presently known gene; we have named these novel genes ala-A, ala-B, and ala-D. The cDNA clone from gene ala-B was named ala-E6.

Molecular biology of enzyme adaptations in higher eukaryotes.

Multicellular eukaryotes have evolved complex homeostatic mechanisms that buffer the majority of their cells from direct interaction with the external environment. Thus, in these organisms long-term adaptations are generally achieved by modulating the developmental profile and tissue specificity of gene expression. Nevertheless, a subset of eukaryotic genes are still involved in direct responses to environmental fluctuations. It is the adaptative responses in the expression of these genes that buffers many other genes from direct environmental effects. Both microevolutionary and macroevolutionary patterns of change in the structure and regulation of such genes are illustrated by the sequences encoding alpha-amylases. The molecular biology and evolution of alpha-amylases in Drosophila and other higher eukaryotes are presented. The amylase system illustrates the effects of both long-term and short-term natural selection, acting on both the structural and regulatory components of a gene--enzyme system. This system offers an opportunity for linking evolutionary genetics to molecular biology, and it allows us to explore the relationship between short-term microevolutionary changes and long-term adaptations.