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Aberrant methylation of tumor suppressor genes can lead to their silencing in many cancers. TSC-22is a gene silenced in several solid tumors, but its function and the mechanism(s) responsible for its silencing are largely unknown. Here we demonstrate that the TSC-22promoter is methylated in primary mouse T or natural killer (NK) large granular lymphocyte (LGL) leukemia and this is associated with down-regulation or silencing of TSC-22expression. The TSC-22deregulation was reversed in vivo by a 5-aza-2′-deoxycytidine therapy of T or NK LGL leukemia, which significantly increased survival of the mice bearing this disease. Ectopic expression of TSC-22 in mouse leukemia or lymphoma cell lines resulted in delayed in vivo tumor formation. Targeted disruption of TSC-22in wild-type mice enhanced proliferation and in vivo repopulation efficiency of hematopoietic precursor cells (HPCs). Collectively, our data suggest that TSC-22normally contributes to the regulation of HPC function and is a putative tumor suppressor gene that is hypermethylated and silenced in T or NK LGL leukemia.

Aberrant methylation of tumor suppressor genes can lead to their silencing in many cancers. TSC-22 is a gene silenced in several solid tumors, but its function and the mechanism(s) responsible for its silencing are largely unknown. Here we demonstrate that the TSC-22 promoter is methylated in primary mouse T or natural killer (NK) large granular lymphocyte (LGL) leukemia and this is associated with down-regulation or silencing of TSC-22 expression. The TSC-22 deregulation was reversed in vivo by a 5-aza-2′-deoxycytidine therapy of T or NK LGL leukemia, which significantly increased survival of the mice bearing this disease. Ectopic expression of TSC-22 in mouse leukemia or lymphoma cell lines resulted in delayed in vivo tumor formation. Targeted disruption of TSC-22 in wild-type mice enhanced proliferation and in vivo repopulation efficiency of hematopoietic precursor cells (HPCs). Collectively, our data suggest that TSC-22 normally contributes to the regulation of HPC function and is a putative tumor suppressor gene that is hypermethylated and silenced in T or NK LGL leukemia.

Vertebrate gastrulation involves complex coordinated movements of cells and cell layers to establish the axial structures and the general body plan. Adhesion molecules and the components of extracellular matrix were shown to be involved in this process. However, other participating molecules and detailed mechanisms of the control of gastrulation movements remain largely unknown. Here, we describe a novel Xenopusgene camello(Xcml) which is expressed in the suprablastoporal zone of gastrulating embryos. Injection of XcmlRNA into dorsovegetal blastomeres retards or inhibits gastrulation movements. Database searches revealed a family of mammalian mRNAs encoding polypeptides highly similar to Xcml protein. Characteristic features of the camello family include the presence of the central hydrophobic domain and the N-acetyltransferase consensus motifs in the C-terminal part, as well as functional similarity to Xcml revealed by overexpression studies in Xenopusembryos. Xcml expression results in the decrease of cell adhesion as demonstrated by the microscopic analysis and the blastomere aggregation assay. Cell fractionation and confocal microscopy data suggest that Xcml protein is localized in the secretory pathway. We propose that Xcml may fine tune the gastrulation movements by modifying the cell surface and possibly extracellular matrix proteins passing through the secretory pathway.

THE terrestrial snail Helix lucorumis a promising model for molecular neurobiology since its central nervous system (CNS) is simple and contains many morphologically and functionally identified large neurones. Among these, the giant interneurones located in pleural and parietal ganglia are especially interesting because they trigger the withdrawal behaviour of the snail and participate in aversive conditioning. Here we describe the identification and characterization of a gene named HCS1which is preferentially expressed in these interneurones. It encodes a putative protein 100 amino acids long containing an N-terminal hydrophobic leader peptide. No sequences with significant homology to HCS1were found in the protein (Swiss-Prot) and nucleotide (EMBLbank) data libraries. We suppose that the product of this gene is a secreted protein, presumably a neuropeptide or a growth factor.

WE report the isolation of a Helix lucorumcDNA clone encoding a precursor of neuropeptides that are closely related to Aplysiaand Tritoniapedal peptides (Pep). The predicted propeptide contains 20 copies of the two variants of HelixPep interspersed with Lys-Arg endopeptidase cleavage sites. Northern blot hybridization revealed multiple Pep-hybridizing species in the HelixCNS RNA. The Pep gene was expressed by several identified serotonergic neurones in pedal and cerebral ganglia, groups of sensory neurones in procerebrum, peripheral neurones in olfactory bulb, mantle and foot, and group of neurones in pedal ganglia presumably involved in locomotion control. Pep mRNA was detected in several neurones at the early stages of nervous system development.

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particle has been determined by a sequencing procedure based on crosslinking histones to the 5'-terminal DNA fragments produced by scission of one DNA strand at the point of crosslinking. The position of histones on DNA has been identified by measuring the length of crosslinked DNA fragments. The results demonstrate that each of the histones is arranged within several adjacent or dispersed DNA segments of a little less than 10 nucleotides in length. Histone-free intervals are located between these segments at the regular distances of about (10)n nucleotides from the 5' end of the DNA and are likely to face one side of the DNA helix. Histones appear to be arranged in a similar manner on both DNA strands and do not form "locks" around DNA. A linearized model of the core particle is proposed.

A novel cDNA encoding a putative secreted protein was isolated from murine bone marrow. The encoded protein named MCLP (urine athelin‐ike rotein) was found to be highly homologous to the pig cathelin, and to four neutrophil antimicrobial polypeptides: CAP 18, indolicidin, Bac 5 and FALL‐39. Secondary structure prediction studies identified a highly cationic region in the C‐terminal part of prepro‐MCLP with a tendency to adopt an amphipathic α‐helical conformation, as observed in many antimicrobial peptides. However, no antibacterial activity was observed with the synthetic peptide corresponding to this region of MCLP.

We report the isolation of a cDNA clone encoding a neuropeptide precursor named preproGFAD from the central nervous system (CNS) of the snail Helix lucorum. Analysis of the expression of this gene shows that it is neurospecific and expressed in several groups of CNS neurons. Most notable is the expression of preproGFAD gene in the right mesocerebrum, where the neurons controlling mating behavior are located. The expression in this particular region is observed in adult animals but not in juvenile ones. The preprohormone is 108 amino acids long and contains a hydrophobic leader peptide and eight Lys-Arg recognition sites for endoproteolysis. The post-translational processing of the prohormone may lead to the generation of seven tetrapeptides, Gly-Phe-Ala-Asp-COOH (GFAD). This peptide has the same sequence as two previously isolated peptides from a related snail, Achatina fulica. The first of them (achatin-I) contains D-Phe; the second (achatin-II) is its L-Phe-containing stereoisomer. Injection of synthetic D-GFAD in nanomolar concentrations into intact animals caused an increase of the heartbeat rate and opening of the genital atrium. In preparations containing CNS with intact innervation of reproductive organs, bath application of D-GFAD caused extensive movements of the penis but not of other reproductive organs. Intracellular activation of individual neurons expressing the preproGFAD gene also elicited penis movements. D-GFAD also suppressed activity of neurons modulating feeding behavior. Our data therefore indicate that the preproGFAD gene encodes the precursor of a neuropeptide that participates in the regulation of male mating behavior. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 183–197, 1998

The binding of isolated high mobility group proteins HMG (1+2) with nucleosomes was studied using gel electrophoresis. The interaction of HMG (1+2) with mononucleosomes could be detected as a new discrete electrophoretic band with a decreased mobility only after cross-linking of HMG (1+2)-nucleosome complex by formaldehyde. Approximately two molecules of the large HMG proteins were bound per nucleosomal particle of a DNA length of ~ 185 base pairs, lacking histones H1 and H5. Using the same techniques, no binding was observed with core particles of a DNA length of ~ 145 base pairs.

Quantitative analysis of distribution of chromosomal proteins on single copy DNA sequences has been further developed. Our approach consists of DNAprotein crosslinking within whole cells or isolated nuclei, specific Immunoaffinity isolation of crosslinked complexes via protein and Identification of crosslinked DNA by hybridisation with single-stranded DNA probes. The present study shows that transcribed chromatin of chicken embryonic erythrocyte β globin gene is characterized by about 1.5 – 2.5-fold higher density of HMG 14/17 and 2-fold lower density of H1 and H5 as compared with non-transcribed chromatin of ovalbumin and lysozyrne genes, whereas HMG 1/2,E proteins were equally distributed between DNA of both transcribed and non-transcribed genes. The depletlon of H1/H5 in β globin sequences was verified by the ‘protein image’ hybridisation technique (1). The DNase I hypersensitive site located 5′ upstream from β globin gene is deficient in all the proteins assayed, what implies a drastic disruption in the nucleosomal array. Minor quantttative changes of protein pattern suggest transient local perturbation of the chromatin on transcription.

The arrangement of histones along DNA in nucleosomal core particles within transcribed heat shock gene (hsp 70) region and repressed insertion within ribosomal genes of Drosophila was analysed by using protein-DNA crosslinking methods combined with hybridization tests In addition, two-dimensional gel electrophoresis was employed to compare the overall nucleosomal shape and the nucleosomal DNA size. The arrangement of histones along DNA and general compactness of nucleosomes were shown to be rather similar in transcriptionally active and inactive genomic regions. On the other hand, nucleosomes with in transcriptionally active chromatin are characterized by a larger size of nucleosomal DNA produced by micrococcal nuclease digestion and some peculiarity in electrophoretic mobility.

A novel method for identification of differentially expressed genes has been developed. It is based on the consecutive restriction digestions of 3′ terminal cDNA fragments to produce a fingerprint of gene expression. cDNA molecules are synthesized using a biotinylated oligo(dT) primer, digested with a frequently cutting restriction endonuclease and the 3′-terminal restriction fragments are isolated using streptavidin microbeads. After amplification by PCR, cDNA fragments are Immobilized again on streptavidin beads, radiolabeled and treated sequentially with a set of restriction endonucleases. The products of individual enzymatic reactions from two or more different RNA populations are resolved by polyacrylamide gel electrophoresis and compared to reveal differentially expressed genes. This strategy enabled us to identify and clone the fragments of five genes expressed differentially In murine thymus and spleen. One of the genes was found to encode terminal deoxynucleotidyl transferase; others are apparently previously unknown genes

A refined map for the linear arrangement of histons along DNA in nucleosomal core particles has been determined by DNA-protein crosslinking. On one strand of 145-bp core DNA, histones are aligned in the following order: (5′) H2B25,36-H455,65-H375,85,95/H495-H2B105,115-H2A115-H3135,145/H2A145 (3′) (the subscripts give approximate distance in nucleotides of the main histone contacts from the 5′-end). Hence, the histone tetramer (H3,H4)2 and two dimers (H2A-H28) are arranged on double-stranded core DNA in a symmetrical and rather autonomous way: H2A/H3-(H2A-H2B)-(H3,H4)2-(H2B-H2A)-H3/H2A. The primary organization was found to be very similar in core particles isolated from repressed nuclei of sea urchin sperm and chicken erythrocytes, from active in replication and transcription nuclei of Drosophila embryos and yeast and from somatic cells of lily. These data show that (i) the core structure is highly conserved in evolution and (ii) the overall inactivation of chromatin does not affect the arrangement of histones along DNA and thus does not seem to be regulated on this level of the core structure.