The type of DNA attachment sites recovered from nuclear matrix depends on isolation procedure used

A large variety of DNA sequences have been described in nuclear matrix attachment regions. It could be most likely a result of the different methods used for their isolation. The idea about how different types of known DNA sequences (strongly attached to the nuclear matrix, weakly attached, or not attached) directly participate in anchoring DNA loops to the nuclear matrices isolated by different experimental procedures was tested in this study. Matrix-attached (M) and matrix-independent or loop (L) fractions as well as nuclear matrices were isolated using extractions of nuclei with 25 mM lithium 3,5-diiodosalicylate (LIS), 2 M NaCl, 0.65 M ammonium sulphate containing buffers followed by DNase I/RNase A digestion, or according to so designated conventional method. Using PCR-based and in vitro binding assays it was established that LIS and ammonium sulphate extractions gave similar results for the type of attachment of sequences investigated. The harsh extraction with 2 M NaCl or the conventional procedure led to some rearrangements in the attachment of DNA loops. As a result a big part of matrix attached sequences were found detached in the loop fractions. However, the in vitro binding abilities of the MARs to the nuclear matrices isolated by different methods did not change

A quantitative method for comparison of expression of alternatively spliced genes using different primer pairs

In this paper we describe a novel two-step method for comparison of expression of alternatively spliced genes by quantitative PCR (QPCR) applying different primer pairs. As a model system we used rat decay accelerating factor (DAF; CD55) mRNA, which comprises three different isoforms: soluble (sDAF), transmembrane (tmDAF) and glycosyl-phosphatidylinositol (GPI) anchored (gpiDAF) forms. The first step was to prepare solid phase specific for each mRNA isoform and purify the three DAF-forms from total RNA. We then assessed amplification efficiency of primer pairs designed to recognise each of the isoforms using equimolar amounts of the three purified DAF mRNAs. The final step in our assay was to compare expression of the three DAF-isoforms in testis by QPCR taking into account the efficiency of their amplification to enable quantification. The RNA capture/QPCR method we described here can be used for quantifying the expression ratios of alternatively spliced mRNAs from a single gene or for direct comparison of expression of different genes

Tightly bound matrix DNA probably plays an important role in organization of chromosome centromeres

A large variety of DNA sequences have been described in matrix attachment regions (MARs), This is a result of the different methods used for their isolation. Here we used the conventional procedure for preparing nuclear matrix from Friend-S cells and found that the fibrogranular network of the matrix is formed by uniform-looking tightly packed granules. The residual DNA extracted from matrix is enriched in highly repetitive sequences. Our previous investigations indicated that these MARs-DNA are probably constitutive in the cell cycle. The DNA was partially cloned to obtain additional information about the nature of these sequences, In sits hybridization with cloned DNAs showed that the formation of clusters in interphase nuclei is a result of close disposition of the centromeric regions of a few chromosomes, Computer analysis of the sequences of two MARs did not reveal any significant homologies with the known sequences in the NCBI database. The comparison of other two clones showed a high degree of similarity to mouse repetitive families, In one of the clones we found four TGGAA motifs which could nucleate stem-loop structures. This unusual structure could explain the unique morphology and function of the mouse centromere in mitosis

Gene reorganisations and expression of c-ras, c-src, c-mos and c-fos oncogenes in Namalwa, Wish and C6 cells.

In a wide variety of neoplasms, proto-oncogenes were found transcriptionally activated by different DNA rearrangements. In the present study we used Namalwa, Wish and C6 cell lines in order to investigate the correlation between gene reorganisations and their expression. According to Southern and fluorescence in situ hybridisation (FISH) analysis the oncogenes c-ras, c-src and c-fos were amplified in Namalwa and Wish cells. reorganisation other than amplification was found for c-mos in the three cell cultures investigated. The amplification levels of the genes studied were assessed by dot blot hybridisation followed by densitometric scanning. c-H-ras and c-src were amplified about 20-fold in the genomes of Namalwa and Wish, while c-fos was amplified approximately 12-fold in the same cell lines. The hybridisation signals in C6 were almost the same as in the control lymphocytes for the four oncogenes investigated. Similar results were obtained for c-mos in the genomes of all cell lines examined. Using RT-PCR, overexpression of proto-oncogenes c-ras, c-src, c-mos and c-fos was found in Namalwa and Wish cells. In C6 cells the expression of the four genes studied was marginal. Overexpression of c-mos was observed in Namalwa and Wish cells, while in C6 it was marginal although existence of reorganisation was found. Hence, it might be suggested that in C6 cells c-mos is down-regulated from other factors and/or genes, or requires for its activation overexpression of other genes

Targeting transcription factors for therapeutic benefit

Transcription factors are a large class of biological molecules that are important for health and disease. Despite that there are challenges to targeting them therapeutically and most approaches alter their activity indirectly. Research at the chemical biology interface has led to the development of new ways of targeting transcription factors including blocking transcription factor dimerisation, targeting specific DNA sequences and DNA decoys. This review discusses these issues with a view to inspiring the development of new agents that could be useful for the treatment of cancer

Catalytic and biocatalytic oxidation of glucose to gluconic acid in a modified three-phase reactor

A comparative study of catalytic and biocatalytic glucose oxidation was carried out. Gluconobacter oxydans NBIMCC 1043 strain was used for biocatalytic glucose conversion. In the case of cell recycle coupled with cross-flow microfiltration the productivity and biomass concentration reached 40% and 3 g l(-1) respectively, in comparison to those of batch fermentation (21% and 2.3 g l(-1), respectively)

Localisation of DNA topoisomerase II alpha in mouse erythroleukemia cells

The presence of DNA topoisomerase II alpha was investigated in interphase and metaphase mouse erythroleukemia (MEL) Friend-S cells, and in extracted with 25 mM lithium diiodosalicylate buffer (Lis) nuclei using indirect immunofluorescence. The results showed that DNA topoisomerase II alpha is localised in the nuclei. In the metaphase cells, we found high concentrations of this enzyme in the mitotic chromosomes. Our results support the idea of the accumulation of DNA topoisomerase II alpha at the end of the cell cycle. The extractions of nuclei with 25 mM Lis led to the complete depletion of DNA topoisomerase II alpha from the residual nuclear matrix. Using a high dilution of the first antibody, we established that the high level of heterochromatin compactisation in the interphase nuclei is caused by the high concentration of DNA topoisomerase II alpha

Interphase Chromosomes of Friend-S Cells Are Attached to the Matrix Structures Through the Centromeric/Telomeric Regions

DNA of the attachment sites of Friend erythroleukemia cells, isolated according to the conventional procedure, represents short, nuclease-resistant fragments with sizes below 400 bp, belonging to the class of mouse satellite. A number of experiments have indicated that their unusual resistance is due to complexing with RNA. By various approaches, it was confirmed that similar fragments might be recovered from total DNA following extensive digestion with DNase I. In situ hybridizations revealed further that at mitosis the sequences of the attachment sites are located at the centromeric/telomeric regions of the chromosomes, while at interphase they are redistributed into 9-13 well-defined clusters spread throughout the entire nuclear area. Parallel biochemical and electronomicroscopic studies have clarified, moreover, that the all three compartments of the matrix harbor such sequences. Thus, it appears that the attachment sites described function only at interphase, anchoring the both ends of each interphase chromosome to the matrix structures

The mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility [RETRACTED]

In mouse, genes encoding complement regulators CD55 and CD59 have been duplicated. The first described form of CD59, CD59a, is broadly distributed in mouse tissues, while the later identified CD59b was originally described as testis specific. Subsequent studies have been contradictory, some reporting widespread and abundant expression of CD59b. Resolution of the distribution patterns of the CD59 isoforms is important for interpretation of disease studies utilising CD59 knockout mice. Here we have performed a comprehensive distribution study of the CD59 isoforms at the mRNA and protein levels. These data confirm that expression of CD59b is essentially restricted to adult testis; trace expression in other tissues is a consequence of contamination with blood cells, shown previously to express CD59b at low level. In testis, onset of expression of CD59b coincided with puberty and was predominant on the spermatozoal acrosome. Ligation of CD59b, but not CD59a, markedly reduced spermatozoal motility, suggesting a specific role in reproductive function