5-Bromo-2'-deoxyuridine induces visible morphological alteration in the DNA puffs of the anterior salivary gland region of Bradysia hygida (Diptera, Sciaridae)

5-Bromo-2'-deoxyuridine (BrdUrd) has long been known to interfere with cell differentiation. We found that treatment ofBradysia hygida larvae with BrdUrd during DNA puff anlage formation in the polytene chromosomes of the salivary gland S1 region noticeably affects anlage morphology. However, it does not affect subsequent metamorphosis to the adult stage. The chromatin of the chromosomal sites that would normally form DNA puffs remains very compact and DNA puff expansion does not occur with administration of 4 to 8 mM BrdUrd. Injection of BrdUrd at different ages provoked a gradient of compaction of the DNA puff chromatin, leading to the formation of very small to almost normal puffs. By immunodetection, we show that the analogue is preferentially incorporated into the DNA puff anlages. When BrdUrd is injected in a mixture with thymidine, it is not incorporated into the DNA, and normal DNA puffs form. Therefore, incorporation of this analogue into the amplified DNA seems to be the cause of this extreme compaction. Autoradiographic experiments and silver grains counting showed that this treatment decreases the efficiency of RNA synthesis at DNA puff anlages

Response of human HT-29 colorectal tumor cells to extended exposure to bromodeoxyuridine

Effects of the extended exposure of a human colorectal tumor-cell line (HT-29) to bromodeoxyuridine (BrdUrd) were studied in anticipation of the clinical use of that agent to treat colorectal cancer, particularly as a regionally delivered radiosensitizer. We found that 72-h exposure to a concentration of BrdUrd that is estimated to be locally maintained in the liver (100 μ M ) was significantly cytotoxic with a 3-log reduction in survival. As measured by GC/MS-SIM method, incorporation of BrdUrd into DNA followed an unexpected time course in that continuous exposure to 10 μ M BrdUrd resulted in maximal incorporation at 3 days, after which the extent of incorporated analog fell significantly (despite daily changes of the medium). This finding was apparently due to a greater rate of loss of BrdUrd from the medium at later time points. Flow cytometric analysis using an anti-BrdUrd antibody (IU-4) revealed that antibody binding also peaked and fell off with time. However, at exposure times of >24 h, the timing and extent of this decline were significantly different than had been indicated by the GC/MS method. These results indicate that the quantitative relationship between antibody staining and BrdUrd incorporation changes as drug-exposure time increases and that quantitative studies of anti-BrdUrd antibody binding must be interpreted with caution, especially when extended drug-treatment protocols have been used.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46921/1/280_2004_Article_BF00694337.pd

In situ reverse transcription: the magic of strength and anonymity

In this study, we describe an approach that enables a highly specific, effective and fast detection of polyadenylated RNA sequences in situ at the light and electron microscopy levels. The method developed is based on the incorporation of 5-bromo-2′-deoxyuridine into the growing cDNA strand by means of the reverse transcriptase. We have shown that unlike the previously used deoxyuridine tagged with biotin or digoxigenin, 5-bromo-2′-deoxyuridine is ‘invisible’ in the DNA–DNA duplex but easily detectable in the DNA–RNA duplex. This feature is an important pre-requisite for the correct interpretation of the data obtained, as our results strongly indicate that reverse transcriptase uses DNA breaks as primers efficiently. We have also shown that the replacement of deoxythymidine by 5-bromo-2′-deoxyuridine considerably stabilizes the growing DNA–RNA duplex, thus enabling the one-step detection of polyadenylated RNA in structurally well-preserved cells. The method developed provides a highly specific signal with the signal/noise ratio higher than 130 for permeabilized cells and 25 for conventional acrylic resin sections under the conditions used. When the high pressure freezing technique followed by the freeze substitution is employed for the cell's preparation, the ratio is higher than 80

Physicochemical characterization of immortal strand DNA

Thesis (Ph. D.)--Massachusetts Institute of Technology, Biological Engineering Division, 2004.Includes bibliographical references.Adult tissue differentiation involves the generation of distinct cell types from adult stem cells (ASCs). Current understanding of tissue differentiation mechanisms is based on studies of protein and RNAs that asymmetrically segregate between daughter cells during embryogenesis. Whether or not other types of biomolecules segregate asymmetrically has not been widely studied. In 1975, John Cairns proposed that ASCs preferentially segregate the oldest parental template DNA strands to themselves and pass on newly replicated DNA strands to their differentiating progeny in order to protect the stem cell from inheriting DNA replication mutations. This laboratory has shown non-random chromosome segregation in murine fetal fibroblasts that model asymmetric self-renewal like ASCs. In these cells, chromosomes that contain the oldest DNA strands co-segregate to the cycling daughter stem-like cells, while chromosomes with more recently replicated DNA segregate to the non-stem cell daughters. Previously, cytological methods were reported to elucidate non-random segregation in these cells. This dissertation research provides additional confirmation of the mechanism using physicochemical methods. Specifically, buoyant density-shift experiments in equilibrium CsCl density gradients were used to detect co-segregated "immortal DNA strands" based on incorporation of the thymidine base analogue bromodeoxyuridine. In addition, DNA from cells undergoing non-random mitotic chromosome segregation was analyzed for unique DNA base modifications and global structural modifications (by HPLC and melting temperature analyses). To date, these studies show no significant differences compared to control randomly segregated DNA. Components of the mitotic chromosome separation(cont.) apparatus that might play a role in the co-segregation mechanism were also evaluated. Two homologous proteins, essential for proper chromosome segregation and cytokinesis, Aurora A kinase and Aurora B kinase, were highly reduced in expression in cells retaining immortal DNA strands and may indicate a role for them in the immortal strand mechanism. These studies independently confirm the immortal strand mechanism and provide methods for its detection in other cell lines. In addition, observed changes in chromosome segregation proteins that are potential candidates for involvement in the mechanism have revealed a new area of investigation in the laboratory. These findings are relevant to understanding normal tissue development, cancer, and aging.y Janice A. Lansita.Ph.D

Cytogenetic and Molecular Analysis of the Channel Catfish (Ictalurus Punctatus) Genome.

This research presents studies on cytogenetics and molecular genetics of channel catfish (Ictalurus punctatus), the most important fish species cultured in the United States. The goals of this project were to analyze the genome of the channel catfish and to develop a direct method of mapping single-locus genes. A series of techniques were developed to facilitate these studies, including culture of fibroblast cells, preparation of chromosomes from catfish of different ages, staining for nucleolus organizer regions (NOR) and heterochromatin (C-banding), restriction enzyme banding, replication R-banding, simultaneous detection of sister-chromatid exchange (SCE) and C-banding, fluorescent in-situ hybridization (FISH) and indirect and direct in-situ polymerase chain reaction (ISPCR). A primary cell line was established from caudal fin tissue. The cell line was fibroblastic, and strongly positive for fibronectin and collagens type I and III in the cytoplasm. These cells maintained a normal karyotype after 42 generations and were positive for the channel catfish gene Ig H that codes for immunoglobulin. Individual chromosomes were identified by location of the NOR and C-banding, and by restriction enzyme and replication R-banding. The 29 chromosome pairs were divided into 8 distinct groups based on morphology and size. Standard C-banding and replication R-banding karyotypes were established, and ideograms were prepared for the first time for this species. A procedure for simultaneous detection of the SCE and C-banding was developed, which may allow measurement of the distance between exchange sites and centromeres. The baseline occurrence of SCE in the absence of mutagenic materials, were measured to be 3.6 $\pm$ 1.6% of chromosomes in each cell. In addition, FISH and ISPCR procedures were developed for analysis of the single-locus Ig H gene on whole-cell, nuclear, and chromosomal preparations. Two copies of the gene were revealed in each positive interphase nucleus. The chromosomal location of the Ig H gene was detected. However, the identity of the chromosome remains unknown because the banding pattern was not analyzable after hybridization. Application of the ISPCR in chromosomal mapping is new for fish species and is only in initial stages for higher vertebrates

In Situ Hybridisation for Human and Comparative Gene Mapping

The overall aim of this project was to develop the technique of in situ hybridisation (ISH) for the localisation of cloned DNA sequences on the Y and other chromosomes in man and other primates using both 3H- and biotin labelling. Using tritium labelling two problems were encountered and circumvented, high non-specific labelling and loss of chromosome morphology following hybridisation. Following this, seven anonymous DNA sequences cloned in plasmid vectors were localised to specific chromosome sites in the human: GMGY10 (DYS59) and GMGY7 (DYS58) to the short of the Y chromosome (Yp), P2F2 (DY25) to chromosome X band q21 and Yp, pY3.4 (DYZ1) to chromosome Y distal band Yq12, GMGY3 (DYS13) to Yp and chromosome 9 region p23-pter, GMGY4b (DYS51) to the pericentromeric region of chromosomes Y, 15, 21, 22, JG73 to pericentromeric region of chromosomes 19, 21, 22 , Y and (14, 15, and 20) and finally JG51 (D21S89) to the pericentromeric region of chromosomes 13, 14, 15, 20, 21, and 22. Under conditions of high stringency the last two probes were localised to chromosome 21, and chromosomes 13, 14 and 22 respectively, contrary to filter hybridisation data where these two sequences appear to be identical. Conventional in situ hybridisation techniques using radioactive probes suffer the serious disadvantages of prolonged autoradiographic exposure times and limited spatial resolution. To overcome these problems several non-isotopic methods have been introduced using a variety of different immunogenic, fluorescent and enzymatic labels. The possibility of using biotin-labelling for gene mapping was explored during this project using a recently described technique that employs a streptavidin-alkaline phosphatase detection system. The method was shown to be simple, reliable, rapid and sufficiently sensitive to detect single copy DNA sequences, as was indicated by the localisation of a 3.2kb DNA sequence, p72 (D21S92), to chromosome 21 proximal band q21. Results were obtained in 24 hours as compared with 1 week autoradiographic exposure for repetitive probe and 3 weeks for single copy probe. Another probe, GMGXY8 (DXYS34) was mapped to chromosome Yp. All probes mentioned above had been regionally mapped by Southern analysis using somatic cell hybrids. ISH not only confirmed but also extended the previous findings by indicating autosomal homologies for probes GMGY3, GMGY4b and regions of extended homology for JG73 and JG51. The homology between short arm of chromosomes Y and 9 revealed for probe GMGY3 is of special interest in view of the fact that GMGY3 resides in the sex-determining region of the Y chromosome. This finding coupled with observations that 9p monosomy is sometimes associated with anomalous sex differentiation could suggest a functional homology between Yp and distal 9p. ISH was also used to determine the chromosomal location of Y-specific sequences in eleven patients with paradoxical sex chromosome complements previously shown to possess such sequences. Eight XX males were studied with 3H-labelled GMGY10 and/or GMGY7 and in all cases it could be clearly demonstrated that these Y-specific sequences were located on distal short arm of the X chromosome (band Xp22.3) as predicted by the X-Y interchange hypothesis (Ferguson-Smith, 1966). The ninth XX male was investigated with probe P2F2 and although signal was recorded on the short arm of the X chromosome it could not be shown conclusively that this was due to an X-Y. interchange. Using GMGY10 or GMGY7 probes in two apparent XO males with an additional minute chromosomal fragment present in their peripheral blood lymphocytes it was demonstrated that the fragment had originated from a Y chromosome thus explaining the male phenotype of these individuals. Apart from its value in gene mapping studies, ISH has an enormous potential as a tool in clinical diagnosis. This was demonstrated in the present study by the confirmation of a suspected Yp:15q translocation in amniotic fluid cells and in paternal lymphocytes with a chromosome 15 polymorphism by using 3H-labelled probe pY3.4. The last part of the project involved comparative mapping in the gorilla (Gorilla gorilla), chimpanzee (Pan troglodytes) and orangutan (Pongo pygmaeus) of a human transfer RNA gene for glutamic acid (tRNA Glu ) and probes GMGY10 and GMGY7. The tRNA Glu gene was localised to distal long arm of chromosome 1 (band 1p36 according to ISCN nomenclature) in all three species, providing further evidence for homology between distal long arm of the ape chromosome 1 and distal short arm of human chromosome 1 where this gene has been previously localised

Activation of Stat3 Signaling in AgRP Neurons Promotes Locomotor Activity

Over the last years, much of the research on obesity has focused on the study of leptin. This adipocyte-derived hormone circulates in proportion to fat mass and functions as an adiposity signal to decrease energy intake and increase energy expenditure in order to maintain energy homeostasis. Leptin signals informations on body energy stores to hypothalamic neurons located in the arcuate nucleus (ARC) of the hypothalamus. One of the leptin-regulated neuronal subtypes in the ARC are the orexigenic agouti-related peptide (AgRP)-producing neurons, which are directly inhibited by leptin. A key pathway downstream of the leptin receptor involves activation of the signal transducer and activator of transcription 3 (Stat3), but the role of Stat3 in the regulation of AgRP neurons remains controversial. In this study, analysis of Stat3-CAgRP mice expressing a constitutively active version of the Stat3 protein (Stat3-C) selectively in AgRP neurons reveals a crucial role for Stat3 in AgRP neurons in the regulation of energy expenditure in vivo. Stat3-CAgRP mice are lean and develop a relative resistance to diet-induced obesity accompanied by improved glucose homeostasis. The lean phenotype of Stat3-CAgRP mice appears in the presence of unaltered AgRP expression and caloric intake as a consequence of increased energy expenditure evoked by elevated locomotor activity. Consistent with the phenotype observed in Stat3-CAgRP mice, expression of Stat3-C in AgRP neurons of leptin deficient ob/ob mice diminishes the obese phenotype of ob/ob mice as a result of increased energy expenditure and locomotor activity in the presence of unaltered food intake. Analysis of brain catecholamines in Stat3-CAgRP mice revealed a trend towards elevated dopamine concentrations in the striatum and frontal cortex, which potentially account for the increased locomotor activity in those mice. Nevertheless, the anatomical interaction of AgRP neurons with neuronal centers that control locomotor activity and the exact molecular mechanism in AgRP neurons leading to Stat3-dependent activation of locomotor activity have to be defined further. Taken together, this thesis introduces a novel model according to which leptin-stimulated Stat3 activation in AgRP neurons directly regulates locomotor activity independent of the regulation of AgRP mRNA expression