Morphological heterogeneity of HeLa cell mitochondria visualized by a modified diaminobenzidine staining technique

The diaminobenzidine (DAB) technique for the ultrastructural localization of sites of cytochrome c oxidase activity in animal tissues has been adapted to the visualization of mitochondria in animal cells growing in culture. The modified technique allows the staining of mitochondria in all cells in coverslip preparatins for light microscopy. Electron microscopy of thin sections of material treated by this method has revealed that all mitochondrial profiles within a cell (and only these) are stained and they exhibit a well preserved size and internal structure. Coverslip cultures of synchronized and unsynchronized HeLa (F-315) cells stained with the DAB reaction were examined under oil immersion. In the majority of the cells, mitochondria were recognized as discrete bodies in the thinner peripheral portion of the cytoplasm. This observation indicates that in a large proportion of HeLa F-315 cells, at least under the growth conditions used here, the mitochondrial complement is dividied into distinct organelles. This examination also revealed a considerable morphological heterogeneity of mitochondria, which exhibited an ovoid or short rod-like or, less frequently, long filamentous shape, with some evidence of branching. The variability in mitochondrial morphology appeared to be far more prounced between different cells than within individual cells; this cellular heterogeneity was not related in any obvious way to cell-cycle-dependent changes

Apropriate Ultrasonic System Components for NDE of Thick Polymer-Composites

In certain marine applications, thick polymer-composite materials may have to endure different operating environments than those experienced in traditional aerospace applications. In particular, structures made of such materials may experience very large compressive and bending forces. To prevent in-service failure, appropriate NDE methods and instrumentation are needed to characterize the state of the material. Specifically, in addition to detecting high-contrast anomalies (cracks and delaminations) it may be of interest to determine the pore content, measure the fiber volume, assess the severity of fiber waviness, and the like [1]

Drosophila microRNAs 263a/b Confer Robustness during Development by Protecting Nascent Sense Organs from Apoptosis

microRNA-263a/b confer robustness to sense organ development by controlling expression of the pro-apoptotic gene hid during apoptotic tissue pruning in Drosophila

C. elegans ATAD-3 Is Essential for Mitochondrial Activity and Development

Contains fulltext : 80701.pdf (publisher's version ) (Open Access)BACKGROUND: Mammalian ATAD3 is a mitochondrial protein, which is thought to play an important role in nucleoid organization. However, its exact function is still unresolved. RESULTS: Here, we characterize the Caenorhabditis elegans (C. elegans) ATAD3 homologue (ATAD-3) and investigate its importance for mitochondrial function and development. We show that ATAD-3 is highly conserved among different species and RNA mediated interference against atad-3 causes severe defects, characterized by early larval arrest, gonadal dysfunction and embryonic lethality. Investigation of mitochondrial physiology revealed a disturbance in organellar structure while biogenesis and function, as indicated by complex I and citrate synthase activities, appeared to be unaltered according to the developmental stage. Nevertheless, we observed very low complex I and citrate synthase activities in L1 larvae populations in comparison to higher larval and adult stages. Our findings indicate that atad-3(RNAi) animals arrest at developmental stages with low mitochondrial activity. In addition, a reduced intestinal fat storage and low lysosomal content after depletion of ATAD-3 suggests a central role of this protein for metabolic activity. CONCLUSIONS: In summary, our data clearly indicate that ATAD-3 is essential for C. elegans development in vivo. Moreover, our results suggest that the protein is important for the upregulation of mitochondrial activity during the transition to higher larval stages

A Cis-Regulatory Map of the Drosophila Genome

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide1, 2 has successfully identified specific subtypes of regulatory elements3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements4, chromatin states5, transcription factor binding sites6, 7, 8, 9, RNA polymerase II regulation8 and insulator elements10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships

A Machine Learning Approach for Identifying Novel Cell Type–Specific Transcriptional Regulators of Myogenesis

Transcriptional enhancers integrate the contributions of multiple classes of transcription factors (TFs) to orchestrate the myriad spatio-temporal gene expression programs that occur during development. A molecular understanding of enhancers with similar activities requires the identification of both their unique and their shared sequence features. To address this problem, we combined phylogenetic profiling with a DNA–based enhancer sequence classifier that analyzes the TF binding sites (TFBSs) governing the transcription of a co-expressed gene set. We first assembled a small number of enhancers that are active in Drosophila melanogaster muscle founder cells (FCs) and other mesodermal cell types. Using phylogenetic profiling, we increased the number of enhancers by incorporating orthologous but divergent sequences from other Drosophila species. Functional assays revealed that the diverged enhancer orthologs were active in largely similar patterns as their D. melanogaster counterparts, although there was extensive evolutionary shuffling of known TFBSs. We then built and trained a classifier using this enhancer set and identified additional related enhancers based on the presence or absence of known and putative TFBSs. Predicted FC enhancers were over-represented in proximity to known FC genes; and many of the TFBSs learned by the classifier were found to be critical for enhancer activity, including POU homeodomain, Myb, Ets, Forkhead, and T-box motifs. Empirical testing also revealed that the T-box TF encoded by org-1 is a previously uncharacterized regulator of muscle cell identity. Finally, we found extensive diversity in the composition of TFBSs within known FC enhancers, suggesting that motif combinatorics plays an essential role in the cellular specificity exhibited by such enhancers. In summary, machine learning combined with evolutionary sequence analysis is useful for recognizing novel TFBSs and for facilitating the identification of cognate TFs that coordinate cell type–specific developmental gene expression patterns

Sequence relationship between long and short repetitive DNA of the rat: a preliminary report.

Long and short repetitive sequences of rat DNA can be isolated and characterized. Long [greater than 1.5 kilobases (kb)] sequences can be separated from short (0.2-0.4 kb) sequences by exclusion chromatography after renaturation of 4-kb DNA fragments to a repetitive Cot and digestion with the single-strand-specific S1 nuclease. (Cot is the initial concentration of DNA in mol of nucleotides/liter multiplied by time in sec.) Long repetitive DNA can be driven by an excess of whole rat DNA can also be used to drive tracer quantities of either long (self-renaturation) or short repetitive DNA. Both the extent and the rate of the renaturations are found to be similar, suggesting that long and short DNA fragments share sequences. When long repetitive DNA is used to drive whole DNA tracers of various lengths, a 3.2-kb interspersion period is found. These data are consistent with the concept that short repetitive sequences are present within long repetitive DNA sequences in the rat genome

Comparison of sea urchin and human mtDNA: evolutionary rearrangement.

Clones of full-length mtDNA have been isolated from a Strongylocentrotus franciscanus recombinant DNA library by screening a cDNA clone of cytochrome oxidase subunit 1 mRNA. Restriction fragment cross-hybridization analysis shows the following difference in gene arrangement between sea urchin and human mtDNA. The 16S rRNA and cytochrome oxidase subunit 1 genes are directly adjacent in sea urchin mtDNA. These two genes are separated in human and other mammalian mtDNAs by the region containing unidentified reading frames 1 and 2. In spite of the difference in gene order, gene polarity appears to have been conserved. We conclude that the difference in gene order reflects a rearrangement that took place in the sea urchin lineage since sea urchins and mammals last shared a common ancestor