Understanding DNA Conformational Dynamics: Answering Questions and Questioning Answers

Abstract

Phosphorus-31 and especially Carbon-13 NMR measurements have recently become primary input to the understanding of DNA solution dynamics. While the ³¹P measurements are inherently easier, the quality of ³¹P dynamics information is suspect and therefore ¹³C measurements are preferred. In fact, it is necessary to obtain several kinds of ¹³C data (T₁s, NOE's, linewidths, integrated peak intensities) over a wide range of magnetic fields (¹³C NMR frequencies) in order to identify major features of DNA internal motions. Further information comes from variation of temperature and DNA fragment length and/or concentration. Most of our ¹³C measurements have been performed at 37.7–90.6 MHz on fully double stranded monomer size (147 base pair) DNA at concentrations in phosphate buffer of < 10 to > 200 mg ml^?1; temperatures studied range from 6 to 55°C. Other measurements have been performed on monomer-size single-stranded DNA at 85 and 92°.

The large data set we have acquired appears to answer some important questions about the nature and extent of DNA overall and internal motional dynamics. However, the picture remains incomplete and a number of questions arise from these results:

1. Overall motion of the double stranded DNA fragments follows expected hydrodynamic behavior;

2. Restricted but rapid internal motion along the DNA structure is well represented by a spaghetti-like wobbling-in-a-cone model;

3. DNA-DNA Interactions and solvent ordering, present at relatively low DNA concentrations, partially quench the internal motion, consistent with hinge-model structural changes (and the spaghetti model above) but not as compatible with in-plane torsional motion models;

4. The deoxyribose C-2′ sites undergo additional motion which is partially uncoupled from the internal wobbling motions;

5. At high DNA concentrations, a phase transition occurs, resulting in ordered structures which drastically affect DNA internal dynamics;

6. DNA interacting with ethidium does not greatly change its conformational mobility;

7. DNA interacting with Hg²⁺ ions shows less than anticipated change in internal DNA dynamics.

The remaining challenge is to interpret our current results in terms of specific conformational processes and to understand why the conformational mobility of double stranded DNA is relatively unhindered by major structural perturbants such as intercalating ethidium and mercury ion.     

The morphology and histology of the endocrine pancreas of the southern hemisphere lamprey, Geotria australis gray

The location and arrangement of the pancreatic endocrine tissue in larval and adult Geotria australis (Geotriidae) differ markedly from those exhibited by the comparable stages of Northern Hemisphere lampreys (Petromyzontidae). In larval Geotria australis, the main zones of islet proliferation are located laterally between the oesophagus and the inner edge of the two large intestinal diverticula unique to this species rather than dorsal and ventral to the oesophagus. In adult Geotria australis, the islet follicles are closely packed into a single discrete capsule which could be easily removed surgically, rather than into cranial, intermediate, and caudal cords. The differences in the adult can be related to a lack of involvement of the bile duct in islet formation during metamorphosis. While B cells were found in both larval and adult islet follicles, the PI acidophilic cells and argyrophilic cells, which appeared respectively at stages 3 and 4 in metamorphosis, were present in all adult stages.     

Chromosome-scale scaffolds for the Chinese hamster reference genome assembly to facilitate the study of the CHO epigenome

The Chinese hamster genome serves as a reference genome for the study of Chinese hamster ovary (CHO) cells, the preferred host system for biopharmaceutical production. Recent re-sequencing of the Chinese hamster genome resulted in the RefSeq PICR meta-assembly, a set of highly accurate scaffolds that filled over 95% of the gaps in previous assembly versions. However, these scaffolds did not reach chromosome-scale due to the absence of long-range scaffolding information during the meta-assembly process. Here, long-range scaffolding of the PICR Chinese hamster genome assembly was performed using high-throughput chromosome conformation capture (Hi-C). This process resulted in a new “PICRH” genome, where 97% of the genome is contained in 11 mega-scaffolds corresponding to the Chinese hamster chromosomes (2n = 22) and the total number of scaffolds is reduced by three-fold from 1,830 scaffolds in PICR to 647 in PICRH. Continuity was improved while preserving accuracy, leading to quality scores higher than recent builds of mouse chromosomes and comparable to human chromosomes. The PICRH genome assembly will be an indispensable tool for designing advanced genetic engineering strategies in CHO cells and enabling systematic examination of genomic and epigenomic instability through comparative analysis of CHO cell lines on a common set of chromosomal coordinates.     

Tight regulation of p53 activity by Mdm2 is required for ureteric bud growth and branching

Mdm2 (Murine Double Minute-2) is required to control cellular p53 activity and protein levels. Mdm2 null embryos die of p53-mediated growth arrest and apoptosis at the peri-implantation stage. Thus, the absolute requirement for Mdm2 in organogenesis is unknown. This study examined the role of Mdm2 in kidney development, an organ which develops via epithelial–mesenchymal interactions and branching morphogenesis. Mdm2 mRNA and protein are expressed in the ureteric bud (UB) epithelium and metanephric mesenchyme (MM) lineages. We report here the results of conditional deletion of Mdm2 from the UB epithelium. UB^mdm2−/− mice die soon after birth and uniformly display severe renal hypodysplasia due to defective UB branching and underdeveloped nephrogenic zone. Ex vivo cultured UB^mdm2−/− explants exhibit arrested development of the UB and its branches and consequently develop few nephron progenitors. UB^mdm2−/− cells have reduced proliferation rate and enhanced apoptosis. Although markedly reduced in number, the UB tips of UB^mdm2−/−metanephroi continue to express c-ret and Wnt11; however, there was a notable reduction in Wnt9b, Lhx-1 and Pax-2 expression levels. We further show that the UB^mdm2−/− mutant phenotype is mediated by aberrant p53 activity because it is rescued by UB-specific deletion of the p53 gene. These results demonstrate a critical and cell autonomous role for Mdm2 in the UB lineage. Mdm2-mediated inhibition of p53 activity is a prerequisite for renal organogenesis.     

TNF-α converting enzyme-mediated ErbB4 transactivation by TNF promotes colonic epithelial cell survival

Disruption of intestinal epithelial homeostasis, including enhanced apoptosis, is a hallmark of inflammatory bowel disease (IBD). We have recently shown that tumor necrosis factor (TNF) increases the kinase activity of ErbB4, a member of the epidermal growth factor receptor family that is elevated in mucosa of IBD patients and that promotes colon epithelial cell survival. In this study, we tested the hypothesis that TNF transactivates ErbB4 through TNF-α converting enzyme (TACE)-mediated ligand release and that this transactivation is necessary to protect colonic epithelial cells from cytokine-induced apoptosis. Using neutralizing antibodies, we show that heparin-binding EGF-like growth factor (HB-EGF) is required for ErbB4 phosphorylation in response to TNF. Pharmacological or genetic inhibition of the metalloprotease TACE, which mediates HB-EGF release from cells, blocked TNF-induced ErbB4 activation. MEK, but not Src or p38, was also required for transactivation. TACE activity and ligand binding were required for ErbB4-mediated antiapoptotic signaling; whereas mouse colon epithelial cells expressing ErbB4 were resistant to TNF-induced apoptosis, TACE inhibition or blockade of ErbB4 ligand binding reversed the survival advantage. We conclude that TNF transactivates ErbB4 through TACE-dependent HB-EGF release, thus protecting colon epithelial cells from cytokine-induced apoptosis. These findings have important implications for understanding how ErbB4 protects the colon from apoptosis-induced tissue injury in inflammatory conditions such as IBD.