Genome Duplications and Other Features in 12 Mb of DNA Sequence from Human Chromosome 16p and 16q

Several publicly funded large-scale sequencing efforts have been initiated with the goal of completing the first reference human genome sequence by the year 2005. Here we present the results of analysis of 11.8 Mb of genomic sequence from chromosome 16. The apparent gene density varies throughout the region, but the number of genes predicted (84) suggests that this is a gene-poor region. This result may also suggest that the total number of human genes is likely to be at the lower end of published estimates. One of the most interesting aspects of this region of the genome is the presence of highly homologous, recently duplicated tracts of sequence distributed throughout the p-arm. Such duplications have implications for mapping and gene analysis as well as the predisposition to recurrent chromosomal structural rearrangements associated with genetic disease.     

Plastic responses of survival and fertility following heat stress in pupal and adult Drosophila virilis

The impact of rising global temperatures on survival and reproduction is putting many species at risk of extinction. In particular, it has recently been shown that thermal effects on reproduction, especially limits to male fertility, can underpin species distributions in insects. However, the physiological factors influencing fertility at high temperatures are poorly understood. Key factors that affect somatic thermal tolerance such as hardening, the ability to phenotypically increase thermal tolerance after a mild heat shock, and the differential impact of temperature on different life stages are largely unexplored for thermal fertility tolerance. Here, we examine the impact of high temperatures on male fertility in the cosmopolitan fruit fly Drosophila virilis. We first determined whether temperature stress at either the pupal or adult life history stage impacts fertility. We then tested the capacity for heat‐hardening to mitigate heat‐induced sterility. We found that thermal stress reduces fertility in different ways in pupae and adults. Pupal heat stress delays sexual maturity, whereas males heated as adults can reproduce initially following heat stress, but become sterile within seven days. We also found evidence that while heat‐hardening in D. virilis can improve high temperature survival, there is no significant protective impact of this same hardening treatment on fertility. These results suggest that males may be unable to prevent the costs of high temperature stress on fertility through heat‐hardening, which limits a species’ ability to quickly and effectively reduce fertility loss in the face of short‐term high temperature events.     

Progesterone stimulates DNA synthesis and lobulo-alveolar development in mammary glands in ovariectomized mice

The objective of this study was to determine whether sustained progesterone (P) use in the absence of estrogen could influence mammary development in mice. Three-week-old intact or ovariectomized mice were primed with subcutaneous (s.c.) cholesterol (C), estrogen (E), P, or estrogen and progesterone (E/P) together. Nine days after priming, mammary glands were removed and incubated as a whole organ in media supplemented with various combinations of lactogenic hormones. After 5 days in whole organ culture, glands were removed and end buds, alveolar buds and lobulo-alveoli were quantified. Glands from mice primed with C or E developed significantly less lobulo-alveoli than glands from mice primed with P or E/P. While the development was greater in animals treated with E/P compared to those treated with P, it was clear that P in the absence of E could still induce lobulo-alveolar development. We have shown in this paper that P, in the absence of E, can stimulate cell proliferation during priming. Subsequently, the P primed glands can differentiate in response to lactogenic hormones. J. Cell. Physiol. 180:298–304, 1999. © 1999 Wiley-Liss, Inc.     

The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis

Endothelial cells play essential roles in maintenance of vascular integrity, angiogenesis, and wound repair. We show that an endothelial cell-restricted microRNA (miR-126) mediates developmental angiogenesis in vivo. Targeted deletion of miR-126 in mice causes leaky vessels, hemorrhaging, and partial embryonic lethality, due to a loss of vascular integrity and defects in endothelial cell proliferation, migration, and angiogenesis. The subset of mutant animals that survives displays defective cardiac neovascularization following myocardial infarction. The vascular abnormalities of miR-126 mutant mice resemble the consequences of diminished signaling by angiogenic growth factors, such as VEGF and FGF. Accordingly, miR-126 enhances the proangiogenic actions of VEGF and FGF and promotes blood vessel formation by repressing the expression of Spred-1, an intracellular inhibitor of angiogenic signaling. These findings have important therapeutic implications for a variety of disorders involving abnormal angiogenesis and vascular leakage.     

Synthesis and immunostimulatory properties of the phosphorothioate analogues of cdiGMP

The synthesis of mono- and bisphosphorothioate analogues of 3',5'-cyclic diguanylic acid (cdiGMP) via the modified H-phosphonate chemistry is reported. The immunostimulatory properties of these analogues were compared with those of cdiGMP.     

Intramolecular Interactions Between the Src Homology 3 Guanylate Kinase Domains of Discs Large Regulate Its Function in Asymmetric Cell Division

Membrane-associated guanylate kinases (MAGUKs) regulate the formation and function of molecular assemblies at specialized regions of the membrane. Allosteric regulation of an intramolecular interaction between the Src homology 3 (SH3) and guanylate kinase (GK) domains of MAGUKs is thought to play a central role in regulating MAGUK function. Here we show that a mutant of the Drosophila MAGUK Discs large (Dlg), dlg^sw, encodes a form of Dlg that disrupts the intramolecular association while leaving the SH3 and GK domains intact, providing an excellent model system to assess the role of the SH3-GK intramolecular interaction in MAGUK function. Analysis of asymmetric cell division of maternal-zygotic dlg^sw embryonic neuroblasts demonstrates that the intramolecular interaction is not required for Dlg localization but is necessary for cell fate determinant segregation to the basal cortex and mitotic spindle alignment with the cortical polarity axis. These defects ultimately result in improper patterning of the embryonic central nervous system. Furthermore, we demonstrate that the sw mutation of Dlg results in unregulated complex assembly as assessed by GukHolder association with the SH3-GK versus PDZ-SH3-GK modules of Dlg^sw. From these studies, we conclude that allosteric regulation of the SH3-GK intramolecular interaction is required for regulation of MAGUK function in asymmetric cell division, possibly through regulation of complex assembly.The membrane-associated guanylate kinase (MAGUK)² superfamily consists of ubiquitous scaffolding proteins that are composed of a common core of contiguously linked modular domains (protein-protein interaction domains, PDZ and SH3 domains, and a domain with homology to the yeast guanylate kinase, GK domain). MAGUKs are concentrated at sites of cell-cell contact (1) and organize a variety of cell adhesion molecules, cytoskeletal proteins, receptors, ion channels, and associated signaling molecules at specialized regions of the membrane (2). Protein complex organization by MAGUKs has been thought to occur, at least in part, through allosteric regulation that arises from an intramolecular interaction between the SH3 and GK domains. This interaction has been shown to regulate binding of numerous MAGUK ligands in vitro (3–5). However, whereas the SH3-GK interaction has been extensively characterized biochemically (6–8), very little data exists regarding its physiological relevance. Here we investigate the role of the SH3-GK intramolecular interaction of Discs large (Dlg), a prototype of the MAGUK superfamily.Loss of dlg activity results in overgrowth of imaginal discs and tumor formation (9). Dlg localizes to septate and neuromuscular junctions and is essential for establishing and maintaining apicobasal polarity of Drosophila epithelia (10). Dlg also plays an important role in regulating the process of asymmetric cell division (ACD) (11–13). ACD is a mechanism for generating cellular diversity via unequal mitotic divisions of progenitor cells. For instance, in wild-type Drosophila neuroblasts, cell signaling networks interact to allow for asymmetric segregation of basal cell fate determinants, followed by alignment of the mitotic spindle along the apical-basal cortical polarity axis (see Refs. 14 and 15) for review). ACD results in the formation of a self-renewing, stem-cell like neuroblast and a smaller ganglion mother cell, which has neuronal or glial fate. In dlg germline clone (dlg^GLC) embryonic metaphase neuroblasts, ganglion mother cell fate determinants are not restricted to the basal cortex (11, 12) and the mitotic spindle does not reliably align with the apical-basal cortical polarity axis (13). Defects in neurogenesis have also been observed for dlg^GLC embryos (16), as well as embryos treated with RNA interference against an alternatively spliced isoform of Dlg (17), providing evidence for the function of Dlg in neuronal differentiation and axon guidance. Such defects in neurogenesis are thought to be attributed to defective localization of basal cell fate determinants during ACD (17). Although Dlg function is important in a broad range of dynamic cellular processes, the role of the Dlg SH3-GK intramolecular interaction in Dlg activity is poorly understood.One potential role for the SH3-GK intramolecular association is to regulate MAGUK complex assembly. In vitro binding assays demonstrated that mutations disrupting this intramolecular interaction allowed mutant SH3-GK modules to associate with SH3 or GK domains of various MAGUK proteins in trans, providing a mechanism of regulating oligomerization of MAGUKs in vivo (6, 8). A role in clustering of ion channels was also observed as mutations that disrupted the intramolecular association, whereas having no effect on association with the potassium channel K_V1.4 or homo-oligomerization of PSD-95, resulted in loss of channel clustering in vivo (18). Furthermore, multiple in vitro studies support its regulation of binding of protein ligands with the GK domain of MAGUKs: examples include interaction of GK domains of Dlg with GukHolder (GukH) (3), SAP97 with guanylate kinase-associated protein (4), and PSD-93 with the microtubule-associated protein 1A (5). These studies suggest that allosteric modulation of the SH3-GK intramolecular interaction is important for regulation of complex assembly, yet little evidence exists that such regulation of MAGUKs is required for their function in vivo.In crystal structures of the SH3-GK module of PSD-95, two β-strands that emerge from the SH3 and GK domains appear to mediate the interaction between the domains (Fig. 1A) (19, 20). Functional studies of the interaction of these β-strands demonstrate that a COOH-terminal truncation of the strand following the GK domain abrogates the intramolecular association of the SH3-GK module. This 13-amino acid truncation of PSD-95 is comparable with a dlg allele that had previously been identified in a genetic screen, dlg^sw (21). Drosophila containing only the sw form of Dlg (refer to COOH-terminal sequences in Fig. 1B) die in the latter stages of embryonic development due to failure of dorsal closure and terminal defects (21). We hypothesized that this mutation disrupts the intramolecular interaction while leaving the SH3 and GK domains largely intact, making the dlg^sw allele an excellent model system for assessing the role of this interaction in MAGUK function.Open in a separate windowFIGURE 1.The dlg^sw allele disrupts the SH3-GK intramolecular interaction formed by interacting E and Fβ-strands. A, a ribbon diagram of the SH3-GK module of PSD-95 (Protein Data Bank 1KJW (19)) created using MOLSCRIPT (58) and RASTER3D (59), highlighting the SH3 (red), HOOK (gray), and GK (blue) domains. The intramolecular interaction between the SH3 and GK domains is mediated by the interaction of two β-strands (shown in black), one strand is contributed by the linker region following the SH3 domain (strand E), whereas the second strand is contributed by a COOH-terminal strand that follows the GK domain (strand F). B, sequence comparison of the COOH-terminal regions of WT Dlg versus Dlg^sw. C, GST pull-downs of the Dlg SH3 domain using GST fusions of WT and mutant Dlg GK domains. Association of protein components was detected by an anti-His antibody that recognized an NH₂-terminal on the SH3 domain of Dlg. D, circular dichroism wavelength scans, monitoring the change in ellipticity as a function of wavelength. Changes in α-helical content are indicated by the change in ellipticity at 222 nm (see black dashed line). Comparison of CD spectra of WT PDZ-SH3-GK (green circles) and PDZ-SH3-GK(sw) (red squares) indicate no significant change in α helical character. E, Sedfit analysis of sedimentation velocity scans of WT PDZ-SH3-GK (green solid line) and PDZ-SH3-GK^sw (red dashed line). Note that the estimated molecular weights provided are based upon the assumption that the protein exists in a globular conformation.In the studies presented here, we utilized the dlg^sw mutant allele to explore the role of the intramolecular interaction in regulating complex assembly and furthermore, to assess the in vivo, physiological significance of this interaction for Dlg function in neuroblast asymmetric cell division. We find that disruption of the SH3-GK intramolecular interaction results in unregulated complex assembly. Such a loss in regulated complex assembly results in loss of Dlg function in the process of asymmetric cell division as assessed by localization of cell fate determinants in a dividing neuroblast and alignment of the mitotic spindle along the apical-basal cortical polarity axis, ultimately resulting in defects in neurogenesis. Thus, these data contribute to our understanding of how the SH3-GK intramolecular interaction regulates MAGUK function in formation and regulation of membrane specializations.     

Curcumin modulates drug metabolizing enzymes in the female Swiss Webster mouse

Curcumin, the yellow pigment found in turmeric, exhibits potent chemopreventative properties in both in vivo and in vitro cancer models. We hypothesized that this effect may occur via curcumin-mediated changes in enzymes involved in both carcinogen bioactivation and estrogen metabolism. Female Swiss Webster mice were treated with either curcumin (200 mg/kg or 400 mg/kg, p.o.) or vehicle control for 1 or 2 weeks. The results demonstrated that curcumin had no effect on the catalytic activities of ovarian aromatase, hepatic catechol-O-methyltransferase or hepatic UDP-glucuronosyltransferase. However, both doses of curcumin caused a 25% decrease in CYP1A catalytic activity, but not polypeptide levels, following 2 weeks of treatment. Additionally, following 2 weeks of curcumin at 400 mg/kg, there was a 20% decrease in the catalytic activity and a 28% decrease in polypeptide levels of CYP3A. While 2 weeks of curcumin treatment (400 mg/kg) caused a 20% increase in glutathione S-transferase activity, there was no parallel increase in hepatic stores of the co-factor glutathione. In conclusion small changes in CYP1A, CYP3A and GST following long term treatment (2 weeks) suggest that the combination of all three metabolic pathways may play a small role in curcumin's chemopreventative action.     

Optimization of primer-specific filter metrics for the assessment of mitochondrial DNA sequence data

Filter metrics are used as a quick assessment of sequence trace files in order to sort data into different categories (i.e. high quality, review, and low quality) without human intervention. The filter metrics consist of two numerical parameters for sequence quality assessment: trace score (TS) and contiguous read length (CRL). Primer-specific settings for the TS and CRL were established using a calibration dataset of 2817 traces and validated using a concordance dataset of 5617 traces. Prior to optimization, 57% of the traces required manual review before import into a sequence analysis program, whereas after optimization only 28% of the traces required manual review. After optimization of primer-specific filter metrics for mitochondrial DNA sequence data, an overall reduction of review of trace files translates into increased throughput of data analysis and decreased time required for manual review.