DNA construction: homemade or ordered out?

Automation and optimization of DNA construction results in the efficient production of large target sequences.     

Genome-wide studies of histone demethylation catalysed by the fission yeast homologues of mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1(+) gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1Delta strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression.     

Rapid method for measuring ScFv thermal stability by yeast surface display

We have characterized a simplified method to determine the relative thermal stability of single-chain antibodies by following the irreversible denaturation of scFv fusions on the surface of yeast by flow cytometry. The method was highly reproducible and correlated well with other methods used to monitor thermal denaturation of the soluble proteins. We found a range of thermal stabilities for wild-type single-chain antibodies with half-maximum denaturation temperatures between 43 and 61 degrees C. The ability to quantitate thermal stability of antibodies or other proteins that are immobilized on the surface of yeast allows rapid comparisons of primary structural information with stability. Thermal denaturation could be a useful parameter to consider in the choice of scFv fragments for various applications.     

ADP-ribosylation of glutamine synthetase in the cyanobacterium Synechocystis sp. strain PCC 6803.

Glutamine synthetase (GS) inactivation was observed in crude cell extracts and in the high-speed supernatant fraction from the cyanobacterium Synechocystis sp. strain PCC 6803 following the addition of ammonium ions, glutamine, or glutamate. Dialysis of the high-speed supernatant resulted in loss of inactivation activity, but this could be restored by the addition of NADH, NADPH, or NADP+ and, to a lesser extent, NAD+, suggesting that inactivation of GS involved ADP-ribosylation. This form of modification was confirmed both by labelling experiments using [32P]NAD+ and by chemical analysis of the hydrolyzed enzyme. Three different forms of GS, exhibiting no activity, biosynthetic activity only, or transferase activity only, could be resolved by chromatography, and the differences in activity were correlated with the extent of the modification. Both biosynthetic and transferase activities were restored to the completely inactive form of GS by treatment with phosphodiesterase.     

Microarray Data Reveal Relationship between Jag1 and Ddr1 in Mouse Liver

Alagille syndrome is an autosomal dominant disorder involving bile duct paucity and cholestasis in addition to cardiac, skeletal, ophthalmologic, renal and vascular manifestations. Mutations in JAG1, encoding a ligand in the Notch signaling pathway, are found in 95% of patients meeting clinical criteria for Alagille syndrome. In order to define the role of Jag1 in the bile duct developmental abnormalities seen in ALGS, we previously created a Jag1 conditional knockout mouse model. Mice heterozygous for the Jag1 conditional and null alleles demonstrate abnormalities in postnatal bile duct growth and remodeling, with portal expansion and increased numbers of malformed bile ducts. In this study we report the results of microarray analysis and identify genes and pathways differentially expressed in the Jag1 conditional/null livers as compared with littermate controls. In the initial microarray analysis, we found that many of the genes up-regulated in the Jag1 conditional/null mutant livers were related to extracellular matrix (ECM) interactions, cell adhesion and cell migration. One of the most highly up-regulated genes was Ddr1, encoding a receptor tyrosine kinase (RTK) belonging to a large RTK family. We have found extensive co-localization of Jag1 and Ddr1 in bile ducts and blood vessels in postnatal liver. In addition, co-immunoprecipitation data provide evidence for a novel protein interaction between Jag1 and Ddr1. Further studies will be required to define the nature of this interaction and its functional consequences, which may have significant implications for bile duct remodeling and repair of liver injury.     

Phylogeographic mitogenomics of Atlantic cod Gadus morhua: Variation in and among trans‐Atlantic,trans‐Laurentian,Northern cod,and landlocked fjord populations

The historical phylogeography, biogeography, and ecology of Atlantic cod (Gadus morhua) have been impacted by cyclic Pleistocene glaciations, where drops in sea temperatures led to sequestering of water in ice sheets, emergence of continental shelves, and changes to ocean currents. High‐resolution, whole‐genome mitogenomic phylogeography can help to elucidate this history. We identified eight major haplogroups among 153 fish from 14 populations by Bayesian, parsimony, and distance methods, including one that extends the species coalescent back to ca. 330 kya. Fish from the Barents and Baltic Seas tend to occur in basal haplogroups versus more recent distribution of fish in the Northwest Atlantic. There was significant differentiation in the majority of trans‐Atlantic comparisons (Φ_ST = .029–.180), but little or none in pairwise comparisons within the Northwest Atlantic of individual populations (Φ_ST = .000–.060) or defined management stocks (Φ_ST = .000–.023). Monte Carlo randomization tests of population phylogeography showed significantly nonrandom trans‐Atlantic phylogeography versus absence of such structure within various partitions of trans‐Laurentian, Northern cod (NAFO 2J3KL) and other management stocks, and Flemish Cap populations. A landlocked meromictic fjord on Baffin Island comprised multiple identical or near‐identical mitogenomes in two major polyphyletic clades, and was significantly differentiated from all other populations (Φ_ST = .153–.340). The phylogeography supports a hypothesis of an eastern origin of genetic diversity ca. 200–250 kya, rapid expansion of a western superhaplogroup comprising four haplogroups ca. 150 kya, and recent postglacial founder populations.     

High-resolution solution structure of human intestinal trefoil factor and functional insights from detailed structural comparisons with the other members of the trefoil family of mammalian cell motility factors

The secreted proteins intestinal trefoil factor (ITF, 59 residues), pS2 (60 residues), and spasmolytic polypeptide (SP, 106 residues) form a small family of trefoil domain-containing mammalian cell motility factors, which are essential for the maintenance of all mucous-coated epithelial surfaces. We have used 1H NMR spectroscopy to determine the high-resolution structure of human ITF, which has allowed detailed structural comparisons with the other trefoil cell motility factors. The conformation of residues 10-53 of hITF is determined to high precision, but the structure of the N- and C-terrminal residues is poorly defined by the NMR data, which is probably indicative of significant mobility. The core of the trefoil domain in hITF consists of a two-stranded antiparallel beta-sheet (Cys 36 to Asp 39 and Trp 47 to Lys 50), which is capped by an irregular loop and forms a central hairpin (loop 3). The beta-sheet is preceded by a short alpha-helix (Lys 29 to Arg 34), with the majority of the remainder of the domain contained in two loops formed from His 25 to Pro 28 (loop 2) and Ala 12 to Arg 18 (loop 1), which lie on either side of the central hairpin. The region formed by the surface of loop 2, the cleft between loop 2 and loop 3, and the adjacent face of loop 3 has previously been proposed to form the functional site of trefoil domains. Detailed comparisons of the backbone conformations and surface features of the family of trefoil cell motility factors (porcine SP, pS2, and hITF) have identified significant structural and electrostatic differences in the loop 2/loop 3 regions, which suggest that each trefoil protein has a specific target or group of target molecules.