Transgene Coplacement and High Efficiency Site-Specific Recombination with the Cre/Loxp System in Drosophila

Studies of gene function and regulation in transgenic Drosophila are often compromised by the possibility of genomic position effects on gene expression. We have developed a method, called transgene coplacement, in which any two sequences can be positioned at exactly the same site and orientation in the genome. Transgene coplacement makes use of the bacteriophage P1 system of Cre/loxP site-specific recombination, which we have introduced into Drosophila. In the presence of a cre transgene driven by a dual hsp70-Mos1 promoter, a white reporter gene flanked by loxP sites is excised with virtually 100% efficiency both in somatic cells and in germ cells. A strong maternal effect, resulting from Cre recombinase present in the oocyte, is observed as white or mosaic eye color in F(1) progeny. Excision in germ cells of the F(1) yields a strong grand-maternal effect, observed as a highly skewed ratio of eye-color phenotypes in the F(2) generation. The excision reactions of Cre/loxP and the related FLP/FRT system are used to create Drosophila lines in which transgenes are at exactly allelic sites in homologous chromosomes.     

The unexpected long period of elevated CH4 emissions from an inundated fen meadow ended only with the occurrence of cattail (Typha latifolia)

Drainage and agricultural use transform natural peatlands from a net carbon (C) sink to a net C source. Rewetting of peatlands, despite of high methane (CH₄) emissions, holds the potential to mitigate climate change by greatly reducing CO₂ emissions. However, the time span for this transition is unknown because most studies are limited to a few years. Especially, nonpermanent open water areas often created after rewetting, are highly productive. Here, we present 14 consecutive years of CH₄ flux measurements following rewetting of a formerly long-term drained peatland in the Peene valley. Measurements were made at two rewetted sites (non-inundated vs. inundated) using manual chambers. During the study period, significant differences in measured CH₄ emissions occurred. In general, these differences overlapped with stages of ecosystem transition from a cultivated grassland to a polytrophic lake dominated by emergent helophytes, but could also be additionally explained by other variables. This transition started with a rapid vegetation shift from dying cultivated grasses to open water floating and submerged hydrophytes and significantly increased CH₄ emissions. Since 2008, helophytes have gradually spread from the shoreline into the open water area, especially in drier years. This process was periodically delayed by exceptional inundation and eventually resulted in the inundated site being covered by emergent helophytes. While the period between 2009 and 2015 showed exceptionally high CH₄ emissions, these decreased significantly after cattail and other emergent helophytes became dominant at the inundated site. Therefore, CH₄ emissions declined only after 10 years of transition following rewetting, potentially reaching a new steady state. Overall, this study highlights the importance of an integrative approach to understand the shallow lakes CH₄ biogeochemistry, encompassing the entire area with its mosaic of different vegetation forms. This should be ideally done through a study design including proper measurement site allocation as well as long-term measurements.     

A review of methods to trace material flows into final products in dynamic material flow analysis: From industry shipments in physical units to monetary input–output tables,Part 1

Dynamic material flow analysis (dMFA) is widely used to model stock-flow dynamics. To appropriately represent material lifetimes, recycling potentials, and service provision, dMFA requires data about the allocation of economy-wide material consumption to different end-use products or sectors, that is, the different product stocks, in which material consumption accumulates. Previous estimates of this allocation only cover few years, countries, and product groups. Recently, several new methods for estimating end-use product allocation in dMFA were proposed, which so far lack systematic comparison. We review and systematize five methods for tracing material consumption into end-use products in inflow-driven dMFA and discuss their strengths and limitations. Widely used data on industry shipments in physical units have low spatio-temporal coverage, which limits their applicability across countries and years. Monetary input–output tables (MIOTs) are widely available and their economy-wide coverage makes them a valuable source to approximate material end-uses. We find four distinct MIOT-based methods: consumption-based, waste input–output MFA (WIO-MFA), Ghosh absorbing Markov chain, and partial Ghosh. We show that when applied to a given MIOT, the methods’ underlying input–output models yield the same results, with the exception of the partial Ghosh method, which involves simplifications. For practical applications, the MIOT system boundary must be aligned to those of dMFA, which involves the removal of service flows, sector (dis)aggregation, and re-defining specific intermediate outputs as final demand. Theoretically, WIO-MFA, applied to a modified MIOT, produces the most accurate results as it excludes massless and waste transactions. In part 2 of this work, we compare methods empirically and suggest improvements for aligning MIOT-dMFA system boundaries.     

PocketOptimizer 2.0: A modular framework for computer-aided ligand-binding design

The ability to design customized proteins to perform specific tasks is of great interest. We are particularly interested in the design of sensitive and specific small molecule ligand-binding proteins for biotechnological or biomedical applications. Computational methods can narrow down the immense combinatorial space to find the best solution and thus provide starting points for experimental procedures. However, success rates strongly depend on accurate modeling and energetic evaluation. Not only intra- but also intermolecular interactions have to be considered. To address this problem, we developed PocketOptimizer, a modular computational protein design pipeline, that predicts mutations in the binding pockets of proteins to increase affinity for a specific ligand. Its modularity enables users to compare different combinations of force fields, rotamer libraries, and scoring functions. Here, we present a much-improved version––PocketOptimizer 2.0. We implemented a cleaner user interface, an extended architecture with more supported tools, such as force fields and scoring functions, a backbone-dependent rotamer library, as well as different improvements in the underlying algorithms. Version 2.0 was tested against a benchmark of design cases and assessed in comparison to the first version. Our results show how newly implemented features such as the new rotamer library can lead to improved prediction accuracy. Therefore, we believe that PocketOptimizer 2.0, with its many new and improved functionalities, provides a robust and versatile environment for the design of small molecule-binding pockets in proteins. It is widely applicable and extendible due to its modular framework. PocketOptimizer 2.0 can be downloaded at https://github.com/Hoecker-Lab/pocketoptimizer .     

Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.     

Genomic regulation of transposable elements in Drosophila

Transposable elements are a major source of genetic change, including the creation of novel genes, the alteration of gene expression in development, and the genesis of major genomic rearrangements. They are ubiquitous among contemporary organisms and probably as old as life itself. The long coexistence of transposable elements in the genome would be expected to be accompanied by host-element coevolution. Indeed, the important role of host factors in the regulation of transposable elements has been illuminated by recent studies of several systems in Drosophila. These include host factors that regulate the P element, a host mutation that renders the genome permissive for gypsy mobilization and infection, and newly induced mutations that affect the expression of transposon insertion mutations. The finding of a type of hybrid dysgenesis in D. virilis, in which multiple unrelated transposable elements are mobilized simultaneously, may also be relevant to host-factor regulation of transposition.     

Ultrastructural Analysis of Spermiogenesis in Segregation Distorter Males of DROSOPHILA MELANOGASTER: The Homozygotes

We have studied spermiogenesis at the ultrastructural level in males of genotype SD(NH)-2/SD-72, which are nearly sterile owing to the dysfunction of virtually all of their sperm. Ultrastructural aspects of spermiogenesis in these homozygous SD males are qualitatively similar to those found among dysfunctional sperm produced by heterozygous SD males. In particular, chromatin condensation and/or compaction has been found to be abnormal. However, major quantitative differences have been noted. Most of the dysfunctional sperm in SD(NH)-2/SD-72 males are individualized and coiled. Then, the sperm evidently undergo degeneration, as few mature sperm can be found in the seminal vesicle. The relevance of these findings to the mechanism leading to near sterility in homozygous SD males is discussed.     

Formation and quantification of protein complexes between peroxisomal alcohol oxidase and GroEL.

We have studied the use of yeast peroxisomal alcohol oxidase (AO) as a model protein for in vitro binding by GroEL. Dilution of denatured AO in neutral buffer leads to aggregation of the protein, which is prevented by the addition of GroEL. Formation of complexes between GroEL and denatured AO was demonstrated by a gel-shift assay using non-denaturing polyacrylamide gel electrophoresis, and quantified by laser-densitometry of the gels. In the presence of MgAMP-PNP or MgADP the affinity of GroEL for AO was enhanced. Under these conditions up to 70% of the purified GroEL formed a complex with this protein. Release was stimulated at room temperature by MgATP, and was further enhanced by addition of GroES.     

A repetitive DNA element,associated with telomeric sequences in Drosophila melanogaster,contains open reading frames

He-T sequences are a complex repetitive family of DNA sequences in Drosophila that are associated with telomeric regions, pericentromeric heterochromatin, and the Y chromosome. A component of the He-T family containing open reading frames (ORFs) is described. These ORF-containing elements within the He-T family are designated T-elements, since hybridization in situ with the polytene salivary gland chromosomes results in detectable signal exclusively at the chromosome tips. One T-element that has been sequenced includes ORFs of 1,428 and 1,614 bp. The ORFs are overlapping but one nucleotide out of frame with respect to each other. The longer ORF contains cysteine-histidine motifs strongly resembling nucleic acid binding domains of gag-like proteins, and the overall organization of the T-element ORFs is reminiscent of LINE elements. The T-elements are transcribed and appear to be conserved in Drosophila species related to D. melanogaster. The results suggest that T-elements may play a role in the structure and/or function of telomeres.by W. Hennig