Sequence and characterization of the Ig heavy chain constant and partial variable region of the mouse strain 129S1

Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particular complex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains. For example, the Igh(b) haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igh(a) haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3' half of the Igh(a) locus of 129S1/SvImJ, covering the C(H) region and approximately half of the V(H) region. This sequence comprises 128 V(H) genes, of which 49 are judged to be functional. The comparison of the Igh(a) sequence with the homologous Igh(b) region from C57BL/6 revealed two major expansions in the germline repertoire of Igh(a). In addition, we found smaller haplotype-specific differences like the duplication of five V(H) genes in the Igh(a) locus. We generated a V(H) allele table by comparing the individual V(H) genes of both haplotypes. Surprisingly, the number and position of D(H) genes in the 129S1 strain differs not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguous genomic sequence of the 3' part of the Igh(a) locus allows a detailed view of the recent evolution of this highly dynamic locus in the mouse.     

Kinetic studies of protein L aggregation and disaggregation

We have investigated the aggregation of protein L in 25% (vol/vol) TFE and 10 mM HCl. Under both conditions, aggregates adopt a fibrillar structure and bind dyes Congo Red and Thioflavin T consistent with the presence of amyloid fibrils. The kinetics of aggregation in 25% TFE suggest a linear-elongation mechanism with critical nucleus size of either two or three monomers. Aggregation kinetics in 10 mM HCl show a prolonged lag phase prior to a rapid increase in aggregation. The lag phase is time-dependent, but the time dependence can be eliminated by the addition of pre-formed seeds. Disaggregation studies show that for aggregates formed in TFE, aggregate stability is a strong function of aggregate age. For example, after 200 min of aggregation, 40% of the aggregation reaction is irreversible, while after 3 days over 60% is irreversible. When the final concentration of the denaturant, TFE, is reduced from 5% to 0, the amount of reversible aggregation doubles. Disaggregation studies of aggregates formed in TFE and 10 mM HCl reveal a complicated effect of pH on aggregate stability.     

Adaptive motor behavior in insects

As insects move through tortuous, unpredictable terrain, their neural system allows them to exhibit striking adaptability and researchers must use every technique at their disposal to unravel the underlying mechanisms. Descending commands from brain centers that process tremendous amounts of information from head sensors work together with local motor control altering their operation to deal with barriers or move toward important targets. By analyzing movements in detail with high-speed video, recording from identified neurons in thoracic ganglia and examining activity in different brain regions, we are beginning to understand how these remarkable animals navigate their environment. Coupled with new and exciting neurogenetic tools, the near future promises an exciting time for studying the neural basis of insect movement.     

Projection maps of three members of the KdgM outer membrane protein family

We present the projection structures of the three outer membrane porins KdgM and KdgN from Erwinia chrysanthemi and NanC from Escherichia coli, based on 2D electron crystallography. A wide screening of 2D crystallization conditions yielded tubular crystals of a suitable size and quality to perform high-resolution electron microscopy. Data processing of untilted samples allowed us to separate the information of the two crystalline layers and resulted in projection maps to a resolution of up to 7 Å. All three proteins exhibit a similar putative β-barrel structure and the three crystal forms have the same symmetry. However, there are differences in the packing arrangements of the monomers as well as the densities of the projections. To interpret these projections, secondary structure prediction was performed using β-barrel specific prediction algorithms. The predicted transmembrane β-barrels have a high similarity in the arrangement of the putative β-strands and the loops, but do not match those of OmpG, a related protein porin whose structure was solved.     

Der1-mediated preprotein import into the periplastid compartment of chromalveolates?

Phototrophic chromalveolates possess plastids surrounded by either 3 or 4 membranes, revealing their secondary endosymbiotic origin from an engulfed eukaryotic alga. In cryptophytes, a member of the chromalveolates, the organelle is embedded within a designated region of the host's rough endoplasmic reticulum (RER). Its eukaryotic compartments other than the plastid were reduced to the mere remains of its former cytosol, the periplastid compartment (PPC, PP space), and its nucleus, the nucleomorph, separated from the RER by its former plasma membrane, the periplast membrane (PPM). In the nucleomorph genome of the cryptophyte Guillardia theta, we identified several genes sharing homology with components of the ER-associated degradation (ERAD) machinery of yeast and higher eukaryotes, namely ORF201 and ORF477, homologs of membrane-bound proteins, Der1p (Degradation in the ER protein 1) and the RING-finger ubiquitin ligase Hrd1, and a truncated version of Udf1, a cofactor of Cdc48, a lumenal ATPase. Exemplarily, studies on the Der1-homolog ORF201 showed that this protein partially rescued a yeast deletion mutant, indicating the existence of a functional PPC-specific ERAD-like system in cryptophytes. With the noninvestigated exception of haptophytes a phylogenetically and mechanistically related system is apparently present in all chromalveolates with 4 membrane-bound plastids because amongst others, PPC-specific Derlins (Der1-like proteins), CDC48 and its cofactor Ufd1 were identified in the nuclear genomes of diatoms and apicomplexa. These proteins are equipped with the required topogenic signals to direct them into the periplastid compartment of their secondary symbionts. Based on our findings, we suggest that all chromalveolates with 4 membrane-bound plastids express an ERAD-derived machinery in the PPM of their secondary plastid, coexisting physically and systematically adjacent to the host's own ERAD system. We propose herewith that this system was functionally adapted to mediate transport of nucleus-encoded PPC/plastid preproteins from the RER into the periplastid space.     

Prediction of herbage yield in grassland: How well do Ellenberg N‐values perform?

Question: How useful are Ellenberg N‐values for predicting the herbage yield of Central European grasslands in comparison to approaches based on ordination scores of plant species composition or on soil parameters? Location: Central Germany (11°00′‐11°37’E, 50°21‐50°34’N, 500–840 m a.s.l.). Methods: Based on data from a field survey in 2001, the following models were constructed for predicting herbage yield in montane Central European grasslands: (1) Linear regression of mean Ellenberg N‐, R‐ and F‐values; (2) Linear regression of ordination scores derived from Non‐metric Multidimensional Scaling (NMDS) of vegetation data; and (3) Multiple linear regression (MLR) of soil variables. Models were evaluated by cross‐validation and validation with additional data collected in 2002. Results: Best predictions were obtained with models based on species composition. Ellenberg N‐values and NMDS scores performed equally well and better than models based on Ellenberg R‐ or F‐values. Predictions based on soil variables were least accurate. When tested with data from 2002, models based on Ellenberg N‐values or on NMDS scores accurately predicted productivity rank order of sites, but not the actual herbage yield of particular sites. Conclusions: Mean Ellenberg N‐values, which are easy to calculate, are as accurate as ordination scores in predicting herbage yield from plant species composition. In contrast, models based on soil variables may be useful for generating hypotheses about the factors limiting herbage yield, but not for prediction. We support the view that Ellenberg N‐values should be called productivity values rather than nitrogen values.     

A proteomic study of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>AAA+ protease FtsH

Background  

The influence of the membrane-bound AAA+ protease FtsH on membrane and cytoplasmic proteins of Corynebacterium glutamicum was investigated in this study. For the analysis of the membrane fraction, anion exchange chromatography was combined with SDS-PAGE, while the cytoplasmic protein fraction was studied by conventional two-dimensional gel electrophoresis.     

New moves in motor control

Motor behaviour results from information processing across multiple neural networks acting at all levels from initial selection of the behaviour to its final generation. Understanding how motor behaviour is produced requires identifying the constituent neurons of these networks, their cellular properties, and their pattern of synaptic connectivity. Neural networks have been traditionally studied with neurophysiological and neuroanatomical approaches. These approaches have been highly successful in particularly suitable 'model' preparations, typically ones in which the numbers of neurons in the networks were relatively small, neural network composition was unvarying across individual animals, and the preparations continued to produce fictive motor patterns in?vitro. However, analysing networks without these characteristics, and analysing the complete ensemble of networks that cooperatively generate behaviours, is difficult with these approaches. Recently developed molecular and neurogenetic tools provide additional avenues for analysing motor networks by allowing individual or groups of neurons within networks to be manipulated in novel ways and allowing experiments to be performed not only in?vitro but also in?vivo. We review here some of the new insights into motor network function that these advances have provided and indicate how these advances might bridge gaps in our understanding of motor control. To these ends, we first review motor neural network organisation highlighting cross-phylum principles. We then use prominent examples from the field to show how neurogenetic approaches can complement classical physiological studies, and identify additional areas where these approaches could be advantageously applied.     

Solubilization of native integral membrane proteins in aqueous buffer by noncovalent chelation with monomethoxy poly(ethylene glycol) (mPEG) polymers

Highly hydrophobic integral membrane proteins (IMPs)are typically purified in excess detergent media, often resulting in rapid inactivation and denaturation of the protein. One promising approach to solve this problem is to couple hydrophilic polymers, such as monomethoxypolyethylene glycol (mPEG) to IMPs under mild conditions in place of detergents. However, the broad application of this approach is hampered by poor reaction efficiencies, low tolerance of detergent stabilized membrane proteins to reaction conditions, and a lack of proper site-specific reversible approaches. Here, we have developed a straightforward, efficient, and mild approach to site-specific noncovalent binding of long-chain polymers to recombinant IMPs. This method uses the hexa-histidine tag (His-Tag) often used for purification of recombinant proteins as an attachment site for mPEGs. Solubility studies performed using five different IMPs confirmed that all tested mPEG-bound IMPs were completely soluble and stable in detergent free aqueous buffer compared to their precipitated native proteins under the identical circumstances. Activity assays and circular dichroism (CD) spectroscopy confirmed the structural integrity of modified IMPs.