Conditional RAG-1 mutants block the hairpin formation step of V(D)J recombination

Hairpin formation serves an important regulatory role in V(D)J recombination because it requires synapsis of an appropriate pair of recombination sites. How hairpin formation is regulated and which regions of the RAG proteins perform this step remain unknown. We analyzed two conditional RAG-1 mutants that affect residues quite close in the primary sequence to an active site amino acid (D600), and we found that they exhibit severely impaired recombination in the presence of certain cleavage site sequences. These mutants are specifically defective for the formation of hairpins, providing the first identification of a region of the V(D)J recombinase necessary for this reaction. Substrates containing mismatched bases at the cleavage site rescued hairpin formation by both mutants, which suggests that the mutations affect the generation of a distorted or unwound DNA intermediate that has been implicated in hairpin formation. Our results also indicate that this region of RAG-1 may be important for coupling hairpin formation to synapsis.     

Two isoforms of trimming glucosidase II exist in mammalian tissues and cell lines but not in yeast and insect cells

We previously cloned glucosidase II and provided in vivo evidence for its involvement in protein folding quality control. DNA-sequencing of different clones demonstrated the existence of two isoforms of glucosidase II which differed by 66 nucleotides due to alternative splicing. The existence of two enzyme isoforms in various organs of pig and rat as well as human, bovine, rat, and mouse cell lines could be demonstrated by RT-PCR and Western blotting. Furthermore, the two isoforms of glucosidase II could be detected in embryonic and postnatal rat kidney and liver. In yeast, Saccharomyces cerevisiae, and in insects, Drosophila S2 cells, only one isoforms of the enzyme was detectable. The ubiquitous occurrence of the two glucosidase II isoforms in mammalian tissues and cell lines might be indicative of a special function of each isoform.     

Stable anterior anchoring of the oocyte nucleus is required to establish dorsoventral polarity of the Drosophila egg

Wnt signals control cell fate decisions and orchestrate cell behavior in metazoan animals. In the fruit fly Drosophila, embryos defective in signaling mediated by the Wnt protein Wingless (Wg) exhibit severe segmentation defects. The Drosophila segment polarity gene naked cuticle (nkd) encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. Nkd antagonizes Wg via a direct interaction with the Wnt signaling component Dishevelled (Dsh). Here we describe two mouse and human proteins, Nkd1 and Nkd2, related to fly Nkd. The most conserved region among the fly and vertebrate proteins, the EFX domain, includes the putative EF hand and flanking sequences. EFX corresponds to a minimal domain required for fly or vertebrate Nkd to interact with the basic/PDZ domains of fly Dsh or vertebrate Dvl proteins in the yeast two-hybrid assay. During mouse development, nkd1 and nkd2 are expressed in multiple tissues in partially overlapping, gradient-like patterns, some of which correlate with known patterns of Wnt activity. Mouse Nkd1 can block Wnt1-mediated, but not beta-catenin-mediated, activation of a Wnt-dependent reporter construct in mammalian cell culture. Misexpression of mouse nkd1 in Drosophila antagonizes Wg function. The data suggest that the vertebrate Nkd-related proteins, similar to their fly counterpart, may act as inducible antagonists of Wnt signals.     

Physicochemical and morphological characterization of two small polluted streams in southwest Germany

As a supporting component of the VALIMARproject, physicochemical investigations wereconducted monthly from 1995 to 1999 at theKrähenbach/Aich stream system (two samplingsites) and at the Körsch stream (sixsampling sites). Several physicochemicalparameters were analysed continuously bydataloggers during the entire sampling period.Moreover, a selection of the most importantmorphological parameters of the stream wererecorded in 1998.The results of these physicochemicalinvestigations demonstrated that theKrähenbach/Aich system could becharacterized as a natural rhithral submontanecarbonate stream system that is welloxygenated. Low temporal variations in pH,conductivity and chloride occurred at allsampling sites in the Krähenbach whichcoincided with the geological conditions atthis test stream. Because of relatively lowconcentrations of organic and inorganiccompounds during the entire study period thesites AB and AC in the Krähenbach/Aichsystem were classified as slightly polluted.The morphological assessment of the streamindicated a moderately affected status atboth of these streams.Physicochemical and morphologicalinvestigations along the Körsch streamsystem (KA to KE including the tributary streamSulzbach) indicated a highly affected rhithralsubmontane carbonate system characterized byhigh water velocity, flow rate and dissolvedoxygen. The Körsch, however, differssignificantly with respect to watertemperature, conductivity, pH, BOD₅,nutrients, and chloride concentrations from theKrähenbach. As a result of differentanthropogenic inputs (e.g. several sewageplants, agriculture activities and waste waterdumping) the Körsch sites KA to KD wereclassified as critically to heavilypolluted. The morphological assessmentindicated a clearly affected to damagedstatus. The upstream site KE could becharacterized as slightly polluted and itsmorphological structure as moderatelyaffected.     

How does a DNA interacting enzyme change its specificity during molecular evolution? A site-directed mutagenesis study at the DNA binding site of the DNA-(adenine-N6)-methyltransferase EcoRV

The EcoRV DNA-(adenine-N6)-methyltransferase (MTase) recognizes GATATC sequences and modifies the first adenine residue within this site. Parts of its DNA interface show high sequence homology to DNA MTases of the dam family which recognize and modify GATC sequences. A phylogenetic analysis of M.EcoRV and dam-MTases suggests that EcoRV arose in evolution from a primordial dam-MTase in agreement to the finding that M.EcoRV also methylates GATC sites albeit at a strongly reduced rate. GATCTC sites that deviate in only one position from the EcoRV sequence are preferred over general dam sites. We have investigated by site-directed mutagenesis the function of 17 conserved and nonconserved residues within three loops flanking the DNA binding cleft of M.EcoRV. M.EcoRV contacts the GATATC sequence with two highly cooperative recognition modules. The contacts to the GAT-part of the recognition sequence are formed by residues conserved between dam MTases and M.EcoRV. Mutations at these positions lead to an increase in the discrimination between GATATC and GATC substrates. Our data show that the change in sequence specificity from dam (GATC) to EcoRV (GATATC) was accompanied by the generation of a second recognition module that contacts the second half of the target sequence. The new DNA contacts are formed by residues from all three loops that are not conserved between M.EcoRV and dam MTases. Mutagenesis at important residues within this module leads to variants that show a decreased ability to recognize the TC-part of the GATATC sequence.     

Evaluation of prenatal diagnosis of cleft lip/palate by foetal ultrasonographic examination

Ultrasound scans in the midtrimester of pregnancy are now a routine part of antenatal care in many countries. This type of screening procedure can detect serious foetal anomalies. Thanks to our registry of congenital anomalies a study was undertaken. The objective of the study was to evaluate prenatal detection of cleft lip (palate)(CL/P) by routine ultrasonographic examination of the foetus in 265679 consecutive pregnancies from 1979 to 1998. The percentage of prenatal detection of CL/P was low. For isolated malformation (foetuses with only CL/P) the detection rate was low: 17.8%; however, this detection rate increased from 5.3% during the period 1979-1988 to 26.5% during the period 1989-1998, for foetuses with associated malformations (foetuses with CL/P and one or more additional major malformations) these detection rates were 34.6, 13. 3 and 50.0%, respectively. In foetuses with associated malformations with CL/P this detection rate was higher for chromosomal abnormalities with CL/P and for non-syndromic, non-chromosomal multiply malformed children with CL/P than for non-chromosomal recognized syndromes with CL/P.     

Functional complementation by wheat eIF2alpha in the yeast GCN2-mediated pathway

Translational control by specific eIF2alpha phosphorylation on serine 51 has been characterized in all eukaryotes with the significant exception of plants. In order to evaluate the capability of plant eIF2alpha to functionally control translation, the wild type (51S) and a nonphosphorylatable mutant (51A) of wheat eIF2alpha were expressed in a yeast genetic system. Expression of either wheat protein did not handicap growth under conditions that repress the eIF2alpha phosphorylation pathway. However, under conditions that induce specific eIF2alpha phosphorylation only strains expressing wheat 51S were able to grow between 2 and 4 days. Growth was dependent upon activity of yeast eIF2alpha kinase GCN2 and resulted in the increased translation of GCN4. The association between plant eIF2alpha and yeast eIF2B is supported by their specific coimmunoprecipitation from transgenic yeast cells. These data support the similarity among eukaryotic translational initiation processes and strengthen the concept that plants may contain an eIF2alpha phosphorylation pathway.