Isolation and characterisation of mutants of GroEL that are fully functional as single rings

A key aspect of the reaction mechanism for the molecular chaperone GroEL is the transmission of an allosteric signal between the two rings of the GroEL complex. Thus, the single-ring mutant SR1 is unable to act as a chaperone as it cannot release bound substrate or GroES. We used a simple selection procedure to identify mutants of SR1 that restored chaperone activity in vivo. A large number of single amino acid changes, mapping at diverse positions throughout the protein, enabled SR1 to regain its ability to act as a chaperone while remaining as a single ring. In vivo assays were used to identify the proteins that had regained maximal activity. In some cases, no difference could be detected between strains expressing wild-type GroEL and those expressing the mutated proteins. Three of the most active proteins where the mutations were in distinct parts of the protein were purified to homogeneity and characterised in vitro. All were capable of acting efficiently as chaperones for two different GroES-dependent substrates. All three proteins bound nucleotide as effectively as did GroEL, but the binding of GroES in the presence of ATP or ADP was reduced significantly relative to the wild-type. These active single rings should provide a useful tool for studying the nature of the allosteric changes that occur in the GroEL reaction cycle.     

Analysis of potential duplicates in barley gene bank collections using re-sampling of microsatellite data

Redundant duplication among putative Nordic spring barley material held at 12 gene banks worldwide was studied using 35 microsatellite primer pairs covering the entire barley genome. These microsatellite markers revealed an average of 7.1 alleles per locus, and a range of 1 to 17 different alleles per locus. Similarity of accession name was initially used to partition the 174 repatriated accessions into 36 potential duplicate groups, and one group containing 36 apparently unique or unrelated accessions. This partitioning was efficient to produce a distribution of mainly small average genetic distances within potential duplicate groups compared to distances from the group of unique accessions. However, comparisons within potential duplicate groups still contained large genetic distances of the same size as distances between unique accessions indicating classification errors. A bootstrap approach based on re-sampling of both microsatellite markers and alleles within marker loci was used to test for homogeneity within potential duplicate groups. The test was used in each group for sequential elimination of accessions with a significantly large average genetic distance to identify a homogeneous group. Such genetically homogeneous groups of two or more accessions were identified in 22 among the 36 potential duplicate groups studied. Results from the genetic analysis of some potential duplicate groups supported previous conclusions based on passport data through inclusion of the historically most-original accession in the genetically homogeneous group. In other potential duplicate groups the apparently most-original accession according to passport data was not included in the homogeneous set of accessions, indicating that this most-original accession does not have duplicate accessions in the group. During the present study the largest average genetic distance accepted in any homogeneous group was smaller than the smallest distance declared significant in any group, with a threshold average genetic distance of approximately 0.14. The results are discussed with respect to the identification of duplicate accessions within potential duplicate groups, as well as the elimination of genetic off types in such groups. Furthermore, large barley gene bank collections may be screened for potential duplicates with genetic distances below the suggested threshold of 0.14.     

A functional overlap of plasminogen and MMPs regulates vascularization during placental development

Both plasminogen activators and matrix metalloproteinases (MMPs) have been implicated in a variety of developmental processes in the mouse during embryo implantation and placentation. We show here that pharmacological treatment of plasminogen-deficient mice with the broad spectrum MMP inhibitor galardin leads to a high rate of embryonic lethality. Implantation sites from plasminogen-deficient galardin-treated mice at 7.5 days post coitus (dpc) showed delay in both decidualization and invasion of maternal vessels into the decidua. At 8.5 dpc, half of the embryos were runted and still at the developmental stage of a 7.5 dpc embryo. Most embryos that escaped these initial defects eventually died, probably from defective vascularization and development of the labyrinth layer of the placenta, although a direct role on embryo development cannot be ruled out. These results demonstrate that the combination of MMPs and plasminogen is essential for the proper development of the placenta. Plasminogen deficiency alone and galardin treatment alone had much less effect and there was a pronounced synergism on both placental vascularization and embryonic lethality, indicating a functional overlap between plasminogen and MMPs.     

tRNA turnaround

Two recently published papers (Takano et al., 2005 and Shaheen and Hopper, 2005) demonstrate that in S. cerevisiae, cytoplasmic tRNAs can be transported into the nucleus. This retrograde movement may expose mature tRNAs to nuclear proofreading or it may regulate tRNA availability in response to amino acid availability.     

Properties of the chaperonin complex from the halophilic archaeon Haloferax volcanii

The halophilic archaeon Haloferax volcanii has three genes encoding type II chaperonins, named cct1, cct2 and cct3. We show here that the three CCT proteins are all expressed but not to the same level. All three proteins are further induced on heat shock. The CCT proteins were purified by ammonium sulphate precipitation, sucrose gradient centrifugation and hydrophobic interaction chromatography. This procedure yields a high molecular mass complex (or complexes). The complex has ATPase activity, which is magnesium dependent, low salt-sensitive and stable to at least 75 degrees C. Activity requires high levels of potassium ions and was reduced in the presence of an increasing concentration of sodium ions.     

Immunisation of mice against neosporosis

In the present study a murine encephalitis model was used to investigate if protection against neosporosis could be achieved by immunisation. Groups of 10 mice were immunised with a sublethal dose of live Neospora caninum tachyzoites, N. caninum antigens incorporated into iscoms, N. caninum lysate mixed with Quil A, or N. caninum lysate in PBS. Control mice were given Quil A only. Challenge infection with 2.5x10(6) N. caninum tachyzoites resulted in clinical symptoms that remained until the end of the experiment in the controls. In contrast, mice immunised with live parasites or parasite lysate in Quil A only showed mild and transient symptoms. Of nine mice immunised with N. caninum iscoms, seven recovered while two died. Most severely affected were the mice immunised with parasite lysate only; all of them died within 28 days post-infection. Histological examination and scoring of brain lesions gave a significantly lower (P<0.0001) lesion score in mice immunised with live parasites than in controls. The groups immunised with iscoms or lysate and Quil A also had reduced lesion scores (P<0.04 and 0.07, respectively) but not the group given parasite lysate alone. The lesions seen in the latter group differed from those in the other groups. There was less cellular reaction and more tachyzoites indicating an active infection. The N. caninum specific antibody responses and cytokine production (IFN-gamma, IL-4 and IL-5) of splenocytes were analysed at the time of challenge infection. The results suggest a correlation between protection and high levels of IFN-gamma. Also, the immune responses recorded in mice immunised with parasite lysate without adjuvant were relatively weak and more towards the Th2 type, when compared with the other immunisation schedules. This is consistent with the weaker inflammatory response observed in the brains of these mice.     

A new splice variant of glial fibrillary acidic protein,GFAP epsilon,interacts with the presenilin proteins

We describe a new human isoform, GFAP epsilon, of the intermediary filament protein GFAP (glial fibrillary acidic protein). GFAP epsilon mRNA is the result of alternative splicing and a new polyadenylation signal, and thus GFAP epsilon has a new C-terminal protein sequence. This provides GFAP epsilon with the capacity for specific binding of presenilin proteins in yeast and in vitro. Our observations suggest a direct link between the presenilins and the cytoskeleton where GFAP epsilon is incorporated. Mutations in GFAP and presenilins are associated with Alexander disease and Alzheimer's disease, respectively. Accordingly, GFAP epsilon should be taken into consideration when studying neurodegenerative diseases.