Humanized mice as a model for rheumatoid arthritis

Genetic susceptibility to rheumatoid arthritis (RA), a common autoimmune disease, is associated with certain HLA-DR4 alleles. Treatments are rarely curative and are often tied to major side effects. We describe the development of a humanized mouse model wherein new, less toxic, vaccine-like treatments for RA might be pretested. This model includes four separate transgenes: HLA-DR*0401 and human CD4 molecules, a RA-related human autoantigenic protein (HCgp-39), and a T-cell receptor (TCRalphabeta) transgene specific for an important HCgp-39 epitope, eliciting strong Th1 responses in the context of HLA-DR*0401.     

Recognition of guanosine by dissimilar tRNA methyltransferases

Guanosines are important for biological activities through their specific functional groups that are recognized for RNA or protein interactions. One example is recognition of N(1) of G37 in tRNA by S-adenosyl-methionine (AdoMet)-dependent tRNA methyltransferases to synthesize m(1)G37-tRNA, which is essential for translational fidelity in all biological domains. Synthesis of m(1)G37-tRNA is catalyzed by TrmD in bacteria and by Trm5 in eukarya and archaea, using unrelated and dissimilar structural folds. This raises the question of how dissimilar proteins recognize the same guanosine. Here we probe the mechanism of discrimination among functional groups of guanosine by TrmD and Trm5. Guanosine analogs were systematically introduced into tRNA through a combination of chemical and enzymatic synthesis. Single turnover kinetic assays and thermodynamic analysis of the effect of each analog on m(1)G37-tRNA synthesis reveal that TrmD and Trm5 discriminate functional groups differently. While both recognize N(1) and O(6) of G37, TrmD places a much stronger emphasis on these functional groups than Trm5. While the exocyclic 2-amino group of G37 is important for TrmD, it is dispensable for Trm5. In addition, while an adjacent G36 is obligatory for TrmD, it is nonessential for Trm5. These results depict a more rigid requirement of guanosine functional groups for TrmD than for Trm5. However, the sensitivity of both enzymes to analog substitutions, together with an experimental revelation of their low cellular concentrations relative to tRNA substrates, suggests a model in which these enzymes rapidly screen tRNA by direct recognition of G37 in order to monitor the global state of m(1)G37-tRNA.     

Characteristics of myotonic dystrophy in Istria: molecular genetic approach. Part II: Analysis of genetic polymorphisms

One of the world highest prevalence estimates of myotonic dystrophy (DM) has been reported in the Croatian region Istria. To analyse the population genetic characteristics of DM locus in Istria, two intragenic and three extragenic polymorphic markers were tested. The Southern blot technique was used for D19S63 locus analysis, whereas PCR analysis was performed for CKMM, Alu polymorphism, DMPK (G/T) intron 9/HinfI polymorphism, and D19S207 genetic markers. The compound haplotypes segregating with DM were established. A complete association between the DM mutation and D19S63, D19S207, intron 9/HinfI polymorphism and Alu polymorphism markers were found. In all DM chromosomes: D19S63 and Alu markers had the allele 1 in common; D19S207 had the allele 3 in common, DMPK (G/T) intron 9/HinfI marker had the allele 2 in common. The analysis of CKMM polymorphism revealed genotype heterogeneity; in DM chromosomes either allele 2 or allele 4 were found. The haplotype analysis in the population of Croatian Istria supports the linkage disequilibrium between the DM mutation and Alu polymorphism, intron 9/HinfI polymorphism, D19S63 and D19S207 markers as reported worldwide. The results of the haplotype analysis suggest a common origin of the mutation in Istrian population.     

B-cell factor 1 is required for optimal expression of the DRA promoter in B cells.

The X box in the DRA promoter of the human histocompatibility complex is required for expression of the DRA gene in B cells. We show that a B-cell factor binds to a sequence that is clearly distinguishable from binding sites for the previously described X box binding nuclear proteins RF-X, NF-X, NF-Xc, NF-S, hXBP, and AP-1. Mutations in the DRA X box that disrupt the binding of this factor result in a lower level of gene expression, as does the presence of Id (a trans-dominant regulatory protein that negatively regulates helix-loop-helix proteins). Furthermore, this factor is recognized by antibodies directed against the helix-loop-helix protein A1, a mouse homolog of the immunoglobulin enhancer binding proteins E12/E47, and it binds to sequences in other genes that were previously shown to bind these proteins. By these criteria, this factor is BCF-1.