The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.

The Dictyostelium discoideum NC4 genome harbors approximately 150 individual copies of a retrotransposable element called the Dictyostelium repetitive element (DRE). This element contains nonidentical terminal repeats (TRs) consisting of conserved building blocks A and B in the left TR and B and C in the right TR. Seven different-sized classes of RNA transcripts from these elements were resolved by Northern (RNA) blot analysis, but their combined abundance was very low. When D. discoideum cells were grown in the presence of the respiratory chain blocker antimycin A, steady-state concentrations of these RNA species increased 10-to 20-fold. The D. discoideum genome contains two DRE subtypes, the full-length 5.7-kb DREa and the internally deleted 2.4-kb DREb. Both subtypes are transcribed, as confirmed by analysis of cloned cDNA. Primary transcripts from the sense strand originate at nucleotide +1 and terminate at two dominant sites, located 21 or 28 nucleotides upstream from the 3' end of the elements. The activity of a reasonably strong polymerase II promoter in the 5'-terminal A module is slightly upregulated by the tRNA gene located 50 ± 4 nucleotides upstream and drastically reduced by the adjacent B module of the DRE. Transcripts from the opposite DNA strand (complementary-sense transcripts) were also detected, directed by an internally located polymerase II promoter residing within the C module. This latter transcription was initiated at multiple sites within the oligo(dA12) stretch which terminates DREs.Retrotransposable Dictyostelium repetitive elements (DREs) are present in approximately 150 to 200 copies in the genomes of different Dictyostelium discoideum strains (28-31).Most remarkably, this element always integrates at a distance of 50 ± 4 nucleotides upstream from tRNA genes but in the opposite transcriptional orientation. Upon integration, a 14 + 2-nucleotide target site duplication is created (29). The 5.7-kb DREs contain an internal coding portion with two open reading frames (ORFs) which is flanked by nonidentical terminal repeats (TRs) (see Fig. IA). These TRs are composed of three distinct modules, termed A, B, and C. The 5' TR consists of one or several A modules followed by a 290-bp B module which provides the AUG translation initiation codon. The 3' TR consists of a B module followed by a terminal C module.Approximately half of the DREs in NC4-derived strains show a variant organization (30). They are only 2.4 kb long and are termed DREbs, as opposed to the 5.7-kb DREas (Fig. IA). DREbs carry an extensive internal deletion encompassing nearly the entire ORF2. They also contain several small deletions within the right TR and a variant A module (termed Ab), which always differs from the Aa module of the 5.7-kb DREas by the same three distinct point mutations (30). A similar situation has been described for jockey elements of Drosophila melanogaster, which occur as full-size (5-kb) copies

CD4+CD25+ regulatory T cells (Tregs) represent a specialized subpopulation of T cells, which are essential for maintaining peripheral tolerance and preventing autoimmunity. The immunomodulatory effects of Tregs depend on their activation status. Here we show that, in contrast to conventional anti-CD4 monoclonal antibodies (mAbs), the humanized CD4-specific monoclonal antibody tregalizumab (BT-061) is able to selectively activate the suppressive properties of Tregs in vitro. BT-061 activates Tregs by binding to CD4 and activation of signaling downstream pathways. The specific functionality of BT-061 may be explained by the recognition of a unique, conformational epitope on domain 2 of the CD4 molecule that is not recognized by other anti-CD4 mAbs. We found that, due to this special epitope binding, BT-061 induces a unique phosphorylation of T-cell receptor complex-associated signaling molecules. This is sufficient to activate the function of Tregs without activating effector T cells. Furthermore, BT-061 does not induce the release of pro-inflammatory cytokines. These results demonstrate that BT-061 stimulation via the CD4 receptor is able to induce T-cell receptor-independent activation of Tregs. Selective activation of Tregs via CD4 is a promising approach for the treatment of autoimmune diseases where insufficient Treg activity has been described. Clinical investigation of this new approach is currently ongoing.

morphism (12, 13). To gain insight into this interesting phenomenon, we analyzed the genomic organization of previously isolated tRNA genes (32). In the course of this study, we repeatedly identified the retrotransposable element DRE as well as the previously characterized transposable Tdd-3 element (30,38). Both elements were found associated with several unrelated tRNA genes in a characteristic position-specific manner.Up to now, DRE has never been found other than in association with tRNA genes in D. discoideum. In all analyzed clones, tRNA genes are separated by 50 ± 3 nucleotides from the associated DRE element, and DRE always occurs in a constant orientation relative to tRNA genes. Since no sequence similarity is apparent at the DRE integration site, a mechanism other than sequence-specific integration must be responsible for this striking position-specific and orientation-specific integration of DRE elements in D. discoideum. Similar results with respect to position specificity but not with respect to orientation specificity were obtained for Ty3, a retrotransposon identified in S. cerevisiae. Ty3 elements or a remnant LTR, termed sigma, were also found in front of various tRNA genes, separated in this case by 16 to 19 nucleotides from the mature coding tDNA sequence (10, 41). Both Ty3 and DRE, therefore, are members of a family of retrotransposons which integrate by a position-specific rather than by sequence-specific or random mechanisms into the chromosome (8, 31). Sequence-specific recombination has been described for many retrotransposable elements in different organisms (1,5,15,21,22,50), but many retrotransposons and most retroviruses are thought to integrate more or less at random (for reviews, see references 3, 42, and 45). In the cases of Ty3 and DRE, a great number and variety of tRNA genes mark the specific integration sites. This is a remarkable fact which puts these elements into a different category from other retrotransposons and

It is important to educate health care providers about the differences between biosimilars and their reference biologics. The adoption of biosimilars is necessary to control long-term costs of biologics, increase patient access to care, and encourage innovation.