20-104 Sigma-AldrichMalachite Green Additive

The (11;19)(q23;p13.1) translocation in acute leukemia results in the formation of an MLL-ELL fusion protein. ELL is an RNA polymerase II elongation factor that interacts with the recently identified EAF1 protein. To characterize the normal functions of ELL and its aberrant activities when fused to MLL, we isolated a second protein that interacts with ELL named EAF2 for ELL Associated Factor 2. EAF2 is highly homologous to EAF1, with 58% identity and 74% amino acid conservation. Using specific antibodies generated to EAF2, we coimmunoprecipitated ELL and EAF2 from multiple cell lines. Confocal microscopy revealed that endogenous EAF2 and ELL colocalized in a nuclear speckled pattern. Database comparisons with EAF2 identified a region with a high content of serine, aspartic acid, and glutamic acid residues that is conserved with EAF1 and exhibited amino acid similarity with several translocation partner proteins of MLL, including AF4 and ENL. We found that EAF2 and EAF1 both contain transcriptional activation domains within this region. Using retroviral bone marrow transduction, we observed that a heterologous fusion of EAF2 to MLL immortalized hematopoietic progenitor cells. In contrast to EAF1, EAF2 does not bind to the carboxy-terminus of ELL. We identified a protein-protein interaction domain within the amino-terminus of ELL that binds to both EAF1 and EAF2. This amino-terminal interaction domain is disrupted in the formation of the MLL-ELL fusion protein. Thus, MLL-ELL retains an interaction domain for EAF1 but not for EAF2. Taken together, these data suggest that MLL-ELL may disrupt the normal protein-protein interactions of ELL.

alpha-Agglutinin of Saccharomyces cerevisiae is a cell wall-associated protein that mediates cell interaction in mating. Although the mature protein includes about 610 residues, the NH2-terminal half of the protein is sufficient for binding to its ligand a-agglutinin. alpha-Agglutinin20-351, a fully active fragment of the protein, has been purified and analyzed. Circular dichroism spectroscopy, together with sequence alignments, suggest that alpha-agglutinin20-351 consists of three immunoglobulin variable-like domains: domain I, residues 20-104; domain II, residues 105-199; and domain III, residues 200-326. Peptide sequencing data established the arrangement of the disulfide bonds in alpha-agglutinin20-351. Cys97 is disulfide-bonded to Cys114, forming an interdomain bond between domains I and II. Cys202 is bonded to Cys300, in an atypical intradomain disulfide bond between the A and F strands of domain III. Cys227 and Cys256 have free sulfhydryls. Sequencing also showed that at least two of three potential N-glycosylation sites with sequence Asn-Xaa-Thr are glycosylated. At least one of three Asn-Xaa-Ser sequences is not glycosylated. No residues NH2-terminal to Ser282 were O-glycosylated, whereas Ser282, and all hydroxy amino acid residues COOH-terminal to this position were modified. Therefore O-glycosylated Ser and Thr residues cluster in the COOH-terminal region of domain III, and the O-glycosylation continues into a Ser/Thr-rich sequence that extends from domain III to the COOH-terminal of the full-length protein.