The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-MLL/FBP17-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

Detecting the conformational change of transmembrane signaling in a bacterial chemoreceptor by measuring effects on disulfide cross-linking in vivo.

Transmembrane signaling by bacterial chemoreceptors is thought to involve relative movement among the four transmembrane helices of the homodimer. We assayed that movement by measuring effects of ligand occupancy on rates of oxidative cross-linking between cysteines introduced into neighboring helices of the transmembrane domain of chemoreceptor Trg from Escherichia coli. Measurements were done on chemoreceptors in their native environment, intact cells that were motile and chemotactically responsive. Receptor occupancy did not appear to cause drastic rearrangement of the four-helix structure since, among 67 cysteine pairs tested, the same 19 exhibited oxidative cross-linking in the presence or absence of saturating chemoattractant. However, occupancy did cause subtle changes that were detected as effects on rates of cross-linking. Among the seven disulfides appropriate for measurements of initial rates of formation, ligand occupancy had significant and different effects on all three cross-links that connected the two helices within a subunit but had minimal effects on the four that spanned the packing interface between subunits. This constitutes direct evidence that the conformational change of transmembrane signaling involves significant movement within a subunit and minimal movement between subunits, a pattern deduced from several previous studies and now documented directly. Among possible modes of movement between the two helices of a subunit, axial sliding of one helix relative to the other was the conformational change that best accounted for the observed effects on cross-linking.

NOMAD: a versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design.

Molecular analysis of complex modular structures, such as promoter regions or multi-domain proteins, often requires the creation of families of experimental DNA constructs having altered composition, order, or spacing of individual modules. Generally, creation of every individual construct of such a family uses a specific combination of restriction sites. However, convenient sites are not always available and the alternatives, such as chemical resynthesis of the experimental constructs or engineering of different restriction sites onto the ends of DNA fragments, are costly and time consuming. A general cloning strategy (nucleic acid ordered assembly with directionality, NOMAD; WWW resource locator http:@Lmb1.bios.uic.edu/NOMAD/NOMAD.htm l) is proposed that overcomes these limitations. Use of NOMAD ensures that the production of experimental constructs is no longer the rate-limiting step in applications that require combinatorial rearrangement of DNA fragments. NOMAD manipulates DNA fragments in the form of "modules" having a standardized cohesive end structure. Specially designed "assembly vectors" allow for sequential and directional insertion of any number of modules in an arbitrary predetermined order, using the ability of type IIS restriction enzymes to cut DNA outside of their recognition sequences. Studies of regulatory regions in DNA, such as promoters, replication origins, and RNA processing signals, construction of chimeric proteins, and creation of new cloning vehicles, are among the applications that will benefit from using NOMAD.

Primordial emergence of the recombination activating gene 1 (RAG1): sequence of the complete shark gene indicates homology to microbial integrases.

The rearrangement of antibody and T-cell receptor gene segments is indispensable to the vertebrate immune response. All extant jawed vertebrates can rearrange these gene segments. This ability is conferred by the recombination activating genes I and II (RAG I and RAG II). To elucidate their origin and function, the cDNA encoding RAG I from a member of the most ancient class of extant gnathostomes, the Carcharhine sharks, was characterized. Homology domains identified within shark RAG I prompted sequence comparison analyses that suggested similarity of the RAG I and II genes, respectively, to the integrase family genes and integration host factor genes of the bacterial site-specific recombination system. Thus, the apparent explosive evolution (or "big bang") of the ancestral immune system may have been initiated by a transfer of microbial site-specific recombinases.

Disruption of epithelial gamma delta T cell repertoires by mutation of the Syk tyrosine kinase.

Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial gamma delta T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial gamma delta T cells are similar to B cells, but distinct from splenic gamma delta T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V gamma/V delta gene rearrangements with specific epithelia are also disrupted by Syk deficiency.

NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of delta 5-3-ketosteroid isomerase.

Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of 10(10). Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4 beta-proton to the 6 beta-position, contribute 10(4.7) and 10(5.6) to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pKa values of the catalytic groups and their targets. Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor, phi = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14. The strength of this hydrogen bond is estimated to be at least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the intermediate, in conjunction with the previously demonstrated tunneling contribution to the proton transfer by Asp-38, provide a plausible and quantitative explanation for the high catalytic power of this isomerase.

The folding mechanism of larger model proteins: role of native structure.

The folding mechanism of a 125-bead heteropolymer model for proteins is investigated with Monte Carlo simulations on a cubic lattice. Sequences that do and do not fold in a reasonable time are compared. The overall folding behavior is found to be more complex than that of models for smaller proteins. Folding begins with a rapid collapse followed by a slow search through the semi-compact globule for a sequence-dependent stable core with about 30 out of 176 native contacts which serves as the transition state for folding to a near-native structure. Efficient search for the core is dependent on structural features of the native state. Sequences that fold have large amounts of stable, cooperative structure that is accessible through short-range initiation sites, such as those in anti-parallel sheets connected by turns. Before folding is completed, the system can encounter a second bottleneck, involving the condensation and rearrangement of surface residues. Overly stable local structure of the surface residues slows this stage of the folding process. The relation of the results from the 125-mer model studies to the folding of real proteins is discussed.

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.

Deletion of the mouse T-cell receptor beta gene enhancer blocks alphabeta T-cell development.

Intrathymic T-cell development requires temporally regulated rearrangement and expression of T-cell receptor (TCR) genes. To assess the role of the TCR beta gene transcriptional enhancer (Ebeta) in this process, mouse strains in which Ebeta is deleted were generated using homologous recombination techniques. We report that mice homozygous for the Ebeta deletion, whether a selectable marker gene is present or not, show a block in alphabeta T-cell development at the CD4-CD8- double-negative cell stage, whereas the number of gammadelta+ T cells is normal, few CD4+CD8+ double-positive thymocytes and no alphabeta+ T cells are produced. DNA-PCR and RNA-PCR analyses of thymic cells from homozygous mutants showed no evidence of TCR beta gene rearrangement although germ-line Vbeta transcripts were detected at a low level, in heterozygous T cells, the targeted allele is not rearranged. Thus, deletion of Ebeta totally prevents rearrangement, but not transcription, of the targeted beta locus. These data formally establish the critical role played by Ebeta in cis-activation of the TCR beta locus for V(D)J recombination during alphabeta T-cell development.

Studies on the biosynthesis of taxol: the taxane carbon skeleton is not of mevalonoid origin.

A cell culture of Taxus chinensis was established to produce the diterpene 2alpha,5alpha,10beta,14beta-tetra-acetoxy4 ++ +(20),11-taxadiene (taxuyunnanine C) in 2.6% (dry weight) yield. The incorporation of [U-13C6]glucose, [1-13C]glucose, and [1,2-13C2]acetate into this diterpene was analyzed by NMR spectroscopy. Label from [1,2-13C2]acetate was diverted to the four acetyl groups of taxuyunnanine C, but not to the taxane ring system. Label from [1-13C]glucose and [U-13C6]glucose was efficiently incorporated into both the taxane ring system and the acetyl groups. The four isoprenoid moieties of the diterpene showed identical labeling patterns. The analysis of long-range 13C13C couplings in taxuyunnanine C obtained from an experiment with [U-13C6]glucose documents the involvement of an intramolecular rearrangement in the biosynthesis of the isoprenoid precursor. The labeling patterns are inconsistent with the mevalonate pathway. The taxoid data share important features with the alternative pathway of isoprenoid biosynthesis operating in certain eubacteria Rohmer, M., Knani, M., Simonin, P., Sutter, B. & Sahm, H. (1993) Biochem. J. 295, 517-524].

Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus.

Mice carrying an ovine beta-lactoglobulin (BLG) transgene secrete BLG protein into their milk. To explore transgene expression stability, we studied expression levels in three BLG transgenic mouse lines. Unexpectedly, two lines exhibited variable levels of transgene expression. Copy number within lines appeared to be stable and there was no evidence of transgene rearrangement. In the most variable line, BLG production levels were stable within individual mice in two successive lactations. Backcrossing demonstrated that genetic background did not contribute significantly to variable expression. Tissue in situ hybridization revealed mosaicism of transgene expression within individual mammary glands from the two variable lines; in low expressors, discrete patches of cells expressing the transgene were observed. Transgene protein concentrations in milk reflected the proportion of epithelial cells expressing BLG mRNA. Furthermore, chromosomal in situ hybridization revealed that transgene arrays in both lines are situated close to the centromere. We propose that mosaicism of transgene expression is a consequence of the chromosomal location and/or the nature of the primary transgene integration event.

Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.

Chorismate mutase (EC 5.4.99.5) catalyzes the intramolecular rearrangement of chorismate to prephenate. Arg-90 in the active site of the enzyme from Bacillus subtilis is in close proximity to the substrate's ether oxygen and may contribute to efficient catalysis by stabilizing the presumed dipolar transition state that would result upon scission of the C--O bond. To test this idea, we have developed a novel complementation system for chorismate mutase activity in Escherichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The codon for Arg-90 was randomized, alone and in combination with that for Cys-88, and active clones were selected. The results show that a positively charged residue either at position 88 (Lys) or 90 (Arg or Lys) is essential. Our data provide strong support for the hypothesis that the positive charge is required for stabilization of the transition state of the enzymatic chorismate rearrangement. The new selection system, in conjunction with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of first generation catalytic antibodies with chorismate mutase activity.

BCL2 regulates neural differentiation.

A main function attributed to the BCL2 protein is its ability to confer resistance against apoptosis. In addition to the constitutively high expression of BCL2, caused by gene rearrangement in follicular lymphomas, elevated expression of the BCL2 gene has been found in differentiating hematopoietic, neural, and epithelial tissues. To address the question of whether the expression of BCL2 is a cause or consequence of cell differentiation, we used a human neural-crest-derived tumor cell line, Paju, that undergoes spontaneous neural differentiation in vitro. The Paju cell line displays moderate expression of BCL2, the level of which increases in parallel with further neural differentiation induced by treatment with phorbol 12-myristate 13-acetate. Transfection of normal human BCL2 cDNA in sense and antisense orientations had a dramatic impact on the differentiation of the Paju cells. Overexpression of BCL2 cDNA induced extensive neurite outgrowth, even in low serum concentrations, together with an increased expression of neuron-specific enolase. Paju cells expressing the anti-sense BCL2 cDNA construct, which reduced the endogenous levels of BCL2, did not undergo spontaneous neural differentiation. These cells acquired an epithelioid morphology and up-regulated the intermediate filament protein nestin, typically present in primitive neuroectodermal cells. The manipulated levels of BCL2 did not have appreciable impact on cell survival in normal culture. Our findings demonstrate that the BCL2 gene product participates in the regulation of neural differentiation.

The C-terminal region of human angiogenin has a dual role in enzymatic activity.

The ribonucleolytic activity of angiogenin (Ang) is essential to Ang's capacity to induce blood vessel formation. Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln-117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate binding and catalysis. As a first step toward structural characterization of this conformational change, additional site-directed mutagenesis and kinetic analysis have been used to examine the intramolecular interactions that stabilize the inactive conformation of the protein. Two residues of this region, Ile-119 and Phe-120, are found to make hydrophobic interactions with the remainder of the protein and thereby help to keep Gln-117 in its obstructive position. Furthermore, the suppression of activity by the intramolecular interactions of Ile-119 and Phe-120 is counterbalanced by an effect of the adjacent residues, Arg-121, Arg-122, and Pro-123 which do not appear to form contacts with the rest of the protein structure. They contribute to enzymatic activity, probably by constituting a peripheral subsite for binding polymeric substrates. The results reveal the nature of the conformational change in human Ang and assign a key role to the C-terminal region both in this process and, presumably, in the regulation of human Ang function.

Function of the pre-T-cell receptor alpha chain in T-cell development and allelic exclusion at the T-cell receptor beta locus.

The pre-T-cell receptor, composed of the T-cell receptor (TCR) beta chain (TCRbeta), pre-Talpha (pTalpha) chain, and CD3 molecules, has been postulated to be a transducer of signals during the early stages of T-cell development. To examine the function of the transmembrane pTalpha chain during tbymocyte development, we generated pTalpha-/- embryonic stem cells and assayed their ability to differentiate into lymphoid cells in vivo after injection into recombination-activating gene (RAG)-2-deficient blastocysts. Thymocytes representing all stages of T-cell differentiation were detected in the thymus of pTalpha-/- chimeric mice, indicating that thymocyte development can occur without pTalpha. However, greatly reduced thymocyte numbers and substantially increased percentages of both CD4-CD8- thymocytes and TCRgammadelta+ thymocytes suggest that pTalpha plays a critical role in thymocyte expansion. To investigate the role of the pTalpha chain in allelic exclusion at the TCRbeta locus, a functionally rearranged TCRbeta minigene was introduced into pTalpha-/- and pTalpha+/- embryonic stem cells, which were subsequently assayed by RAG-2-deficient blastocyst complementation. In the absence of pTalpha, expression of the transgenic TCRbeta inhibited rearrangement of the endogenous TCRbeta locus to an extent similar to that seen in normal TCRbeta transgenic mice, suggesting that pTalpha may not be required for signaling allelic exclusion at the TCRbeta locus.