Backtracking leukemia to birth: Identification of clonotypic gene fusion sequences in neonatal blood spots

Epidemiological evidence has suggested that some pediatric leukemias may be initiated in utero and, for some pairs of identical twins with concordant leukemia, this possibility has been strongly endorsed by molecular studies of clonality. Direct evidence for a prenatal origin can only be derived by prospective or retrospective detection of leukemia-specific molecular abnormalities in fetal or newborn samples. We report a PCR-based method that has been developed to scrutinize neonatal blood spots (Guthrie cards) for the presence of numerically infrequent leukemic cells at birth in individuals who subsequently developed leukemia. We demonstrate that unique or clonotypic MLL-AF4 genomic fusion sequences are present and detectable in neonatal blood spots from individuals who were diagnosed with acute lymphoblastic leukemia at ages 5 months to 2 years and, therefore, have arisen during fetal hematopoiesis in utero. This result provides unequivocal evidence for a prenatal initiation of acute leukemia in young patients. The method should be applicable to other fusion genes in children with common subtypes of leukemia and will be of value in attempts to unravel the natural history and etiology of this major subtype of pediatric cancer.

Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification

Macromolecular transport systems in bacteria currently are classified by function and sequence comparisons into five basic types. In this classification system, type II and type IV secretion systems both possess members of a superfamily of genes for putative NTP hydrolase (NTPase) proteins that are strikingly similar in structure, function, and sequence. These include VirB11, TrbB, TraG, GspE, PilB, PilT, and ComG1. The predicted protein product of tadA, a recently discovered gene required for tenacious adherence of Actinobacillus actinomycetemcomitans, also has significant sequence similarity to members of this superfamily and to several unclassified and uncharacterized gene products of both Archaea and Bacteria. To understand the relationship of tadA and tadA-like genes to those encoding the putative NTPases of type II/IV secretion, we used a phylogenetic approach to obtain a genealogy of 148 NTPase genes and reconstruct a scenario of gene superfamily evolution. In this phylogeny, clear distinctions can be made between type II and type IV families and their constituent subfamilies. In addition, the subgroup containing tadA constitutes a novel and extremely widespread subfamily of the family encompassing all putative NTPases of type IV secretion systems. We report diagnostic amino acid residue positions for each major monophyletic family and subfamily in the phylogenetic tree, and we propose an easy method for precisely classifying and naming putative NTPase genes based on phylogeny. This molecular key-based method can be applied to other gene superfamilies and represents a valuable tool for genome analysis.

Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA–protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites.

DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a delayed ion extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.