In Saccharomyces cerevisiae chromosomal DNA replication initiates at intervals of ϳ40 kb and depends upon the activity of autonomously replicating sequence (ARS) elements. The identification of ARS elements and analysis of their function as chromosomal replication origins requires the use of functional assays because they are not sufficiently similar to identify by DNA sequence analysis. To complete the systematic identification of ARS elements on S. cerevisiae chromosome III, overlapping clones covering 140 kb of the right arm were tested for their ability to promote extrachromosomal maintenance of plasmids. Examination of chromosomal replication intermediates of each of the seven ARS elements identified revealed that their efficiencies of use as chromosomal replication origins varied widely, with four ARS elements active in Յ10% of cells in the population and two ARS elements active in Ն90% of the population. Together with our previous analysis of a 200-kb region of chromosome III, these data provide the first complete analysis of ARS elements and DNA replication origins on an entire eukaryotic chromosome. INTRODUCTIONThe replication of eukaryotic chromosomes initiates at multiple replication origins spaced at intervals of 40 -100 kb. In the budding yeast, Saccharomyces cerevisiae, replication origins depend upon cis-acting replicators called autonomously replicating sequence (ARS) elements, which are recognized by their ability to maintain extrachromosomal plasmids. The initiation of replication at individual replicators is a tightly regulated process. Replication initiations are confined to S phase, and individual replicators initiate at reproducible times during S phase, some early and some late (Reynolds et al., 1989, and references therein;Friedman et al., 1997;Donaldson et al., 1998b). Moreover, the efficiencies of initiation vary from one replicator to another, the extreme case being ARS elements that are not active as replication origins in their normal chromosomal positions (Dubey et al., 1991;Friedman et al., 1997;Yamashita et al., 1997).Replicators become competent to initiate replication during the G1 phase of the cell cycle through the stepwise assembly of prereplicative complexes on replicators that are bound by origin recognition complex (ORC) (reviewed by Dutta and Bell, 1997). The actual initiation events require the activities of at least two protein kinases, the cyclin-dependent kinase (CDK) Cdc28p associated with cyclin B (Clb5p or Clb6p) and the Cdc7p kinase associated with its regulatory subunit Dbf4p. The assembly of prereplicative complexes is prevented by the activity of cyclin B-associated CDK, effectively preventing reinitiation at origins during a single S phase (reviewed by Diffley, 1996). Timing determinants also appear to be specified during G1 of the cell cycle (Raghuraman et al., 1997), although the relationship between establishment of the prereplication complex and the specification of initiation timing is unclear. In the case of a latereplicating region of chromosome XIV, DN...

Cell-based fluorescence assays in leukemia/lymphoma evaluations typically rely on qualitative approaches for the identification and enumeration of the target cell population(s), but other equally important diagnostic assays are quantitative or semiquantitative. Qualitative assays usually give an overall picture of the composite phenotype based on the expression level of a set of antigens on particular cell lineages that render diagnostic patterns. Conversely, quantitative flow cytometry precisely measures the antigen density or absolute target cell count, and semi-quantitative assays quantify the abnormal target cell population above a certain threshold relative to its normal counterpart or total cells. The following sections attempt to provide an overview of the different types of flow cytometric evaluation of normal and pathological specimens, providing details of

No abstract

Anaplastic large cell lymphoma (ALCL) is a distinct type of T/null-cell non-Hodgkin lymphoma that commonly involves nodal and extranodal sites. The World Health Organization of lymphoid neoplasms recognizes two types: anaplastic lymphoma kinase (ALK) positive or ALK negative, the former as a result of abnormalities involving the ALK gene at chromosome 2p23. Patients with ALCL rarely develop a leukemic phase of disease, either at the time of initial presentation or during the clinical course. Described herein is a patient with ALK+ ALCL, small cell variant, associated with the t(2;5)(p23;q35), who initially presented with leukemic involvement and an extraordinarily high leukocyte count of 529 x 10(9)/L, which subsequently peaked at 587 x 10(9)/L. Despite chemotherapy the patient died 2(1/2) months after diagnosis. In the literature review 20 well-documented cases are identified of ALCL in leukemic phase reported previously, with a WBC ranging from 15 to 151 x 10(9)/L. Leukemic phase of ALCL occurs almost exclusively in patients with ALK+ ALCL, most often associated with the small cell variant and the t(2;5)(p23;q35), similar to the present case. Patients with leukemic phase ALK+ ALCL appear to have a poorer prognosis than most patients with ALK+ ALCL.

No abstract

An important goal in medicine is the development of methods for cell-specific targeting of therapeutic molecules to pathogens or pathogen-infected cells. However, little progress has been made in cell-specific targeting of bacterially infected cells. Using a phage display approach, we have isolated a 20-mer peptide that binds to Mycoplasma arginini infected pancreatic beta-cells in tissue culture. This peptide binds to M. arginini infected beta-cells 200 times better than a control phage and is specific for the infected cells. Furthermore, transferring the M. arginini contamination to another cell line renders the newly infected cell line susceptible to peptide binding. Immunolocalization experiments suggest that the peptide is binding to M. arginini adhered to the cell surface. The free synthetic peptide retains its binding in the absence of the phage vehicle and tetramerization of the peptide increases its affinity for the infected cells. Efforts have been made to use this peptide to eliminate Mycoplasma from infected cell lines using ferromagnetic beads coated with the selected peptide. A ten-fold reduction of infection was accomplished with one fractionation via this approach. Our results suggest that this peptide, isolated from an unbiased selection, may be of utility for the detection and reduction of Mycoplasma infection in cultured cells. Furthermore, a general implication of our findings is that phage display methods may be useful for identifying peptides that target a broad array of other biological pathogens in a specific fashion.

By screening a solid-phase combinatorial peptide library, a short peptide ligand, YYWLHH, has been discovered that binds with high affinity and selectivity to staphylococcal enterotoxin B (SEB), but only weakly to other SEs that share sequence and structural homology with SEB. Using column affinity chromatography with an immobilized YYWLHH stationary phase, it was possible to separate SEB quantitatively from Staphylococcus aureus fermentation broth, a complex mixture of proteins, carbohydrates and other biomolecules. The immobilized peptide was also used to purify native SEB from a mixture containing denatured and hydrolyzed SEB, and showed little cross-reactivity with other SEs. To our knowledge this is the first report of a highly specific short peptide ligand for SEB. Such a ligand is a potential candidate to replace antibodies for detection, removal and purification strategies for SEB.

No abstract