Mutation identification DNA analysis system (MIDAS) for detection of known mutations.

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA detection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/ apurinic (AP) site is cleaved by AP endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32p- or FITC-labeled oligonucleotides as mutation probes. Generally between 20-50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full-length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser-induced fluorescence-capillary electrophoresis (LIF-CE) of fluorophore-labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3'-endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG-MRE). MIDAS differentiated between a clinical sample BRCA 1 wild-type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF-CE should make detection of known point mutations, deletions, and insertions a rapid and cost-effective technique well suited for automation.

GATA-6 stimulates a cell line-specific activation element in the human lactase promoter.

Lactase-phlorizin hydrolase (LPH) synthesis is restricted to differentiated small intestinal enterocytes and is highly regulated during development. Analysis of expression of LPH promoter segments fused with luciferase transfected in Caco-2 cells, a line that uniquely expresses LPH mRNA, mapped an 18-base pair (bp) segment 100 bp upstream of the transcription start site that is required for transactivation. Remarkably, the LPH upstream element (LUE) has no stimulatory activity in both human intestinal and nonintestinal lines in which LPH mRNA is absent. Electrophoretic analysis of sequence-specific DNA-nuclear protein complexes demonstrated the presence of a Caco-2 cell-specific protein(s) (CCP), which is uniformly absent in LPH nonproducer cell lines. Mutational analysis of the LUE demonstrated that bases contained within a GATA consensus motif are critical for both CCP binding and transcription from the LPH promoter. Caco-2 cells express high levels of GATA-6 mRNA in a cell line-specific manner, suggesting that GATA-6 is a CCP that complexes with the LUE. When expressed by a plasmid, GATA-6 transactivated the LPH promoter. The stimulation was abrogated with mutations in the GATA consensus motif as well as mutations in a flanking downstream element. These studies are consistent with an important role of an intestinal GATA binding protein in cell type-specific transactivation of the LPH promoter.

The use of dynamic size-sieving capillary electrophoresis and mismatch repair enzymes for mutant DNA analysis.

The detection of base pair mismatches in limiting amounts of DNA is important in the early diagnosis of cancer and other genetic diseases. The specific type and exact location of a bp mismatch in certain genes can yield information on the likelihood of a patient developing a genetic disease, as well as the severity of the disease. We demonstrate two methods of specific DNA point mutation detection and identification that involve the integration of mismatch repair cleavage enzyme analyses with dynamic size-sieving capillary electrophoresis. The mismatch repair cleavage enzymes employ the very mechanism that a cell uses in its own mismatch recognition and repair systems. One analysis employs an isothermal signal amplification process, and the other involves polymerase chain reaction (PCR) (Hoffmann-LaRoche, Nutley, NJ, U.S.A.) for amplification of the DNA. Separation of the DNA fragments using dynamic size-sieving CE yields a cleaved fragment, providing definitive evidence of a bp mismatch. The specificity and sensitivity of the assay are facilitated by the detection of fluorescently labeled DNA fragments using laser-induced fluorescence detection; picogram quantities of a target DNA can be analyzed reproducibly.

A transactivator of c-myc is coordinately regulated with the proto-oncogene during cellular growth.

A recently cloned nuclear protein, which binds a far upstream element (FUSE) of the human c-myc proto-oncogene, stimulates promoter driven expression in undifferentiated cells. In concert with a loss of c-myc expression, both FUSE binding protein (FBP) mRNA and protein levels disappeared in HL60 cells after PMA-induced differentiation, due to a drop in the rate of transcription that was measured by nuclear runoff. During the differentiation of these cells, the brief half-lives of FBP mRNA (3 h) and protein (1.5 h) did not change, allowing for the rapid down-regulation of nuclear protein levels, as detected by immunohistochemical staining. Like c-myc, FBP is expressed in proliferating cells from a variety of lineages, but not in quiescent cells. When T cells and fibroblasts were stimulated to transit from G0 into the cell cycle, there was a dramatic rise of both FBP mRNA and DNA sequence specific nuclear FBP binding activity, which correlated with the appearance of c-myc mRNA. In contrast to the transient expression of many other immediate early growth response genes, both FBP and c-myc expression were sustained for more than 24 h. In fibroblasts, the coordinate expression of FBP and c-myc throughout all phases of the cell cycle is consistent with FBP's role as a growth-dependent regulator of c-myc expression.

Targeted melting and binding of a DNA regulatory element by a transactivator of c-myc.

A far upstream element (FUSE) of c-myc stimulates promoter activity when bound by a newly identified trans-acting protein, which is expressed in cycling cells. Since FUSE binding protein (FBP) binds only the noncoding strand (NCS) of its regulatory element in a sequence-specific manner, and not double-stranded (ds) DNA, formation of the protein DNA complex in vivo first requires unwinding of the DNA helix. In this report, we show evidence that FBP forces strand separation of short stretches of linear dsDNA. Because FUSE is contained within a region of helical instability that is partially unwound in negatively supercoiled DNA, it is a target for more extensive duplex strand separation by FBP, which first exposes and then selectively binds its NCS cognate sequence. In contrast, other single-stranded DNA binding proteins (SSBs) do not demonstrate this FUSE targeting activity. The novel linkage of regional dsDNA melting with cis-element binding by a transcriptional activator has broad implications in the regulation of eukaryotic gene expression.

A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif.

The far upstream element (FUSE) of the human c-myc proto-oncogene stimulates expression in undifferentiated cells. A FUSE-binding protein (FBP) is present in undifferentiated but not differentiated cells. Peptide sequences from the purified protein allowed cloning of cDNAs encoding FBP. Expression of FBP mRNA declined upon differentiation, suggesting transcriptional regulation of FBP. Features in the FBP cDNA suggest that FBP is also regulated by RNA processing, translation, and post-translational mechanisms. Both cellular and recombinant FBP form sequence-specific complexes with a single strand of FUSE. Transfection of FBP into human leukemia cells stimulated c-myc-promoter-driven expression from a reporter plasmid in a FUSE-dependent manner. Deletion and insertion mutagenesis of FBP defined a novel single-strand DNA-binding domain. Analysis of the primary and predicted secondary structure of the amino acid sequence reveals four copies of a reiterated unit comprised of a 30-residue direct repeat and an amphipathic alpha-helix separated by an 18- to 21-residue spacer. The third and fourth copies of this repeat-helix unit constitute the minimum single-stranded DNA-binding domain. To determine whether the FUSE site, in vivo, possesses single-strand conformation, and therefore could be bound by FBP, cells were treated with potassium permanganate (KMnO4) to modify unpaired bases. Modification of genomic DNA in vivo revealed hyperreactivity associated with single-stranded DNA in the FUSE sequence and protection on the strand that binds FBP in vitro. The role of single-stranded DNA and single-strand binding proteins in c-myc regulation is discussed.

Expression of pregnancy-specific beta 1-glycoprotein genes in hematopoietic cells.

The presence of PSG in blood cells has been demonstrated by immunohistochemical staining. However, the origin of those proteins is not known. This report examines the expression of the PSG genes in different types of freshly isolated blood cells. RNA isolated from bone marrow and peripheral blood cells of healthy individuals was analyzed for PSG transcripts by reverse transcriptase-polymerase chain reaction using synthetic oligonucleotide primers specific for the PSG genes. The level of expression of the PSG genes in different types of cells exhibited significant individual variation. Trace amounts of PSG transcripts could be detected in polymorphonuclear cells (PMN), monocytes and B lymphocytes while T lymphocytes always contained the highest level of transcript. The expression of PSG genes in the blood cells apparently was not affected by the method of isolation nor by overnight culturing of these cells except in the case when lymphocytes were separated by rosetting with sheep red blood cells. All reported PSG transcripts were detected in blood cells. Both type I and type II transcripts of the PSG genes were detected in blood cells with the exception of type II transcript of PSG5 and PSG11 which were only found in the placenta. Tissue specificity in the expression or alternative splicing of some of the PSG family members was implicated.

Autoimmune hemolytic anemia presenting in Sézary syndrome. Report of a case and review of the literature.

A 52-year-old man, who presented with Sézary syndrome with autoimmune hemolytic anemia (AIHA) and was successfully treated with corticosteroids is reported. Helper function assay determining immunoglobulin confirmed inducer capability of this clonal population. This patient brings to 4 the number of cases of T cell cutaneous lymphoma and AIHA now reported in the English literature, and is the first case of Sézary syndrome and AIHA thus far.

Ultraviolet B-induced DNA fragmentation (apoptosis) in activated T-lymphocytes and Jurkat cells is augmented by inhibition of RNA and protein synthesis.

UVB (280-320 nm) light profoundly affects cellular immunity and immunogenicity. To further characterize the interaction of UVB light with T cells, we examined UVB-induced DNA fragmentation patterns in normal T cells and the T-cell line Jurkat. Resting or preactivated peripheral blood T cells from normal donors or Jurkat cells were exposed to various doses of UVB or gamma irradiation. Cells were sampled at 0-48 h after exposure, and DNA fragmentation was analyzed by electrophoresis in agarose gels. UVB and gamma irradiation, in a dose- and time-dependent manner, induced DNA fragmentation in Jurkat cells and in T cells preactivated with phytohemagglutinin (PHA; 10 micrograms/ml) and phorbol myristate acetate (PMA; 10 ng/ml), but not in resting T cells. The presence of RNA or protein synthesis inhibitors such as actinomycin D or cycloheximide neither inhibited nor delayed DNA fragmentation; in fact, DNA fragmentation was augmented above control values. Similarly, DNA fragmentation increased in the presence of the calcium-chelating agent ethylene-glycol-bis-tetraacetic acid (EGTA) and decreased in the presence of the calcium ionophore A23187. In the presence of ethylenediaminetetraacetic acid (EDTA), DNA fragmentation decreased. In summary, these data show that UVB-induced DNA fragmentation strongly depends upon the cell activation status, and upon the presence of divalent cations other than calcium, possibly magnesium. The data indicate furthermore that in this model, inhibition of RNA or protein synthesis can induce rather than inhibit apoptosis, suggesting that the synthesis inhibitors disrupted primarily the synthesis or action of enzymes ordinarily aimed at repairing DNA fragmentation.

Isolation of hemopoietic precursor cells from human marrow by negative selection using monoclonal antibodies and immunomagnetic beads.

A purified or enriched population of human marrow hemopoietic precursor cells may be produced by using a monoclonal antibody to specifically bind to the desired cell population (positive selection), or by removing undesired cell populations contaminating the mixture (negative selection). We describe a simple negative selection technique using commercially available monoclonal antibodies and immunomagnetic beads to produce a mononuclear marrow cell fraction which comprises no more than 2% of the initial cell population yet contains all the BFU-E and CFU-GM present originally. This procedure is an efficient method for producing hemopoietic precursor cells appropriate for studying cell-substrate adhesions, homing mechanisms, and response to growth factors without interference by contaminating accessory cells.

Human B cell lines can be triggered to secrete an interleukin 2-like molecule.

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt's and non-Burkitt's types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 ng/ml) triggered IL-2 secretion in 7/8 (87%) EBV-negative lymphoma cell lines of American origin and in 6/6 (100%) normal lymphoblastoid cell lines, but in only 1/6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5/5 (0%) African Burkitt's cell lines. IL-2 secretion was not detected in control supernatants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS-1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tac monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti-tac monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation.

Ultraviolet B light inactivates bone marrow T lymphocytes but spares hematopoietic precursor cells.

Bone marrow cells from ten normal donors were exposed to ultraviolet (UV)C or UVB light for total exposures of 0.1 to 100 mJ/cm2, and assayed for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and phytohemagglutinin (PHA)-stimulated proliferative responses. After exposure to UVC CFU-GM, BFU-E and PHA responses showed a UV dose-dependent sharp decrease to levels less than 1% of controls with 0.5, 2.0, and 10 mJ/cm2, respectively. With UVB, PHA responses were most sensitive, declining to less than 1% at 5 mJ/cm2. BFU-E decreased to less than 1% of control with 15 mJ/cm2 UVB. CFU-GM, at UVB doses of 0.1 to 2.0 mJ/cm2, increased to 125% to 130% of control and decreased to less than 1% only at exposures greater than 20 mJ/cm2. Thus, these studies show that UVB, but not UVC light, can be used to inactivate bone marrow T lymphocytes selectively while sparing hematopoietic precursor cells. The data suggest that UVB irradiation can be used for T-lymphocyte purging for allogeneic marrow transplantation.

Heterogeneity of B-cell growth factor receptor reactivity in healthy donors and in patients with chronic lymphatic leukemia: relationship to B-cell-derived lymphokines.

Twenty-five long-term B-cell lines were studied for B-BCGF activity. The cell lines were cultured in the presence or absence of the new tumor promoter teleocidin, and control and teleocidin-treated derived supernatants were cocultured with purified B cells obtained from healthy donors and patients with B chronic lymphatic leukemia (B-CLL), in the presence of anti-mu. In attempt to delineate the role of other B-cell lymphokines in promoting proliferation of activated B cells, the supernatants were also studied for interleukin 1 (IL-1), interleukin 2 (IL-2), and interferon gamma (IFN-gamma). The effect of B-cell-derived lymphokines on the proliferation of activated B cells obtained from the 14 donors was heterogeneous, and three types of response were observed: In four healthy donors there was induction of B-cell proliferation by B-cell lymphokines derived from both control cells and teleocidin-treated cells. In cells obtained from the other five healthy donors there was induction of B-cell proliferation by B-cell lymphokines derived from teleocidin-treated cells. In the five B-CLL patients, B-cell proliferative response to B-cell lymphokines derived from both control cells and teleocidin-activated cells was absent. Comparison of B-BCGF reactivity to T-BCGF reactivity demonstrated that B-CLL B lymphocytes did not respond to either B-BCGF or T-BCGF, whereas normal B cells responded to T-BCGF and may proliferate upon stimulation with B-cell-derived IL-2 and/or B-BCGF. These results suggest heterogeneity of B-BCGF receptor reactivity in B lymphocytes derived from healthy donors, and lack of both B-BCGF and T-BCGF receptor reactivities in B lymphocytes derived from B-CLL patients; B-cell-derived lymphokines influence normal B-cell response but not leukemic B cells; B-BCGF optimal effect is in large part due to other B-cell lymphokines, especially B-cell-derived IL-2; the possible existence of various B-BCGFs.

Burkitt's cells can be triggered by teleocidin to secrete interferon-gamma.

The secretion of interferon (IFN)-gamma by T lymphocytes is mediated by the synthesis of interleukin 2 (IL-2) and the availability of IL-2 receptors. Since some Burkitt's lymphoma lines express Tac antigen and can be triggered to secrete IL-2 following activation with the new tumor promoter teleocidin, we addressed the question of whether the induction of IL-2 by B lymphocytes is accompanied by the induction of IFN-gamma. IFN-gamma has not been detected in any of the 25 cell lines studied, and following stimulation with teleocidin, we triggered the synthesis of IFN-gamma in JLP(C), a pre-Burkitt's cell line. The mechanism of IFN-gamma secretion by B lymphocytes is not clear. Our findings demonstrate that the synthesis of IL-2 by B cells is not accompanied by IFN-gamma and suggest that the synthesis of IFN-gamma is not mediated by IL-2 or IL-1 or B-cell growth factor. Neutralization studies have shown that IFN-gamma secretion is not accompanied by the induction of IFN-alpha or IFN-beta. Our data imply that B cells can be triggered to secrete IFN-gamma under certain circumstances. Whether similar function occurs in vivo is not known.

Fish gonadotropin(s). I. Bioassay of salmon gonadotropin(s) in vitro with immature trout gonads.

1. The effect of salmon gonadotropin(s) (SG) on cyclic AMP (cAMP) levels in immature trout gonadal tissue of both sexes was measured by radioimmunoassay. 2. A dose-response line was obtained to SG in gonads of both male and female trout. 3. As little as 0.45 SG units (1 SG unit = 1 mug NIH-LH-S18 in the chick bioassay) significantly increased cAMP formation in the presence of 8 mM theophylline; mammalian LH, FSH, LTH, ACTH, TSH and HCG were inactive. 4. The assay for SG was investigated with respect to time of incubation and two phosphodiesterase inhibitors; some conditions for the cAMP radioimmunoassay (cAMP-RIA) were compared.

Fish gonadotropin(s). II. Isolation of gonadotropin(s) from chum salmon pituitary glands using affinity chromatography.

A highly purified gonadotropic hormone preparation has been obtained from chum salmon (Oncorhynchus keta) pituitaries by extraction with ethanolic or aqueous buffer, affinity chromatography on Con A Sepharose and gel filtration on Sephadex G-75 superfine. A purified fraction from Sephadex G-75 averaged 448 mug NIH-LH-S18/mg glycoprotein as measured by the uptake of radiophosphate into chick testes. A total of 1.1 g of salmon gonadotropin (s) (SG)/kg fresh tissue was recovered when the isolation began with an aqueous extraction. Analytical polyacrylamide gel electrophoresis (P.A.G.E.) of the purified fraction from Sephadex G-75 displayed a single broad zone in non-dissociating conditions and two bands in 8 M urea. Polyacrylamide gel electrofocusing yielded six sharp bands with an isoelectric point range of 4.38 to 5.05, and four bands with an isoelectric point range of 4.31 to 4.95 in 8 M urea. A molecular weight of 41,000 was determined by gel filtration. A subunit molecular weight of 17,800 +/- 10% was found by P.A.G.E. in 0.1% sodium dodecyl sulphate (SDS), suggesting that native SG consists of two subunits. Purified preparations were highly stable in Tris-Cl buffers and retained their activity for several months when stored at -73 degrees C.

Fish gonadotropin(s). III. Evidence for more than one gonadotropin in chum salmon pituitary glands.

Two proteins with gonadotropin activity have been isolated from a highly purified chum salmon (Oncorhynchus keta) gonadotropin preparation (G-75 Fraction II) by chromatography on DEAE Bio Gel A. These gonadotropins exhibited distinct behaviour in polyacrylamide gel electrophoresis, chromatography on Sephadex G-75 superfine, and ratios of cAMP stimulation in immature rainbow trout ovaries and testes. Rechromatography of G-75 Fraction II on Sephadex G-75 superfine gave a symmetrical protein peak with a coincident cAMP activity profile. Repeated freezing and thawing elicited a shift in the cAMP activity profile toward the trailing edge of the protein peak. Data are discussed in terms of two gonadotropin molecules which respond differently to phase changes. Charge polymorphism was exhibited by isoelectric focusing in polyacrylamide gels of one of the DEAE fractions. Five UV absorbing bands were observed which stimulated cAMP production in immature rainbow trout gonads. Three of these bands increased adenyl cyclase activity in trout ovaries and testes. One of the bands stimulated cAMP production primarily in trout testes and the other stimulated trout ovaries, providing evidence for two gonadotropins, each of which is sex specific.