Nucleoprotein gene tracking: localization of specific HIV-1 genes to subchromatin nucleoprotein complexes containing endonuclease activity in HIV-1-infected human cells.

We developed a technique with which to isolate specific subchromatin deoxyribonucleoprotein/ribonucleoprotein precursor complexes containing discrete genes from intact mammalian nuclei by direct restriction enzyme treatment with MspI. These nucleoprotein complexes can be further fractionated and purified by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electroelution and removal of detergent, virtually thousands of nucleoprotein complexes can be examined for the presence of tightly bound genes and enzymatic activities. Nucleoprotein gene tracking procedures were applied to study the acidic nucleoprotein complexes from steady-state human H9 cells uninfected or infected with human immunodeficiency virus type 1 (HIV-1) virus. The purified nucleoprotein complexes were screened for the presence of loosely and tightly associated HIV-1 gene sequences (pol, env, tat, and rev) using a DNA hybridization protocol. In HIV-1-infected cells, four specific nucleoprotein complexes out of several hundred were found to contain tightly bound HIV-1 pol gene sequences after purification. By contrast, the other HIV-1 gene sequences (env, tat, and rev) were not tightly bound to any of the nucleoprotein complexes in HIV-infected cells. The observations suggest that certain HIV-1 genes associate with specific chromatin nucleoprotein complexes, regardless of their pattern of DNA integration into the human genome. At least two of the HIV-1 pol-containing nucleoprotein complexes of apparent M(r) approximately 94,000, pI approximately 6.5, and M(r) approximately 47,000, pI approximately 5.1 contain DNA endonuclease activity. This was confirmed in the present study, using linearized pUC19 plasmid substrate. The technique can be used to study a variety of problems concerning the association of specific genes and enzymes with specific nucleoprotein complexes J. Cell. Biochem. Suppls. 32/33:158-165, 1999.

Gulf War illnesses: complex medical, scientific and political paradox.

Gulf War illnesses are a collection of disorders that for the most part can be diagnosed and treated, if effective programmes exist to assist veterans, and in some cases their immediate family members. Although these illnesses are complex and have multi-organ signs and symptoms, a proportion of these patients can be identified as having Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) and/or Fibromyalgia Syndrome (FMS). Although there are many possible causes of CSF/ME/FMS, chronic infections can explain, at least in a subset of patients, the apparent transmission of these illnesses to family members and the appearance of chronic, multi-organ and auto-immune signs and symptoms. Unfortunately, many veterans who have been diagnosed with chronic infections, such as mycoplasmal infections, cannot obtain adequate treatment for their condition, resulting in their reliance on private physicians and clinics for assistance. This lack of response may ultimately be responsible for the transmission of the illness to non-veterans.

The eight myths of Operation 'Desert Storm' and Gulf War syndrome.

Several conventional claims regarding Gulf War Syndrome are criticized: that Gulf War veterans are no sicker than the civilian population as a whole; that Gulf War Syndrome is a myth invented by the press; that GWS cannot be defined as a legitimate medical syndrome; that since its cause cannot be determined, it is not a problem associated with Operation 'Desert Storm'; that the US and UK governments are doing all they can to investigate and treat illness in veterans or deny existence of over 100,000 cases in veterans and their families; that GWS will settle without treatment; that the armed forces were well prepared for integrated conflict involving chemical and biological warfare in the Middle East, increasing the risk of this in the future.

Chromatin nucleoprotein complexes containing tightly bound c-abl, p53 and bcl-2 gene sequences: correlation with progression of chronic myelogenous leukemia.

We previously developed a technique to isolate subchromatin nucleoprotein complexes (NPC) that contain tightly bound genes and enzymatic activities. NPC fractions (NPCF) were prepared by directly treating isolated nuclei with MspI to generate six NPCF (S1, M1, S2, M2, 0.1K and R). The NPCF have been used to predict the potential efficacy of interferon-alpha (IFN-alpha) treatment in patients with chronic myelogenous leukemia (CML) [Nicolson et al., Gene 159 (1995) 105-111]. Here the NPCF were probed for the presence of tightly bound c-abl, p53 and bcl-2 genes. We found that the NPCF isolated from the nuclei of leukocytes of normal individuals rarely contained detectable quantities of tightly-bound c-abl, p53 or blc-2 genes or gene sequences, whereas in CML nuclei these genes were often found in tight association with multiple NPCF. Examination of NPCF isolated from the leukocyte nuclei from patients with highly progressive CML for the presence of the three genes revealed that more NPCF contained the three tightly-bound genes than leukocyte NPCF from patients with stable or less progressed CML. These data suggest that as CML progresses to more malignant states, oncogenes, suppressor genes and apoptosis-associated genes become tightly associated with NPCF.

Interferon-alpha directly inhibits DNA polymerase activity in isolated chromatin nucleoprotein complexes: correlation with IFN-alpha treatment outcome in patients with chronic myelogenous leukemia.

We have developed an in vitro assay to assess and predict the potential efficacy of in vivo interferon-alpha (IFN-alpha) treatment (5 x 10(6) units/m2 per day) for patients with chronic myelogenous leukemia (CML). Although determining the numbers and affinities of IFN-alpha receptors on CML cells has been developed as a method for predicting treatment response to IFN-alpha, it fails to predict response in CML. Previously, we and others observed that mitogens, toxins and lectins that bind to cell-surface receptors are endocytosed, escaping endosomes in order to act directly on cellular targets. Therefore, we tested the ability of low concentrations of IFN-alpha (5-10 units) to act directly on DNA polymerase (Pol) in purified chromatin nucleoprotein complexes (NPC). NPC were prepared by a methodology that uses direct treatment of leukocyte nuclei with MspI to generate six NPC-containing fractions (S1, M1, S2, M2, 0.1K and R). We found three general categories of in vitro DNA synthesis response for the six different NPC fractions isolated from the white blood cells of patients with CML (n = 19) before their treatment with IFN-alpha. IFN-alpha induced either stimulation, inhibition or had no apparent effect on Pol activity in the six different NPC fractions in a blind assay. In most of the NPC fractions isolated from the leukocytes of patients with progressive CML and in those from CML patients who failed to show a clinical response to IFN-alpha, this cytokine stimulated or had no effect on Pol activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleoprotein complexes from metastatic cells containing oncogenes and tissue-specific genes: a novel method to track genes associated with specific nucleoproteins.

Intact nuclei derived from murine metastatic large-cell lymphoma and human chronic myelogenous leukemia cells were digested to discrete subchromatin deoxyribonucleoprotein/ribonucleoprotein precursor complexes by treatment with Msp-I. The resultant complexes were composed of nucleoproteins (NPs) that were isolated and purified by two-dimensional isoelectric focusing/sodium dodecylsulfate polyacrylamide gel electrophoresis (2D-SDS-PAGE), electroelution from the gel, and removal of SDS by extractigel chromatography. Various NPs purified by 2D-SDS-PAGE were examined for the presence of oncogenes and tissue-specific genes using a dot-blot hybridization technique. The RNA polymerase products of NPs were labeled, purified, and subsequently used in a back-hybridization assay to identify transcripts for particular genes. By utilizing a 2D-SDS-PAGE Southwestern technique in parallel with the dot-blot and RNA back-hybridization assays, we assessed whether it is possible to "track" a gene and its associations in particular NPs. In patients with chronic myelogenous leukemia, we screened approximately 1000 NPs for bcl-2 sequences and found them present in a single NP of apparent M(r) approximately 19,000, pI approximately 5.5. In murine RAW117-H10 cells transformed by the abl oncogene, we found by Western analysis that an antigen cross-reacting with abl antigen was localized to a p53 gene-containing NP of apparent M(r) approximately 22,000, pI approximately 7.2. A coincident Southwestern experiment using the same blot showed that the abl gene was bound by the same NP. The techniques described present the basis for "tracking" a particular gene to individual NPs and studying its relationship to other genes, their respective gene products, and its binding properties with particular NPs.

Nucleoproteins derived from subnuclear RNA polymerase complexes of metastatic large-cell lymphoma cells possess transcription activities and regulatory properties in vitro.

Intact nuclei derived from poorly or highly liver-metastatic murine large-cell lymphoma cell line RAW117 were digested to discrete subchromatin deoxyribonucleoprotein/ribonucleoprotein (DNP/RNP) complexes with Msp-I. The DNP/RNP complexes were composed of DNP/RNPs which were derived from the DNP/RNP complexes by incubation in the presence or absence of DNase-I and subsequent isolation by two-dimensional [isoelectric focusing/sodium dodecylsulfate (SDS)] polyacrylamide gel electrophoresis (PAGE), electroelution from the gel, and removal of SDS. Approximately 450 DNP/RNPs in the two-dimensional gels corresponding to discrete spots or in some cases streaks were analyzed for the presence of v-abl, p53, c-neu, c-H-ras, beta-casein, 18s rDNA, and mu-chain immunoglobulin genes using a hybridization technique. Ten DNP/RNP complexes contained tightly associated p53 DNA, whereas six contained c- or v-abl, four contained mu-chain gene, two contained c-H-ras, one contained dot-blot beta-casein, two contained 18s rDNA, and c-neu was found in one of the DNP/RNPs. The DNP/RNPs were also analyzed for in vitro RNA polymerase and primase activities. To assess the potential transcription abilities of the isolated DNP/RNPs, individual DNP/RNPs or DNP/RNP mixtures (reconstituted after SDS-PAGE separation) were examined for RNA polymerase initiation and synthesis. When RNA products were formed, these were purified by extracellulose chromatography and used as back-hybridization probes for the genes of interest. The RNA products were also analyzed by RNA gel electrophoresis. RNA formation was inhibitable by actinomycin D, and the RNAs formed ranged in size from approximately 80 kbp to approximately 1 kbp. By mixing various DNP/RNP complexes together, different patterns of RNA synthesis were found. For example, one DNP/RNP of M(r) approximately 140,000, isoelectric point (pl) approximately 5.8 synthesized a high molecular weight RNA in vitro that hybridized with beta-casein cDNA, but beta-casein is not expressed in RAW117 cells, suggesting that the silencing of the beta-casein gene was negated by isolation of the DNP/RNP. Mixing this DNP/RNP with two other specific DNP/RNPs again inhibited the synthesis of beta-casein RNA, suggesting that interactions between DNP/RNP complexes can result in differential RNA expression or regulation of RNA polymerases in vitro.

Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities.

We report on the discovery and isolation of DNA- and RNA-containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of approximately 90 and approximately 25 kbp. The deoxyribonucleoprotein-ribonucleoprotein complexes contain RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large-cell lymphoma cells by restriction digestion with Msp-I, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two-dimensional (isoelectric focusing/M(r)) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of approximately 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific activities. Using the DNP/RNP complexes a discrete DNA polymerase alpha product of approximately 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase alpha inhibitor aphidicolin. RNA polymerase assays in the presence of excess alpha-amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex-associated enzyme activities to be cell-cycle dependent. The DNA and RNA polymerase-related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors.(ABSTRACT TRUNCATED AT 250 WORDS)

Probing nucleosome core secondary structure before and after alpha-chymotrypsin treatment by Raman spectroscopy and thermal denaturation.

Nucleosome cores were digested with alpha-chymotrypsin until histone H3 was degraded to a partial histone, CP1. As we reported previously, cleavage occurred at leucine 20 to H3 and resulted in an increase in circular dichroism between 265 to 285 nm. Some modest core unfolding was also observed as determined by a small decrease in the sedimentation coefficient. Studies reported here deal with the analysis of core secondary structure and subsequent perturbation caused by treatment with alpha-chymotrypsin. Raman spectroscopy indicated that chymotryptic treatment promoted a change in the conformational environment of a population of core histone tyrosines. In addition, a shift from B-form to an intermediate B- or A-form was observed for core DNA. High-resolution thermal denaturation was used to determine alterations in the stabilization of core DNA related to perturbation of the core histones. Brief chymotryptic treatment indicated changes in both pre-melt and irreversible transitions.