Biomedical subjects

H R Hubbell

Publications and source records attributed to H R Hubbell.

At least 19 recordsLinked to original sources

PubMed1992

Cellular and enzymatic activities of a synthetic heteropolymer double-stranded RNA of defined size.

We have synthesized a novel heteropolymer double-stranded RNA (dsRNA) molecule of defined length and strandedness (dsRNA309) and evaluated its ability to induce cytokine gene expression, activate dsRNA-dependent enzymes, and inhibit both tumor cell growth and virus replication. Unlike the conventionally studied synthetic homopolymer dsRNAs, polyinosinic acid:polycytidylic acid (poly(I-C)) and its mismatched analogue polyinosinic:polycytidylic, uridylic acid (poly(I-C12,U), dsRNA309 possessed restricted biological activity. dsRNA309 was unable to inhibit tumor cell growth or efficiently induce cytokine (i.e. interferon-beta and interleukin-1 alpha) gene expression. However, dsRNA309 was able to inhibit virus replication and activate dsRNA-dependent intracellular enzymes, 2'-5' oligoadenylate synthetase (2'-5' A synthetase) and the dsRNA-activated inhibitor kinase in in vitro assay systems. Overall, dsRNA309 provided a means for examining the mechanisms governing the dsRNA-regulated antiviral and antiproliferative responses, and studies with dsRNA309 demonstrated that the ability of a synthetic dsRNA to activate dsRNA-dependent intracellular enzymes does not necessarily predict the same gene inducing capacity.

2',5'-Oligoadenylate Synthetase↗

PubMed1992

Potentiated lymphokine-activated killer cell activity generated by low-dose interleukin-2 and mismatched double-stranded RNA.

Lymphokine-activated killer (LAK) cell activity was measured in human peripheral blood mononuclear cells (PBMC) treated in vitro for 3 days with recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA). Lytic activity was measured utilizing K562 (NK-sensitive) and 786-0 (NK-resistant) target cells. PBMC cultured with rIL-2 (10-1000 BRMP U/ml) alone showed concentration-dependent lytic activity against the 786-0 target cells, while cells cultured in unsupplemented medium or medium supplemented with mismatched dsRNA (200 micrograms/ml) alone could not lyse the 786-0 targets. The combination of mismatched dsRNA with suboptimal concentrations of rIL-2 (10-30 U/ml) showed enhancement of both natural killer (NK) and LAK cell activities. The uptake of [3H]thymidine by treated effector cells was dependent on time and rIL-2 concentration and was not increased in the cells treated with low-dose rIL-2/mismatched dsRNA, compared to those treated with low-dose rIL-2 or mismatched dsRNA alone. Similarly, changes in the expression of CD3, CD4, CD8, CD57, CD16 and CD25 cell surface antigens were independent or rIL-2 concentration and not altered by the presence of mismatched dsRNA. These results indicate that mismatched dsRNA can potentiate rIL-2-induced LAK cell activity by increasing the functional activity per cell, rather than by increasing the number of activated cells.

Cytotoxicity, Immunologic↗

PubMed1992

Antitumor effects of interleukin-2 and mismatched double-stranded RNA, individually and in combination, against a human malignant melanoma xenograft.

The antitumor effects of recombinant interleukin-2 (rIL-2) and mismatched double-stranded RNA (dsRNA) were assessed in tissue culture and in a nude mouse model. Mismatched dsRNA did not show a direct antiproliferative effect against the human malignant melanoma cell line, BRO, in tissue culture. However, treatment of the BRO cells with up to 1000 units/ml rIL-2 in culture showed a slight increase in growth rate. Combined rIL-2/mismatched dsRNA treatment also demonstrated a similar slight enhancement of growth. Nude mice bearing subcutaneous tumors were treated by intraperitoneal injection of low doses (5000-20,000 units) of rIL-2 and mismatched dsRNA (500 micrograms). The in vivo tumor growth was significantly inhibited by the combined treatments (P less than 0.05) and survival was significantly increased (P less than 0.05). Measurement of cytotoxicity using splenocytes from treated animals showed significant augmentation of lytic activity against natural killer (NK)-sensitive YAC-1 cells in all rIL-2/mismatched dsRNA treatment groups, compared to the individual treatments or controls (P less than 0.05). Cytotoxicity of the splenocytes against the NK-resistant BRO cells was also augmented in animals treated with mismatched dsRNA and the highest rIL-2 dose utilized here (P less than 0.01). Renal, liver, and hematological toxicity was evaluated by measurement of blood urea nitrogen, creatinine, serum asparrtate aminotransferase, and a complete blood count with differential. There were no significant differences in these parameters in any of the treatment groups. Similarly, no differences in weight of the animals was seen in any treatment group. These results indicate that the combination of low-dose rIL-2 and mismatched dsRNA can potentiate host-mediated antitumor effects, yielding increased survival, without significant toxicity.

Animals↗

PubMed1991

Cyclic AMP mediates the direct antiproliferative action of mismatched double-stranded RNA.

Previous experiments have demonstrated that double-stranded RNAs (dsRNAs) can exert an antiproliferative effect on human tumor cells, independent of interferon (IFN) induction. However, the mechanism by which dsRNAs inhibit tumor growth has not been elucidated. As a first step in determining the molecular events responsible for growth arrest, we have explored the role of signal transduction through the cAMP system in the antiproliferative effect of the mismatched dsRNA, r(I)n.r(C12,U)n (Ampligen). These studies utilized the human glioma cell line A1235, which does not produce detectable levels of IFN-alpha, -beta, or -gamma in response to mismatched dsRNA treatment. Treatment of A1235 cells with mismatched dsRNA in combination with either 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), which inhibits cAMP-dependent protein kinase and protein kinase C, or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), which preferentially inhibits the cAMP-dependent protein kinase, yielded an antagonism of the mismatched dsRNA-induced antiproliferative effect. Measurement of adenylate cyclase activation showed a dose-dependent increase in activity at antiproliferative mismatched dsRNA concentrations, but not at lower, nonantiproliferative doses. This increase in activity was rapid, seen as early as 30 sec after initiation of treatment, and it was sustained at peak levels for 1-2 hr. Analysis of the intracellular cAMP concentration gave similar kinetics of induction. Exposure of cells to the stable cAMP analogue dibutyryl cAMP yielded dose-dependent inhibition of cell growth. The cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also inhibited proliferation. In contrast, neither H-7 nor HA1004 had an effect on growth inhibition induced by human natural IFN-alpha treatment. In addition, antiproliferative doses of IFN-alpha did not increase cAMP concentrations. These results indicate that the cAMP system is utilized by mismatched dsRNA as an early signal transduction mechanism for growth control. Furthermore, the antiproliferative effects induced by mismatched dsRNA and IFN can occur by different mechanisms of action.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

Mismatched double-stranded RNA, Ampligen (poly(I): poly(C12U), demonstrates antiviral and immunostimulatory activities in HIV disease.

Mismatched double-stranded RNA (Ampligen) has broad spectrum antiviral and immunomodulatory activities. These activities generate stabilization or improvement in three important surrogate markers of HIV disease progression. Patients with HIV disease treated with Ampligen do not become positive for p24 antigen, in contrast to patients treated with AZT or placebo. Viral burden can also be decreased in patients receiving Ampligen/AZT therapy. In vitro studies indicate that both AZT sensitive and AZT resistant viruses can be inhibited by Ampligen alone and are synergistically inhibited by Ampligen in combination with AZT. The immunomodulatory effects of Ampligen are manifested as a stabilization of CD4 counts. When Ampligen is combined with AZT, an increase in CD4 count is seen. Furthermore, a return or increase in delayed type hypersensitivity to mumps, Candida, and trichophyton was seen in approximately 70% of patients treated with Ampligen. The activity of Ampligen in HIV disease is due to its multifunctional activity as an antiviral and immune stimulating agent. The antiviral effect directly inhibits HIV-infection and other viruses which have been implicated in HIV disease acceleration and progression. The immunomodulatory activity can stabilize, increase, or restore immune function. This enhanced immune function can also lead to the further inhibition of additional infections associated with disease progression. Thus, Ampligen has multiple mechanisms of action against HIV disease.

Adjuvants, Immunologic↗

PubMed1987

Sensitivities of human glioma cell lines to interferons and double-stranded RNAs individually and in synergistic combinations.

The antiproliferative effects of human interferons (IFNs) and double-stranded RNAs (dsRNAs) were studied in five human glioma cell lines. Dose response curves were generated over a 72 hour treatment period. The concentration of interferon or double-stranded RNA necessary to produce a 50% antiproliferative response (GI50) was calculated by linear regression analysis. Two cell lines were more sensitive to IFN-beta than to IFN-alpha, one cell line was more sensitive to IFN-alpha than to IFN-beta and two cell lines had approximately equal sensitivities to both interferons. All cell lines showed some sensitivity to either IFN-alpha or IFN-beta. IFN-gamma had no antiproliferative effect on any of the cell lines. In addition, only one of the cell lines displayed sensitivity to dsRNA, in which the response to poly(I).poly(C) was greater than that to a mismatched analogue of poly(I).poly(C), r(I)n.r(C12,U)n (Ampligen). There was no correlation between the sensitivities to type I IFNs (alpha and beta), type II IFN (gamma) or the dsRNAs. The antiproliferative effect of combinations of IFNs, or IFNs and Ampligen, was studied in one of the cell lines. A significant synergistic antitumor effect was seen with all of the IFN/Ampligen combinations (p less than 0.02), including IFN-gamma/Ampligen, even though these cells were resistant to IFN-gamma alone. Synergy was also seen in the IFN-alpha/IFN-gamma (p less than 0.02) and IFN-beta/IFN-gamma (p less than 0.05) combinations. The IFN-alpha/IFN-beta combination gave an additive antitumor effect. These results indicate that IFN-alpha and IFN-beta alone or combinations of type I IFNs, type II IFNs and Ampligen can be effective in inhibiting the growth of glioma cells.

Cell Division↗

PubMed1987

Cloning of a non-c-myc DNA fragment from the double minutes of a human colon carcinoid cell line.

The cell line COLO 320 DM, derived from an untreated human colon carcinoid tumor, was subcloned to obtain a population (Cl 11) with an average of 37 double minutes (DM) per cell. Fractionation of the chromosomes by differential centrifugation yielded a fraction enriched in DM. DNA isolated from the DM-enriched fraction was inserted into the Pst I site of pBR322. One clone, p446, representative of a number of similar clones, contained a region complementary to genomic unique sequences (region p446U). Southern blot analysis using COLO 320 DNA, and DNA from two other cell lines derived from the same biopsy, COLO 320 HSR and COLO 321 HSR, demonstrated amplification and rearrangement of sequences complementary to p446U when compared with 28 different tumor and normal cell lines, some of which contained DM or homogeneously staining regions (HSR). COLO 320 DM Cl 11 had approximately 110 copies per cell of the p446U sequence, or three copies per DM. COLO 320 HSR, which contained one HSR, had 35 copies per cell, while COLO 321 HSR, which contained two HSR, had 700 copies. In addition, p446U did not hybridize with insert sequences of recombinant plasmid pHM(E + H), which includes the human c-myc coding region, 3 kb of upstream flanking sequences and 0.5 kb of downstream flanking sequences, or with an exon 3 probe, pMYC RI-CLA. Amplification of p446U was also not seen in cell lines containing amplified c-myc or N-myc genes. These results indicate that more than one sequence may be amplified in DM or HSR containing tumor cells, but that they need not be amplified together in other tumors.

Base Sequence↗

PubMed1987

Implications of retroviral and oncogene activity in chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a stem cell disease which, on a clinical level, progresses from the release from growth control of normally differentiated cells (a preleukemic state) to an acute leukemia. On a molecular level, the evolution of CML to acute leukemia is a multistep process. We propose that an early step, at the stem cell level, is acquisition of the ability for gene movement, which allows subsequent submicroscopic and chromosomal rearrangements that cause changes in the growth characteristics and regulation of the stem cell. A specific platelet DNA polymerase (PDP - reverse transcriptase) may play a role in gene movement. The characteristic reciprocal translocation of chromosomes #9 and #22, causing the activation of the c-abl oncogene, appears to be responsible for the uncontrolled cellular growth. Yet, other growth factors (e.g., platelet derived growth factor) and activated oncogenes (e.g., c-sis) must be responsible for the stimulation, progression, and variability seen during the course of the disease. Because CML is a progressive disease with clinically definable stages, CML appears to be a model system for the study of the molecular basis of the progression of preleukemia to leukemia specifically, and preneoplasia to aggressive neoplasia in general.

Blood Platelets↗

PubMed1987

Synergistic antiproliferative effect of recombinant alpha-interferons with recombinant gamma-interferon.

Two human tumor cell lines were studied for their response to the antiproliferative effect of recombinant human interferons (IFNs) alpha 2, alpha 4, a hybrid alpha (delta 4 alpha 2 Bgl II alpha 1), and gamma, individually and in combination. Natural human alpha-IFN was used as a reference point for all experiments. RT4 (bladder carcinoma) cells were overall more sensitive to the antiproliferative effects of the IFNs than A2182 (lung adenocarcinoma) cells. Three-way analysis of variance indicated that the relative effectiveness of the alpha-IFNs was alpha 2 less than alpha 4 less than hybrid alpha less than natural alpha-IFN. On an international reference unit per milliliter basis, gamma-IFN was 50- and 75-fold more effective than natural alpha-IFN and hybrid alpha-IFN in RT4 cells and 5.6-, 12.1-, and 14.9-fold more effective than alpha 4-, hybrid alpha-, and natural alpha-IFN in A2182 cells. In contrast, when recalculated on a nanogram per milliliter basis, gamma-IFN was only threefold more effective than the hybrid alpha-IFN in RT4 and approximately twofold less effective than alpha 4 and the hybrid alpha in A2182. Combinations of alpha-IFNs gave additive or antagonistic effects. When any of the alpha-IFNs were combined with the gamma-IFN, however, a synergistic antiproliferative effect was seen. The magnitude of the synergy was dependent upon the concentration of gamma-IFN used and the type of alpha-IFN in the combination. Antagonistic effects were seen at the lowest gamma-IFN concentration studied (0.2 IRU/ml). Synergy also varied according to the potency of the alpha-IFN used.

Carcinoma↗

PubMed1987

Augmented antitumor effect of combined human natural interferon-alpha and mismatched double-stranded RNA treatment against a human malignant melanoma xenograft.

The antitumor effect of combined natural human interferon-alpha (IFN) and mismatched double-stranded RNA (dsRNA) treatment against the human malignant melanoma cell line, BRO, was studied. In vitro results, using a tissue culture antiproliferative assay, indicated that these cells were moderately sensitive to IFN-alpha. In contrast, mismatched dsRNA had no antitumor effect, and a minimal stimulation of cell growth, over part of the concentration range tested, was observed. Mismatched dsRNA did not potentiate the antitumor effect of IFN-alpha in cells receiving combination treatment. Xenografts of BRO cells, inoculated subcutaneously into nude mice, were used to evaluate the antitumor effects of IFN-alpha and mismatched dsRNA. Growth of the primary tumor was inhibited by both drugs alone or in combination (p less than 0.001), but the combined treatment was most effective and appeared to be additive. The number of spontaneous lung metastases was also inhibited (p less than 0.02) in all treatment groups. Survival, however, was significantly increased only in the IFN-alpha/mismatched dsRNA group (p less than 0.02 compared to controls, p less than 0.05 compared to mismatched dsRNA alone). Determination of splenic natural killer (NK) cell activity against BRO cells demonstrated that significantly augmented NK activity to the same extent, but that the IFN-alpha alone had no effect. These results indicate that IFN-alpha worked through direct antiproliferative mechanisms while mismatched dsRNA stimulated host immunomodulatory effects. The increased tumor growth inhibition and survival in the dual treatment group appears to result from the combined direct antiproliferative and indirect immunomodulatory effects.

Animals↗

PubMed1986

Synergistic antiproliferative effect of human interferons in combination with mismatched double-stranded RNA on human tumor cells.

Four human tumor cell lines were studied for their response to antiproliferative effects of various interferons (IFNs) alone and in combination with the novel mismatched dsRNA, r(I)n r(C12,U)n (Ampligen). RT4 cells (bladder carcinoma) were resistant to Ampligen alone, while A2182 (lung carcinoma), HT 1080 C14 (fibrosarcoma) and RT112 (bladder carcinoma) cells were inhibited in a dose-dependent manner. In contrast, RT4 cells were sensitive to the antitumor effects of IFNs as were HT1080 C14 and RT112 cells, while A2182 cells were resistant. In 3 of 4 cell lines, the recombinant IFNs were less effective than the corresponding natural IFNs when compared by analysis of variance on an IRU/ml basis over a range of concentrations. In all cell lines, a synergistic antiproliferative effect was seen with all IFN preparations studied in combination with Ampligen, as calculated by the isobole method according to Berenbaum (1981). The antiproliferative effect of IFN was potentiated greater than 3.3- to greater than 250-fold, depending on the cell lines, IFN, and concentrations used. Varying the concentration of beta ser-IFN while holding the Ampligen concentration constant gave synergy at all of the physiologically achievable concentrations tested in RT4 cells. These results indicate that: Ampligen worked synergistically with all IFNs in all cell lines studied; growth inhibition of cells resistant to IFNs can be potentiated by low doses of Ampligen; the antiproliferative effect of IFNs can be potentiated by Ampligen in Ampligen-resistant cells; and Ampligen may work by a mechanism other than, or in addition to, the induction of IFNs.

Cell Division↗

PubMed1985

Differential antiproliferative actions of 2',5' oligo A trimer core and its cordycepin analogue on human tumor cells.

The antiproliferative effect of 2',5' A3 core and 2',5'-3'dA3 (cordycepin trimer) core was measured in 8 human tumor cell lines. Cells were treated in a dose-response manner for 72 hr and the concentration of drug necessary to inhibit cell growth 50% (GI50) was determined. A wide range of sensitivities to these drugs was found, even among tumors of the same histological type. The cell lines showed different sensitivities and dose-response curves to the 2',5'A3 and 2',5'-3'dA3 cores. Uptake studies of the 2',5'A3 and 2',5'-3'dA3 cores, using high-pressure liquid chromatography, demonstrated that both cores were rapidly degraded in the tissue culture medium and taken up as adenosine or cordycepin, respectively. There was a direct correlation between the uptake of cordycepin and the antiproliferative effect. In contrast, there was no correlation between cell sensitivity and the uptake of the 2',5'A3 core degradation products. Analysis of intracellular nucleosides and nucleotides indicated that differences in intracellular metabolism of adenosine might explain the different sensitivities of the various cell lines to 2',5'A3 core. Molar equivalent concentrations of adenosine and cordycepin inhibited cell growth; however, equimolar concentrations of these nucleosides were not effective. In addition, the antiproliferative effect of both core compounds and their corresponding nucleosides could be potentiated by the addition of the adenosine deaminase inhibitor, deoxycoformycin. The results indicate that these cores act as prodrugs and that the active metabolites are their corresponding nucleosides.

Adenine Nucleotides↗

PubMed1985

Silver staining as an indicator of active ribosomal genes.

Silver nitrate has been used as a cytological stain since the late 1800s. A modification of the Bielschowsky technique preferentially stains nucleoli and chromosomal nucleolus organizer regions (NORs). The specificity of staining is related to the method of preparation of the cytological specimens. The silver binds proteins and may be associated with the phosphate groups of certain phosphoproteins. Biochemical analyses of nucleolar proteins indicate that a limited array of specific proteins bind silver. A number of investigations have demonstrated that silver staining is indicative of active ribosomal RNA transcription, although a minor component may be associated with the fibrillar centers of cells in which ribosomal genes are inactive. Silver staining is a simple, reliable cytological method for the demonstration of ribosomal gene activity.

Animals↗

PubMed1985

Comparative studies of ampligen (mismatched double-stranded RNA) and interferons.

Historically, it has been assumed that double-stranded (ds) RNAs function at a cellular level exclusively via an interferon (IFN) induction mechanism. However, current studies conducted both in the laboratory and at the clinical level reveal that this assumption is incorrect and, indeed, underestimates the intrinsic antitumor activity of certain dsRNAs. A specific dsRNA (Ampligen) shows strong antiproliferative activity against human carcinoid tumor cells in a clonogenic assay when natural alpha- and beta-IFNs were inactive. Similarly, in vivo studies in which human renal cancer cells were transplanted into athymic mice demonstrate a strong antitumor effect of Ampligen whereas such tumors are largely unaffected by alpha-IFN treatment. In a comparative study including many fresh human tumors of various histological types (breast, ovarian, melanoma, renal, and carcinoid) numerous examples were uncovered of Ampligen sensitivity (antiproliferative effect) in the face of relative or complete insensitivity to IFNs. Synergistic effects of Ampligen plus IFN overcame the resistance of some human tumor cells to either biological modifier given alone. It can also be demonstrated that the antitumor action of Ampligen on certain human lung tumor cells is not shared by polyinosinic . polycytidylic acid, thus indicating that different dsRNAs may themselves exhibit dissimilar effects on various human tumors.

Animals↗

PubMed1985

Antiproliferative and immunomodulatory actions of beta-interferon and double-stranded RNA, individually and in combination, on human bladder tumor xenografts in nude mice.

A cell line, RT4, derived from a human transitional cell carcinoma of the bladder, was grown as a xenograft in athymic mice. The growth of the xenografts was inhibited by beta-interferon (IFN-beta), polyriboinosinic acid.polyribocytidilic acid, the mismatched double-stranded RNA analogue r(I)n . r(C12,U)n, and to a lesser extent recombinant IFN-beta, when treatment was initiated at the time of tumor inoculation. In contrast, the growth rate of established tumors, approximately 6 mm in diameter at the initiation of therapy, was inhibited by both double-stranded RNAs, but not natural IFN-beta, indicating a possible tumor size dependence on the effectiveness of IFN-beta. Combinations of natural or recombinant IFN-beta with either polyriboinosinic acid.polyribocytidilic acid or r(I)n.r(C12,U)n gave an antagonistic effect regardless of tumor mass at the initiation of treatment. This antagonism could be overcome by alternating r(I)n. r(C12,U)n and natural IFN-beta treatment. Natural killer cell activity against RT4 cells in culture was augmented in the spleens of mice treated with r(I)n.r(C12,U)n, but not in those treated with natural IFN-beta. RT4 cells treated in culture with IFN-beta, however, were significantly less efficient as targets for natural killer cells from r(I)n.r(C12,U)n-treated and control spleens. These results indicate that: the effectiveness of IFN-beta may be related to the tumor mass; double-stranded RNAs appear to work, at least partially, in an indirect, immunomodulatory manner; combination therapy can yield an antagonistic rather than an additive or synergistic antitumor effect; and strategic scheduling can overcome the antagonistic effect of combination therapy.

Animals↗

PubMed1985

Preclinical studies with Ampligen (mismatched double-stranded RNA).

Historically, double-stranded (ds) RNAs have been largely over-looked as potentially valuable anticancer/antiviral drugs, primarily because of the many clinical toxicities and lack of efficacy associated with the first clinically tested dsRNA--polyinosinic-polycytidylic acid (rIn X rCn). However, studies summarized herein demonstrate that the therapeutic ratio of dsRNAs can be greatly enhanced by purposeful mispairing of bases. For example, a mispaired dsRNA, termed Ampligen (rIn X r(C12,U)n), shows strong antitumor activity in a variety of relevant test systems with little or none of the toxicities associated with rIn X rCn. Furthermore, Ampligen demonstrates a much wider therapeutic spectrum than that displayed to date by any single type of interferon (natural or recombinant DNA-derived). Importantly, Ampligen, the product of a straight-forward enzymatic synthesis, shows excellent lot-to-lot biological and biophysical specifications, which is often not the case with biologically derived new compounds. Furthermore, a significant fraction of human solid tumors, which are largely unresponsive to conventional chemotherapy or interferon (IFN), is sensitive to Ampligen in a fresh human tumor clonogenic assay. Indeed, whereas 50% of untreated and IFN-treated athymic mice engrafted with human renal cancer cells die within 20-22 weeks, mice treated with Ampligen survive a minimum of 32 weeks (p less than 0.0003). A summary of all animal models tested and human clinical trials to date demonstrates that Ampligen exerts a greater antitumor activity than IFN and has a superior therapeutic ratio compared to rIn X rCn.

Animals↗

PubMed1979

S1 nuclease definition of highly repeated DNA sequences in the Guinea pig, Cavia porcellus.

Native DNA of the Guinea pig, Cavia porcellus, purified from liver or tissue culture cells, was heat denatured and reassociated to a Cot value of 0.01 (equivalent Cot value of 7.2 x 10(-2)). The reassociated DNA was isolated by digestion with the single-strand DNA specific enzyme S1 nuclease. Spectrophotometric and radioactivity assays demonstrated that 24% of the total DNA was resistant to S1 nuclease treatment. Zero-time reassociation indicated that approximately 3% of the DNA was inverted repeat sequences. Thus, highly repeated sequences comprised 21% of the total genome. CsCl buoyant density ultracentrifugation indicated that this fraction was composed of both main band and satellite sequences. Although actinomycin D - CsCl density gradients failed to give significant separation of the repetitive sequences, distamycin A - CsCl gradients were able to fractionate the DNA into several overlapping bands. The heterogeneity of the repetitive DNA was further demonstrated by the first derivative plots calculated from their thermal denaturation profiles. This analysis revealed six major thermalytes which indicate that there may be at least six discrete components in the repetitive DNA.

Animals↗

PubMed1979

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.

Animals↗