Biomedical subjects

C Patriotis

Publications and source records attributed to C Patriotis.

At least 19 recordsLinked to original sources

PubMed2001

Tpl-2 induces apoptosis by promoting the assembly of protein complexes that contain caspase-9, the adapter protein Tvl-1, and procaspase-3.

The Tpl-2 proto-oncoprotein promotes cellular proliferation when overexpressed in a variety of tumor cell lines. Here, we present evidence that when overexpressed in immortalized non-transformed cells, Tpl-2 induces apoptosis by promoting the activation of caspase-3 via a caspase-9-dependent mechanism, and that apoptosis is enhanced when Tpl-2 is co-expressed with the newly identified ankyrin repeat protein Tvl-1. The activation of caspase-3 by caspase-9 is known to depend on the assembly of a multimolecular complex that includes Apaf-1 and caspase-9. Data presented here show that co-expression of Tpl-2 with Tvl-1 promotes the assembly of a complex that involves several proteins that bind Apaf-1 including Tvl-1, itself, Tpl-2 and phosphorylated procaspase-9. More important, procaspase-3, which under normal growth conditions is not associated with the complex, binds Tvl-1 conditionally in response to Tpl-2-generated apoptotic signals. The conditional association of procaspase-3 with Tvl-1 promotes the in vivo proteolytic maturation of procaspase-3 by caspase-9, a process casually linked to apoptosis.

Adaptor Proteins, Signal Transducing↗

PubMed2001

ArrayExplorer, a program in Visual Basic for robust and accurate filter cDNA array analysis.

Determining the dynamics in the global regulation of gene expression holds the promise of bringing a better understanding of the processes that govern physiological cell growth regulation and its disruption during the development of disease. The advent for cDNA arrays has created the possibility for the parallel analysis of expression of thousands of genes in a given cell population, simultaneously. The level of expression of a given set of genes within the studied tissue corresponds to the intensity of a labeled cDNA probe synthesized from the studied tissue RNA and bound specifically to the cDNAs of the genes spotted on the array. The accurate extraction of gene expression intensity values is essential for further data analysis and the interpretation of the obtained results. Here, we describe a new array image-processing software developed in Microsoft Visual Basic, the ArrayExplorer, which provides a user-friendly, multiple-window interface and a number of automatic and manual features that facilitate a reliable, robust, and accurate extraction of gene intensity values from filter-array images.

Biotechnology↗

PubMed2000

TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway.

Tpl2 knockout mice produce low levels of TNF-alpha when exposed to lipopolysaccharide (LPS) and they are resistant to LPS/D-Galactosamine-induced pathology. LPS stimulation of peritoneal macrophages from these mice did not activate MEK1, ERK1, and ERK2 but did activate JNK, p38 MAPK, and NF-kappaB. The block in ERK1 and ERK2 activation was causally linked to the defect in TNF-alpha induction by experiments showing that normal murine macrophages treated with the MEK inhibitor PD98059 exhibit a similar defect. Deletion of the AU-rich motif in the TNF-alpha mRNA minimized the effect of Tpl2 inactivation on the induction of TNF-alpha. Subcellular fractionation of LPS-stimulated macrophages revealed that LPS signals transduced by Tpl2 specifically promote the transport of TNF-alpha mRNA from the nucleus to the cytoplasm.

3' Untranslated Regions↗

PubMed1999

The ankyrin repeat-containing adaptor protein Tvl-1 is a novel substrate and regulator of Raf-1.

Tvl-1 is a 269-amino acid ankyrin repeat protein expressed primarily in thymus, lung, and testes that was identified by screening a murine T-cell two-hybrid cDNA library for proteins that associate with the serine-threonine kinase Raf-1. The interaction of Tvl-1 with Raf-1 was confirmed by co-immunoprecipitation of the two proteins from COS-1 cells transiently transfected with Tvl-1 and Raf-1 expression constructs as well as by co-immunoprecipitation of the endogenous proteins from CV-1 and NB2 cells. Tvl-1 interacts with Raf-1 via its carboxyl-terminal ankyrin repeat domain. The same domain also mediates Tvl-1 homodimerization. Tvl-1 was detected by immunofluorescence in both the cytoplasm and the nucleus suggesting that in addition to Raf-1 it may also interact with nuclear proteins. Activated Raf-1 phosphorylates Tvl-1 both in vitro and in vivo. In baculovirus-infected Sf9 insect cells, Tvl-1 potentiates the activation of Raf-1 by Src and Ras while in COS-1 cells it potentiates the activation of Raf-1 by EGF. These data suggest that Tvl-1 is both a target as well as a regulator of Raf-1. The human homologue of Tvl-1 maps to chromosome 19p12, upstream of MEF2B with the two genes in a head to head arrangement.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-kappaB.

The Tpl-2 kinase activates the nuclear factor of activated T cells (NFAT) and induces IL-2 expression in T-cell lines. Here we show that the activation of the IL-2 promoter by Tpl-2 is inhibited by mutant signaling molecules that inhibit the mitogen-activated protein kinase (MAPK) or the calcineurin/NFAT pathways and is promoted by combinations of signaling molecules that activate these pathways. We, therefore, conclude that signals generated by the convergence of the MAPK and the calcineurin/NFAT pathway are necessary and sufficient for the activation of the IL-2 promoter by Tpl-2. The activation of both the IL-2 promoter and an NFAT-driven minimal promoter were shown to depend on signals transduced by Raf1. However, it was only the IL-2 promoter whose activation by Tpl-2 was fully blocked by the dominant negative mutant MEK1S218/222A and the MEK1/MEK2 inhibitor PD098059. Since the activation of NFAT is MAPK-dependent these findings suggested that the activation of MAPK by Tpl-2 is either independent or only partially dependent on MEK1 and MEK2. In addition, they suggested that the activation of the IL-2 promoter is under the control of not only NFAT but also a second factor whose activation is MEK-dependent. Experiments in COS-1 and EL-4 cells confirmed both hypotheses and revealed that the second factor activated by Tpl-2 is NF-kappaB. While the activation of the IL-2 promoter and an NFAT-driven minimal promoter by Tpl-2 was fully blocked by the dominant negative mutant NFAT delta418, it was only partially blocked by the calcineurin inhibitor cyclosporin A suggesting that the Tpl-2-mediated NFAT activation is under the control of a combination of calcineurin-dependent and independent pathways. Both pathways were fully blocked by Bcl-2 or Bcl-X(L).

Animals↗

PubMed1998

The Tpl-2 protooncoprotein activates the nuclear factor of activated T cells and induces interleukin 2 expression in T cell lines.

Tpl-2 expression is induced within 30-60 min after ConA stimulation of rat splenocytes, suggesting that it may contribute to the induction of IL-2 during T cell activation. Herein we show that wild-type and carboxyl-terminally truncated (activated) Tpl-2 activate the nuclear factor of activated T cells (NFAT) and induce interleukin 2 (IL-2) expression in EL4 cells. In Jurkat cells the truncated Tpl-2 activates NFAT and induces IL-2, whereas wild-type Tpl-2 activates NFAT only when cotransfected with NFAT expression constructs, suggesting that Tpl-2 may induce NFAT activation signals. Experiments in NIH 3T3 cells revealed that the NFATp isoform, but not the NFATc or NFATx isoform, undergoes nuclear translocation when coexpressed with wild-type Tpl-2 and confirmed this hypothesis. Activation of NFAT by anti-CD3 stimulation but not by phorbol 12-myristate 13-acetate and ionomycin in Jurkat cells was inhibited by the kinase-dead Tpl-2K167M, suggesting that Tpl-2 contributes to the transduction of NFAT activation signals originating in the T cell receptor. The Tpl-2-mediated induction of IL-2 was not observed in T cell lymphoma lines other than EL4 and Jurkat, as well as in normal T cells. NFAT activation by Tpl-2, however, was observed in several cell lines including some of nonhematopoietic origin. The activation of NFAT by Tpl-2 in different cell types defines a molecular mechanism that may contribute to its oncogenic potential.

3T3 Cells↗

PubMed1994

Tpl-2 acts in concert with Ras and Raf-1 to activate mitogen-activated protein kinase.

Mitogenic signals initiated at the plasma membrane by extracellular factors acting on receptor tyrosine kinases or G protein-coupled receptors are transmitted to the nucleus through an intricate signaling network. Components of this network participate, upon stimulation, in a complex array of phosphorylation-dependent protein-protein interactions which leads to the formation of transient multimolecular complexes. Complexes containing products of the protooncogenes ras and raf-1 and the protein kinase MEK-1 activate the mitogen-activated protein kinases (MAPKs), which play a central role in the integration of different mitogenic signals by directly phosphorylating cytoplasmic and nuclear targets. In this report we present evidence that the kinase encoded by the tumor progression locus 2 gene (Tpl-2) contributes to the activation of the MAPK cascade. MAPK activation induced by the Tpl-2 protein is blocked by dominant negative mutants of Ras and Raf-1, whereas a kinase-deficient Tpl-2 mutant down-regulates mitogenic signals induced by v-Ha-Ras or v-Raf. These data suggest that Tpl-2 activates the MAPK cascade, perhaps through its participation in the assembly of Ras/Raf-1-containing multimolecular complexes.

3T3 Cells↗

PubMed1994

Interphase chromosomes of Friend-S cells are attached to the matrix structures through the centromeric/telomeric regions.

DNA of the attachment sites of Friend erythroleukemia cells, isolated according to the conventional procedure, represents short, nuclease-resistant fragments with sizes below 400 bp, belonging to the class of mouse satellite. A number of experiments have indicated that their unusual resistance is due to complexing with RNA. By various approaches, it was confirmed that similar fragments might be recovered from total DNA following extensive digestion with DNase I. In situ hybridizations revealed further that at mitosis the sequences of the attachment sites are located at the centromeric/telomeric regions of the chromosomes, while at interphase they are redistributed into 9-13 well-defined clusters spread throughout the entire nuclear area. Parallel biochemical and electronmicroscopic studies have clarified, moreover, that the all three compartments of the matrix harbor such sequences. Thus, it appears that the attachment sites described function only at interphase, anchoring the both ends of each interphase chromosome to the matrix structures.

Animals↗

PubMed1994

The activated Mlvi-4 locus in Moloney murine leukemia virus-induced rat T-cell lymphomas encodes an env/Mlvi-4 fusion protein.

A genomic DNA probe derived from the region immediately 3' of the clusters of integrated proviruses in the Mlvi-4 locus detects a 5.5-kb mRNA transcript which is specifically expressed in normal rat thymus and spleen. The same probe detects two tumor-specific mRNA transcripts 2.5 and 10 kb long, both of which are expressed only in tumors carrying a provirus in the Mlvi-4 locus. Sequence analysis of two cDNA clones (LE3a and B1.1) of the 2.5-kb tumor-specific mRNA, obtained from two independent tumors (6889 and B1), revealed that they are both derived from hybrid env/Mlvi-4 mRNA transcripts. The splicing of env to Mlvi-4 sequences linked a cryptic splice donor site at nucleotide position 6397 of the viral genome with a splice acceptor site in the region immediately 3' of the integrated provirus. The mRNA that gives rise to cDNA clone B1.1 terminates 1,005 bases 3' of the splice acceptor site without additional splicing. The mRNA that gives rise to cDNA clone LE3a terminates in the same site but undergoes differential splicing of an 81-base-long intron. The resulting mRNAs contain 247-amino-acid (clone B1.1) or 226-amino-acid (clone LE3a) open reading frames sharing 221 N-terminal amino acids, of which 207 are derived from the viral env gene and 14 are derived from Mlvi-4. RNase protection assays using 6889 tumor cell RNA and a probe derived from the cDNA clone LE3a detected both mRNA transcripts. More abundant of the two, however, was the one encoding the putative 247-amino-acid protein. Transient transfections of a construct expressing the RNA transcript defined by clone B1.1 into D17 cells led to the expression of an Env/Mlvi-4 fusion protein with an apparent molecular mass of 33 kDa. Given that cells with provirus insertions in the Mlvi-4 locus are selected and that retroviral env gene products may have profound effects in the biology of hematopoietic cells, we suggest that the detected fusion proteins may contribute to the growth of T-cell lymphomas.

Amino Acid Sequence↗

PubMed1994

Effects of provirus integration in the Tpl-1/Ets-1 locus in Moloney murine leukemia virus-induced rat T-cell lymphomas: levels of expression, polyadenylation, transcriptional initiation, and differential splicing of the Ets-1 mRNA.

The Tpl-1 locus was defined as a genomic DNA region which is targeted by provirus insertion during progression of Moloney murine leukemia virus-induced rat T-cell lymphomas. Using a panel of 156 (Mus musculus x Mus spretus) x Mus musculus interspecific backcross mice, we mapped Tpl-1 to mouse chromosome 9 at a distance of 1.2 +/- 0.9 centimorgans from the Ets-1 proto-oncogene (S.E. Bear, A. Bellacosa, P.A. Lazo, N.A. Jenkins, N.G. Copeland, C. Hanson, G. Levan, and P.N. Tsichlis, Proc. Natl. Acad. Sci. USA 86:7495-7499, 1989). In this report, we present evidence that all the known Tpl-1 provirus insertions occurred immediately 5' of the first exon of Ets-1 (exon A) and that the earlier detected distance between Tpl-1 and Ets-1 was due to the high frequency of meiotic recombination in the region between the site of provirus integration and exon III. Northern (RNA) blot analysis of polyadenylated RNA from normal adult rat tissues and Moloney murine leukemia virus-induced T-cell lymphomas and hybridization to a Tpl-1/Ets-1 probe derived from the 5' end of the gene revealed two lymphoid cell-specific RNA transcripts, of 5.5 and 2.2 kb. Sequence analysis of a near-full-length (4,991-bp) cDNA clone of the 5.5-kb RNA revealed a 441-amino-acid open reading frame encoding a protein identical to the human and mouse Ets-1 proteins with the exception of five and nine species-specific conservative amino acid differences, respectively. The steady-state level of the Tpl-1/Ets-1 RNA and of the Ets-1 protein was modestly elevated in tumors carrying a provirus in the Tpl-1 locus. The relative ratio of the two Ets-1 transcripts, which were shown to arise by differential polyadenylation, was not affected by provirus insertion. Moreover, the major site of transcriptional initiation, which was localized by primer extension 250 bp upstream of the 5' end of the Ets-1 cDNA clone, was shown to be identical in normal cells and tumors carrying a provirus in the Tpl-1 locus. Finally, the differential splicing of Ets-1 exon VII was shown by RNase protection to occur at a rate of 15 to 26% and to remain unaffected by provirus insertion. The subtlety of these effects, in contrast to the strong growth selection of cells with a provirus in the Tpl-1/Ets-1 locus, suggests that provirus insertion may affect the fine regulation of the gene, perhaps during cell cycle progression.

Alternative Splicing↗

PubMed1993

Identification of the gene encoding the mitochondrial elongation factor G in mammals.

Protein synthesis in cytosolic and rough endoplasmic reticulum associated ribosomes is directed by factors, many of which have been well characterized. Although these factors have been the subject of intense study, most of the corresponding factors regulating protein synthesis in the mitochondrial ribosomes remain unknown. In this report we present the cloning and initial characterization of the gene encoding the rat mitochondrial elongation factor-G (rEF-Gmt). The rat gene encoding EF-Gmt (rMef-g) maps to rat chromosome 2 and it is expressed in all tissues with highest levels in liver, thymus and brain. Its DNA sequence predicts a 752 amino acid protein exhibiting 72% homology to the yeast Saccharomyces cerevisiae mitochondrial elongation factor-G (YMEF-G), 62% and 61% homology to the Thermus thermophilus and E. coli elongation factor-G (EF-G) respectively and 52% homology to the rat elongation factor-2 (EF-2). The deduced amino acid sequence of EF-G contains characteristic motifs shared by all GTP binding proteins. Therefore, similarly to other elongation factors, the enzymatic function of EF-Gmt is predicted to depend on GTP binding and hydrolysis. EF-Gmt differs from its cytoplasmic homolog, EF-2, in that it contains an aspartic acid residue at amino acid position 621 which corresponds to the EF-2 histidine residue at position 715. Since this histidine residue, following posttranslational modification into diphthamide, appears to be the sole cellular target of diphtheria toxin and Pseudomonas aeruginosa endotoxin A, we conclude that EF-Gmt will not be inactivated by these toxins. The severe effects of these toxins on protein elongation in tissues expressing EF-Gmt suggest that EF-Gmt and EF-2 exhibit nonoverlapping functions. The cloning and characterization of the mammalian mitochondrial elongation factor G will permit us to address its role in the regulation of normal mitochondrial function and in disease states attributed to mitochondrial dysfunction.

Animals↗

PubMed1993

Tumor progression locus 2 (Tpl-2) encodes a protein kinase involved in the progression of rodent T-cell lymphomas and in T-cell activation.

The Tpl-2 locus, cloned by provirus tagging from one of three sublines of the Moloney leukemia virus-induced rat thymoma 2769, defines a gene encoding a protein kinase associated with progression in 22.5% of the tumors. Tpl-2 is expressed primarily in spleen, thymus, liver, and lung. Provirus integration occurs in the last intron of the gene, leading to the expression of a truncated mRNA that terminates in the proviral long terminal repeat and encodes a protein with an altered C-terminal domain. Strong evidence that this genetic change confers growth advantage to affected cell clones was provided by the finding that, during cultivation of all three sublines derived from tumor 2769, cells were selected that harbored independent provirus insertions in the Tpl-2 locus. Exposure of normal rat spleen cells to Con A induces the expression of enhanced levels of Tpl-2 within the first 60 min from the time of exposure suggesting that, in normal splenocytes, Tpl-2 may be involved in the transition from a quiescent to the G1 phase of the cell cycle.

Amino Acid Sequence↗

PubMed1993

Structure of a Moloney murine leukemia virus-virus-like 30 recombinant: implications for transduction of the c-Ha-ras proto-oncogene.

Tumor progression locus 2 (Tpl-2) encodes a novel serine-threonine protein kinase which is activated by provirus integration in the late stages of oncogenesis in Moloney leukemia virus (MoMuLV) induced rat T-cell lymphomas. In this report, we present evidence that the provirus integrated in the Tpl-2 locus in 1 of 10 T-cell lymphomas harboring a Tpl-2 rearrangement (2779) is a recombinant between MoMuLV and virus-like 30 (VL30) sequences (Mo-VL30). Recombination between MoMuLV and VL30 may contribute to the transduction of ras, as suggested by the finding that VL30 flanks the ras oncogene in all of the ras transducing viruses isolated from rats to date. The Mo-VL30 recombinant described here represents evidence that recombination between MoMuLV and VL30 can be uncoupled from the transduction of ras, and it may precede the transduction. Sequence comparison between clones of Mo-VL30, Harvey sarcoma virus (Ha-MSV), and genomic c-Ha-ras revealed that all three share a 124-bp region of 87.3% homology. This region was detected at nucleotide positions -1845 to -1720 of c-Ha-ras and 20 bp 5' of the recombination breakpoint between VL30 and ras in Ha-MSV. On the basis of the sequence comparison between VL30, Ha-MSV, and c-Ha-ras, we are proposing a model which explains how VL30 may have facilitated the transduction of c-Ha-ras and perhaps the other ras proto-oncogenes. According to this model, the sequence homology between VL30 and c-Ha-ras targets this gene for transduction by promoting the integration of the provirus in this locus through homologous recombination.

Animals↗

PubMed1993

Genomic organization and expression of Tpl-2 in normal cells and Moloney murine leukemia virus-induced rat T-cell lymphomas: activation by provirus insertion.

Tpl-2 is a gene encoding a protein kinase which is primarily expressed in normal spleen, thymus, and lung tissue and is activated by provirus insertion in Moloney murine leukemia virus-induced T-cell lymphomas during the late stages of oncogenesis. Tpl-2 is composed of eight exons and spans a 35-kb genomic DNA region. The provirus integrates reproducibly in the last intron and in the same transcriptional orientation as the Tpl-2 gene. This genetic change leads to the expression of enhanced steady-state levels of a truncated Tpl-2 RNA transcript which is predicted to encode a protein with an altered C-terminal domain. Tpl-2 is transcribed from two alternating promoters, P1 and P2. The RNA transcripts originating in the two promoters harbor different 5' untranslated regions derived from the alternate noncoding exons IA and IB. Utilization of the P2 promoter, which gives rise to exon IB containing Tpl-2 RNA transcripts, was detected primarily in tumor cells. The Tpl-2 protein was expressed in COS-1 cells as an N-terminal fusion with a 12-amino-acid hemagglutinin tag. Immunoprecipitation of transfected COS-1 cell lysates with antihemagglutinin or anti-Tpl-2 antibodies, followed by incubation with [gamma-32P]ATP, confirmed that Tpl-2 possesses protein kinase activity.

Animals↗

PubMed1993

New markers, D16FC1 and Tp12, differentiate between rat chromosomes 16 and 17.

Problems in differentiating rat chromosomes 16 and 17 cytogenetically can be resolved with unique probes mapped to these chromosomes. Using somatic cell hybridization and nonisotopic in situ hybridization, probes D16FC1 and Tp12 were localized to 16p16-->p15 and 17q12.1-->q12.2, respectively. The locations of these probes can serve as reference points to facilitate mapping of future probes to rat chromosomes 16 and 17.

Animals↗

PubMed1992

A molecular genetic linkage map of mouse chromosome 18 reveals extensive linkage conservation with human chromosomes 5 and 18.

An interspecific backcross between C57BL/6J and Mus spretus was used to generate a molecular genetic linkage map of mouse chromosome 18 that includes 23 molecular markers and spans approximately 86% of the estimated length of the chromosome. The Apc, Camk2a, D18Fcr1, D18Fcr2, D18Leh1, D18Leh2, Dcc, Emb-rs3, Fgfa, Fim-2/Csfmr, Gnal, Grl-1, Grp, Hk-1rs1, Ii, Kns, Lmnb, Mbp, Mcc, Mtv-38, Palb, Pdgfrb, and Tpl-2 genes were mapped relative to each other in one interspecific backcross. A second interspecific backcross and a centromere-specific DNA satellite probe were used to determine the distance of the most proximal chromosome 18 marker to the centromere. The interspecific map extends the known regions of linkage homology between mouse chromosome 18 and human chromosomes 5 and 18 and identifies a new homology segment with human chromosome 10p. It also provides molecular access to many regions of mouse chromosome 18 for the first time.

Animals↗

PubMed1990

Activation of multiple genes by provirus integration in the Mlvi-4 locus in T-cell lymphomas induced by Moloney murine leukemia virus.

Moloney murine leukemia virus-induced rat T-cell lymphomas harbor proviruses integrated near c-myc and near Mlvi-1/Mis-1/Pvt-1, another locus of common integration which maps 270 kilobases 3' of c-myc. In this report, we present the characterization of a new locus of common integration in Moloney murine leukemia virus-induced T-cell lymphomas (Mlvi-4) which maps 30 kilobases 3' of c-myc, between c-myc and Mlvi-1. The Mlvi-4 locus, whose chromosomal map location is conserved in rats, mice, and humans, is also the target of chromosomal rearrangements in a variety of animal and human tumors. Evidence presented elsewhere shows that provirus integration in Mlvi-4 enhances the expression of c-myc and Mlvi-1 by cis-acting mechanisms operating over long distances of genomic DNA. In this manuscript, we show that provirus integration in the Mlvi-4 locus activates, by promoter insertion, one additional gene which maps immediately 3' to the cluster of the Mlvi-4 proviruses and which is transcribed in the same orientation as c-myc, giving rise to 3- and 10-kilobase mRNA transcripts. The Mlvi-4 gene is also expressed in normal thymus and spleen at very low levels, giving rise to 3- and 5.5-kilobase messages. Although Mlvi-4 is expressed in normal thymus, it is not expressed in Moloney murine leukemia virus-induced T-cell lymphomas corresponding to several stages of T-cell differentiation, but lacking a provirus in this locus. This suggests that Mlvi-4 may be expressed only in a subpopulation of T cells. We conclude that provirus insertion in Mlvi-4 activates c-myc and two additional genes, Mlvi-1 and Mlvi-4, whose expression is restricted to, and may be developmentally regulated in, T cells. Since Mlvi-4 is the target of genetic changes in a great variety of human and animal neoplasms, these results are critical for our understanding of oncogenesis.

Animals↗

PubMed1990

DNA-RNA complexes that might represent transient attachment sites of nuclear DNA to the matrix.

In this study we describe DNA-RNA complexes in matrix DNA of Friend cells. The presence of such unusual structures is confirmed by the following evidence. When a preparation of matrix DNA is electrophoresed in agarose an RNA component always migrates together with DNA. There should be a close interaction between DNA and RNA in such a preparation because the presence of the RNA component causes resistance of DNA to DNase I and Exo III. An intimate, hybrid-type association of part of the RNA component with DNA is indicated also by the fact that about 20% of this RNA is sensitive to RNase H. By specific inhibition of the RNA synthesis with alpha-amanitin and actinomycin D it was shown that the bulk of associated RNA is transcribed by RNA polymerase III. Hybridization experiments showed similarity between the DNA sequences isolated from the complexes and those from the base of dehistonized DNA loops obtained by high-salt extraction of nuclei. This observation suggests that the complexes might represent attachment sites of nuclear DNA to the matrix: possibly, the attachment is mediated via the RNA component. Experiments with induction of erythroid differentiation indicated that a profound reorganization of the nucleus, accompanying terminal differentiation, leads to a striking reduction in the number of complexes and thus in the number of attachment sites. This suggests that the complexes should function as transient attachment sites.

Amanitins↗