ETO and AML1 phosphoproteins are expressed in CD34+ hematopoietic progenitors: implications for t(8;21) leukemogenesis and monitoring residual disease.

To study acute myelogenous leukemia 1 (AML1) transcription factor, ETO protein, and t(8;21) AML chimeric AML1/ ETO protein in normal hematopoiesis and in leukemia, we raised rabbit antisera to a bacterially expressed polypeptide containing amino acid residues 1 to 220 of ETO and to synthetic peptides extending from residues 528 to 548 of ETO and 32 to 50 of AML1. The latter was selected to have little chance of cross-reactivity with other members of the PEBP2 alpha family. With affinity-purified reagents, we observed immunofluorescent staining for both AML1 and ETO in the nucleus of HEL, K562, and Kasumi-1 leukemic cell lines, the last from a t(8;21) AML.

Nuclear envelope structure.

The past 18 months have seen significant advances in our knowledge of the constituents of the nuclear envelope, their interactions during interphase and the mechanisms involved in their mitotic dynamics. Although most of the new data are in general agreement with, and contribute detail to, our traditional image of the nuclear envelope, a few observations appear to mark the beginning of new and important directions in research.

A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro.

Cell-free extracts prepared from activated clam oocytes contain factors which induce phosphorylation of the single 67-kD lamin (L67), disassemble clam oocyte nuclei, and cause chromosome condensation in vitro (Dessev, G., R. Palazzo, L.

Dynamic aspects of cytoskeletal and karyoskeletal intermediate filament systems during the cell cycle.

IF are major cytoskeletal and karyoskeletal components of eukaryotic cells (Steinert and Roop 1988). In numerous instances, their constituent protein subunits have been shown to be substrates for a variety of kinases such as A-kinase, C-kinase, and Ca++/calmodulin kinase (Geisler and Weber 1988; Inagaki et. 1988; Ando et al.

Lamin dimers. Presence in the nuclear lamina of surf clam oocytes and release during nuclear envelope breakdown.

The nuclear lamina of surf clam oocytes contains dimers of 67-kDa lamin which are stabilized by both noncovalent interactions and disulfide bonds. The latter can be reduced but re-form when the reducing agent is removed. The cysteine residues involved in these disulfide bonds are inaccessible to alkylating agents unless the protein is unfolded in urea.

Effect of Calcium on the Stability of the Vitelline Envelope of Surf Clam Oocytes.

Fertilization and parthenogenic activation of oocytes of the surf clam, Spisula solidissima, require the presence of calcium in the extracellular medium. Here we report that the depletion of calcium causes a dramatic increase in the stability of the vitelline envelopes (VE). On the basis of this effect, we have developed a method of isolating intact VE and have studied their morphology, composition, and properties.

The oocyte lamin persists as a single major component of the nuclear lamina during embryonic development of the surf clam.

Nuclei and nuclear lamina-enriched fractions, isolated from 1 to 5-day-old embryos of the surf clam, Spisula solidissima, contain only one major lamin protein, which appears to be identical to the oocyte lamin (L67), as judged by 2D IEF/SDS PAGE, reactivity with a polyclonal antibody directed against L67 and 125I tryptic peptide mapping. The same protein is also present in liver, muscle, nerve and testis from adult animals. No proteins--recognized by several poly- and monoclonal antibodies, specific for somatic lamins from different vertebrate species or the oocyte lamin LIII of Xenopus- have been detected in nuclei or NL-enriched preparations, isolated from embryos or adult tissues.

Disassembly of the nuclear envelope of spisula oocytes in a cell-free system.

Nuclei isolated from oocytes of the surf clam Spisula solidissima are disassembled when exposed to extracts from maturing oocytes. In the course of this process the nuclear lamina undergoes a marked reduction in size and the nuclear membrane appears to be fragmented into vesicles. These events are accompanied by extensive phosphorylation of the oocyte 67-kDa lamin and its solubilization.

Meiotic breakdown of nuclear envelope in oocytes of Spisula solidissima involves phosphorylation and release of nuclear lamin.

During meiotic nuclear envelope breakdown (NEBD) in maturing oocytes of the surf clam, Spisula solidissima, the 67-kDa lamin is extensively phosphorylated, concurrently with its solubilization. This is accompanied by a reduction of the nuclear diameter. Quercetin, a protein kinase inhibitor, does not affect lamin phosphorylation and release, nor NEBD per se, but specifically inhibits the early phosphorylation of a set of proteins, on which NEBD seems to depend.

Protein kinase activity associated with the nuclear lamina.

A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea.

Crosslinking of DNA to nuclear lamina proteins by UV irradiation in vivo.

We have been able to demonstrate that a fraction of DNA becomes crosslinked to nuclear lamina shells isolated from Ehrlich ascites tumour cells irradiated with UV light. Terminal labeling of short DNA fragments covalently attached to proteins reveals that DNA has become crosslinked to all three lamins and to a protein comigrating with vimentin.

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells.

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA.

Isolation and characterization of nuclear lamina from Ehrlich ascites tumor cells.

We have developed a simple and rapid method for isolation of purified nuclear lamina from Ehrlich ascites tumor cells. The procedure employs chromatin structures prepared from whole cells at low ionic strength and is carried out under conditions that minimize the formation of artifactual protein-DNA complexes. When the isolation is performed in the presence of EDTA, nuclear lamina without distinct pore complexes is obtained.

Formation of single-stranded regions in the course of digestion of DNA with DNAase II and micrococcal nuclease.

In the course of digestion of DNA with DNAase II or micrococcal nuclease, considerable amounts of single-stranded (ss) regions are formed, as determined by a second digestion with ss-specific nucleases, hyperchromicity measurements, and electron microscopy. Most of the ss stretches are located internally in the DNA molecules. The effect appears to be related to regions of decreased stability arising around single-stranded cuts in the double helix.

Removal of histone H1 exposes linker DNA in chromatin to DNAse I.

After removal of histone H1 about 40% of DNA in chromatin acquires the sensitivity of naked DNA to DNAse I. Digestion of H1-depleted chromatin with DNAse I leads to a qualitative change in the digestion pattern, generating DNA fragments of approx. 200 b.

Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2-mercaptoethanol.

Mercaptoethanol, when present in the sample buffer during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is responsible for the appearance of two nonprotein bands (electrophoretic mobilities corresponding to 68 and 54 kdalton) stainable with silver and with Coomassie blue. After iodination in vitro of DNA preparations isolated by alkaline phenol extraction using chloramine-T procedure, part of the radioactive label is found in these bands, provided the reaction is terminated by mercaptoethanol, whereas only a diffuse background is present in this area if the reaction is stopped by sodium metabisulfite. Similar results are obtained with highly purified total cytoplasmic RNA.

Effect of chromatin decondensation on the intranuclear matrix.

We have studied the effect of chromatin condensation on the morphology of the residual structures isolated from rat liver nuclei. DNAse I digestion followed by high salt extraction of nuclei in the presence of Mg++ yields residual structures consisting of a dense peripheral layer surrounding an internal network, similar to those described by Berezney and Coffey [6]. These structures are stable at low ionic strength in the presence of EDTA.

Interaction between lysine-rich histones and DNA.

Using a membrane filter retention technique we have studied the interaction between DNA and lysine rich histone H5 in vitro. It is found that, depending on the ionic conditions, H5 can bind DNA in a random or cooperative manner and exhibits a preference to DNA with high molecular weight and/or high A + T content, as also observed with H1. The presence of 6 M urea in the assay mixture does not impair the selectivity of H5 to A + T rich DNA but partly affects its selectivity to DNA size.

Digestion of chromatin with deoxyribonuclease II.

The action of DNAse II on DNA in chromatin was studied. The formation of acid-soluble products followed a two-phase kinetic curve. At the end of the first more rapid phase about 25% of DNA was degraded.

Isolation and properties of structured chromatin from Guerin ascites tumour and rat liver.

The method proposed by Hancock for isolation of structured chromatin from tissue culture cells is modified and used for isolation of chromatin from Guerin ascites tumour and rat liver. Micrococcal nuclease digestion patterns and thermal denaturation of these chromatins are studied and compared wiith those of chromatins prepared by precipitation and extraction with salts (salt chromatins). In contrast to the multiphasic melting profiles and salt chromatins, the structured chromatins exhibit relatively homogeneous denaturation patterns under a variety of conditions, suggesting thhat their DNA is uniformly stabilized by histones and there are no free independently melting DNA stretches.