DNA-binding proteins of mammalian mitochondria

Acid-soluble proteins were isolated from liver and spleen mitochondria and their ability to form complexes with DNA was investigated. According to electrophoresis data, acid-soluble proteins include about 20 polypeptides ranging in the molecular mass from 10 to 120 kDa. It was found that acid-soluble proteins form stable DNA-protein complexes at a physiological NaCl concentration. Different polypeptides possess different degrees of DNA affinity. There is no significant difference between DNA-binding proteins of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In the presence of 5 microg of DNA most polypeptides were bound to DNA, and further increase in DNA amount affected little the binding of proteins to DNA. There was no distinct difference in DNA-protein complex formation of liver mitochondrial acid-soluble proteins with nDNA or mtDNA. Also, it was detected that with these mitochondrial acid-soluble proteins, proteases that specifically cleave these proteins are associated. It was shown for the first time that these proteases are activated by DNA. DNA-binding proteins including DNA-activated mitochondrial proteases are likely to participate in the regulation of the structural organization and functional activity of mitochondrial DNA.     

Composition and DNA-binding properties of the nuclear matrix proteins from mammalian cell nuclei

A rapidly sedimenting DNA-protein complex was isolated from nuclear lysates in 2 M NaCl and characterized with regard to its polypeptide composition and the DNA-binding properties of the purified proteins. The complex consists of the nuclear matrix with attached DNA. Electrophoresis in SDS-polyacrylamide gels revealed two major and five minor polypeptide bands, mainly in the 60 to 75 kDa molecular weight region. The DNA-matrix complex dissociated into free DNA and proteins in the presence of 2 M NaCl and 5 M urea. The proteins could be purified by chromatography on hydroxyapatite and showed a strong tendency to reassociate at 0.15 M NaCl concentration in the absence of urea. DNA was bound to the reassociated proteins at 0.15 M NaCl concentration. Part of the DNA-protein complex was stable at 1 M NaCl concentration. The binding appeared to be random with regard to the DNA sequence.     

Differential responses of proliferating versus quiescent cells to adriamycin.

The relative sensitivity of proliferating and quiescent cells to DNA-damaging agents is a key factor for cancer chemotherapy. Here we undertook a reevaluation of the way that proliferating and quiescent cells differ in their responses and fate to adriamycin-induced damage. Distinct types of assays that measure membrane integrity, metabolic activity, cell size, DNA content, and the ability to proliferate were used to compare growing and quiescent Swiss3T3 fibroblasts after adriamycin treatment. We found that immediately after adriamycin treatment of growing cells, p53 and p21(Cip1/Waf1) were induced but the cells remained viable. In contrast, less p53 and p21(Cip1/Waf1) were induced in quiescent cells after adriamycin treatment, but the cells were more prone to immediate cell death, possibly involving apoptosis. Adriamycin induced a G2/M cell cycle arrest in growing cells and a concomitant increase in cell size. In contrast, adriamycin induced an increase in sub-G1 DNA content in quiescent cells and a decrease in cell size. In contrast to the short-term responses, adriamycin-treated quiescent cells have a better long-term survival and proliferation potential than adriamycin-treated growing cells in colony formation assays. These data suggest that proliferating and resting cells are remarkably different in their short-term and long-term responses to adriamycin.     

Phosphorylation of nuclear proteins

Many nuclear proteins are phosphorylated: they range from enzymes to several structural proteins such as histones, non-histone chromosomal proteins and the nuclear lamins. The pattern of phosphorylation varies through the cell cycle. Although histone H1 is phosphorylated during interphase its phosphorylation increases sharply during mitosis. Histone H3, chromosomal protein HMG 14 and lamins A, B and C all show reversible phosphorylation during mitosis. Several nuclear kinases have been characterized, including one that increases during mitosis and phosphorylates H1 in vitro. Factors have been demonstrated in maturing amphibian oocytes and mitotic mammalian cells that induce chromosome condensation and breakdown of the nuclear membrane. The possibility that they are autocatalytic protein kinases is considered. The location of histone phosphorylation sites within the nucleosome is consistent with a role for phosphorylation in modulating chromatin folding.     

Nonchromatin nuclear proteins of mammalian lens epithelial cells

The nuclear matrix (NM) proteins of six tissue cultured lens epithelial cell lines and one embryonic rabbit epidermal cell line were analyzed to determine possible tissue and species specificity of these proteins. The NM proteins were isolated by the modified Penman technique. The tissue cultured cells were pulsed with [³⁵S] methionine and nuclear matrix proteins were fractionated by two-dimensional (2-D) gel electrophoresis. The 2-D gels were dried and autoradiographed. The relative abundance of spot patterns of nuclear matrix proteins of different cells were compared. The data from these experiments revealed that all the examined cell lines have distinct spot patterns, however, all of NM profile showed a spot pattern in the 45 kDa region with acidic pH. Some of these spots cross-reacted with anti-vimentin antibodies, whereas a prominent protein spot in this region did not cross react with either vimentin or actin antibodies. The observed variations in the NM protein patterns of lens epithelial cells may reflect tissue and species specificity and also a role in the regulatory properties of these nuclear proteins in the eye tissue development. J. Cell. Biochem. 64:644–650. © 1997 Wiley-Liss, Inc.     

H1 variant synthesis in proliferating and quiescent human cells

The synthesis of histone H1 isoprotein species in human cells of several different types and in several different physiological states was studied. Up to five H1 and two H1 degrees isoprotein species could be resolved by two-dimensional electrophoresis. All five H1 isoprotein species were synthesized in exponentially growing cultures of IMR-90 human fibroblasts; in quiescent IMR-90 cells the synthesis of three H1 isoprotein species was greatly decreased while the synthesis of two others was much less affected. When DNA synthesis in exponentially growing cultures of IMR-90 was inhibited, the pattern of H1 isoprotein synthesis became similar to that found in quiescent cultures. Other human cells, isolated from blood, yielded similar results. These results suggest that the pattern of H1 synthesis is the same for cells in non-S phases of the cell cycle and in quiescent cells. Thus for histone H1 in human cells the relationship of the variant synthesis pattern to the growth state and DNA replication is similar to that of the core histone H3 but not that of H2A.     

Phosphorylation of nuclear proteins in rat regenerating liver.

In studies of the phosphorylated proteins in rat liver and Walker-256, it was established that the ratio of various fractions of P-N linkages to P-O linkages varies from 0.6 to 3.1. In rat regenerating liver nuclei, the ratio of P-N and P-O varies with time after partial hepatectomy. Using [3H]-lysine and 32Pi, it is shown that phosphoryllysine forms in some new and, presumably, some preexisting H1 molecules. Using [3H]histidine and 32Pi, it is shown that phosphohistidine forms exclusively in preexisting H4. The half-life of H4 phosphohistidine appears to be about 2 h.     

Tissue-specific distribution of DNA-binding nuclear proteins

The DNA-binding capacity of nuclear proteins of mouse cells was examined by the protein-blotting method. Under conditions in which the lac repressor specifically binds to the lac operator, the DNA-binding nuclear proteins from different tissues showed a tissue-specific distribution, suggesting that the species and amounts of nuclear proteins with DNA binding activity differ in different tissues. When cloned eukaryotic genes were used for binding, eukaryotic DNA showed stronger binding than prokaryotic DNA. Competition experiments suggested that many nuclear proteins have different DNA binding properties from that of the prokaryotic repressor.     

Levels of synthesis of primate-specific nuclear proteins differ between growth-arrested and proliferating cells

A monoclonal antibody that reacts specifically with the proliferation-sensitive nuclear proteins, isoelectric focusing (IEF) 8Z30 and 8Z31 (molecular weight (MW), 76,000 charge variants, HeLa protein catalogue number) has been characterized. As determined by indirect immunofluorescence, the antibody stains the nucleolus and nucleoplasm of interphase-cultured cells of primate origin, but does not react with cells of other species. Proteins having similar MWs and isoelectric points as the human or monkey (primates) proteins were not observed in cultured cells of the following species: aves, bat, dog, dolphin, goat, hamster, mink, mouse, pisces, potoroo, rabbit and rat. Quantitative two-dimensional (2D) gel electrophoretic analysis of [35S]methionine-labeled proteins synthesized by normal (quiescent, proliferating) and SV40-transformed human MRC-5 fibroblasts revealed significant differences in the levels of synthesis of both IEF 8Z30 and 8Z31. In quiescent cells the main labelled product corresponded to IEF 8Z31 (ratio IEF 8Z31/8Z30, 2.3), while in the transformed cells the major product was IEF 8Z30 (ratio, 0.62). Normal proliferating fibroblasts exhibited similar levels of both proteins (ratio, 1.21). Combined levels of synthesis of both proteins were 1.50 and 1.20 times as high in the transformed cells as in the quiescent and proliferating cells, respectively. Similar results were observed in other pairs of normal and transformed human cells, such as WI38/WI38 SV40 and amnion/AMA. Modulation of the levels of synthesis of these proteins may play a role in cell proliferation.     

DNA-binding proteins present in varicella-zoster virus-infected cells.

DNA-binding proteins present in varicella-zoster virus-infected cells were identified by DNA-cellulose chromatography of radioactively labeled cell extracts. Seven virus-specific proteins, ranging in molecular weight from approximately 175,000 to 21,000, showed affinity for single- or double-stranded DNA or both. These proteins include the varicella-zoster virus major capsid protein, a phosphorylated tegument protein, and a 125,000-molecular-weight species which may be analogous to the major DNA-binding protein of herpes simplex virus. We also identified a number of DNA-binding phosphoproteins by these procedures. Finally, protein blot studies were carried out to determine whether these proteins bind preferentially to virus rather than to host cell DNA.     

Biochemical investigations of the nuclear proteins during the transition of root meristems from quiescent to proliferating state in Pisum sativum

Abstract

The electrophoretic analysis of nuclear proteins extracted from root meristems at different times of germination puts in evidence the variations of content of specific proteins. Several nuclear proteins are phosphorylated by endogenous protein kinase and often the maximum rate of phosphorylation it has been observed in proteins present in the nucleus at low concentrations. Moreover also the phosphorylation rate of specific proteins changes at different times of germination. It is interesting the fact that both variations of concentration and phosphorylation in nuclear proteins occurr at the time when root meristems leave the quiescence to enter a proliferating state. We suggest that these variations play a role in this physiological event.     

Comparison of the phosphorylation events in membranes from proliferating vs. quiescent endothelial cells

In an attempt to elucidate the intracellular events regulating the proliferation of endothelial cells (EC), we have compared the phosphorylation events in membranes prepared from proliferating (sparse) and quiescent (confluent) EC. Triton-solubilized membranes from sparse and confluent EC were incubated at pH 6.5 in the presence of divalent cations and [32P]ATP. Membrane proteins were then separated by SDS-PAGE and the radiolabeled phosphoproteins visualized by autoradiography. The overall kinase activity per milligram protein was 1.7 +/- 0.2-fold greater in membranes prepared from proliferating than from quiescent cells. The extent of phosphorylation was dramatically elevated in sparse over confluent samples for four phosphoproteins having the following approximate molecular masses: 180, 100, 97, and 55 kDa. The 180 and 100 kDa phosphoproteins exhibited 3.6- and 7.4-fold higher labeling, respectively, in sparse than in confluent membranes and both were phosphorylated on serine residues exclusively. The 97 kDa phosphoprotein was 11.6-fold higher in sparse membranes and contained both phosphoserine (p-ser) and phosphotheronine (p-thr), the latter comprising 61% of the radioactivity. The 55 kDA phosphoprotein contained 62% p-ser, 16% p-thr, and 22% phosphotyrosine (p-tyr) and was 2.3-fold higher in sparse membranes. Of these four phosphoproteins, only the 55 kDa protein was phosphorylated in confluent samples to an appreciable degree. Whereas the p-ser and p-thr content of the 55 kDa band increased moderately in sparse vs. confluent sample (1.8-fold increase), the tyrosine residues of this protein in sparse membranes were radiolabeled to a much greater extent relative to confluent membranes (5.4-fold increase). Analysis of the cofactor requirements of the FC membrane kinase(s) revealed that Mn2+ is the optimum cofactor and that Mg2+ can replace Mn2+ only for the kinase acting on the 100 kDa band. This suggests the presence of multiple EC membrane kinases. In the presence of both cofactors, the phosphorylation pattern is similar to the pattern obtained with Mn2+ alone. The kinase activity acting on all four phosphoproteins was independent of Ca2+, cAMP, cGMP, and phorbol 12-myristate 13-acetate. The mechanism responsible for the difference in kinase activity of proliferating vs. quiescent cells was not due to an inhibitor or enhanced phosphatase activity in confluent cells; the phosphorylation patterns obtained with sparse solubilized membranes and a mixture of sparse and confluent solubilized membranes were similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

The sensitivity of quiescent and proliferating mouse spermatogonial stem cells to X irradiation.

The radiosensitivity of spermatogonial stem cells to X rays was determined in the various stages of the cycle of the seminiferous epithelium of the CBA mouse. The numbers of undifferentiated spermatogonia present 10 days after graded doses of X rays (0.5-8.0 Gy) were taken as a measure of stem cell survival. Dose-response relationships were generated for each stage of the epithelial cycle by counting spermatogonial numbers and also by using the repopulation index method. Spermatogonial stem cells were found to be most sensitive to X rays during quiescence (stages IV-VII) and most resistant during active proliferation (stages IX-II). The D0 for X rays varied from 1.0 Gy for quiescent spermatogonial stem cells to 2.4 Gy for actively proliferating stem cells. In most epithelial stages the dose-response curves showed no shoulder in the low-dose region.     

High efficiency protein transduction of quiescent and proliferating primary hematopoietic cells

Primary hematopoietic cells are relatively refractory to DNA transfection methodologies. This is particularly so when they are quiescent or terminally differentiated and no longer able to divide. However, whole proteins can be introduced into such cells by protein transduction. We have modified the protein transduction domain (PTD) from the HIV-TAT protein used by other investigators. Using green fluorescent protein (GFP) as a reporter, we show that this new sequence allows more efficient transduction of recombinant fusion protein into a variety of hematopoietic cells tested compared with the native HIV TAT domain. This is true for peripheral blood CD34+ cells, dendritic cells, granulocytes, monocytes and lymphocytes all of which are quiescent or terminally differentiated. Furthermore, we were able to transduce myeloblasts from patients with acute myeloid leukemia (AML). In all cell types tested transduction efficiency was almost 100%. Transduction is maximal 15-30 s after addition of PTD or TAT-GFP fusion proteins as tested on quiescent T lymphocytes. This method will allow us to study of the effects of a variety of gene products in cell types that were previously resistant to gene transfection studies.