Complete murine cDNA sequence, genomic structure, and tissue expression of the high mobility group protein HMG-I(Y)

A cDNA coding for the non-histone chromosomal protein HMG-I, or its isoform HMG-Y, was isolated from a murine Friend cell library using synthetic oligonucleotide hybridization probes. Sequence analysis showed that the 1670-base pair full length cDNA insert consists of a 201-base pair, G/C-rich (74%), 5'-untranslated region, a 288-base pair amino acid coding sequence, and an unusually long 1182-base pair 3'-untranslated region. The deduced 96-residue amino acid coding sequence of the murine HMG-I(Y) cDNA is very similar to the reported amino acid sequence of human HMG-I, except that it lacks 11 internal amino acids reported in the human protein. Based on Southern blot hybridization analysis of genomic DNA, there appear to be fewer than five copies of HMG-I(Y) genes in the haploid murine genome. These murine HMG-I(Y) genes contain a large (at least 890 base pairs) exon that includes most, or all, of the 3'-untranslated region; whereas the much shorter 5'-untranslated region and amino acid coding sequences are interrupted by at least one intron. A single size class (approximately 1700 nucleotides in murine cells and 2000 nucleotides in human cells) of HMG-I(Y) mRNAs was detected at high levels in total RNA extracts from rapidly dividing, transformed cells, but to a lesser extent, or not at all, in extracts from slowly or non-dividing cells.     

Two genes for the high mobility group protein HMG-Y are present in the genome of Canavalia gladiata D.C.

Two cDNAs encoding high-mobility-group (HMG) proteins that correspond to animal HMG-Y proteins were isolated from maturing seeds of Canavalia gladiata D.C. The deduced amino acid sequences of these cDNAs showed similarity to other plant HMG-I/Y proteins reported to date. The mRNAs for the HMG-Y proteins were detected in leaves, stems, roots, pods and seeds of C. gladiata. The level of the mRNA was high in the maturing seeds of 30 days after flowering and 2-day germinated seeds. Two genomic clones were isolated from DNA of C. gladiata and both were shown to represent single-copy genes consisting of two exons and one intron. This is the first report of the genomic sequences for HMG-I/Y protein in plants.     

An antigen expressed in proliferating cells at late G1-S phase

A monoclonal antibody Pr-28 was prepared, which recognized an antigen present only in proliferating cells. Immunofluorescence analysis of Pr-28 antigen showed that the antigen was localized mainly in perinuclear cytoplasm. Although Pr-28 antibody was produced against a chicken cell antigen, it reacts not only with chicken cells but also other cells of murine origin, such as L-cells and NIH 3T3 cells. The molecular weight (Mr) of the antigen recognized by Pr-28 antibody was 45,000 D as determined by SDS-PAGE run under reducing conditions. The antigen disappeared in NIH 3T3 quiescent cells, reappearing in quiescent cells stimulated by fetal calf serum (FCS). The synthesis of Mr 45,000 protein occurred at late G1 phase, just before DNA synthesis in serum-stimulated quiescent NIH 3T3 cells and ceased in S phase.     

The roles of the MCM, ORC, and Cdc6 proteins in determining the replication competence of chromatin in quiescent cells

Most eukaryotic cell types can withdraw from proliferative cell cycles and remain quiescent for extended periods. Intact nuclei isolated from quiescent murine NIH3T3 cells fail to replicate in vitro when incubated in Xenopus egg extracts, although intact nuclei from proliferating cells replicate well. Permeabilization of the nuclear envelope rescues the ability of quiescent nuclei to replicate in the extract. We show that origin replication complex (ORC), minichromosome maintenance (MCM), and Cdc6 proteins are all present in early quiescent cells. Immunodepletion of Cdc6 or the MCM complex from Xenopus egg extract inhibits replication of permeable, quiescent, but not proliferating, NIH3T3 nuclei. Immunoblotting results demonstrate that mouse homologues of Mcm2, Mcm5, and Cdc6 are displaced from chromatin in quiescent cells. However, this absence of chromatin-bound Cdc6 and MCM proteins from quiescent cells appears not to be due to the absence of ORC subunits as murine homologues of Orc1 and Orc2 remain chromatin-bound in quiescent cells. Surprisingly, intact quiescent nuclei fail to bind exogenously added XCdc6 or to replicate in Xenopus egg extracts immunodepleted of ORC, even though G1- or S-phase nuclei still replicate in these extracts. Our results identify Cdc6 and the MCM complex as essential replication components absent from quiescent chromatin due to nonfunctional chromatin-bound ORC proteins. These results can explain why quiescent mammalian nuclei are unable to replicate in vivo and in Xenopus egg extracts.     

High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing the lowest PiT1 mRNA levels. The mechanism behind the role of PiT1 in increased cell proliferation is not known. We, however, found that compared to control cells, cultures of NIH3T3 cells overexpressing PiT1 upon seeding showed increased cell number after 24 h and had shifted more cells from G0/G1 to S+G2/M within 12 h, suggesting that an early event may play a role. We here show that expression of human PiT1 in NIH3T3 cells led to faster cell adhesion; this effect was not cell type specific in that it was also observed when expressing human PiT1 in MC3T3-E1 cells. We also show for NIH3T3 that PiT1 overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby contribute to their proliferative advantage within the first 24 h after seeding.     

A conformational study of the sequence specific binding of HMG-I (Y) with the bovine interleukin-2 cDNA

The DNA sequence specific interaction of the high mobility group non-histone protein HMG-I (Y) with the 3' untranslated region of the bovine interleukin-2 cDNA has been studied. Circular dichroism and thermal denaturation studies suggest that HMG-I (Y) alters the conformational state and increases the thermal stability of the DNA. Additionally, amino acid sequence analysis suggests that the previously identified non-histone protein HMG-Y is an isoform of HMG-I.     

Expression and synthesis of high mobility group chromosomal proteins in different rat skeletal cell lines during myogenesis.

The synthesis, turnover, and expression of all the major high mobility group (HMG) chromosomal proteins was studied in different rat skeletal myogenic cell lines. Whereas pulse-chase experiments revealed a similar half-life (greater than 2 cell generations) for all the HMG proteins in both L8 myoblasts and myotubes, [3H]lysine incorporation data indicated a 2- to 4-fold greater incorporation of the label in the HMG proteins in proliferating myoblasts relative to the nondividing myotubes. Analysis of the HMG-1, -14, and -17 mRNAs during myogenesis showed a significant down-regulation in L6 and L8 myotubes compared to the myoblasts. However, the timing of the shift and the extent of down-regulation was cell type-dependent, being more pronounced in L6 myotubes at fusion compared to 4 days postfusion in L8 myotubes. By contrast, L8-derived fusion-defective fu-1 cells over the same period of growth showed no change in HMG-14/17 mRNA levels. HMG-I(Y) protein isoforms, noted for the first time in rat myoblasts, like their counterparts, seemed to be stable and showed a precipitous reduction in their mRNAs during myogenesis. The results suggest a cell type-specific correlation between HMG expression and cell proliferation; they also argue for their role in maintenance of the cell's state of differentiation.     

Sequence-specific binding of HMG-I(Y) to the proximal promoter of the gp91-phox gene.

Screening of a cDNA expression library with a CCAAT-box element derived from the myelomonocyte-specific gp91-phox promoter resulted in the isolation of three independent HMG-I(Y) cDNA clones. Filter binding competition studies reveal that HMG-Y binds to this promoter element in a sequence-specific manner and exhibits a gradient of binding affinities for various A/T-rich sequences. Two adjacent A/T-rich regions within the gp91-phox promoter CCAAT-box element are required for maximal binding. In addition, competition experiments demonstrate that the binding affinity of HMG-Y is influenced by sequences that flank A/T-rich core binding sites.     

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of Ej-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.     

Induction of Syncytia by Neuropathogenic Murine Leukemia Viruses Depends on Receptor Density, Host Cell Determinants, and the Intrinsic Fusion Potential of Envelope Protein

Infection by the neuropathogenic murine leukemia virus (MLV) TR1.3 results in hemorrhagic disease that correlates directly to in vivo syncytium formation of brain capillary endothelial cells (BCEC). This phenotype maps to amino acid 102 in the envelope (Env) protein of TR1.3. Substitution of glycine (G) for tryptophan (W) at this position (W102G Env) in the nonpathogenic MLV FB29 induces both syncytium formation and neurologic disease in vivo. Using an in vitro gene reporter cell fusion assay, we showed that fusion either with murine NIH 3T3 cells or with nonmurine target cells that expressed receptors at or below endogenous murine levels mirrored that seen in BCEC in vivo. In these instances only TR1.3 and W102G Env induced cell fusion. In contrast, when receptor levels on nonmurine cells were raised above endogenous murine levels, FB29 Env was as fusogenic as the neuropathogenic TR1.3 and W102G Env. These results indicate that TR1.3 Env and W102G Env are intrinsically more fusogenic than FB29 Env, that the induction of fusion requires a threshold number of receptors that is greater for FB29 Env than for TR1.3 or W102G Env, and that receptor density on murine NIH 3T3 cells and BCEC is below the threshold for FB29-dependent fusion. Surprisingly, receptor density on NIH 3T3 cells could not be increased by stable expression of exogenous receptors, and FB29-dependent fusion was not observed in NIH 3T3 cells that transiently expressed elevated receptor numbers. These results suggest that an additional undefined host cell factor(s) may limit both receptor expression and fusion potential in murine cells.     

Role of c-myc and other genes in interleukin 2 regulated CT6 T lymphocytes and their malignant variants

The cloned murine cytotoxic T cell line CT6 solely requires interleukin 2 (IL 2) for viability and cell cycle progression. Treatment of G arrested cultures of CT6 cells with recombinant IL 2 induces the rapid sequential expression of the nuclear proto-oncogenes c-fos, c-myc, and c-myb but does not affect the expression of several cytosolic or membrane-associated proto-oncogenes. A comparison of early genes induced by growth factor treatment of quiescent NIH/3T3 fibroblasts and CT6 cells demonstrated that only c-fos and c-myc induction is shared in the two different lineages. Factor-independent lines derived from CT6 cells show no mitogenic response to IL 2, yet binding of IL 2 with its receptor in the cells was capable of inducing the expression of c-fos and c-myc. In factor-independent cell lines, c-myc was uniformly expressed at high constitutive levels, suggesting that c-myc abrogates growth factor requirements of these cells. The levels of c-myc expression in the factor-independent lines was not due to an autocrine production of IL 2 but may be a consequence of constitutively activated IL 2 receptors.     

The role of cell differentiation state and HMG-I/Y in the expression of transgenes flanked by matrix attachment regions

The tobacco nuclear matrix attachment region (MAR), RB7, has been shown to have a much greater effect on transgene expression in cultured cells than in transgenic plants. This is comparable to work in mouse systems showing that MARs have a positive effect on transgene expression in embryonic tissues but not adult tissues. There are several possible explanations for these observations. One is that cell differentiation state and proliferation rate can affect MAR function. We tested this possibility by initiating suspension cell cultures from well-characterized transgenic plants transformed with 35S::GUS with and without flanking MARs and then comparing GUS specific activity in the cell lines to those of the transgenic plants from which the cell lines were derived. If cell differentiation state and proliferation rate do affect MAR function, we would expect the ratio of transgene expression (cell suspensions : plants) to be greater in MAR lines than in control lines. This turned out not to be the case. Thus, it appears that MAR function is not enhanced simply because cells in culture divide rapidly and are not differentiated. Because in animal systems the chromosomal protein HMG-I/Y has been shown to be upregulated in proliferating cells and may have a role in MAR function, we have also examined the levels of the tobacco HMG-I/Y homolog by immunoblotting. The level of this protein does not differ between primary transformant cultured cells (NT-1) and Nicotiana tabacum plants (SR-1). However, a higher molecular weight cross-reacting polypeptide was found in nuclei from the NT-1 cell suspensions but was not detected in SR-1 leaf nuclei or cell suspensions derived from the SR-1 plants.