Few quantitative studies addressing immunofluorescence histogram analysis have been published. One study by Overton (Cytornetry 9619-626, 1988) has shown threshold and histogram subtraction methods to be accurate for analysis of well-separated immunofluorescence distributions of positive and negative cells. An evaluation of methods to analyze immunofluorescence histograms when positive and negative immunofluorescence distributions overlap has not, to our knowledge, been reported. In this paper, data obtained from flow cytometry of immunofluorescently stained cells infected with recombinant retroviruses that produce a range of simian virus 40 large T antigen levels were analyzed by threshold, histogram subtraction, and distribution modeling methods. This analysis showed that as the separation between the immunofluorescence distributions of positive and negative cell populations decrease the best methods for histogram analysis are modeling followed, in order, by histogram subtraction, and threshold analysis.

Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G 1 /S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G 1 arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells. NGF,1 a neurotrophic polypeptide, belongs to a closely related family of neurotrophins composed of brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5. These paracrine hormones activate the development, maintenance, and regeneration of neurons in the nervous system (1). NGF signals the development of sympathetic, sensory, and a population of central nervous system neurons through its high affinity receptor, TrkA.The induction of neuronal differentiation invokes two interrelated cellular processes: progression through the stages of neurite outgrowth and cell cycle arrest (2). The rat pheochromocytoma cell line PC12, derived from a transplantable chromaffin tumor, provides a model system for the NGF-mediated conversion to a neuronal phenotype (3). PC12 cells contain both the tyrosine kinase (TrkA) and low affinity (p75 NTR ) NGF receptors (4, 5). Differentiation requires the TrkA receptor and proceeds through the Ras/MAPK pathway (6, 7). NGF decreases the growth rate of PC12 cells (8) and, in the short term, causes synchronized PC12 cells to accumulate in the G 1 phase of the cell cycle with a decrease in DNA synthesis (9). Continued exposure to NGF arrests the population in G 1 with an increased number in the G 2 /M phase also (10). Long-term treatment of PC12 cells with NGF promotes terminal differentiation, in which the PC12 cells resemble symp...

Flow cytometry was used to detect cells infected with retroviral vectors encoding both simian virus 40 large T antigen and G418 resistance after indirect immunofluorescence staining using a T-antigen-specific monoclonal antibody and a fluorescein-conjugated secondary antibody. Titers of viral stocks determined by flow cytometry were equivalent to those determined by quantitation of G418-resistant colonies.

Mycoplasma bacteriophage L51 single-stranded DNA and L2 double-stranded DNA are host cell modified and restricted when they transfect Acholeplasma laidlawii JAl and K2 cells. The L51 genome has a single restriction endonuclease MboI site (recognition sequence GATC), which contains 5-methylcytosine when the DNA is isolated from L51 phage grown in K2 cells but is unmethylated when the DNA is from phage grown in JAl cells. This GATC sequence is nonessential, since an L51 mutant in which the MboI site was deleted was still viable. DNA from this deletion mutant phage was not restricted during transfection of either strain K2 or JAl. Therefore, strain K2 restricts DNA containing the sequence GATC, and strain JAI restricts DNA containing the sequence GAT 5-methylcytosine. We conclude that K2 cells have a restriction system specific for DNA containing the sequence GATC and protect their DNA by methylating cytosine in this sequence. In constrast, JAl cells (which contain no methylated DNA bases) have a newly discovered type of restrictionmodification system. From results of studies of the restriction of specifically methylated DNAs, we conclude that JAl cells restrict DNA containing 5-methylcytosine, regardless of the nucleotide sequence containing 5-methylcytosine. This is the first report of a DNA restriction activity specific for a single (methylated) base. Modification in this system is the absence of cytosine methylating activity. A restriction-deficient variant of strain JAl, which retains the JAl modification phenotype, was isolated, indicating that JAl cells have a gene product with restriction specificity for DNA containing 5-methylcytosine.Mycoplasmas are procaryotes that have no cell walls: each cell is bounded only by a lipid-protein cell membrane (12). They are the smallest known free-living cells and have genomes 20 to 40%o the size of those of typical eubacteria (12, 16). These organisms arose by degenerative evolution as a branch of the gram-positive eubacteria (16,30).Some strains of the mycoplasma Acholeplasma laidlawii are hosts for four different bacteriophages (4, 18). Mycoplasma phage L51 is a bullet-shaped virion containing circular, single-stranded (SS) DNA of 4.5 kilobases (kb). Mycoplasma phage L3 is a T7-like virion containing linear, doublestranded (DS) DNA of 39 kb. Mycoplasma phage L2 is an enveloped virion containing circular, superhelical, DS DNA of 11.8 kb. Mycoplasma phage L172 is an enveloped virion containing circular, SS DNA of 14 kb.Bacteria have restriction-modification systems, which may have evolved to protect cells against infection by foreign DNA (for a review, see reference 27). Restriction involves endonucleases that recognize specific nucleotide sequences (.4 bases in length [11,24]) and cleave the DNA molecule at or near the sequence. A cell protects its DNA from endonuclease cleavage with a methylase that recognizes the same DNA sequence as the endonuclease and methylates a specific nucleotide within that sequence (2, 10). Specifically modified (methylated) cellular DNA ...