Bruce H. Howard

Histone-like TAFs within the PCAF Histone Acetylase Complex

PCAF histone acetylase plays a role in regulation of transcription, cell cycle progression, and differentiation. Here, we show that PCAF is found in a complex consisting of more than 20 distinct polypeptides. Strikingly, some polypeptides are identical to TBP-associated factors (TAFs), which are subunits of TFIID. Like TFIID, histone fold-containing factors are present within the PCAF complex. The histone H3- and H2B-like subunits within the PCAF complex are identical to those within TFIID, namely, hTAF(II)31 and hTAF(II)20/15, respectively. The PCAF complex has a novel histone H4-like subunit with similarity to hTAF(II)80 that interacts with the histone H3-like domain of hTAF(II)31. Moreover, the PCAF complex has a novel subunit with WD40 repeats having a similarity to hTAF(II)100.

The centromeric nucleosome of budding yeast is perfectly positioned and covers the entire centromere

The centromeres of budding yeast are ∼120 bp in size and contain three functional elements: an AT-rich region flanked by binding sites for Cbf1 and CBF3. A specialized nucleosome containing the H3 variant Cse4 (CenH3) is formed at the centromere. Our genome-wide paired-end sequencing of nucleosomal DNA reveals that the centromeric nucleosome contains a micrococcal nuclease-resistant kernel of 123–135 bp, depending on the centromere, and is therefore significantly shorter than the canonical nucleosome. Unlike canonical nucleosomes, the centromeric nucleosome is essentially perfectly positioned. The entire centromere is included, together with at least 1 bp of DNA upstream of the Cbf1 site and at least 4 bp downstream of the CBF3 site. The fact that the binding sites for Cbf1 and CBF3 are included within the centromeric nucleosome has important implications for models of the centromeric nucleosome and for kinetochore function.

Purification of transiently transfected cells by magnetic affinity cell sorting

A method was developed to purify transiently transfected HeLa cells or African green monkey kidney CV-1 cells by magnetic affinity cell sorting. Monolayer cultures were transfected with mammalian expression vectors coding for either of two novel cell surface antigens, the Tac subunit of the human IL-2 receptor or vesicular stomatitis virus G protein. During the transient expression phase, cell populations were placed in suspension and mixed with monoclonal-antibody-coated magnetic particles in the presence of a sorting solution designed to minimize nonspecific cell/cell and cell/particle interactions. Transfected cells expressing the vector-encoded cell surface antigen were then isolated by application of a magnetic field. Reconstruction experiments indicated that IL-2 receptor-positive cells were bound about 100-fold more efficiently than receptor-negative cells. In transient transfection experiments, populations of greater than 90% antigen-positive cells were reproducibly obtained.

ISOLATION OF A POPULATION OF TRANSIENTLY TRANSFECTED QUIESCENT AND SENESCENT CELLS BY MAGNETIC AFFINITY CELL SORTING

Rous sarcoma virus (RSV) and cytomegalovirus (CMV) promoters were tested for activity in proliferating and nonproliferating (quiescent or senescent) human embryo fibroblasts. These promoters were cloned upstream of the coding sequence for the Tac subunit of the interleukin 2 receptor, and activity was calculated from the fraction of Tac antigen positive cells detected in a coupled transient transfection/magnetic affinity cell sorting assay. Differences in promoter activities are substantial in quiescent cells: the efficiency of the RSV promoter is no greater than background whereas the CMV promoter is equally active in serum concentrations ranging from 0.5 to 20%. While both promoters are functional in growing cells (WI-38 and HeLa), the CMV promoter exhibits twofold greater activity. Surprisingly, in senescent cells both promoters exhibit the same degree of activity.

Efficient gene transfer by sequential treatment of mammalian cells with DEAE-dextran and deoxyribonucleic acid.

A variation of the classical DEAE-dextran method of gene transfer was developed for efficient transfection of HeLa cells with plasmid DNA. A brief exposure of the cells to medium containing DEAE-dextran was found to be sufficient for subsequent uptake of pRSVcat and to be superior to cocultivation of the cells with DEAE-dextran plus DNA. This sequential method of gene transfer is nontoxic and yielded up to 60% of HeLa cells positive for a surface protein encoded by the transfected sequence. The implications of this sequential transfection technique regarding the mechanisms of gene transfer are discussed.

Expression of GTP-binding proteins and prostaglandin E2 receptors during human T cell activation

GTP-binding proteins (G-proteins) are a family of closely related, yet structurally distinct signal transducing proteins. In this study the presence and relative abundance of several G-proteins and of their corresponding mRNAs were measured in resting and activated human T lymphocytes. We found that T lymphocytes contain RNA coding for Gs, Gi2, and Gi3. No Gi1- and Go-specific RNA could be detected. Membrane fractions of resting and activated lymphocytes were studied in immunoblot experiments. Again, Gs, Gi2, and Gi3, but not Gi1 and Go, were detected. Upon mitogenic activation, a relative increase in mRNA for Gs and Gi3, but not for Gi2 could be demonstrated in Northern blot experiments. Immunoblotting indicated an increase in Gs and Gi3 density in membrane fractions of T cells as well. Paralleling the increase in Gs, we found that activated T cells produce five to seven times more cAMP per cell in response to prostaglandin E2 (PGE2) than resting lymphocytes. Finally, PGE2 binding studies showed that the number of receptors for this hormone increased from 435 +/- 322 to 1035 +/- 357 per cell following in vitro stimulation. We propose that in vitro T cell activation is paralleled by an increase in sensitivity to PGE2-induced cAMP generation. This sensitization is accompanied by both an increase in cell surface PGE2 receptor numbers as well as by increased expression of the signal transducing protein Gs and may physiologically be important for limiting an immune response.

Magnetic affinity cell sorting to isolate transiently transfected cells, multidrug-resistant cells, somatic cell hybrids, and virally infected cells

Publisher Summary Many techniques have been developed to introduce foreign DNA into eukaryotic cells for stable expression of genes, transient analysis of gene products, and gene regulation. Several methods of transfection have been used, such as calcium phosphate coprecipitation, diethylaminoethyl (DEAE)-dextran, protoplast fusion, microinjection, electroporation, and lipofection. With the exception of DEAE-dextran transfection, which can be used only for transient assays, other techniques are employed to study both transiently and stably transfected cell populations. Isolation of stably transfected cells generally requires the expression of a dominant selectable marker gene, which often confers a drug resistance phenotype. Selection of stable transfectant is time consuming and at times may result in the selection of cells with rearrangements or the insertional mutagenesis of cellular genes. The chromatin structure at the integration sites may influence the expression of the gene of interest, so such studies may not be suitable for the analysis of gene expression.

Sequence-specific toxicity of transfected retroviral DNA.

Experimental gene transfer and viral infections can result in the accumulation of unintegrated DNA in target cells. The effects of such accumulation on target cell metabolism have not been directly studied. The experiments reported in this paper show that transfection of cloned retroviral long-terminal-repeat (LTR) DNA, or of a variety of eukaryotic promoters, into proliferating HeLa cells results in rapid, sequence-specific, and dose-dependent cell death. Plasmids containing the Rous sarcoma virus LTR or the human immunodeficiency virus LTR cloned in pUC-related plasmids are 5 to 10 times more toxic than pUC19. The demonstrated sensitivity of eukaryotic cells to exogenously introduced DNA has important implications for the interpretation of gene transfer experiments and may be relevant to the pathogenic mechanisms in the course of retroviral infections such as AIDS.

Vectors for introducing genes into cells of higher eukaryotes

Abstract Numerous animal viruses have been adapted to serve as vehicles for transferring genetic material into mammalian and avian cells. These vectors will be vital in efforts to elucidate mechanisms of gene regulation of higher eukaryotes.

PURIFICATION OF TRANSIENTLY TRANSFECTED CELLS BY MAGNETIC‐AFFINITY CELL SORTING

A variety of techniques have been developed to introduce genes into eukaryotic cells to investigate the molecular mechanisms underlying gene expression and regulation. Of these techniques, one of the most useful is DNA-mediated gene transfer, where expression of the gene of interest can be studied by either stable transformation or transient expression. In general, mammalian cells can be stably transformed by co-transfection of the gene of interest and a vector that carries a dominant selectable marker such as Eco-gpt (Mulligan & Berg, 1981) or neo (Southern & Berg, 1982), followed by selection for drug resistance. Thus, stable transformation gives rise to subclones of cells in which the gene under investigation is integrated into chromatin, allowing a continuous study of the expression of the gene in those cells. However, stable transformation has several drawbacks. First, selection for drugresistant cells requires several weeks. Second, the chromatin structure at the integration site is likely to influence the expression of the gene. Third, long-term selection of cells after cotransfection with a selectable marker may cause rearrangements of cellular genes. This last aspect is a potentially serious problem if the gene of interest interferes with host cell proliferation. In addition, this method tends to select only clones expressing high levels of the marker, which may not be the representative of the optimum expression of the gene of interest. Transient expression, on the other hand, can be studied within 24-72 h after transfection. No selection for drug resistance is required. Moreover, the expression of the gene is independent of its integration site in the chromatin. Therefore, transient expression has been extensively used to study promoter function, as well as translational control in gene