A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast.

Polyethylene glycol (PEG) can induce genetic transformation in both bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) without cell wall removal. PEG-mediated transformation of E. coli is technically simple and yields transformants with an efficiency of 10(6)-10(7) transformants/microgram DNA. Detailed analysis of the parameters involved in PEG-mediated transformation of E. coli reveals basic differences between the PEG and standard CaCl2 methods for transformation of E. coli. PEG-mediated transformation of yeast is far simpler than existing protoplast methods and is comparable in efficiency. The new methods described here for PEG-mediated genetic transformation may prove to be of general utility in performing genetic transformation in a wide variety of organisms.

A technically simple "non-lethal" vital staining procedure for viral plaque and cell transformation assays. Brief report.

The widely used tetrazolium dye, MTT, has several advantages as a vital stain in the identification of viral plaques. First, since the yellow colored dye MTT stains live cells dark blue, viral plaques can be counted without removal of the phenol red containing agar overlay. Secondly, the high contrast between live and dead cells afforded by MTT permits one to readily detect small plaques at an early time after infection. Thirdly, since cells intensely stained with MTT remain viable, MTT vital staining can be used to isolate cells which are either transformed or resistant to viruses. Thus, MTT vital staining should be useful in several types of viral plaque assays.

Structure and expression of the Lyt-3a gene of C.AKR mice.

The mouse Lyt-3a gene, which encodes the Lyt-3.1 T-cell surface alloantigen of the C.AKR strain, has been cloned, and the nucleotide sequence of its exons and more than 2 kb of 5' flanking sequence have been determined. The gene extends over approximately 16 kb of DNA and consists of six exons encoding leader, leader plus V-like domain, membrane-proximal, transmembrane, and cytoplasmic domains. The only difference between the coding region of the Lyt-3a gene and the cDNA sequences reported for Lyt-3b (Nakauchi et al. 1987. Panaccio et al. 1987) is at position 77 of the mature protein where Lyt-3a encodes serine and Lyt-3b encodes arginine. This substitution must therefore be the basis for the serological distinction between the Lyt-3.1 and Lyt-3.2 alloantigens. Potential TATA and CAAT sequences, two Sp1 protein binding sites, two extended repeats of the dinucleotide, CA, a number of short inverted repeats, and an inverted segment of the mouse B1 repetitive sequence are found 5' to the Lyt-3a gene. Two consensus poly-A addition signals and a complete copy of the mouse B1 sequence are found 3' to the gene. Both B1-related regions are flanked by short direct repeats suggesting that they arose by an insertional mechanism. Cotransfection of the Lyt-3a gene together with a cloned Lyt-2a gene resulted in expression of both Lyt-2 and Lyt-3.1 on the surface of Ltk- and BW5147 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence analysis of the C.AKR Lyt-2a gene: structural polymorphism in alleles encoding the Lyt-2.1 T-cell surface alloantigen.

The Lyt-2a allele of the C.AKR strain of mice (genotype Lyt-2a, Lyt-3a) was cloned, and its complete nucleotide sequence as well as that of 2 kb of 5' flanking DNA was determined. The sequence was compared with the partial sequence of the Lyt-2a allele of DBA/2 (genotype Lyt-2a, Lyt-3b) and the nearly complete sequence of the B10.CAS2 Lyt-2b allele reported by Liaw and coworkers (1986). The coding regions of the two Lyt-2a alleles differ from each other by two nucleotide substitutions in the three exons over which they could be compared, resulting in two amino acid substitutions in the leader and transmembrane segments. The coding region of the C.AKR Lyt-2a allele differs from the Lyt-2b allele by two nucleotide substitutions in the extracellular V-like domain, one of which is silent and the second of which leads to substitution of valine for methionine at amino acid position 78 giving rise to the Lyt-2.1 allotypic specificity. The coding region of the DBA/2 Lyt-2a allele shares with C.AKR the allotypic substitution at position 78 and differs from Lyt-2b by three additional nucleotide substitutions in the coding regions, two of which lead to amino acid substitutions in the leader and transmembrane segments. It would therefore appear that the Lyt-2 alleles of the three strains analyzed are distinct, and the nomenclature Lyt-2a1 and Lyt-2a2 is suggested to distinguish the alleles of C.AKR and DBA/2, respectively. These alleles share a common difference from the Lyt-2b gene product at position 78, and since the amino acid substitutions which distinguish them from each other are in the leader and transmembrane segments, their mature Lyt-2 gene products appear antigenically identical.

High-efficiency polyethylene glycol-mediated transformation of mammalian cells.

A new, high-efficiency method for transformation of mammalian cells with nucleic acids is described which yields 10(5)-10(6) plaques/micrograms poliovirus infectious RNA (iRNA). The optimized procedure consists of two steps: (1) exposure of cells to iRNA in a high ionic-strength buffer followed by (2) a brief exposure to a 35% polyethylene glycol (PEG) solution. Optimized conditions for each variable in the procedure are described. Under optimized conditions for PEG-mediated transformation with RNA, large numbers of transformants are recovered with plasmid DNA as well. The procedure presented is similar to other high-efficiency PEG-mediated methods previously described for the genetic transformation of both nonprotoplasted Escherichia coli and yeast.

Uptake by cells of nucleic acids promoted by compounds sharing the pleiotropic effects of poly(ethylene glycol).

Poly(ethylene glycol) (PEG) is a member of a group of membrane active compounds that have pleiotropic effects on cells, eg, promotion of cell fusion, induction of erythroleukemia cell differentiation, and protection of cells from freezing damage. Since PEG has recently been shown to be an efficient promoter of genetic transformation in bacteria, yeast, and mammalian cells, studies were carried out to determine whether other PEG-related compounds could also promote genetic transformation. In this study, 24 compounds, which behave like PEG in other biological systems, are shown to promote transfection of human cells with isolated poliovirus RNA. That PEG and other commercially important compounds promote transfection indicates that such compounds may represent a biohazard to man.

Induction by concanavalin A of specific mRNAs and cytolytic function in a CD8-positive T cell hybridoma.

A previous report from this laboratory described the production of CD8+, class I-specific T cell hybridomas which developed specific cytolytic activity and the ability to secrete IL-2 upon Con A or specific Ag stimulation. Unlike normal lymphocytes or long-term CTL lines for which exposure to Ag triggers both differentiation and proliferation, T cell hybridoma lines can be activated functionally against a background of continuous proliferation. They therefore provide a unique system with which to study the molecular events involved in the induction of cytolytic function. The expression of mRNA from a series of genes was evaluated by Northern hybridization at various times after Con A stimulation of the H-2Ld-specific CD8+ 3D9 hybridoma. Induction of the c-fos proto-oncogene by 45 min poststimulation was followed shortly by c-myc induction. Perforin mRNA was expressed at a low level in the unstimulated hybridomas, but was down-regulated upon Con A stimulation to levels undetectable by PCR. Interestingly, production of granzyme A mRNA was strongly induced by 45 min after Con A stimulation. In the CD8+ RT-1.3G3 hybridoma, which is nonlytic and specific for the HIV-1 envelope glycoprotein, c-fos but not granzyme A mRNA was induced by 45 min poststimulation, and no granzyme A mRNA was detectable at any time. Thus, a significant role for granzyme A in the induction of cytolytic activity is suggested. Cytolysis by the 3D9 hybridoma involved both target cell membrane damage and DNA fragmentation, and both Ca(2+)-dependent and Ca(2+)-independent cytolysis were observed. Although TNF-alpha mRNA was induced by 4 h poststimulation, Ab to TNF-alpha failed to inhibit the Ca(2+)-independent lysis observed, leaving the basis for the observed Ca(2+)-independent lysis unexplained.

Cis-acting DNA elements and cell type-specific nuclear proteins which may play a role in regulation of mouse CD8 alpha (Lyt-2) gene transcription.

Fusion of mouse CD8+ class I MHC-restricted T cells with the BW5147 thymoma invariably yields CD8- hybridomas in which RNA transcribed from the CD8 alpha (Lyt-2) gene is undetectable. To determine whether cis-acting DNA sequences may negatively regulate transcription of the Lyt-2 gene in BW5147 cells, one possible explanation for the above observation, BW5147 cells were stably transfected with the Lyt-2 gene containing 1 - 11,000 nucleotides of 5' flanking DNA and surface expression of Lyt-2 was monitored by flow microfluorometry. Initial results suggested the presence of a negative element between 1400 and 5000 nucleotides upstream of the site of transcription initiation. Further studies suggested the presence of two potential negative regulatory elements in this region, one of which includes a 269 nucleotide Accl - SstI fragment comprised of nucleotides -4700 to -4431 which bound nuclear proteins from CD8+ and CD8- cell lines in electrophoretic mobility shift assays (EMSA). EMSA studies performed using nuclear extracts from a variety of cell lines and tissues demonstrated that unique retarded complexes, called bands 1 and 2, correlated significantly with expression or non-expression of Lyt-2 respectively. EMSA analysis of proteins fractionated by SDS-PAGE from nuclear extract of the CD8+ VL3 T lymphoma cell line revealed proteins of approximately 110-130 kDa (called L2a-P1) and > 200 kDa (called L2a-P2) which bind within a 100 nucleotide region of this fragment (called L2a) to yield band 1 and 2 respectively, and which may play a role in regulation of Lyt-2 gene transcription.

Interaction of the nuclear matrix-associated region (MAR)-binding proteins, SATB1 and CDP/Cux, with a MAR element (L2a) in an upstream regulatory region of the mouse CD8a gene.

Matrix-associated regions (MARs), AT-rich DNA segments that have an affinity for the nuclear matrix, have been shown to play a role in transcriptional regulation of eukaryotic genes. The present study demonstrates that a DNA element, called L2a, which has been implicated in the transcriptional regulation of the mouse CD8a gene encoding an important T cell coreceptor, is a MAR. Moreover, the identities of two nuclear proteins, L2a-P1 and L2a-P2, previously shown to bind to the L2a element, have been determined. The L2a-P1 protein found to be present in all CD8-positive T cell lines tested is SATB1, a known MAR-binding protein. The widely expressed L2a-P2 protein is CDP/Cux, a MAR-binding protein that has been associated with repression of gene transcription. Interaction of both proteins with the L2a element was studied using the missing nucleoside approach, DNase I footprinting, and electrophoretic mobility shift assays with wild type and mutant L2a elements. The data suggest that CDP/Cux bound to the L2a element is displaced by binding of SATB1 and the accompanying conformational change in the DNA lying between the primary binding sites of SATB1 and CDP/Cux. We suggest that displacement of CDP/Cux by SATB1 favors transcription of the CD8a gene, possibly by enhancing or altering its association with the nuclear matrix.