Quantification of active caspase 3 in apoptotic cells.

We describe an enzyme-linked immunosorbent assay (ELISA) for quantifying relative amounts of active caspase 3 in apoptotic cells. Covalent modification of caspase 3 active sites with a biotinylated inhibitor differentiates active from latent caspases. Capture on an ELISA plate with an antibody specific for caspase 3 makes the assay specific for caspase 3.

Production of fertile transgenic maize by electroporation of suspension culture cells.

Fertile, transgenic maize plants were generated by electroporation of suspension culture cells that were treated with a pectin-degrading enzyme. Electroporation of cells from two different suspension cultures, one derived from A188 X B73 and one derived from a B73-related inbred, with a plasmid containing the bar gene, resulted in high-frequency recovery of stably transformed callus lines. Plants were regenerated from thirteen transformed callus lines and transmission of bar to progeny was demonstrated.

High-level expression in Escherichia coli of biologically active bovine growth hormone.

High-level synthesis of bovine growth hormone (bGH) in Escherichia coli was achieved by maximizing gene transcription and optimizing the translational efficiency of bGH mRNA. Nearly all of the recombinant hormone was found in the pellet fraction after bacterial cell lysis. This property allowed the purification of bGH nearly to homogeneity.

Loss of tumorigenicity correlates with a reduction in pp60src kinase activity in a revertant subclone of avian sarcoma virus-infected field vole cells.

We have recently isolated an interesting revertant subclone (revertant 866-4) of ESV-infected field vole cells that is indistinguishable from uninfected vole cells with respect to its lack of transformed cell properties. These revertants are not only normal morphologically, but they do not grow in soft agar and are nontumorigenic in athymic nude mice. Despite this lack of transformed cell properties, we have found that this cell line still contains pp60src at concentrations (0.

Karyotype analysis and quantitation of viral transforming genes in Rous sarcoma virus transformed, revertant, and retransformed field vole cells.

Comparative studies of the number of cellular chromosomes and viral genes, including the gene required for malignant transformation, were performed on several clones of Rous sarcoma virus-transformed, revertant, and spontaneously retransformed field vole cells. The results of these studies indicate that no appreciable differences in either total viral gene equivalents or transforming gene sequences can be detected between transformed and revertant cell types, even though considerable differences in the number of certain chromosomes exist among the clones tested. Furthermore, no increase in the amount of total genes or transforming gene sequences accompanies retransformation of revertant clones, including clones that exhibited significant increases in chromosome number following retransformation.

Association of pp60src and src protein kinase activity with the plasma membrane of nonpermissive and permissive avian sarcoma virus-infected cells.

The intracellular localization of pp60src and src protein kinase activity in avian sarcoma virus (ASV)-infected chicken embryo fibroblasts and transformed and morphologically reverted field vole cells was examined by subcellular fractionation procedures. Fractionation by differential centrifugation of Dounce-homogenized cellular extracts prepared from vole cells showed that 83 to 91% of pp60src sedimented with particulate subcellular components from both transformed and revertant vole cells. A slightly lesser amount (60 to 70%) of pp60src was found associated with the particulate fraction from ASV-infected chicken embryo fibroblasts.

Anogenital warts contain several distinct species of human papillomavirus.

Anogenital warts from 26 patients were examined for the presence of human papillomavirus (HPV). Although no whole, intact virus could be identified, varying amounts of nonintegrated HPV DNA were detected in 18 tissue specimens (70%) by employing both an agarose gel-ethidium bromide staining method and the Southern blot hybridization procedure. When hybridization analysis was performed under stringent conditions, six anogenital warts were observed to contain HPV genomic sequences related to either of the cutaneous viruses HPV type 1 (HPV-1) or HPV-2.

Relationship between condylomata and laryngeal papillomata. Clinical and molecular virological evidence.

In a survey of 49 papilloma patients accurate maternal condyloma history was obtained in 31 instances and of these, 21 were positive for the presence of condyloma during pregnancy or parturition. Molecular virological studies indicate that positive hybridization could be demonstrated to human papilloma virus 2 in both laryngeal papilloma and condyloma by the Southern blot technique. Immunoperoxidase staining illustrated the presence of virus-related particles only near the surface of the mucous membrane papilloma, which is in contrast to the definite staining of the stratum granulosum and stratum corneum of verrucae.

The src gene product of transformed and morphologically reverted ASV-infected mammalian cells.

Morphological revertants of avian sarcoma virus transformed vole cells contain the sarcoma gene product (pp60src) in an enzymatically active form, suggesting that the presence of pp60src protein kinase activity is infussicient to induce morphological transformation. Structural analyses of pp60src from infected vole cell clones suggest that in one of the revertant clones on alteration in pp60src may be responsible for morphological reversion while in a second clone, reversion may result from an alteration in a cell gene product with which pp60src must interact. As these morphological revertant cells are tumorigenic, different cell components are required to interact with pp60src to facilitate the two events.

Morphological revertants of an avian sarcoma virus-transformed mammalian cell line exhibit tumorigenicity and contain pp60src.

The biological and biochemical properties of Rous sarcoma virus-transformed and revertant field vole cells were investigated. Revertant vole cells appear morphologically similar to normal, uninfected cells, yet, like transformed vole cells, they are fully capable of growing in agar suspension and producing tumors in athymic nude mice. These highly tumorigenic, yet morphologically normal appearing, vole cells express viral-specific antigens such as the gag gene product (Pr76) but lack the env gene protein (gp85).

Nature of Rous sarcoma virus-specific RNA in transformed and revertant field vole cells.

Cytoplasmic and polyribosomal RNAs from Rous sarcoma virus-transformed and phenotypically reverted field vole cells were fractionated by rate-zonal sedimentation and hybridized with a (3)H-labeled complementary DNA viral probe to determine the size classes of virus-specific RNA present in these cell types. In contrast to Rous sarcoma virus-infected permissive avian cells, only two of three discrete species of virus-specific RNA were detected in the cytoplasm of these vole cells. These included genome-length 35S RNA and a 21S RNA.

Evidence for splicing of avian sarcoma virus 5'-terminal genomic sequences into viral-specific RNA in infected cells.

The 5'-terminal nucleotide sequences of the avian sarcoma virus (ASV) genome are transcribed by the reverse transcriptase in vitro into a DNA transcript that represents the entire distance ( approximately 100 nucleotides) between the tRNA(Trp) primer molecule and the 5' terminus. We have used these DNA(100) transcripts in hybridization reactions with ASV-specific RNA from infected avian cells and find nucleotide sequences complementary to these transcripts on all of the various size classes of viral mRNA identified. Similar hybridization results were obtained with a specific DNA transcript complementary to viral genomic nucleotide sequences between the tRNA(Trp) primer molecule and up to, but not including, the terminal redundant sequences (DNA(70)), indicating that the observed hybridization of DNA(100) to all size classes of viral RNA in infected cells did not reflect hybridization of DNA(100) to the terminal redundant sequences at the 3' end of the viral genome.

Quantitation and localization of Rous sarcoma virus-specific RNA in transformed and revertant field vole cells.

Hybridization analysis of RNA from transformed clones of Rous sarcoma virus (RSV)-infected field vole cells and revertant subclones indicated the presence of similar amounts of viral-specific RNA in both cell types. Employing both a relatively uniform and representative complementary DNA probe and genomelength complementary DNA, we have demonstrated that the majority of RSV proviral DNA is transcribed into viral-specific RNA in both transformed and revertant clones. The viral-specific RNA is present in several size classes, the largest of which is genome-length 35S RNA.

Effect of arginine on the stability and size of argECBH messenger ribonucleic acid in Escherichia coli.

The chemical stability of argECBH messenger ribonucleic acid (mRNA) produced by Escherichia coli was found to be unaltered during steady-state repression by arginine. During extreme arginine deprivation, the increase in argECBH mRNA stability was related to general effects of amino acid starvation on mRNA stability. Thus a mechanism whereby argECBH gene expression is regulated by altering the decay rate of this mRNA is not consistent with our data.

Arginine control of transcription of argECBH messenger ribonucleic acid in Escherichia coli.

The level of messenger ribonucleic acid specific for the argECBH gene cluster (arg-mRNA) of Escherichia coli was measured by deoxyribonucleic acid-ribonucleic acid hybridization in a number of strains. During the first 10 min after removal of arginine (derepression), the rate of arg-mRNA accumulation was six to ten times greater than that found in arginine-repressed argR(+) cells. In the absence of arginine, l-canavanine (200 mug/ml) repressed arg-mRNA synthesis to a level only 20 to 30% lower than that found after arginine deprivation.