Melvin H. Green

Sarkosyl activation of RNA polymerase activity in mitotic mouse cells.

The transition from the G2 phase of the eukaryotic cell cycle to the mitotic phase produces dramatic morphological alterations, among which are the condensation of chromatin to form chromosomes, and the disappearance of the nuclear membrane and nucleoli. These changes are accompanied by a nearly complete inhibition of cellular RNA synthesis [ 11. Recent work by Simmons et al. [2] indicates that inhibitors of protein synthesis did not prevent the resumption of RNA synthesis when mitotic cells entered the G1 phase, suggesting that RNA polymerase is present but not active during mitosis.

Inactivation of the prophage lambda repressor without induction

The DNA-RNA hybridization technique was used to demonstrate that the repressor of prophage λ greatly inhibits the rate of transcription of both the prophage and superinfecting λ genomes in Escherichia coli (λ). The repressor of the prophage λ c I,tl mutant, but not that of λ + , was inactivated at 45°C, thereby permitting a 40-fold increase in the rate of synthesis of λ messenger RNA. Since de-repression occurred in the absence of protein synthesis, at least part of the λ genome can be transcribed by the E. coli RNA polymerase. Heat inactivation of the λ c I,tl repressor was a necessary, but not a sufficient cause for the eventual killing (induction) of the lysogenic cell. Induction of cells carrying this mutant prophage also required the synthesis of protein while at the elevated temperature. Since other c I mutants of λ can be induced by exposure to the elevated temperature even in the absence of protein synthesis, it is postulated that the necessary “inducing protein” is always present in the lysogenic cell as an integral part of the λ repressor. Depending then on the site of the c I mutation, the inducing protein may or may not become activated upon thermal inactivation of the repressor. Following inactivation of the c I,tl repressor at 45°C, its renewed synthesis at 37°C was shown to be inhibited by chloramphenicol. It is therefore concluded that this repressor is, at least in part, a protein.

Properties of the polyoma virus transcription complex obtained from mouse nuclei

Abstract At a late stage of polyoma virus development in cultures of mouse 3T3 Balb/C, most of the replicated viral DNA which can be solubilized by the SDS method (1) can also be extracted as a 55S DNA-protein complex (2) by the Triton method. However, the Triton pellet fraction obtained from nuclei of infected cells was at least 50 times more active in the synthesis of polyoma-specific RNA than was the Triton supernatant upon addition of nucleoside triphosphates. This finding suggests that the active template for polyoma RNA synthesis in vivo is not the polyoma 55S complex, but rather a minor fraction of the viral DNA which is not solubilized by the Triton method. The in vitro synthesis of polyoma-specific RNA was totally sensitive to inhibition by α-amanitin. Unlike E. coli RNA polymerase, the mouse RNA polymerases were found to be resistant to inactivation by Sarkosyl.

Isolation of two forms of SV40 nucleoprotein containing RNA polymerase from infected monkey cells.

Abstract Triton extraction of SV40 infected monkey cells separates the viral transcription complex (VTC) into two fractions. Both forms of VTC are comprised of viral DNA associated with RNA polymerase which had initiated RNA synthesis prior to extraction. Approximately 20% of the total VTC can be solubilized by a variety of extraction conditions, while the remainder is pelleted together with the cellular chromatin or DNA. The proportion of VTC in the pellet fraction is not altered by extraction at a 10-fold lower cell concentration or under conditions which release most of the protein from chromatin and solubilize nearly all of the nonintegrated SV40 DNA. The predominant template for SV40 transcription at a late stage of infection is apparently associated with cellular DNA in vivo , or else it becomes selectively associated during the extraction procedure. Most of the Triton-soluble VTC, detected by endogenous RNA polymerase activity, sediments somewhat faster than the DNA-labeled SV40 chromatin, suggesting that a minor fraction of the DNA is engaged in transcription. Treatment of the VTC with Sarkosyl reduces its sedimentation rate from 50–55 S to 21–25 S and enhances the endogenous RNA polymerase activity by a factor of 3–5. It is thus likely that some cellular and/or viral proteins associated with SV40 DNA affect the rate of viral transcription in vivo .

Early-late transcription switch: Isolation of a lambda DNA-RNA polymerase complex active in the synthesis of late RNA

Abstract Certain regions of bacteriophage DNA are transcribed at late but not at early times after infection of a bacterial cell ( Hall et al., 1963 , Hall et al., 1964 ). The mechanism by which these “late” regions become available to prime for RNA synthesis at late time is unknown. Escherichia coli RNA polymerase will transcribe in vitro only the “early” genes of T4 DNA ( Khesin et al ., 1963 ; Geiduscheck et al. , 1966) and of λ DNA ( Maitra et al. , 1966 ; Naono & Gros, 1966 ). Recent evidence suggests that the T4 gene 55 product allows E . coli RNA polymerase to transcribe late regions of T4 DNA in vitro (Snyder & Geiduschek, 1968). It has also been demonstrated that T4 infection alters part of the E . coli RNA polymerase ( Walter et al ., 1968 ). The present report describes the isolation of a λ DNA-RNA polymerase complex from E . coli at a late stage after infection. In vitro the complex synthesizes early and late λ mRNA in the same proportion as do the cells from which the complex was purified.

Characterization of polycistronic late lambda messenger RNA.

Abstract The kinetics of inhibition of RNA synthesis in Escherichia coli by rifampicin permitted the selective labeling of lambda (λ) late RNA (A-J region of the λ genome). This RNA sedimented heterogeneously, with rates as great as 60 S. The proportion of labeled 40–60 S RNA increased from 1–2% in the absence of rifampicin to approximately 40% in its presence. Based on sucrose gradient sedimentation and gel electrophoresis analyses, the molecular weight of 60 S λ late RNA was estimated at 5–9 × 106 daltons. From the known length of the A-J segment, it is postulated that this entire region can be transcribed as a single polycistronic message.

Hyaluronan: a potential carrier for growth factors for the healing of ligamentous tissues

The anterior cruciate ligament has little capacity to heal after injury, in contrast to the medial collateral ligament. Cell migration and cell proliferation are essential steps in the wound healing of connective tissue, and we have developed an in vitro assay to study cell outgrowth (i.e., both cell migration and cell proliferation) from tissue explants. Using this assay, we have previously shown a synergistic effect of four growth factors on the early stages of cell outgrowth (i.e., at days 3 and 6) from these explants. Hyaluronan, a linear polysaccharide with a molecular weight of 3 × 106 daltons, has been shown to enhance the anterior cruciate ligament healing capacity in vivo by over 50%. The purpose of this study was to assess in vitro whether hyaluronan and the combination of growth factors (transforming growth factor‐β1 25 ng/ml, platelet‐derived growth factor‐BB 100 ng/ml, basic fibroblast growth factor 100 ng/ml, and insulin 25 µg/ml) would enhance cell outgrowth from explants at 3 and 6 days. At 3 days the mean cell outgrowth from the explants treated with hyaluronan and the combination of growth factors exceeded the growth in the explants treated with growth factors alone for the anterior cruciate ligament (mean cell count ± standard error: 19 ± 4 and 2 ± 1 cells, respectively) and for the medial collateral ligament (297 ± 40 and 87 ± 18 cells, respectively). At 6 days, the mean cell outgrowth in the presence of growth factors was enhanced sixfold by hyaluronan for the anterior cruciate ligament (160 ± 27 compared with 26 ± 15 cells) and fourfold for the medial collateral ligament (1363 ± 219 compared with 330 ± 74 cells). As previously shown, little cell outgrowth occurred from the anterior cruciate or medial collateral ligament explants when no growth factors were added to the modified Eagles media at 3 and 6 days (<20 cells). For the medial collateral ligament only, hyaluronan had a small stimulatory effect on cell outgrowth in minimal essential media in the absence of growth factors. At 3 days the stimulation by hyaluronan was fourfold (31 ± 8 cells compared with 7 ± 4 cells), and at 6 days the stimulation by hyaluronan was nearly fourfold (73 ± 17 compared with 20 ± 12 cells). These results suggest that hyaluronan could be used together with growth factors in efforts to enhance the healing of ligamentous tissue injuries.

Selective cytotoxicity of L-canavanine in tumorigenic Madin-Darby canine kidney T1 cells

L-canavanine, an analog of L-arginine, was examined for toxicity in a normal canine kidney epithelial cell line, Madin-Darby canine kidney (MDCK), and in its chemically transformed derivative MDCK-T1. Under conditions where the analog reversibly arrested growth of MDCK cells, more than 90% of the tumorigenic cells were killed. This selective cytotoxicity was not due to any difference in growth rate (both lines doubled every 24 h). Nor was it caused by the inhibition of protein synthesis or DNA replication, since amino acid deprivation and drugs which inhibited replication directly and indirectly did not kill the transformed cells preferentially. To the contrary, cycloheximide killed MDCK cells preferentially. Although the mechanism for the selective cytotoxicity of canavanine in the tumorigenic cells remains obscure, one clue was afforded by the observation that the analog caused a loss in plating efficiency of the T1 cells prior to their detachment from plates. Selective sensitivity to canavanine may therefore reside in cell surface proteins, which are known to differ both qualitatively and quantitatively between normal and transformed cells. The present results support our previous findings suggesting the possible value of using canavanine as an agent for cancer chemotherapy.

Properties of phage λ DNA-RNA polymerase complexes isolated from Escherichia coli (λ)

Abstract 1. 1. A method is described for the isolation of highly purified λ DNA-RNA polymerase complex from λ-infected Escherichia coli (λ). The method selects for the covalently closed circular form of λ DNA. Nearly all of the RNA polymerase (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) is in the initiated state. The complex is essentially free of ribonuclease and endonuclease. There appears to be little, if any, nonspecific binding of RNA polymerase to λ DNA during the course of isolation of the complex. It is thus demonstrated that the closed circular form of λ DNA serves as a template in vivo for RNA synthesis. 2. 2. Several properties pertaining to RNA synthesis by the complex are described The RNA chain length increases linearly with time, attaining a maximum molecular weight of approx. 3·106. The majority of the growing RNA chains extends into genes that were repressed in the lysogenic cells from which the complex was obtained. The closed circular form of the λ DNA template persists throughout the reaction. Some of the newly synthesized RNA remains associated with the template in a non-covalent form, presumably hydrogen bonded. 3. 3. Complex isolated from derepressed lysogens synthesizes 6–8 times more-RNA per unit of λ DNA than does complex from repressed lysogens. The RNA originates from λ genes that were transcribed in the cell immediately prior to lysis. The proportion of RNA synthesized from the cI-rex region was 60% for complex from repressed lysogens, but only 14% for complex isolated from lysogens following heat inactivation of the λ repressor. It is suggested that a region other than cI-rex is constitutively expressed.

Alteration of RNA polymerase during transcription of phage λ DNA “in vivo”

Abstract λ DNA-RNA polymerase complex was isolated from λ-infected E. coli at a late stage of phage development. The polymerase was separated from the λ DNA and characterized with regard to the presence of σ-like initiation factor. The data support the hypothesis that initiation factor dissociates from RNA polymerase following the initiation of RNA synthesis.