Arthur B. Pardee

The genetic control and cytoplasmic expression of “Inducibility” in the synthesis of β-galactosidase by E. coli

A number of extremely closely linked mutations have been found to affect the synthesis of β -galactosidase in E. coli . Some of these ( z mutations) are expressed by loss of the capacity to synthesize active enzyme. Others ( i mutations) allow the enzyme to be synthesized constitutively instead of inducibly as in the wild type. The study of galactosidase synthesis in heteromerozygotes of E. coli indicates that the z and i mutations belong to different cistrons. Moreover the constitutive allele of the i cistron is recessive over the inducible allele. The kinetics of expression of the i + (inducible) character suggest that the i gene controls the synthesis of a specific substance which represses the synthesis of ( β -galactosidase. The constitutive state results from loss of the capacity to synthesize active repressor.

Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation

Cellular oncogenes are DNA sequences implicated in the genesis of cancer, but their functions in the transformation process are not understood. Our experiments provide data linking expression of two well-studied proto-oncogenes, c-myc and c-rasKi, to current knowledge of proliferation control and its perturbation by differentiation and chemical transformation. Growth stimulation of quiescent cells by serum elevates expression of the myc proto-oncogene in Balb/c 3T3 (A31) cells. In two chemically transformed A31 derivatives (BPA31 and DA31), c-myc expression is constitutive. The levels of c-myc mRNA in quiescent and growing transformed cells are nearly the same, and are only slightly elevated compared to the level found in growing A31 cells. By contrast, c-rasKi expression is cell-cycle-dependent in BPA31 cells. The relative abundance of c-rasKi mRNA begins to increase in mid- to late G0/G1. During terminal differentiation of teratocarcinoma stem cells (F9) into nonproliferating endoderm, relative mRNA abundance is diminished more markedly for c-myc than for c-rasKi. These results demonstrate that expression of the myc and rasKi proto-oncogenes is dependent upon the cellular growth state, and that growth control exhibits growth-factor-dependent, cell-cycle-timed oncogene expression. In the case of the BPA31 cells, c-myc is not rearranged, amplified, or overexpressed. However, the oncogene has lost its cycle-dependent regulation in the chemically transformed cells.

The restriction point of the cell cycle.

As formulated in 1974, the concept of the restriction point of the cell cycle was based on cell biological experiments, yet allowing accurate molecular predictions and spurring a search for the restriction factor. Although cyclin D meets the criteria of the R-factor, the picture as outlined here is more interesting and far more complex. We discuss the relationship between the restriction knot and DNA damage-checkpoints. Finally, we discuss how loss of the restriction point in cancer leads to loss of checkpoint control and to insensitivity to antimitogens including some mechanism-based anticancer therapeutics. Key Words: Cell cycle, Cyclins, Growth factors, Oncogenes

Recent advances in differential display

Differential display and RNA arbitrary primed polmerase chain reaction are methods recently designed to identify and isolate differentially expressed genes. Methodological modifications have since been introduced to streamline the techniques. The major effort has centered on how to eliminate false positives as approached from a variety of angles, ranging from RNA sample preparation, northern blot confirmation, primer length variation, to better experimental design.

Penicillin-binding proteins and cell shape in E. coli

β-LACTAM antibiotics (penicillins and cephalosporins) have attracted considerable attention as probes of cell growth and division1,2. Although extensive studies have been made on both penicillin-sensitive enzymes and penicillin binding proteins3, there has been no clear indication of the role of any of these components in the effects of β-lactam antibiotics on cell growth. We report the identification of a minor penicillin binding protein which we believe to be the target at which the amidinopenicillanic acid designated FL1060 (ref. 4) acts to affect the shape of Escherichia coli. This is the first example of the identification of a penicillin binding protein with an essential and defined role in bacterial cell growth.

Studies on the macromolecular organization of microbial cells

Five different methods of preparing extracts from bacterial cells yielded similar distributions of macromolecular constituents as indicated by ultracentrifugal patterns. Extracts from a number of different bacteria showed essentially similar patterns. Three principal components were found, with sedimentation constants of approximately 40, 29, and 5 S. In many extracts, it was evident that the most slowly moving peak consisted of two components, a broad diffuse boundary and a sharp spike containing desoxypentose nucleic acid. Extracts from the photosynthetic bacterium Rhodospirillum rubrum contained, in addition to the above-mentioned components, a component of very large size (sedimentation constant approximately 190 S), with which the entire pigment complement of the cells was associated. Chemical analyses of crudely fractionated extracts showed that the bulk of the pentose nucleic acid of the cells was associated with the particles of sedimentation constants from 20 to 40 S, while the desoxypentose nucleic acid was almost all present in the fraction of lower sedimentation rate.

Activation of the p21WAF1/CIP1 promoter independent of p53 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through the Sp1 sites

Suberoylanilide hydroxamic acid (SAHA) is a novel histone deacetylase inhibitor with high potency in inducing differentiation of cultured murine erythroleukemia cells. We have recently demonstrated that SAHA induces cell cycle arrest and apoptosis in human breast cancer cells, accompanied by up-regulation of the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, via a p53-independent mechanism. In this study, we used p21 gene expression as a model system to elucidate the molecular mechanism(s) underlying SAHA-mediated gene activation. Treatment of human breast cancer cell line MCF7 cells with SAHA induced p21 mRNA as a consequence of an immediate-early gene activation. Moreover, SAHA activated the p21 promoter primarily through two Sp1 sites located at −82 and −69 relative to the transcription start site. Furthermore, Sp1 and Sp3 proteins were the major factors binding to the Sp1 site of the p21 promoter. However, SAHA did not alter their DNA binding activities, suggesting that SAHA mediates p21 promoter activity by a mechanism other than altering the DNA binding activities of Sp1 and Sp3. Further studies using the GAL4 luciferase assay system demonstrated that both GAL4-Sp1 and GAL4-Sp3 fusion proteins supported SAHA-mediated gene activation from a promoter driven by five GAL4 DNA binding sites, and that GAL4-Sp3 fusion protein was suppressive in the absence of SAHA treatment. Collectively, our results suggest that SAHA activates the p21 promoter through the Sp1 sites, and that both Sp1 and Sp3 proteins can mediate SAHA-induced gene activation.

The initial kinetics of enzyme induction

Abstract Kinetics of induction of several enzymes of Escherichia coli have been investigated under conditions where non-specific nutrient effects and permeability mechanisms are not important. Measurements made over time intervals of a few minutes permitted detection of initial events brought about by addition or removal of inducers or inhibitors. With each enzyme a lag of about 3 min between addition of inducer and appearance of enzyme at 37° was noted. This lag was dependent on temperature and independent of inducer concentration, in contrast to an inhibitor which showed the opposite behavior. The induction lag, therefore, does not represent the time required for penetration of the inducer. Studies with inhibitors suggest that some metabolism involving the synthesis of a ribonucleic acid is required before enzyme synthesis can proceed. The formation of enzyme ceased about 5 min after inducer was removed or after glucose was supplied to the culture, suggesting that the enzyme-forming system is unstable. A model for the enzyme forming apparatus of the cell is suggested which is consistent with the kinetic data and with other information regarding induction. A repressor prevents function of the genetic material of the bacteria. The inducer is thought to prevent the repressor from acting. This freed genetic material forms a special, unstable ribonucleic acid which interacts with the ribosomes to provide an active template for enzyme synthesis. The time required for enzyme formation to commence is attributed principally to the interval during which the repressor is lost and the unstable ribonucleic acid is formed. A possible similarity between repressor and unstable ribonucleic acid is suggested. Instability appears to be spontaneous at temperatures where metabolism goes on, rather than being caused by use of the system for enzyme production.

DNA synthesis inhibition and the induction of protein X in Escherichia coli

Abstract The synthesis of protein X (Inouye & Pardee, 1970) is switched on by the inhibition of DNA synthesis. This induction of protein X is controlled at transcription, since it can be blocked by either chloramphenicol or rifampicin. Approximately 90% of the total protein X in the cell is found in the cytoplasm and 10% in the membrane. After one generation of growth in the absence of DNA synthesis, protein X is 3 to 4% of the total cell protein. The rate of synthesis of protein X showed a gradual increase over about 25 min to a maximal rate following inhibition of DNA synthesis by nalidixic acid (40 μg/ml). Thymine starvation of an auxotroph or transfer of a dnaB mutant to its restrictive temperature resulted in different kinetics of induction of protein X, suggesting that induction is an indirect consequence of DNA synthesis inhibition. In synchronous cultures, protein X was induced by nalidixic acid only when DNA replication was in progress. Moreover, a dnaA initiation mutant at the restrictive temperature could not be induced to synthesize protein X after DNA replication was completed. DNA degradation is involved in the induction of protein X as shown by its induction in a recC− mutant by the antibiotic bleomycin which degrades DNA, but not by nalidixic acid. Thus, a stalled replication fork and subsequent DNA degradation are required for the induction of protein X by nalidixic acid or thymine starvation. Protein X has affinity for single-stranded DNA specifically, when Mg2+ is present. We suggest that production of protein X is part of a feedback loop that limits DNA degradation and allows DNA repair.

Metastasis and AKT activation

Metastasis is responsible for 90% of cancer patient deaths. More information is needed about the molecular basis for its potential detection and treatment. The activated AKT kinase is necessary for many events of the metastatic pathway including escape of cells from the tumors environment, into and then out of the circulation, activation of proliferation, blockage of apoptosis, and activation of angiogenesis. A series of steps leading to metastatic properties can be initiated upon activation of AKT by phosphorylation on Ser-473. These findings lead to the question of how this activation is connected to metastasis. Activated AKT phosphorylates GSK-3β causing its proteolytic removal. This increases stability of the negative transcription factor SNAIL, thereby decreasing transcription of the transmembrane protein E-cadherin that forms adhesions between adjacent cells, thereby permitting their detachment. How is AKT hyperactivated in metastatic cells? Increased PI3K or TORC2 kinase activity- or decreased PHLPP phosphatase could be responsible. Furthermore, a positive feedback mechanism is that the decrease of E-cadherin lowers PTEN and thereby increases PIP3, further activating AKT and metastasis.