ftsZ is an essential cell division gene in Escherichia coli.

The ftsZ gene is thought to be an essential cell division gene in Escherichia coli. We constructed a null allele of ftsZ in a strain carrying additional copies of ftsZ on a plasmid with a temperature-sensitive replication defect. This strain was temperature sensitive for cell division and viability, confirming that ftsZ is an essential cell division gene. Further analysis revealed that after a shift to the nonpermissive temperature, cell division ceased when the level of FtsZ started to decrease, indicating that septation is very sensitive to the level of FtsZ. Subsequent studies showed that nucleoid segregation was normal while FtsZ was decreasing and that ftsZ expression was not autoregulated. The null allele could not be complemented by lambda 16-2, even though this bacteriophage can complement the thermosensitive ftsZ84 mutation and carries 6 kb of DNA upstream of the ftsZ gene.     

An Arabidopsis homolog of the bacterial cell division inhibitor SulA is involved in plastid division

Plastids have evolved from an endosymbiosis between a cyanobacterial symbiont and a eukaryotic host cell. Their division is mediated both by proteins of the host cell and conserved bacterial division proteins. Here, we identified a new component of the plastid division machinery, Arabidopsis thaliana SulA. Disruption of its cyanobacterial homolog (SSulA) in Synechocystis and overexpression of an AtSulA-green fluorescent protein fusion in Arabidopsis demonstrate that these genes are involved in cell and plastid division, respectively. Overexpression of AtSulA inhibits plastid division in planta but rescues plastid division defects caused by overexpression of AtFtsZ1-1 and AtFtsZ2-1, demonstrating that its role in plastid division may involve an interaction with AtFtsZ1-1 and AtFtsZ2-1.     

A gene encoding an SOS inhibitor is present in different conjugative plasmids.

In 9 of 20 conjugative plasmids of different incompatibility groups, including F and R100 (or R6-5), coexist two sequences which are homologous, respectively, to the gene psiB, which encodes an inhibitor of SOS induction, and to the gene ssb, which encodes a single-stranded-DNA-binding protein.     

A new Escherichia coli cell division gene, ftsK.

A mutation in a newly discovered Escherichia coli cell division gene, ftsK, causes a temperature-sensitive late-stage block in division but does not affect chromosome replication or segregation. This defect is specifically suppressed by deletion of dacA, coding for the peptidoglycan DD-carboxypeptidase, PBP 5. FtsK is a large polypeptide (147 kDa) consisting of an N-terminal domain with several predicted membrane-spanning regions, a proline-glutamine-rich domain, and a C-terminal domain with a nucleotide-binding consensus sequence. FtsK has extensive sequence identity with a family of proteins from a wide variety of prokaryotes and plasmids. The plasmid proteins are required for intercellular DNA transfer, and one of the bacterial proteins (the SpoIIIE protein of Bacillus subtilis) has also been implicated in intracellular chromosomal DNA transfer.     

Crystal structure of the bacterial cell division inhibitor MinC

Bacterial cell division requires accurate selection of the middle of the cell, where the bacterial tubulin homologue FtsZ polymerizes into a ring structure. In Escherichia coli, site selection is dependent on MinC, MinD and MINE: MinC acts, with MinD, to inhibit division at sites other than the midcell by directly interacting with FTSZ: Here we report the crystal structure to 2.2 A of MinC from Thermotoga maritima. MinC consists of two domains separated by a short linker. The C-terminal domain is a right-handed beta-helix and is involved in dimer formation. The crystals contain two different MinC dimers, demonstrating flexibility in the linker region. The two-domain architecture and dimerization of MinC can be rationalized with a model of cell division inhibition. MinC does not act like SulA, which affects the GTPase activity of FtsZ, and the model can explain how MinC would select for the FtsZ polymer rather than the monomer.     

Identification and sequence of gene dicB: translation of the division inhibitor from an in-phase internal start.

The dicA1 mutation, located in the replication termination region of Escherichia coli at 34.9 min, confers a temperature-sensitive, division defective phenotype to its hosts. Previous analysis had suggested that dicA codes for a repressor of a nearby division inhibition gene dicB. We show now that gene dicB is part of a complex operon. Five open reading frames (ORFs 1 to 5) preceeded by a promoter sensitive to dicA repression are found within a 1500 bp segment, and are organized into two clusters separated by a long untranslated region. Evidence for expression of these ORFs was obtained from in vitro or in vivo translation of plasmid-coded genes. IPTG-dependent cell filamentation was obtained when either the entire or the C-terminal part of the fourth ORF was placed under control of the lac promoter. In both cases, a 7 KD protein corresponding to translation from an in-frame ATG of ORF4 (dicB) was made. We propose that this C-terminal protein is the division inhibitor synthesized in dicA1 mutants.     

The rapamycin analog CCI-779 is a potent inhibitor of pancreatic cancer cell proliferation

We present immunohistochemical evidence that the mTOR/p70s6k pathway is activated in pancreatic tumors and show that the mTOR inhibitor and rapamycin analog CCI-779 potently suppresses the proliferation of pancreatic cancer cells. Consistent with a recent study, CCI-779 increased c-Jun phosphorylation (Ser63) in a dose- and time-dependent manner, and induced apoptosis in p53-defective BxPC-3 cells. In contrast to the study, however, we observed that CCI-779 concomitantly increased c-Jun protein levels and that its ability to induce apoptosis might not require the activated c-Jun. Furthermore, CCI-779 neither induced c-Jun phosphorylation in other p53-defective pancreatic cancer cells (MiaPaCa-2) nor inhibited their proliferation. c-Jun, in fact, appeared to be partly responsible for the resistance of MiaPaCa-2 cells to CCI-779. Together, these results indicate a complex role for c-Jun in cellular responses to CCI-779 and provide an important basis for investigating CCI-779 further as a potential therapeutic agent for pancreatic tumors.     

Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell division gene ftsQ.

We report the identification, cloning, and mapping of a new cell division gene, ftsQ. This gene formed part of a cluster of three division genes (in the order ftsQ ftsA ftsZ) which itself formed part of a larger cluster of at least 10 genes, all of which were involved in some step in cell division, cell envelope synthesis, or both. The ftsQAZ group was transcribed from at least two independent promoters.     

A new essential gene of the 'minimal genome' affecting cell division

The complete sequencing of bacterial genomes has offered new opportunities for the identification of essential genes involved in the control and progression of the cell cycle. For this purpose, we have disrupted ten E. coli genes belonging to the so-called 'minimal genome'. One of these genes, yihA, was necessary for normal cell division. The yihA gene possesses characteristic GTPase motifs and its homologues are present in eukaryotes, archaea and most prokaryotes. Depletion of YihA protein led to a severe reduction in growth rate and to extensive filamentation, with a block beyond the stage of nucleoid segregation. Filamentation was correlated with reduced FtsZ levels and could be specifically suppressed by overexpression of ftsQI, ftsA and ftsZ, and to some extent by ftsZ alone. We hypothesize that YihA, like the Era GTPase, may participate in a checkpoint mechanism that ensures a correct coordination of cell cycle events.     

Coupling of DNA replication and cell division: sulB is an allele of ftsZ.

Treatments that damage DNA in Escherichia coli result in the inhibition of cell division. This inhibition is controlled by the lexA-recA regulatory circuit and can be specifically uncoupled by the mutations sulA (sfiA) and sulB (sfiB), which map at 21 and 2 min, respectively. Presently it is thought that sulA codes for an inducible inhibitor of cell division, the expression of which is controlled directly by the lexA repressor. In this report, it is shown that sulB is an allele of ftsZ, an essential cell division gene. A sulB mutation leads to an altered ftsZ gene product which is slightly thermosensitive and has an altered mobility on polyacrylamide gels. It is suggested that the altered ftsZ gene product is resistant to the sulA inhibitor, thus permitting cell division after induction of the SOS response. It is also shown that an increase in the gene dosage of ftsZ delays the onset of filamentation after SOS induction.     

Chloramphenicol is an inhibitor of photosynthesis.

Chloramphenicol inhibited significantly but incompletely photosynthesis in leaf segments of rice. Fluorescence and polarographic experiments indicated that chloramphenicol competes with the CO2 reducing cycle for electrons from photosystem I because it serves as an electron acceptor of photosystem I and its reduction intermediate transfers its electron to molecular oxygen.     

Mitogen-inducible gene 6 is an endogenous inhibitor of HGF/Met-induced cell migration and neurite growth

Hepatocyte growth factor (HGF)/Met signaling controls cell migration, growth and differentiation in several embryonic organs and is implicated in human cancer. The physiologic mechanisms that attenuate Met signaling are not well understood. Here we report a mechanism by which mitogen-inducible gene 6 (Mig6; also called Gene 33 and receptor-associated late transducer) negatively regulates HGF/Met-induced cell migration. The effect is observed by Mig6 overexpression and is reversed by Mig6 small interfering RNA knock-down experiments; this indicates that endogenous Mig6 is part of a mechanism that inhibits Met signaling. Mig6 functions in cells of hepatic origin and in neurons, which suggests a role for Mig6 in different cell lineages. Mechanistically, Mig6 requires an intact Cdc42/Rac interactive binding site to exert its inhibitory action, which suggests that Mig6 acts, at least in part, distally from Met, possibly by inhibiting Rho-like GTPases. Because Mig6 also is induced by HGF stimulation, our results suggest that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.     

SOS induction of the gene sulA is partly inhibited in Escherichia coli K12 cells overproducing the RecA protein

A study was made of the SOS induction of the gene sulA of Escherichia coli K12 in relation to the gene dosage of the gene recA. In experiments the sulA::lacZ fusion strain PQ37 and derivatives of PQ37 with the multi-copy plasmids pDR1453 or pBR322 were used. The SOS response was induced with nitrofurantoin, SOS induction of the gene sulA was determined on the basis of the amount of beta-galactosidase synthesized, i.e. by the SOS chromotest (Quillardet et al., 1982a). It was found in this work that cells with the plasmid pDR1453, which contain the gene recA of E. coli K12 (Sancar and Rupp, 1979), have a decreased SOS induction of the gene sulA. Cells with the plasmid pBR322 do not exhibit this decrease. Inactivation of the gene recA in the plasmid pDR1453 with preservation of the functional gene recA in the chromosome leads to a restoration of 'standard' SOS induction of the gene sulA. The results show that the amount of the gene product of the gene recA affects the SOS induction of the gene sulA.     

A survey of essential gene function in the yeast cell division cycle

Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO(7) promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication.     

A system for the measurement of gene targeting efficiency in human cell lines using an antibiotic resistance-GFP fusion gene

Gene targeting in a broad range of human somatic cell lines has been hampered by inefficient homologous recombination. To improve this technology and facilitate its widespread application, it is critical to first have a robust and efficient research system for measuring gene targeting efficiency. Here, using a fusion gene consisting of hygromycin B phosphotransferase and 3'-truncated enhanced GFP (HygR-5' EGFP) as a reporter gene, we created a molecular system monitoring the ratio of homologous to random integration (H/R ratio) of targeting vectors into the genome. Cell clones transduced with a reporter vector containing HygR-5' EGFP were efficiently established from two human somatic cell lines. Established HygR-5' EGFP reporter clones retained their capacity to monitor gene targeting efficiency for a longer duration than a conventional reporter system using an unfused 5' EGFP gene. With the HygR-5' EGFP reporter system, we reproduced previous findings of gene targeting frequency being up-regulated by the use of an adeno-associated viral (AAV) backbone, a promoter-trap system, or a longer homology arm in a targeting vector, suggesting that this system accurately monitors H/R ratio. Thus, our HygR-5' EGFP reporter system will assist in the development of an efficient AAV-based gene targeting technology.