Molecular organization of plasmid R906 (Inc P-1)

Genetic and restriction (for enzymes EcoRI, BamHI and HindIII) maps of the relatively broad host range plasmid R906 are constructed. There are two non-essential regions on the R906 DNA which can be deleted and cloned. Non-essential regions confer a resistance to different agents and restriction sites are clustered in these regions. Essential and conjugativity genes are located in two other DNA regions approximately at 0-23 and 29-44 kb of the R906 map. These large regions share a high level of homology with Inc-1 group plasmids R751 and RP4 according to Southern-blot hybridization and heteroduplex analyses. A transposon-like structure is found on the R751 DNA among R751/R906 heteroduplex molecules. This transposon of total length 5.1 kb has 1.4 kb inverted repeats at the ends. Bla genes of R906 and RP4 plasmids do not have homologous sequences. Data evidence that IncP-1 group plasmids irrespective to their original bacterial source and range of coded antibiotic resistance have very similar molecular organization. The role of possible factors which are responsible for the broad host range property of the IncP-1 group plasmids is discussed.     

Molecular genetic studies of the DNA promotor regions in Bacillus thuringiensis subsp. galleriae 69-6. I. Structural and functional analysis

Three recombinant plasmids pPBT9, pPBT10 and pPBT74 carrying promoter-containing regions of DNA of Bacillus thuringiensis which are responsible for the expression of the promoterless tet gene, were studied. In the in vitro experiments, it had been shown that these promoter-active HindIII fragments of bacillar DNA contained RNA polymerase binding sites. The AluI subfragments that specifically bind to Escherichia coli RNA polymerase promote the tet gene expression, similar to the whole HindIII fragments. Sequence analysis revealed that the approximately 220 base pair AluI subfragment of the bacillar insertion of the pPBT10 plasmid contained sites typical for "-10" and "-35" homology regions of promoters specific for sigma 55-RNA polymerase from Bac. subtilis. The 1.45 kb HindII bacillar fragment of the plasmid pPBT9 had three AluI subfragments that bind to E. coli RNA polymerase. Only approximately 400 base pair AluI subfragment among these restored the tet gene expression in vivo. Bireplicon pBP plasmids were constructed that promoted the expression of the enterobacterial antibiotic resistance gene under the control of Bac. thuringiensis promoters in Bac. subtilis cells.     

Control of plasmid incompatibility: characteristics of the bireplicon hybrid pAS8 and its deletion mutants]

The phenomenon of incompatibility has been investigated using deletion mutants of hybrid bireplicon plasmid pAS8. The hybrid pAS8 displays incompatibility specific for both components of its structure. In contrast to P-specificity of pAS8, functions of ColE1-specificity are not effectively expressed. Expression of ColE1-specificity in pAS8 plasmid and its derivatives is characterized by different directions and this is due to the presence or absence of genes of RP4 replication machinery in the plasmid DNA. Mutant plasmids show different efficiency of P-specificity depending on the extension of deletion in the region of essential genes of the RP4 component. Some of the mutants, in spite of the loss of replication genes, including origin of vegetative replication, are incompatible with the representatives of the Inc P group in both directions of testing. Different character and the level of expression of ColE1- and P-specificity in the pAS8 hybrid and its deletion derivatives are not associated with change in the number of plasmid DNA copies, for all of them are subjects to stringent control of replication. The data suggest the existence of incompatibility functions control mechanism which does not seem to include replication genes. Possible ways of realization of the inc genes functions are discussed.     

Comparative analysis of the P-1-group plasmids of incompatibility

Wide host range plasmids (IncP-1) R906, R751 and R702 have several cleavage sites for BamHI, HindIII and EcoRI enzymes, in contrast to RP4 plasmid. Using these enzymes, deletion mutants of R906 plasmid have been obtained in vitro which only lost short DNA fragments (1 to 14 kb). A narrow host range pAV1 plasmid of the same incompatibility group has been transformed into the cells of Escherichia coli. pAV1 is stably maintained in the new host and retains its narrow host range in the course of conjugation. Different restriction fragments of R702, R751, R906 and R906-derived deletion mutants hybridize with the nick-translated probe of RP4 DNA. It is suggested that the wide host range plasmids have a similarity in structural and functional organization.     

Pteridine–sulfonamide conjugates as dual inhibitors of carbonic anhydrases and dihydrofolate reductase with potential antitumor activity

Recent evidences suggest that cancer treatment based on combination of cytostatic and conventional chemostatic therapeutics, which are usually cytotoxic, can provide an improved curative option. On the sequence of our previous work on methotrexate (MTX) derivatives, we have developed and evaluated novel MTX analogues, containing a pteridine moiety conjugated with benzenesulfonamide derivatives, thus endowed with the potential capacity for dual inhibition of dihydrofolate reductase (DHFR) and carbonic anhydrases (CA). These enzymes are often overexpressed in tumors and are involved in two unrelated cellular pathways, important for tumor survival and progression. Their simultaneous inhibition may turn beneficial in terms of enhanced antitumor activity.Herein we report the design and synthesis of several diaminopteridine–benzenesulfonamide and -benzenesulfonate conjugates, differing in the nature and size of the spacer group between the two key moieties. The inhibition studies performed on a set of CAs and DHFR, revealed the activities in the low nanomolar and low micromolar ranges of concentration, respectively. Some inhibitors showed selectivity for the tumor-related CA (isozyme IX). Cell proliferation assays using two tumor cell lines (the non-small cell lung carcinoma, A549, and prostate carcinoma, PC-3) showed activities only in the millimolar range. Nevertheless, this fact points out the need of improving the cell intake properties of these new compounds, since the general inhibitory profiles revealed their potential as anticancer agents.     

Internal EcoRI-generated deletion in prophage Mu DNA

The hybrid plasmid consisting of the plasmid pRP1.2 (derivative of RP4) genome and deleted prophage Mucts 62 genome which lost the central EcoRI fragment of DNA was constructed. The ability of deleted Mu phage to carry out E. coli chromosomal genes transposition was still retained.     

Binding of human sex hormone-binding globulin-androgen complexes to the placental syncytiotrophoblast membrane

We have found that human SHBG complexed with androgens binds specifically to the plasma membrane of human placental syncytiotrophoblast. Apparent equilibrium association constants were 5.3.10(11) M-1 for SHBG-testosterone complex and 1.1.10(11) M-1 for SHBG-5 alpha-dihydrotestosterone. Devoid of steroid, SHBG did not bind to the membrane. This suggests that the specific membrane binding of SHBG-androgen complexes is a step of the mechanism of androgen action on syncytiotrophoblast.     

Modular organization of FDH: Exploring the basis of hydrolase catalysis

An abundant enzyme of liver cytosol, 10-formyltetrahydrofolate dehydrogenase (FDH), is an interesting example of a multidomain protein. It consists of two functionally unrelated domains, an aldehyde dehydrogenase-homologous domain and a folate-binding hydrolase domain, which are connected by an approximately 100-residue linker. The amino-terminal hydrolase domain of FDH (Nt-FDH) is a homolog of formyl transferase enzymes that utilize 10-formyl-THF as a formyl donor. Interestingly, the concerted action of all three domains of FDH produces a new catalytic activity, NADP+-dependent oxidation of 10-formyltetrahydrofolate (10-formyl-THF) to THF and CO2. The present studies had two objectives: First, to explore the modular organization of FDH through the production of hybrid enzymes by domain replacement with methionyl-tRNA formyltransferase (FMT), an enzyme homologous to the hydrolase domain of FDH. The second was to explore the molecular basis for the distinct catalytic mechanisms of Nt-FDH and related 10-formyl-THF utilizing enzymes. Our studies revealed that FMT cannot substitute for the hydrolase domain of FDH in order to catalyze the dehydrogenase reaction. It is apparently due to inability of FMT to catalyze the hydrolysis of 10-formyl-THF in the absence of the cosubstrate of the transferase reaction despite the high similarity of the catalytic centers of the two enzymes. Our results further imply that Ile in place of Asn in the FDH hydrolase catalytic center is an important determinant for hydrolase catalysis as opposed to transferase catalysis.     

A Novel Tumor Suppressor Function of Glycine N-Methyltransferase Is Independent of Its Catalytic Activity but Requires Nuclear Localization

Glycine N-methyltransferase (GNMT), an abundant cytosolic enzyme, catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to glycine generating S-adenosylhomocysteine and sarcosine (N-methylglycine). This reaction is regulated by 5-methyltetrahydrofolate, which inhibits the enzyme catalysis. In the present study, we observed that GNMT is strongly down regulated in human cancers and is undetectable in cancer cell lines while the transient expression of the protein in cancer cells induces apoptosis and results in the activation of ERK1/2 as an early pro-survival response. The antiproliferative effect of GNMT can be partially reversed by treatment with the pan-caspase inhibitor zVAD-fmk but not by supplementation with high folate or SAM. GNMT exerts the suppressor effect primarily in cells originated from malignant tumors: transformed cell line of non-cancer origin, HEK293, was insensitive to GNMT. Of note, high levels of GNMT, detected in regenerating liver and in NIH3T3 mouse fibroblasts, do not produce cytotoxic effects. Importantly, GNMT, a predominantly cytoplasmic protein, was translocated into nuclei upon transfection of cancer cells. The presence of GNMT in the nuclei was also observed in normal human tissues by immunohistochemical staining. We further demonstrated that the induction of apoptosis is associated with the GNMT nuclear localization but is independent of its catalytic activity or folate binding. GNMT targeted to nuclei, through the fusion with nuclear localization signal, still exerts strong antiproliferative effects while its restriction to cytoplasm, through the fusion with nuclear export signal, prevents these effects (in each case the protein was excluded from cytosol or nuclei, respectively). Overall, our study indicates that GNMT has a secondary function, as a regulator of cellular proliferation, which is independent of its catalytic role.