Antal Kiss

Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti

SummaryR. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRmc41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod-) or nitrogen fixation (Fix-) have been readily obtained. In some Nod- mutants the deletion of a segment of plasmid pRme41b was found.Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. 32P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digests from the wild-type bacterium and from a series of Nod- mutants revealed that at least a 24 kb DNA fragment including the nif structural genes was missing from most of the Nod- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.

The number of rRNA genes in Escherichia coli

The three different ribosomal RNA species of Escherichia colt’ (5 S, 16 S and 23 S) originate from a single 30 S precursor RNA. The DNA coding for this 30 S rRNA is called an rRNA gene set (rm). It has long been known that these genes are redundant in bacteria, their number has been estimated on the basis of saturation hybridization experiments as being 510 (for a review see [ 1 ] ). The most reliable measurements suggested the existence of six rm genes [2]. Plasmids and transducing phages helped to map unambiguously five of these, which are now designated rrnA (85 min), rrnB (88 min), rrnC (83 min), rmD (71 min) and rmE (89 min) [3]. In this paper we present evidence strongly suggesting that the actual number of rRNA gene sets is seven in E. coli K12.

Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli

The gene coding for the sequence-specific modification methylase methM . BspI of Bacillus sphaericus R has been cloned in Escherichia coli by means of plasmid pBR322. The selection was based on the expression of the cloned gene which rendered the recombinant plasmid resistant to BspI restriction endonuclease cleavage. The gene is carried by a 9 kb BamHI fragment and by a smaller 2.5 kb EcoRI fragment derived from the BamHI fragment. The Bsp-specific methylase level was found to be higher in the recombinant clones than in the parental strain. The methylase gene is probably located on the Bacillus sphaericus chromosome, and not on a plasmid known to be carried by this strain. The recombinant clones do not exhibit an BspI restriction endonuclease activity.

Construction and characterization of R-prime plasmids carrying symbiotic genes of R. meliloti

SummaryR-prime plasmids carrying 100–200 kb regions of R. meliloti DNA coding for symbiotic functions were isolated from a Km8 derivative of R68.45. The R-primes were obtained from matings between R. meliloti strains containing Tn5-induced symbiotic mutations and E. coli recipients by selecting for the kanamycin resistance marker of Tn5. It was demonstrated that the regions inserted into the R-primes were identical with those DNA segments where Tn5 was located in the respective parental R. meliloti.R-primes were generated from both the R. meliloti chromosome and from the megaplasmid pRme41b. A set of R-primes, carrying the nitrogen fixation (nif) gene cluster, also carried genes required for nodulation (nod genes) of alfalfa. Transfer of these R-primes into different Nod- mutants restored the Nod+ phenotype. When they were introduced into A. tumefaciens the transconjugants formed small nodules on alfalfa. This indicates that nodulation and nitrogen fixation genes of the R. meliloti megaplasmid are clustered on a relatively short DNA segment.

Structure of the Bacillus sphaericus R modification methylase gene

A 2.5 X 10(3) base-pair segment of Bacillus sphaericus R DNA cloned in Escherichia coli has previously been shown to carry the functional BspRI modification methylase gene. The approximate location of the gene on this DNA segment and its direction of transcription were established by subcloning experiments. The nucleotide sequence of the relevant region was determined by the Maxam-Gilbert procedure. An open reading frame that can code for a 424 amino acid protein was found. The calculated molecular weight (48,264) of this protein is in fair agreement with previous estimates (50,000 to 52,000). The synthesis of this protein was demonstrated in E. coli minicells. The initiation point of transcription by E. coli RNA polymerase was localized by in vitro transcription experiments. The open reading frame starts 29 base-pairs downstream from the transcription initiation site and it is preceded by a sequence showing extensive Shine-Dalgarno complementarity. Subcloning experiments and translation in minicells suggest that after removal of this translational initiation site, a secondary start site 29 amino acids downstream can also start translation in E. coli, and this shorter protein retains the methylase activity. The overall base composition of the gene and the codon usage indicate a strong preference for A.T base-pairs.

DNA nicks inflicted by restriction endonucleases are repaired by a RecA‐ and RecB‐dependent pathway in Escherichia coli

Two mutants of the EcoRI endonuclease (R200K and E144C) predominantly nick only one strand of the DNA substrate. Temperature sensitivity of the mutant enzymes allowed us to study the consequences of inflicting DNA nicks at EcoRI sites in vivo. Expression of the EcoRI endonuclease mutants in the absence of the EcoRI methyltransferase induces the SOS DNA repair response and greatly reduces viability of recA56, recB21 and lexA3 mutant strains of Escherichia coli. In parallel studies, overexpression of the EcoRV endonuclease in cells also expressing the EcoRV methyltransferase was used to introduce nicks at non‐cognate EcoRV sites in the bacterial genome. EcoRV overproduction was lethal in recA56 and recB21 mutant strains and moderately toxic in a lexA3 mutant strain. The toxic effect of EcoRV overproduction could be partially alleviated by introduction into the cells of multiple copies of the E. coli DNA ligase gene. These observations suggest that an increased number of DNA nicks can overwhelm the repair capacity of DNA ligase, resulting in the conversion of a proportion of DNA nicks into DNA lesions that require recombination for repair.

Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence the EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.SssI was coupled to a triple-helix-forming oligonucleotide (TFO−C141S) specifically designed for the EpCAM gene. Reporter plasmids encoding the green fluorescent protein under control of different EpCAM promoter fragments were treated with the TFO−C141S conjugate to determine the specificity of targeted DNA methylation in the context of a functional EpCAM promoter. Treatment of the plasmids with TFO−C141S resulted in efficient and specific methylation of the targeted CpG located directly downstream of the triple helix forming site (TFS). No background DNA methylation was observed neither in a 700 bp region of the EpCAM promoter nor in a 400 bp region of the reporter gene downstream of the TFS. Methylation of the target CpG did not have a detectable effect on promoter activity. This study shows that the combination of a specific TFO and a reduced activity methyltransferase variant can be used to target DNA methylation to predetermined sites with high specificity, allowing determination of crucial CpGs for promoter activity.

Reversible inactivation of the CG specific SssI DNA (cytosine-C5)-methyltransferase with a photocleavable protecting group.

Caging of proteins by conjugation with a photocleavable group is a powerful approach for reversibly blocking enzymatic activity. Here we describe the covalent modification of the bacterial SssI DNA methyltransferase (M.SssI) with the cysteine‐specific reagent 4,5‐dimethoxy‐2‐nitrobenzylbromide (DMNBB). M.SssI contains two cysteine residues; replacement of the active‐site Cys141 with Ser resulted in an approximately 100‐fold loss of enzymatic activity; this indicates an important role for this residue in catalysis. However, replacement of Cys368 with Ala did not affect methyltransferase activity. Treatment of the Cys368Ala mutant enzyme with DMNBB led to an almost complete loss of activity. Irradiation of the inactivated enzyme with near‐ultraviolet light (320–400 nm) restored 60 % of the catalytic activity. This indicates that caging by DMNBB can be used for the reversible inactivation of M.SssI.

Identification of two new promoters probably involved in the transcription of a ribosomal RNA gene of Escherichia coli

The DNA sequence in the region preceding the rrnB gene of Escherichia coli was determined up to the 1821st nucleotide upstream from the beginning of the sequence coding for mature 16 S rRNA. In vitro transcription experiments indicated the presence of two new promoters in this region, located more than 1 kb upstream from the known P1 and P2 promoters of rrnB. Previous electron microscopic studies demonstrated that these sites bind RNA-polymerase very strongly. In vitro transcription, starting at these sites reads through the entire region into the rrnB gene without termination. A similar uninterrupted transcription into rrnB in vivo can be demonstrated by S1-mapping, and by fusing the DNA containing the new promoters (but not P1 and P2) to the lacZ gene. Thus it seems likely that these promoters (P3 and P4) belong functionally to the rrnB gene and play some role in its regulation of expression.

Persistent downregulation of the pancarcinoma-associated epithelial cell adhesion molecule via active intranuclear methylation

The epithelial cell adhesion molecule (EpCAM) is expressed at high levels on the surface of most carcinoma cells. SiRNA silencing of EpCAM expression leads to reduced metastatic potential of tumor cells demonstrating its importance in oncogenesis and tumor progression. However, siRNA therapy requires either sequential delivery or integration into the host cell genome. Hence we set out to explore a more definite form to influence EpCAM gene expression. The mechanisms underlying the transcriptional activation of the EpCAM gene, both in normal epithelial tissue as well as in carcinogenesis, are poorly understood. We show that DNA methylation plays a crucial role in EpCAM expression, and moreover, active silencing of endogenous EpCAM via methylation of the EpCAM promoter results in a persistent downregulation of EpCAM expression. In a panel of carcinoma derived cell lines, bisulfite analyses showed a correlation between the methylation status of the EpCAM promoter and EpCAM expression. Treatment of EpCAM‐negative cell lines with a demethylating agent induced EpCAM expression, both on mRNA and protein level, and caused upregulation of EpCAM expression in an EpCAM‐positive cell line. After delivery of the DNA methyltransferase M.SssI into EpCAM‐positive ovarian carcinoma cells, methylation of the EpCAM promoter resulted in silencing of EpCAM expression. SiRNA‐mediated silencing remained for 4 days, after which EpCAM re‐expression increased in time, while M.SssI‐mediated downregulation of EpCAM maintained through successive cell divisions as the repression persisted for at least 17 days. This is the first study showing that active DNA methylation leads to sustained silencing of endogenous EpCAM expression.