Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA

We demonstrate that S1 nuclease converts supercoiled plasmid DNA to unit-length, linear dsDNA through the creation of a single, double-stranded break in a plasmid molecule. These double-stranded breaks occur not only in the origin of replication near inverted repeats but also at a wide variety of locations throughout the plasmid. S1 nuclease exhibits this activity under conditions typically employed for the nuclease's single-stranded nuclease activity. Thus, S1 nuclease digestion of plasmid DNA, unlike analogous digestion with DNaseI, effectively halts after the first double-stranded break. This property makes easier the construction of large domain insertion libraries in which the goal is to insert linear DNA at a variety of locations throughout a plasmid. We used this property to create a library in which a circularly permuted TEM1 β-lactamase gene was inserted throughout a plasmid containing the gene encoding Escherichia coli ribose binding protein. Gene fusions that encode allosteric switch proteins in which ribose modulates β-lactamase catalytic activity were isolated from this library using a combination of a genetic selection and a screen.     

Isolation and characteristics of clones complementary to mRNA of the murine cellular tumor antigen p53

43 cDNA clones specific for murine cellular tumor antigen p53 were isolated from a library constructed using 17S fraction of mRNA from the SV40 transformed murine fibroblasts (SVT2). These clones contain the whole coding region of p53 mRNA and the most of non-translated sequences. A plasmid containing 1.8 kb insert of p53 cDNA was constructed. The p53 specific insert in this plasmid was colinear with p53 mRNA, as revealed by S1 nuclease analysis. 5'-region of the p53 gene comprising non-translated and promoter areas was cloned from the mouse genomic library. A combined clone containing promoter and the whole region, corresponding to p53 mRNA has been constructed.     

Cloning in Escherichia coli of the gene specifying the DNA-entry nuclease of Bacillus subtilis

With the aim of cloning genes involved in transformation of Bacillus subtilis, a set of transformation-deficient mutants was isolated by means of insertional mutagenesis with plasmid pHV60 (Vosman et al., 1986). Analysis of these mutants showed that those mapping in the aroI region lacked the DNA-entry nuclease activity. Plasmid pHV60 derivatives, containing flanking chromosomal DNA fragments, were isolated from these mutants and were used to screen a library of B. subtilis chromosomal DNA in phage lambda EMBL4. In Escherichia coli lysates, prepared with the phages that hybridized to the pHV60-based probe, a prominent nuclease activity could be detected. The nuclease encoded by the phage DNA had the same Mr as the B. subtilis DNA-entry nuclease and its activity was strongly stimulated by Mn2+, which is also characteristic for the B. subtilis DNA-entry nuclease. From these results it was concluded that the gene specifying the B. subtilis DNA-entry nuclease had been cloned. It was shown that the nuclease activity was specified by a 700-bp EcoRI-PstI fragment.     

Non-specific DNAases from the rumen bacterium Prevotella bryantii

Extracellular non-specific nucleases were observed in some strains belonging to the ruminal species of the genus Prevotella, mostly P. brevis and P. bryantii. The nuclease from P. bryantii appeared to be extracellular; it mediates the degradation of the supercoiled plasmid DNA via an open circle intermediate. The cleavage is not site specific although a preference for certain cleavage sites does seem to exist. Our attempts to clone the wild-type P. bryantii B(1)4 nuclease in E. coli strain ER1992 that reports on the DNA damage sustained, were unsuccessful probably due to excessive intracellular nuclease activity that killed the cells bearing the gene for the nuclease. On the other hand, the nuclease from a related strain TCl-1, which has a less active enzyme of the same type, was successfully cloned.     

A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB.

It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1. A suppressor of this thermosensitive phenotype was isolated from a wild-type plasmid library by transforming a pif1 null strain to growth on glycerol at the non-permissive temperature. This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA. We therefore named this gene RIM1 for replication in mitochondria. The primary structure of the RIM1 protein is homologous to the single-stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis. The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T-antigen. Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB. Antibodies against this purified protein localize RIM1 to mitochondria. The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus.     

Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli.

The secretion of a Serratia marcescens nuclease was followed by fermentation with Escherichia coli. A plasmid, p403-SD2, carrying a 1.3-kilobase-pair insert with a 0.4-kilobase-pair region upstream of the nuclease gene caused a growth-phase-regulated expression of nuclease in E. coli in the same way as that seen in S. marcescens. Deletion of the regulatory gene generating plasmid p403-Rsa1 resulted in a constitutive expression of the nuclease. Anaerobiosis stimulated the expression from p403-SD2 in stationary growth phase by a factor of 10 compared with expression stimulated by cultivation in aerobic conditions; no such effect was found for plasmid p403-Rsa1. Different nutritional factors caused the expression level and the amount of extracellular nuclease to vary more when nuclease was expressed from plasmid p403-SD2 than when it was expressed from plasmid p403-Rsa1. A correlation between the regulatory gene and the extracellular secretion of nuclease is proposed.     

Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli

The secretion of a Serratia marcescens nuclease was followed by fermentation with Escherichia coli. A plasmid, p403-SD2, carrying a 1.3-kilobase-pair insert with a 0.4-kilobase-pair region upstream of the nuclease gene caused a growth-phase-regulated expression of nuclease in E. coli in the same way as that seen in S. marcescens. Deletion of the regulatory gene generating plasmid p403-Rsa1 resulted in a constitutive expression of the nuclease. Anaerobiosis stimulated the expression from p403-SD2 in stationary growth phase by a factor of 10 compared with expression stimulated by cultivation in aerobic conditions; no such effect was found for plasmid p403-Rsa1. Different nutritional factors caused the expression level and the amount of extracellular nuclease to vary more when nuclease was expressed from plasmid p403-SD2 than when it was expressed from plasmid p403-Rsa1. A correlation between the regulatory gene and the extracellular secretion of nuclease is proposed.     

Cloning and expression of the H chain V region of antibody OVB3 that reacts with human ovarian cancer

Hybridoma OVB3 produces an antibody (IgG2b kappa) that reacts with an Ag present on the surface of almost all human ovarian carcinomas. An EcoRI fragment of genomic DNA containing the rearranged H chain V region of monoclonal OVB3 was cloned from a lambda gtWES library and then sub-cloned into a pGEM4 vector. To show that the cloned DNA sequence did encode the V region of a functional antibody, the DNA fragment was inserted into plasmid pSV-VNP gamma SNase in place of VNp to produce pSV-VOVB3 gamma SNase. This plasmid was then transfected into variant OVB3 hybridoma cells, which no longer produced the H chain of antibody OVB3, and functional antibody was secreted by the recipient cells. The recombinant chimeric antibody bound to ovarian cancer cells and contained Staphylococcus aureus nuclease activity, proving that a functional V region gene had been cloned.     

Chromatin structure in a region of a yeast transposable element regulating adjacent gene expression.

The chromatin structure of a portion of yeast transposable elements known to be responsible for regulation of the expression of the adjacent HIS4 gene has been investigated, using the nuclease probe micrococcal nuclease. Yeast strains containing Ty917 or derivatives of this element that possess either a His-, weak His+, or strong His+ phenotype were examined. The chromatin at the Ty/HIS4 junction region was accessible to micrococcal nuclease. A partial nucleosome ladder was observed upon digestion with micrococcal nuclease indicating the presence of three phased nucleosomes located in Ty sequences upstream of the HIS4 gene. Phased nucleosomes could not be detected upstream of the HIS4 gene in wild-type cells. These data suggest that nucleosomal structure is not a major contributor to Ty917-regulated adjacent gene expression at HIS4.     

CRISPR/Cas9 system in Plasmodium falciparum using the centromere plasmid

The CRISPR/Cas9 nuclease system is a powerful method to genetically modify the human malarial parasite, Plasmodium falciparum. Currently, this method is carried out by co-transfection with two plasmids, one containing the Cas9 nuclease gene, and another encoding the sgRNA and the donor template DNA. However, the efficiency of modification is currently low owing to the low frequency of these plasmids in the parasites. To improve the CRISPR/Cas9 nuclease system for P. falciparum, we developed a novel method using the transgenic parasite, PfCAS9, which stably expresses the Cas9 nuclease using the centromere plasmid. To examine the efficiency of genetic modification using the PfCAS9 parasite, we performed site-directed mutagenesis of kelch13 gene, which is considered to be involved in artemisinin resistance. Our results demonstrated that the targeted mutation could be introduced with almost 100% efficiency when the transfected PfCAS9 parasites were treated with two drugs to maintain both the centromere plasmid containing the Cas9 nuclease and the plasmid having the sgRNA. Therefore, the PfCAS9 parasite is a useful parasite line for the genetic modification of P. falciparum.     

RNA1 is sufficient to mediate plasmid ColE1 incompatibility in vivo

The multicopy plasmid ColE1 specifies a small RNA designated RNA1 that has been implicated in copy number control and incompatibility. We have inserted a 148 base-pair ColE1 DNA fragment containing a promoter-less RNA1 gene into a plasmid vector downstream from the tryptophan promoter of Serratia marcesens. The ColE1 RNA1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5′-terminus of the wild-type RNA1 sequence. Deletions of these sequences by Bal3I nuclease in vitro and genetic selection for ColE1 incompatibility function in vivo permitted isolation of a plasmid expressing wild-type ColE1 RNA1 initiated properly from the S. marcesens trp promoter. These experiments demonstrate that RNA1 is sufficient to mediate ColE1 incompatibility in vivo. In addition, several plasmids were isolated that contain altered RNA1 genes. These alterations consist of additions or deletions of sequences at the 5′-terminus of RNA1. Analysis of the ability of these altered RNA1 molecules to express incompatibility in vivo suggests that the 5′-terminal region of RNA1 is crucial for its function.     

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the leu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by S1 nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the leu-5 mutation had been mapped before. We show that the leu-5 strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the leu-5 strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in leu-5 extracts, the leu-5 strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.     

The development of TnNuc and its use for the isolation of novel secretion signals in Lactococcus lactis

We have previously used Tn917 for the identification and characterization of regulated promoters from Lactococcus lactis [Israelsen et al., Appl. Environ. Microbiol. 61 (1995) 2540-2547]. We describe here the construction of a new Tn917-transposon derivative, termed TnNuc, which includes the Staphylococcus aureus nuclease gene (nuc) as a reporter for secretion. Transposition of TnNuc into the L. lactis chromosome allows the generation of fusions in-frame with the nuc gene. TnNuc includes also lacZ, a reporter used for identification of relevant clones from the library, i.e. clones with Lac+ phenotype result from transposition of TnNuc into a functional gene on the L. lactis chromosome. The presence of a functional signal sequence at the upstream flanking region of the left repeat of the transposed element results in the detection of nuclease activity using a sensitive plate assay. TnNuc was used for the identification of novel secretion signals from L. lactis. The sequences identified included known and unknown lactococcal-secreted proteins containing either a signal peptidase-I or -II recognition sequence. In one case, the gene identified codes for a transmembrane protein. The sequences identified were used to study functionality when located in a plasmid under the control of the pH and growth phase-dependent promoter P170 [Madsen et al., Mol. Microbiol. 32 (1999) 75-87]. In all cases, concurrent secretion of nuclease was observed during induction of P170 in a fermentor.     

Supercoil-dependent recognition of specific DNA sites by chromosomal protein HMG 2

The ability of the chromosomal high mobility group protein HMG 2 to recognize supercoil-dependent structures within the chicken adult beta-globin gene was investigated by examining its ability to protect such sites from digestion by S1 nuclease. Low molar ratios of HMG 2 were found to be sufficient for complete inhibition of S1 cleavage of a supercoiled plasmid containing the globin gene. Furthermore, HMG 2 protected an S1 cleavage site within the 5'-flanking region of the globin gene, in preference to a palindromic S1 site within the plasmid vector.