Sequence specificity of alternating hydroyprolyl/phosphono peptide nucleic acids against zebrafish embryo mRNAs.

Morpholino phosphorodiamidate (MO) DNA mimics display excellent water solubility and hybridization properties toward DNA and RNA, and have been utilized in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. Peptide nucleic acids (PNAs) exhibit excellent mismatch discrimination, nuclease resistance, and protease resistance, but low solubility. Negatively charged DNA mimics composed of alternating residues of trans-4-hydroxy-L-proline peptide nucleic acid monomers and phosphono peptide nucleic acid monomers (HypNA-pPNA) combine all of the positive features of both MOs and PNAs. Thus, we evaluated PNA oligomers and HypNA-pPNA oligomers as an alternative to MOs for oligonucleotide inhibition of gene expression in zebrafish embryos. We observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail and uroD, with greater mismatch stringency. Furthermore, we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. These observations validate HypNA-pPNAs as an alternative to MO oligomers for reverse genetic studies. The stronger hybridization and greater specificity of HypNA-pPNAs enable knockdown of mRNAs unaffected by MO oligomers.

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression.

The design of potent systems for the delivery of charged and noncharged molecules that target genes of interest remains a challenge. We describe a novel technology that combines a new generation of peptide nucleic acids (PNAs), or HypNA-pPNAs, with a new noncovalent peptide-based delivery system, Pep-2, which promotes efficient delivery of PNAs into several cell lines. We have validated the potential of this technology by showing that Pep2-mediated delivery of an antisense HypNA-pPNA chimera directed specifically against cyclin B1 induces rapid and robust downregulation of its protein levels and efficiently blocks cell cycle progression of several cell lines, as well as proliferation of cells derived from a breast cancer. Pep-2-based delivery system was shown to be 100-fold more efficient in delivering HypNA-pPNAs than classical cationic lipid-based methods. Whereas Pep-2 is essential for improving the bioavailability of PNAs and HypNA-pPNAs, the latter contribute significantly to the efficiency and specificity of the biological response. We have found that Pep-2/HypNA-pPNA strategy promotes potent antisense effects, which are approximately 25-fold greater than with classical antisense oligonucleotide directed specifically against the same cyclin B1 target. Taken together, these data demonstrate that peptide-mediated delivery of HypNA-pPNAs constitutes a very promising technology for therapeutic applications.

Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach.

An effective procedure for specific determination of the cap structure at the 5'-terminus of mRNA and for isolation of the corresponding full-length cDNA has been developed. The procedure involves covalent attachment of an oligonucleotide template extender to the 5'-cap structure of mRNA followed by RT-PCR using M-MLV SuperScript II reverse transcriptase. In the course of reverse transcription, the enzyme 'jumps over' the cap structure and includes the sequence complementary to the oligonucleotide template extender into the 3'-end of the first cDNA strand. The cap-jumping method was successfully tested using some mammalian cellular mRNAs, genomic RNAs of tobacco mosaic virus (TMV) U1 and the recently isolated crucifer-infecting tobamovirus. Moreover, cDNA products corresponding to the genomic tobamovirus RNA were obtained from total RNA extracted from tobacco plants infected by crucifer-infecting tobamovirus or tobacco mosaic virus. Using the cap-jumping method, we have shown for the first time that genomic crucifer-infecting tobamovirus (crTMV) RNA contains a 5'-cap structure. This improved method can be recommended for the construction of full-length and 5'-end enriched cDNA libraries, identification of capped RNAs and determination of their 5'-terminal sequences.

A single amino acid substitution beyond the C2H2-zinc finger in Ros derepresses virulence and T-DNA genes in Agrobacterium tumefaciens.

Ros is a chromosomally-encoded repressor containing a novel C2H2 zinc finger in Agrobacterium tumefaciens. Ros regulates the expression of six virulence genes and an oncogene on the Ti plasmid. Constitutive expression of these genes occurs in the spontaneous mutant 4011R derived from the octopine strain Ach-5, resulting in T-DNA processing in the absence of induction, and in the biosynthesis of cytokinin. Interestingly, the mutation in 4011R is an Arg to Cys conversion at amino acid residue 125 near the C-terminus well outside the zinc finger of Ros. Yet, Ros bearing this mutation is unable to bind to the Ros-box and is unable to complement other ros mutants.

Agrobacterium transcriptional regulator Ros is a prokaryotic zinc finger protein that regulates the plant oncogene ipt.

Virulence genes of Agrobacterium tumefaciens are under the control of positive and negative transcriptional regulators. We found that the transcriptional regulator Ros controls expression of the plant oncogene ipt, which encodes isopentenyl transferase, in A. tumefaciens. This enzyme is involved in biosynthesis of the plant growth hormone cytokinin in the host plant. An ipt promoter::cat reporter gene fusion showed a 10-fold increase in ipt promoter activity in A. tumefaciens ros mutant strains when compared with wild type. Also, increased levels (10- to 20-fold) of isopentenyl adenosine, the product of the reaction catalyzed by isopentenyl transferase, were detected in ros mutant strains. In vitro studies using purified Ros showed it binds directly to the ipt promoter. Analysis of the deduced Ros amino acid sequence identified a novel type of C2H2 zinc finger. In Ros the peptide loop spacing of the zinc finger is 9 amino acids as opposed to the invariant 12 amino acids in the classical C2H2 motif. Site-directed mutagenesis of Cys-82 and His-92 in this motif showed that these residues are essential for Zn2+ and DNA binding activities of Ros. The existence of such a regulator in Agrobacterium may be due to horizontal interkingdom retrotransfer of the ros gene from plant to bacteria.

Modular structure of the Rhizobium meliloti DctB protein.

To investigate the modular structure of the Rhizobium meliloti dicarboxylic acid sensor protein, DctB, three truncated DctB proteins (DctB4, DctB5 and DctB4G) were constructed, overproduced in Escherichia coli and purified. The DctB4G protein was composed of 446 amino acids of the DctB C-terminus and displayed strong autophosphorylation activity in vitro. This activity was sustained when a further 120 amino acids at the N-terminus of the polypeptide were deleted (DctB5). This protein which has an intact transmitter domain exhibits specific but inefficient phospho-transfer capabilities. Removal of 58 amino acids from the DctB4G C-terminus which included blocks F and G2 of the transmitter domain, rendered the resultant protein (DctB4) incompetent in autophosphorylation. Phosphorylation activity was restored to DctB4 through intramolecular complementation with DctB. Therefore, it would appear that the R. meliloti DctB protein is active as a dimer (or higher order oligomer). Furthermore, the intramolecular complementation experiments indicate that the amino acids 171-291, a predicted periplasmic stretch, play an important role in the dimerization process.

Regulation of dct genes in the Rhizobium meliloti-alfalfa interaction.

In order to support symbiotic N2 fixation, Rhizobium meliloti must be able to utilize the C4-dicarboxylic acids provided by its legume host, alfalfa. These compounds are taken up via a single transport protein, DctA. Transcription from the dctA promoter is positively regulated by the DctB/DctD two-component system. In response to dicarboxylic acids, the transmembrane sensor DctB, activates the transcriptional activator DctD, which together with σ(54) holoenzyme initiates transcription of dctA. In bacteroids an alternative mode of activation has also been implicated in dctA expression and the exact nature of this system remains to be elucidated. Evidence also suggests that expression of the dctA promoter can be influenced negatively by other DNA regulatory proteins.

Cloning of the second adenylate cyclase gene (cya2) from Rhizobium meliloti F34: sequence similarity to eukaryotic cyclases.

A second adenylate cyclase (cya2) gene was isolated from a Rhizobium meliloti F34 gene bank. Complemented E. coli delta cya mutants were capable of utilizing a number of, but not all, carbon sources known to be regulated by cAMP. DNA hybridization studies showed cya2 to be unique to R. meliloti strains. The cya2 nucleotide sequence was determined and found to encode a protein of 363 amino acids. Residues were identified within the C-terminal domain which are conserved in both eukaryotic adenylate and guanylate cyclases, including a putative ATP binding site. Similar residues were also found in the prokaryotic R. meliloti Cya1 protein. A R. meliloti cya1/cya2 double mutant was constructed and characterized; however, cAMP production was still observed in this strain indicating the presence of a third cya gene.

Plasma and tissue iron changes in the rat after acute administration of ethionine.

1. Ethionine administered acutely to the adult female rat markedly elevates and then lowers plasma iron concentration over several days. Liver iron undergoes a reverse cycle. 2. Ethionine does not cause changes in the blood parameters, including total plasma iron-binding capacity and plasma iron clearance. Erythrocytes of rats injected with ethionine show altered responses to hypertonicity. 3. Increased reticulo-endothelial activity of the spleen, indicated by increased uptake of 59Fe-labelled erythrocytes by liver and spleen, apparently contributes to plasma iron elevation. Also the liver releases iron which further raises plasma iron.

Accumulation of iron in the rabbit erythroid cell as affected by ouabain, sodium and potassium ions, and temperature.

Reticulocytosis was induced in rabbits with phenylhydrazine. The accumulation of a small part of (59)Fe in blood cells of these animals was inhibited by ouabain and related to changes in extracellular sodium and potassium concentrations. Sodium increases movement from the cell surface into the cell, whereas potassium and ouabain decrease this movement. (59)Fe movement was found to be temperature-dependent. Thus, the Na-K ATPase system appears to be important in the movement of iron from the cell membrane (stroma) to the cell interior, but influences only a small part of the total iron transport.