Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes.

Mitochondrial diseases are not uncommon, and may result from mutations in both nuclear and mitochondrial DNA (mtDNA). At present, only palliative therapies are available for these disorders, and interest in the development of efficient treatment protocols is high. Here, we demonstrate that in cells heteroplasmic for the T8993G mutation, which is a cause for the NARP and MILS syndromes, infection with an adenovirus, which encodes the mitochondrially targeted R.XmaI restriction endonuclease, leads to selective destruction of mutant mtDNA. This destruction proceeds in a time- and dose-dependent manner and results in cells with significantly increased rates of oxygen consumption and ATP production. The delivery of R.XmaI to mitochondria is accompanied by improvement in the ability to utilize galactose as the sole carbon source, which is a surrogate indicator of the proficiency of oxidative phosphorylation. Concurrently, the rate of lactic acid production by these cells, which is a marker of mitochondrial dysfunction, decreases. We further demonstrate that levels of phosphorylated P53 and gammaH2ax proteins, markers of nuclear DNA damage, do not change in response to infection with recombinant adenovirus indicating the absence of nuclear DNA damage and the relative safety of the technique. Finally, some advantages and limitations of the proposed approach are discussed.

Protection of human keratinocyte mtDNA by low-level nitric oxide.

This study was designed to evaluate the DNA damaging effects of nitric oxide and to determine whether the endogenous generation of nitric oxide at low levels in the cell exerts a protective effect against this damage. Damage to mitochondrial and nuclear DNA in normal human epidermal keratinocytes (NHEK) was assessed after treatment of these cells with varying concentrations of S-nitroso-N-acetylpenicillamine, which decomposes to release nitric oxide. The results showed that mitochondrial DNA was more vulnerable to nitric oxide-induced damage than was a similarly sized fragment of the beta-globin gene. To evaluate the effects on DNA damage by pretreatment of cells with low-levels of nitric oxide, NHEK cells were treated with the prodrug V-PYRRO/NO. This agent is metabolized inside these cells and releases small quantities of nitric oxide. The cells then were exposed to damaging amounts of nitric oxide produced by S-nitroso-N-acetylpenicillamine. The results of these studies showed that pretreatment of NHEK cells with V-PYRRO/NO attenuated the mtDNA damage and loss of cell viability produced by exposure to S-nitroso-N-acetylpenicillamine.

Peptide nucleic acid (PNA) binding-mediated induction of human gamma-globin gene expression.

Peptide nucleic acids (PNAs) can bind to homopurine/homopyrimidine sequences of double-stranded DNA targets in a sequence-specific manner and form [PNA]2/DNA triplexes with single-stranded DNA D-loop structures at the PNA binding sites. These D-loop structures have been found to have a capacity to initiate transcription in vitro. If this strategy can be used to induce transcription of endogenous genes, it may provide a novel approach for gene therapy of many human diseases. Human [beta] globin disorders such as sickle cell anemia and beta-thalassemia are very common genetic diseases that are caused by mutations in the beta-globin gene. When gamma-globin genes are highly expressed in sickle cell patients, the presence of high levels of fetal hemoglobin (HbF, alpha2gamma2) can compensate for the defective beta-globin gene product and such patients have much improved symptoms or are free of disease. However, the gamma-globin genes are developmentally regulated and normally expressed at very low levels (>1%) in adult blood cells. We have investigated the possibility of inducing gamma-globin gene expression with PNAs. Using PNAs designed to bind to the 5' flanking region of the gamma-globin gene, induction of expression of a reporter gene construct was demonstrated both in vitro and in vivo. More importantly, PNA-mediated induction of endogenous gamma-globin gene expression was also demonstrated in K562 human erythroleukemia cells. This result suggests that induction of gamma-globin gene expression with PNAs might provide a new approach for the treatment of sickle cell disease. PNA-induced gene expression strategy also may have implications in gene therapy of other diseases such as genetic diseases, cancer and infectious diseases.

A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome.

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211-213]. A family of related plasmids containing different antibiotic-resistance markers (CmR or GmR or KmR) and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.

New mini-Tn5 derivatives for insertion mutagenesis and genetic engineering in gram-negative bacteria.

Five mini-Tn5 derivatives encoding resistance to Km, Cm, Gm, Tc, and Sm, coupled with the polylinker of the pBluescriptII plasmid, were constructed. These derivatives are carried by an ampicillin-resistant plasmid that has a conditional origin of replication from plasmid R6K and origin of conjugal transfer from the broad host range plasmid RP4. The new vectors are smaller than those previously described and possess numerous unique restriction sites inside the minitransposons for gene cloning in addition to SfiI and NotI sites found in their predecessors.

Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of gram-negative bacteria.

Four mini-Tn10 derivatives bearing the nptII, cat, aacC1 and tet genes along with MCS of the pBluescriptII plasmid were constructed. These derivatives are embedded into the gamma-ori R6K-based suicide plasmid pBSL177, which can be mobilized into a broad range of hosts by the RP4 plasmid. The relatively small size and the presence of the mutant ATS Tn10 transposase gene, fused to Ptac promoter, as well as the versatile MCS of the pBluescriptII plasmid make these vectors a reliable tool for insertion mutagenesis and chromosomal insertion of the cloned DNA fragments.

Improved antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis.

Several antibiotic-resistance gene cassettes and omega elements for Escherichia coli vector construction include the aacC1, aadA+, bla, cat, nptII and tet gene cassettes, and also the omega-Gm, omega-Sm, omega-Ap, omega-Cm, omega-Km and omega-Tc elements. Both cassettes and elements are flanked by pBluescriptII plasmid multiple cloning sites (MCS) duplicated in inverted (symmetric MCS) or direct (tandem MCS) orientation. Genes that were modified in order to remove sites for the most common restriction endonucleases from their coding regions (except aacC1 and aadA+) were used for cassette and omega-element construction.

[Cycle dependent expression of the gene coding for B2mRNA]. / Tsikolzavisimaia ékspressiia gena, kodiruiushchego B2mRNK.

The relative contents of individual tissue specific RNA B2mRNAx was studied in the population of nuclear and poly(A)+ cytoplasmic RNA from the livers of intact, falsely operated and having suffered the partial liver resection rats. Dot-hybridization technique was used to study this transcript containing the transcribed copy of the B2 repetitive genetic element of rats. The expression of the gene coding for B2mRNAx takes place at the lower level in the liver induced to proliferation as compared with the one in the liver of intact animals. It is changed reproducibly in antiphase with the c-fos RNA and with major inclinations at the moments of cellular phases switch.