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At least 1,153 records · Page 64Linked to original sources
PubMed2003

Expression analysis and mitochondrial targeting properties of the chicken manganese-containing superoxide dismutase.

Manganese-containing superoxide dismutase (MnSOD) is a major detoxifying enzyme that functions in cellular oxygen metabolism by converting O(2)(-) to H(2)O(2). A cDNA encoding the chicken MnSOD (cMnSOD) has been isolated from a chicken embryo fibroblast (CEF) cell cDNA library. The cloned cMnSOD is 1102 bp in length with an open reading frame (ORF) of 224 amino acids that includes a 26-amino-acid 5'-proximal mitochondrial targeting sequence (MTS). The mature 198-amino-acid region of the cMnSOD is highly conserved among various mammalian species. Two cMnSOD mRNA species (1.2 and 1.0 kb) were expressed in most of the tissues and organs analyzed, with the highest expression levels found in brain, kidney, and heart tissues. Compared to earlier stages of development, expression of cMnSOD was highest in day 13 embryonic heart tissue, and was maintained until post-hatch. Exogenously introduced cMnSOD-GFP fusion constructs (which included the MTS) clearly accumulated in the mitochondria of chicken cells, as expected. Surprisingly, the cMnSOD MTS signal, which displays little similarity to mammalian MTS sequences, enabled cMnSOD-GFP fusion proteins to target mitochondria not only from different cell types (fibroblastic and epithelial), but from a number of mammalian species (human, mouse, and pig). This suggests that specific amino acid motifs within the MTS domain may be more important than the overall sequence similarities for mitochondrial targeting.

Amino Acid Sequence↗
PubMed2002

The gene encoding Arabidopsis thaliana mitochondrial ribosomal protein S13 is a recent duplication of the gene encoding plastid S13.

A gene encoding mitochondrial S13 is generally present in the mitochondrial genome of higher plants, but is lacking from the Arabidopsis thaliana mitochondrial genome. Previous research has failed to identify a nuclear gene capable of encoding a mitochondrial S13 protein or the protein itself. Doubts have even been raised as to whether a mitochondrial S13 exists in Arabidopsis. Here, we show that the nuclear gene encoding the plastid S13 has been partially duplicated in A. thaliana, such that the copy has lost the exon encoding the plastid transit peptide and acquired a sequence capable of encoding a mitochondrial targeting sequence. The two S13 sequences were fused to green fluorescent protein and shown to be targeted to plastids and mitochondria respectively.

Amino Acid Sequence↗
PubMed1997

Mobilization of a Minos transposon in Drosophila melanogaster chromosomes and chromatid repair by heteroduplex formation.

Transposase-mediated mobilization of the element Minos has been studied in the Drosophila melanogaster genome. Excision and transposition of a nonautonomous Minos transposon in the presence of a Minos transposase gene was detected with a dominant eye color marker carried by the transposon. Frequencies of excision in somatic tissues and in the germ line were higher in flies heterozygous for the transposon than in homozygotes or hemizygotes. Transposition of a X chromosome-linked insertion of Minos into new autosomal sites occurred in 1-12% of males expressing transposase, suggesting that this system is usable for gene tagging and enhancer trapping in Drosophila. Sequence analysis of PCR-amplified donor sites after excision showed precise restoration of the original target sequence in approximately 75% of events in heterozygotes and the presence of footprints or partially deleted elements in the remaining events. Most footprints consisted of the four terminal bases of the transposon, flanked by the TA target duplication. Sequencing of a chromosomal donor site that was directly cloned after excision showed a characteristic two-base mismatch heteroduplex in the center of the 6-bp footprint. Circular extrachromosomal forms of the transposon, presumably representing excised Minos elements, could be detected only in the presence of transposase. A model for chromatid repair after Minos excision is discussed in which staggered cuts are first produced at the ends of the inverted repeats, the broken chromatid ends are joined, and the resulting heteroduplex is subsequently repaired. The model also suggests a simple mechanism for the production of the target site duplication and for regeneration of the transposon ends during reintegration.

Animals↗
PubMed1999

Effects of homology length and donor vector arrangement on the efficiency of double-strand break-mediated recombination in human cells.

We use an Epstein-Barr virus (EBV) plasmid model chromosome system to study how different donor plasmid constructs affect recombination stimulated by an I-SceI-induced double-strand break in the target sequence in human cells. The entire 3.5 kb lacZ gene was efficiently recombined into a target EBV vector lacking lacZ sequences, but having limited homology to the donor plasmid. A donor plasmid with lacZ flanked by sequence homologous to the target consistently generated gene conversion events and was more effective than a donor carrying lacZ outside the same sequence homology. Reducing the length of homology between the target and donor from 5.5 kb to 1 kb caused only a 3-fold drop in recombination frequency, contrasting with the exponential dependence on homology length seen when no DSB is present in the target. These results document a DSB-induced 175-fold increase in recombination of a heterologous gene into a target, requiring only limited flanking homology.

Cells, Cultured↗
PubMed2000

The human PEX3 gene encoding a peroxisomal assembly protein: genomic organization, positional mapping, and mutation analysis in candidate phenotypes.

In yeasts, the peroxin Pex3p was identified as a peroxisomal integral membrane protein that presumably plays a role in the early steps of peroxisomal assembly. In humans, defects of peroxins cause peroxisomal biogenesis disorders such as Zellweger syndrome. We previously reported data on the human PEX3 cDNA and its protein, which in addition to the peroxisomal targeting sequence contains a putative endoplasmic reticulum targeting signal. Here we report the genomic organization, sequencing of the putative promoter region, chromosomal localization, and physical mapping of the human PEX3 gene. The gene is composed of 12 exons and 11 introns spanning a region of approximately 40 kb. The highly conserved putative promoter region is very GC rich, lacks typical TATA and CCAAT boxes, and contains potential Sp1, AP1, and AP2 binding sites. The gene was localized to chromosome 6q23-24 and D6S279 was identified to be the closest positional marker. As yeast mutants deficient in PEX3 have been shown to lack peroxisomes as well as any peroxisomal remnant structures, human PEX3 is a candidate gene for peroxisomal assembly disorders. Mutation analysis of the human PEX3 gene was therefore performed in fibroblasts from patients suffering from peroxisome biogenesis disorders. Complementation groups 1, 4, 7, 8, and 9 according to the numbering system of Kennedy Krieger Institute were analyzed but no difference to the wild-type sequence was detected. PEX3 mutations were therefore excluded as the molecular basis of the peroxisomal defect in these complementation groups.

ATP-Binding Cassette Transporters↗
PubMed2001

Spatially localized generation of nucleotide sequence-specific DNA damage.

Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have been used in conjunction with laser-induced two-photon excitation (TPE) to damage a specific DNA target sequence. To demonstrate that TPE can initiate photochemistry resulting in psoralen-DNA photoadducts, target DNA sequences were incubated with psoTFOs to form triple-helical complexes and then irradiated in liquid solution with pulsed 765-nm laser light, which is half the quantum energy required for conventional one-photon excitation, as used in psoralen + UV A radiation (320-400 nm) therapy. Target DNA acquired strand-specific psoralen monoadducts in a light dose-dependent fashion. To localize DNA damage in a model tissue-like medium, a DNA-psoTFO mixture was prepared in a polyacrylamide gel and then irradiated with a converging laser beam targeting the rear of the gel. The highest number of photoadducts formed at the rear while relatively sparing DNA at the front of the gel, demonstrating spatial localization of sequence-specific DNA damage by TPE. To assess whether TPE treatment could be extended to cells without significant toxicity, cultured monolayers of normal human dermal fibroblasts were incubated with tritium-labeled psoralen without TFO to maximize detectable damage and irradiated by TPE. DNA from irradiated cells treated with psoralen exhibited a 4- to 7-fold increase in tritium activity relative to untreated controls. Functional survival assays indicated that the psoralen-TPE treatment was not toxic to cells. These results demonstrate that DNA damage can be simultaneously manipulated at the nucleotide level and in three dimensions. This approach for targeting photochemical DNA damage may have photochemotherapeutic applications in skin and other optically accessible tissues.

Base Sequence↗
PubMed1999

The molecular basis of malonyl-CoA decarboxylase deficiency.

We characterized a 2.1-kb human cDNA with a 1362-bp (454-amino acid) open reading frame showing 70.3% amino acid identity to goose malonyl-CoA decarboxylase (MCD). We have identified two different homozygous mutations in human MCD (hMCD) by using RT-PCR analysis of fibroblast RNA from two previously reported consanguineous Scottish patients with MCD deficiency. The first mutation is a 442C-->G transversion resulting in a premature stop codon (S148X) in the N-terminal half of the protein. The second is a 13-bp insertion in the mature RNA, causing a frameshift with predicted protein truncation. This insertion is the result of an intronic mutation generating a novel splice acceptor sequence (IVS4-14A-->G). Both mutations were found to segregate appropriately within the families and were not found in 100 normal unrelated individuals. These mutations would be predicted to cause MCD deficiency, thus confirming this transcript as the hMCD ortholog. The peptide sequence of hMCD revealed a C-terminal peroxisomal targeting sequence (-SKL). This targeting signal appears to be functional in vivo, since the distribution of MCD enzymatic activity in rat liver homogenates-as measured by means of subcellular fractionation-strongly suggests that MCD is localized to peroxisomes in addition to the mitochondrial localization reported elsewhere. These data strongly support this cDNA as encoding human MCD, an important regulator of fatty acid metabolism.

Adolescent↗
PubMed2003

Widespread occurrence of Mycobacterium tuberculosis DNA from 18th-19th century Hungarians.

A large number (265) of burials from 1731-1838 were discovered in sealed crypts of the Dominican Church, Vác, Hungary in 1994. Many bodies were naturally mummified, so that both soft tissues and bones were available. Contemporary archives enabled the determination of age at death, and the identification of family groups. In some cases, symptoms before death were described and, occasionally, occupation. Initial radiological examination of a small number of individuals had indicated calcified lung lesions and demonstrable acid-fast bacteria suggestive of tuberculosis infection. Tuberculosis was endemic in 18th-19th century Europe, so human remains should contain detectable Mycobacterium tuberculosis complex (MTB) DNA, enabling comparisons with modern isolates. Therefore, a comprehensive examination of 168 individuals for the presence of MTB DNA was undertaken. Specific DNA amplification methods for MTB showed that 55% of individuals were positive and that the incidence varied according to age at death and sampling site in the body. Radiographs were obtained from 27 individuals and revealed an association between gross pathology and the presence of MTB DNA. There was an inverse relationship between PCR positivity and MTB target sequence size. In some cases, the preservation of MTB DNA was excellent, and several target gene sequences could be detected from the same sample. This information, combined with MTB DNA sequencing data and molecular typing techniques, will enable us to study the past epidemiology of TB infection, and extends the timeframe for studying changes in molecular fingerprints.

Adolescent↗
PubMed2005

Examination of mitochondrial protein targeting of haem synthetic enzymes: in vivo identification of three functional haem-responsive motifs in 5-aminolaevulinate synthase.

The initial and the terminal three enzymes of the mammalian haem biosynthetic pathway are nuclear encoded, cytoplasmically synthesized and post-translationally translocated into the mitochondrion. The first enzyme, ALAS (5-aminolaevulinate synthase), occurs as an isoenzyme encoded on different chromosomes and is synthesized either as a housekeeping protein (ALAS-1) in all non-erythroid cell types, or only in differentiating erythroid precursor cells (ALAS-2). Both ALAS proteins possess mitochondrial targeting sequences that have putative haem-binding motifs. In the present study, evidence is presented demonstrating that two haem-binding motifs in the leader sequence, as well as one present in the N-terminus of the mature ALAS-1 function in vivo in the haem-regulated translocation of ALAS-1. Coproporphyrinogen oxidase, the antepenultimate pathway enzyme, possesses a leader sequence that is approx. 120 residues long. In contrast with an earlier report suggesting that only 30 residues were required for translocation of the coproporphyrinogen oxidase, we report that the complete leader is necessary for translocation and that this process is not haem-sensitive in vivo. PPO (protoporphyrinogen oxidase) lacks a typical mitochondrial targeting leader sequence and was found to be effectively targeted by just 17 N-terminal residues. Bacillus subtilis PPO, which is very similar to human PPO at its N-terminal end, is not targeted to the mitochondrion when expressed in mammalian cells, demonstrating that the translocation is highly specific with regard to both the length and spacing of charged residues in this targeting region. Ferrochelatase, the terminal enzyme, possesses a typical N-terminal leader sequence and no evidence of a role for the C-terminus was found in mitochondrial targeting.

5-Aminolevulinate Synthetase↗
PubMed1995

Gene-targeted inhibition of transactivation of human immunodeficiency virus type-1 (HIV-1)-LTR by antisense oligonucleotides.

We have used an in vitro approach to study the efficiency of antisense oligonucleotides in inhibiting LTR-(HIV-1)-directed CAT expression catalyzed by tat protein, the functional protein of the transactivator gene. We selected the target sequence localized near the 5' end of the tat mRNA. The following conclusions can be drawn from the data presented here: a) Antisense oligonucleotides modified by conjugation of cholesterol at the 3' end have a severalfold higher inhibitory response, b) inhibitory response is dependent on the mode of introducing oligonucleotides, and c) the inhibition by antisense oligonucleotides is sequence specific and directed towards the targeted region. This approach could be useful for targeting functional regions of regulatory gene products and designing gene-targeted inhibitors of virus replication.

Animals↗
PubMed2006

Inhibition of infectious human immunodeficiency virus type 1 virions via lentiviral vector encoded short antisense RNAs.

During the life cycles of most retroviruses and lentiviruses, dimerization and packaging of two copies of viral genomic RNA is required for the subsequent conversion of RNA into double stranded DNA by reverse transcriptase. For human immunodeficiency virus type 1 (HIV-1), dimerization is mediated by interactions of the stem-loop structures in the dimerization-packaging, or psi (Psi) domain. We have tethered anti-HIV gag ribozymes and small antisense RNAs to the HIV Psi domain in an HIV-1 lentiviral vector to facilitate copackaging of these replication inhibitors with HIV genomic RNAs during HIV infectious challenge. In order to maximize the base pairing of the ribozymes or antisense segments to the HIV-1 genomic target, sequences in HIV-1 were identified that are highly accessible to antisense pairing. Ribozymes or antisense RNAs designed to target these sequences were inserted in the lentiviral vector at the same relative distance to the Psi element as the HIV-1 target sites. Packaged vectors were transduced into CEM cells followed by challenges with HIV-1. Only the constructs that harbored short antisense segments complementary to HIV-1 gag produced replication incompetent HIV-1. These results demonstrate that a short stretch of antisense pairing downstream of the dimerization domain in an HIV-based vector can drive dimerization and provide a powerful approach for inhibition of HIV-1.

Base Sequence↗
PubMed2004

Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs.

Persistent infection with hepatitis C virus (HCV) is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. It has recently been shown that HCV RNA replication is susceptible to small interfering RNAs (siRNAs), but the antiviral activity of siRNAs depends very much on their complementarity to the target sequence. Thus, the high degree of sequence diversity between different HCV genotypes and the rapid evolution of new quasispecies is a major problem in the development of siRNA-based gene therapies. For this study, we developed two alternative strategies to overcome these obstacles. In one approach, we used endoribonuclease-prepared siRNAs (esiRNAs) to simultaneously target multiple sites of the viral genome. We show that esiRNAs directed against various regions of the HCV coding sequence as well as the 5' nontranslated region (5' NTR) efficiently block the replication of subgenomic and genomic HCV replicons. In an alternative approach, we generated pseudotyped retroviruses encoding short hairpin RNAs (shRNAs). A total of 12 shRNAs, most of them targeting highly conserved sequence motifs within the 5' NTR or the early core coding region, were analyzed for their antiviral activities. After the transduction of Huh-7 cells containing a subgenomic HCV replicon, we found that all shRNAs targeting sequences in domain IV or nearby coding sequences blocked viral replication. In contrast, only one of seven shRNAs targeting sequences in domain II or III had a similar degree of antiviral activity, indicating that large sections of the NTRs are resistant to RNA interference. Moreover, we show that naive Huh-7 cells that stably expressed certain 5' NTR-specific shRNAs were largely resistant to a challenge with HCV replicons. These results demonstrate that the retroviral transduction of HCV-specific shRNAs provides a new possibility for antiviral intervention.

Antiviral Agents↗
PubMed1998

Isoforms of hepatocyte nuclear factor-6 differ in DNA-binding properties, contain a bifunctional homeodomain, and define the new ONECUT class of homeodomain proteins.

Hepatocyte nuclear factor-6 (HNF-6) contains a single cut domain and a homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. We describe here two isoforms of HNF-6 which differ by the linker that separates these domains. Both isoforms stimulated transcription. The affinity of HNF-6alpha and HNF-6beta for DNA differed, depending on the target sequence. Binding of HNF-6 to DNA involved the cut domain and the homeodomain, but the latter was not required for binding to a subset of sites. Mutations of the F48M50 dyad that did not affect DNA binding reduced the transcriptional stimulation of constructs that do not require the homeodomain for DNA binding, but did not affect the stimulation of constructs that do require the homeodomain. Comparative trees of mammalian, Drosophila, and Caenorhabditis elegans proteins showed that HNF-6 defines a new class, which we call ONECUT, of homeodomain proteins. C. elegans proteins of this class bound to HNF-6 DNA targets. Thus, depending on their sequence, these targets determine for HNF-6 at least two modes of DNA binding, which hinge on the homeodomain and on the linker that separates it from the cut domain, and two modes of transcriptional stimulation, which hinge on the homeodomain.

Amino Acid Sequence↗
PubMed2001

A new technique for cyclic in situ amplification and a case report about amplification of a single copy gene sequence in human metaphase chromosomes through PCR-PRINS.

Since the introduction of PRimed IN Situ labeling (PRINS) as a rapid and extremely sensitive alternative method to conventional fluorescence in situ hybridization (FISH), its application in clinical cytogenetics has been limited to the detection of highly repeated sequences, such as centromeric and telomeric regions. In the original PRINS method, unlabeled oligonucleotide probes are annealed to their repeated complementary target sequences in fixed human metaphase chromosomes on a slide. The probes serve as primers for subsequent in situ chain elongation with Taq DNA polymerase and labeled nucleotides. In contrast to conventional PCR, cyclic in situ amplification of the chromosomal target DNA with paired primers remained both difficult and strictly limited to highly repeated sequences, since the maintenance of constant reaction conditions on the slide during temperature and pressure shifts presents a major problem. We developed a new system for in situ PCR that allows the amplification of target sequences analogous to PCR in the test tube. We applied this method successfully for the detection of highly repeated sequences, for the detection of low copy repeats, and in one case, for the detection of a single-copy DNA sequence. The significance of this development for further in situ PCR applications will be discussed.

Apolipoproteins A↗
PubMed1990

Mitochondrial translocation and processing of the precursor to the alpha-subunit of rat liver succinyl-CoA synthetase.

Succinyl-CoA synthetase functions in the mitochondrial matrix as an alpha beta-dimer. Its constitutive subunits are thus expected to be encoded in the nucleus and synthesized in the cytoplasm as precursors containing signal sequences for mitochondrial translocation. We have previously reported the isolation and sequence of a rat liver cDNA clone (lambda SCS19) that apparently encodes the cytoplasmic precursor to the alpha-subunit. Here we report the preparation of mRNA transcripts of this cDNA insert and their in vitro translation to produce labeled protein that can be translocated across the membranes of subsequently added rat liver mitochondria. Translocation is accompanied by proteolytic processing to convert the 34.5-kilodalton precursor to the 32-kilodalton mature form of the subunit. The N-terminal sequence of the mature alpha-subunit from the GTP-specific isozyme has been determined by sequential Edman degradation and compared with the amino acid sequence deduced from the cDNA. This confirms that the cloned sequence encodes the GTP-specific alpha-subunit, and establishes that the point of cleavage is between histidyl and glycyl residues and that the signal sequence consists of 27 residues. The signal sequence shares characteristics of other mitochondrial targeting sequences that have been elucidated (largely of yeast mitochondrial precursors), including the potential to form an amphiphilic helix. Import is dependent upon the presence of ATP and is inhibited by compounds that diminish mitochondrial membrane potential. Translocation of the precursor is effective for precursor produced by the reticulocyte translation system, but is not seen for the product that is translated by a wheat germ extract, indicating that the latter may lack a factor or component that is necessary for the targeting and import process.

Amino Acid Sequence↗
PubMed2006

Targeted cDNA differential display (TcDD).

Targeted cDNA differential display (TcDD) was developed to study expression of a different selected gene families especially those at low copy numbers per cell. This method is an adaptation of our previously described targeted genomic differential display method (TGDD). In TcDD, the expression of genes containing target sequences such as CAG repeating sequences or genes encoding for zinc-finger binding proteins were followed in an experimental rat model with salt-induced hypertension. DNA sequencing experiments demonstrated that the effectiveness of targeting was greater than 99%.

Animals↗
PubMed2001

A single gene produces mitochondrial, cytoplasmic, and peroxisomal NADP-dependent isocitrate dehydrogenase in Aspergillus nidulans.

NADP-dependent isocitrate dehydrogenase enzymes catalyze the decarboxylation of isocitrate to 2-oxoglutarate accompanied by the production of NADPH. In mammals two different genes encode mitochondrial and cytoplasmic/peroxisomal located enzymes, whereas in Saccharomyces cerevisiae three separate genes specify compartment specific enzymes. We have identified a single gene, idpA, in the filamentous fungus Aspergillus nidulans that specifies a protein with a high degree of identity to mammalian and S. cerevisiae enzymes. Northern blot analysis and reverse transcription-polymerase chain reaction revealed the presence of two idpA transcripts and two transcription start points were identified by sequencing cDNA clones and by 5'-rapid amplification of cDNA ends. The shorter transcript was found to be inducible by acetate and by fatty acids while the longer transcript was present in higher amounts during growth in glucose containing media. The longer transcript is predicted to encode a polypeptide containing an N-terminal mitochondrial targeting sequence as well as a C-terminal tripeptide (ARL) as a potential peroxisomal targeting signal. The shorter transcript is predicted to encode a polypeptide lacking the mitochondrial targeting signal but retaining the C-terminal sequence. Immunoblotting using antibody raised against S. cerevisiae Idp1p detected two polypeptides consistent with these predictions. The functions of the predicted targeting sequences were confirmed by microscopic analysis of transformants containing fluorescent protein fusion constructs. Using anti-Idp1p antibodies, protein localization to mitochondria and peroxisomes was observed during growth on glucose whereas cytoplasmic and peroxisomal localization was found upon acetate or fatty acid induction. Therefore, we have established that by the use of two transcription start points a single gene is sufficient to specify localization of NADP-dependent isocitrate dehydrogenase to three different cellular compartments in A. nidulans.

Amino Acid Sequence↗
PubMed2004

Looking into DNA recognition: zinc finger binding specificity.

We present a quantitative, theoretical analysis of the recognition mechanisms used by two zinc finger proteins: Zif268, which selectively binds to GC-rich sequences, and a Zif268 mutant, which binds to a TATA box site. This analysis is based on a recently developed method (ADAPT), which allows binding specificity to be analyzed via the calculation of complexation energies for all possible DNA target sequences. The results obtained with the zinc finger proteins show that, although both mainly select their targets using direct, pairwise protein-DNA interactions, they also use sequence-dependent DNA deformation to enhance their selectivity. A new extension of our methodology enables us to determine the quantitative contribution of these two components and also to measure the contributions of individual residues to overall specificity. The results show that indirect recognition is particularly important in the case of the TATA box binding mutant, accounting for 30% of the total selectivity. The residue-by-residue analysis of the protein-DNA interaction energy indicates that the existence of amino acid-base contacts does not necessarily imply sequence selectivity, and that side chains without contacts can nevertheless contribute to defining the protein's target sequence.

Amino Acids↗