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PubMed1993

SREBP-1, a basic-helix-loop-helix-leucine zipper protein that controls transcription of the low density lipoprotein receptor gene.

Sterol regulatory element 1 (SRE-1), a decamer (5'-ATC-ACCCCAC-3') flanking the low density lipoprotein (LDL) receptor gene, activates transcription in sterol-depleted cells and is silenced by sterols. We report the cDNA cloning of human SREBP-1, a protein that binds SRE-1, activates transcription, and thereby mediates the final regulatory step in LDL metabolism. SREBP-1 contains a basic-helix-loop-helix-leucine zipper (bHLH-ZIP) motif, but it differs from other bHLH-ZIP proteins in its larger size (1147 amino acids) and target sequence. Instead of an inverted repeat (CANNTG), the target for all known bHLH-ZIP proteins, SRE-1 contains a direct repeat of CAC. Overexpression of SREBP-1 activates transcription of reporter genes containing SRE-1 in the absence (15-fold) and presence (90-fold) of sterols, abolishing sterol regulation. We suggest that SREBP-1 is regulated by an unknown factor that is overwhelmed when SREBP-1 is overexpressed. Understanding the regulation of SREBP-1 may be crucial for understanding the control of plasma cholesterol in humans.

Amino Acid Sequence↗
PubMed1992

Multitarget-ribozyme directed to cleave at up to nine highly conserved HIV-1 env RNA regions inhibits HIV-1 replication--potential effectiveness against most presently sequenced HIV-1 isolates.

Several mono-, di-, tetra-, penta- and nonaribozymes were developed. These multitarget-ribozymes were targeted to cleave HIV-1 env RNA at up to nine different conserved sites. Each multitarget-ribozyme consisted of a chain of up to nine hammerhead motifs, each flanked by a different targeting sequence. The multitarget-ribozymes were functional in vitro and gave rise to multiple, specific partial and/or complete RNA digestion products. Per RNA copy, multitarget-ribozymes were more efficient than monoribozymes or ribozymes targeting a subset of the same sites. In contrast to monoribozymes, a 400nt nonaribozyme, targeted to cleave at nine different sites within a 1.3kb HIV-1 env RNA substrate, was active and showed the same specificity of cleavage when it was part of a large 3.3kb transcript. We conclude that multitarget-ribozymes retain the specificity of monoribozymes, but they are more efficient per ribozyme RNA copy and they remain active when they are part of a large transcript. A tetra-, penta- or nonaribozyme under control of the SV40 late promoter, the beta-actin gene promoter or the HIV-1 LTR, respectively, were cotransfected with the infectious HIV-1 DNA clone pNL4-3 into permissive HeLa T4 cells. Each cotransfection resulted in a specific inhibition of HIV-1 replication as determined by syncytia formation and p24 antigen release. In addition, coexpression of the nonaribozyme with an HIV-1 env RNA transcript resulted in the specific dramatic reduction of the env transcript. We conclude that the multitarget-ribozymes are also functional intracellularly. A nucleotide sequence comparison of the target sites indicates that the multitarget-ribozymes could potentially be effective against all thirty HIV-1 isolates presently sequenced. Their use may help to slow the selection of viral escape mutants and thereby prolong their effectiveness. We anticipate that multitarget-ribozymes will also be more effective in the successful targeting of less variable cellular RNAs.

Base Sequence↗
PubMed1988

Retrovirus-like features and site specific insertions of a transposable element, tom, in Drosophila ananassae.

The tom element, putatively associated with optic morphology (Om) mutations in Drosophila ananassae, was identified as a retrovirus-like transposable element. The tom element was found to terminate with 475 (or 474) base pair direct repeats which are identical in sequence to each other. Southern blot and heteroduplex analyses showed the tom element to have high homology to 297 and 17.6, two retrotransposons found in D. melanogaster. As in the cases of 297 and 17.6, tom includes nucleotide sequences coding for a presumptive protease and reverse transcriptase, similar in amino acid sequence to those of the Moloney murine leukaemia virus. At the tom insertion site of the sn9g locus, a host DNA sequence (T)ATAT was found to be duplicated on each side of the tom insertion and all other tom elements examined were also flanked by (T)ATAT. In each of six cases, the 5' flanking host sequence was TATAT. These results indicate that the target sequence of the tom element may be TATAT and that the entire region or a part of this sequence was duplicated on insertion of the tom element.

Animals↗
PubMed2005

Effects of oxalate exposure on Madin-Darby canine kidney cells in culture: renal prothrombin fragment-1 mRNA expression.

It has been suggested that renal tubular cell damage induced by oxalic acid, one of the components of urinary calculi, may be involved in a variety of ways in the development of urolithiasis. During our study on a calculus related protein, renal prothrombin fragment-1 (RPTF-1), we noted that this is an inflammation related substance that mediates an acute inflammatory reaction, one of the original roles of prothrombin. RPTF-1 is a part of prothrombin that is a coagulation factor known to be expressed in the renal tubule. We examined whether oxalic acid may cause cytotoxic effects on tubular epithelial cells and whether such chemical stimulation may promote the translation of RPTF-1 mRNA into RPTF-1 proteins. We used Madin-Darby canine kidney (MDCK) cells derived from the distal tubule of a dog kidney. In this study, the effects of oxalic acid in culture solution at different concentrations on cytotoxicity were assessed using a MTT assay. The location of active oxygen species was identified using dichlorofluorescein diacetate. After the prothrombin sequence of RPTF-1 was confirmed in MDCK cells, RPTF-1 mRNA expression was determined by RT-PCR. The gene sequence of the same promoter area was ligated, and a luciferase sequence was inserted downstream of the vector. The target sequence was transfected into MDCK cells and the relation between oxalic acid and prothrombin promoter was examined. In addition, the variable expression of RPTF-1 mRNA was quantitatively compared depending on oxalic acid concentrations using real-time PCR. When cytotoxicity was investigated, cells were not damaged but, by contrast, were stimulated and activated under oxalic acid below a certain concentration. The relation between cytotoxicity on the cultured MDCK cell membrane and active oxygen species was confirmed. Luminescence in MDCK cells containing the luciferase gene was detected by the addition of oxalic acid, which activated the prothrombin promoter. A part of the prothrombin gene sequence in the MDCK cells was detected and an increase in the expression of RPTF-1 mRNA in MDCK cells by the addition of oxalic acid was confirmed using real-time PCR. Increased expression of prothrombin by adding oxalic acid has already been demonstrated in previous studies. In this study, however, RPTF-1 mRNA was promoted by oxalic acid and a direct association between oxalic acid and RPTF-1 is indicated. This finding shows that increased oxalic acid in urine induces the expression of RPTF-1 in tubular epithelial cells and thereby causes the generation of active oxygen species.

Animals↗
PubMed2000

Multiple forms of alpha2-macroglobulin from a bony fish, the common carp (Cyprinus carpio): striking sequence diversity in functional sites.

Unlike mammals, bony fish possess multiple genes encoding the complement component C3, a member of the alpha2-macroglobulin (alpha2M) protein family, presumably expanding the diversity of immune recognition. To examine whether the alpha2M gene has also duplicated and diverged in the bony fish lineage, cDNA cloning of alpha2M from a pseudotetraploid teleost, the common carp (Cyprinus carpio), was conducted and resulted in the isolation of three distinct alpha2M sequences from a single individual, indicating the presence of multiple alpha2M genes in this species. The deduced amino acid sequences contained a post-translational cleavage signal, predicting a C3-like two-chain structure, as in lamprey alpha2M. Two distinct alpha2M proteins were purified from carp serum; both proved to be Mr 380,000 dimers, the subunits of which are composed of disulfide-linked alpha chains (Mr 93,000) and beta chains (Mr 85,000), as reported for the alpha2M from plaice, another teleost species. The presence of an internal thioester in the alpha chain was demonstrated by its autolytic fragmentation and direct incorporation of [14C]methylamine. Interestingly, the three forms of carp alpha2M exhibited outstanding sequence diversity in the bait region which displays target sequences for various proteases, and in the C-terminal region of the alpha chain assigned as the receptor-binding domain, while an Asn residue at the position corresponding to the catalytic His in C3 was completely conserved in the carp alpha2Ms, as in most alpha2Ms of other animals. The possible functional significance of the sequence diversity is discussed.

Amino Acid Sequence↗
PubMed2005

Improved detection of grapevine leafroll-associated virus 1 by magnetic capture hybridisation RT-PCR on a conserved region of viral RNA.

We report the development of a sensitive diagnostic method for the detection of the grapevine leafroll-associated virus 1 (GLRaV-1). We have considered the current shortcoming in detection of GLRaV-1 to be linked to two factors, sequence variation in the viral RNA and low template concentration. Sequence information available allowed the selection of optimal target sequences for detection by RT-PCR, having high copy number and low levels of sequence variation. This was combined with the use of magnetic capture hybridisation to allow the removal of RT-PCR inhibitors and the addition of 100-fold excess template RNA to a single RT-PCR. The reproducibility of the technique was confirmed using field samples.

Closteroviridae↗
PubMed1991

Distamycin inhibits the binding of a nuclear factor to the -278/-256 upstream sequence of the human HLA-DR alpha gene.

In this study we analyse the effects of the anti-tumor compound distamycin on the binding of nuclear factor(s) to a synthetic oligonucleotide (GTATA/IFN-gamma) mimicking a putative regulatory region of the human HLA-DR alpha gene. This region contains the sequence (GTATA), that is required for nuclear protein binding and is likely to interact with distamycin. The present results, by showing that distamycin inhibits the interaction between nuclear factors and the GTATA/IFN-gamma oligonucleotide, suggest that distamycin might alter the binding of transacting factors to cis-elements containing AT/TA sequences. Alterations of nuclear protein binding to specific target sequences could be one of the molecular mechanism(s) by which distamycin exerts its antiproliferative activity on living cells.

Antineoplastic Agents↗
PubMed2006

2-Cys peroxiredoxins from Schistosoma japonicum: the expression profile and localization in the life cycle.

Peroxiredoxin (Prx) is known to be an antioxidant protein that protects the organisms against various oxidative stresses and functions as a signal transductor. Here, we determined the full-length cDNA sequences of three types of Prx from an Asian blood fluke, Schistosoma japonicum: Prx-1, Prx-2 and Prx-3. According to the deduced amino acid sequences, only Prx-3 had a mitochondria-targeting sequence. Using RT-PCR, it was shown that these Prx genes were constitutively expressed in the eggs, cercariae and adult worms of the schistosome. Western blot analysis using antisera specific for each Prx revealed that all the three Prx proteins existed in these developmental stages. By immunolocalization analysis, Prx-1 existed on the surface of a miracidium and in the space between a miracidium and an eggshell. Furthermore, Prx-1 was deposited in the host tissues around the eggs. In adult worms, Prx-1 was not only expressed in the tegument, but also contained in their excretory/secretory products. The surface of the 7 day-schistosomula was stained with anti-Prx-1 antiserum. On the other hand, Prx-2 only existed inside the miracidia in eggs. In addition, Prx-2 was mainly detected in the sub-tegumental tissues, parenchyma, vitelline gland and gut epithelium of the adult worms, but was not detected in the tegument of adults and schistosomula. Taken together with previous reports by other investigators, these data suggest that Prx-1 acts to protect the parasite against the ROS produced by host immune cells, and that Prx-2 plays important roles in intracellular redox signaling and/or in the reduction of ROS generated through the hemoglobinolytic process in the digestive tract.

Amino Acid Sequence↗
PubMed2003

Mutation analysis of feline Niemann-Pick C1 disease.

Niemann-Pick C (NPC) disease is an autosomal recessive neurovisceral lysosomal storage disorder that results in defective intracellular transport of cholesterol. The major form of human NPC (NPC1) has been mapped to chromosome 18, the NPC1 gene (NPC1) has been sequenced and several mutations have been identified in NPC1 patients. A feline model of NPC has been characterized and is phenotypically, morphologically, and biochemically similar to human NPC1. Complementation studies using cultured fibroblasts from NPC affected cats and NPC1 affected humans support that the gene responsible for the NPC phenotype in this colony of cats is orthologous to human NPC1. Using human-based PCR primers, initial fragments of the feline NPC cDNA were amplified and sequenced. From these sequences, feline-specific PCR primers were generated and designed to amplify six overlapping bands that span the entire feline NPC1 open reading frame. A single base substitution (2864G-C) was identified in NPC1 affected cats. Obligate carriers are heterozygous at the same allele and a PCR-based assay was developed to identify the geneotype of all cats in the colony. The mutation results in an amino acid change from cysteine to serine (C955S). Several of the mutations identified in people occur in the same region. Marked similarity exists between the human and feline NPC1 cDNA sequences, and is greater than that between the human and murine NPC1 sequences. The human cDNA sequence predicts a 1278aa protein with a lysosomal targeting sequence, several trans-membrane domains and extensive homology with other known mediators of cholesterol homeostasis.

Amino Acid Sequence↗
PubMedDate not supplied

A targeted peptide nucleic acid to down-regulate mouse microsomal triglyceride transfer protein expression in hepatocytes.

Peptide nucleic acids (PNA's) have shown to hold potential as antisense drugs. In this study we have designed PNA drugs for the microsomal triglyceride transfer protein (MTP), which is known to play a critical role in the assembly of atherogenic lipoproteins, and have converted the most potent drug into a liver-targeted prodrug. First, we have synthesized three PNA sequences targeting domains on the mouse MTP mRNA, which were not involved in intrastrand base-pairing interactions as jugded from its secondary structure. Only one of the PNA's, PNA569, showed dose-dependent inhibition of MTP expression in a cell-free system for coupled transcription/translation of MTP. Second, to improve the cellular uptake of this PNA drug, we have conjugated PNA569 to a high affinity ligand for the asialoglycoprotein receptor, K(GalNAc)(2). As compared to the parent PNA, the prodrug PNA-K(GalNAc)(2) was found to display to a markedly improved capacity to inhibit MTP mRNA expression in parenchymal liver cells. A glycoconjugated nonsense control appeared to be ineffective. In conclusion, the design of a targeted PNA is described to reduce MTP expression in parenchymal liver cells by 70%. The presented approach for targeted tissue-specific down-regulation of genes by PNA's may be valid for other genes as well.

Animals↗
PubMed1996

Dysfunction of the Orleans reeler gene arising from exon skipping due to transposition of a full-length copy of an active L1 sequence into the skipped exon.

We examined the genomic structure of the reeler gene in Orleans reeler mouse mutant. Exon skipping of the reeler gene caused a 220 bp deletion in the transcript, resulting in a frame shift of the reeler gene which disrupts the 8th EGF-like motif of the reeler product. Surprisingly, the skipped exon was inserted by the 7104 bp L1 element which carried the full-length stretch of the mouse L1 sequence, consisting of a 212 bp F-type tandem repeat, open reading frame 1 (ORF1), ORF2, the polyadenylation signal and a poly A stretch. The transposed L1 sequence was flanked by 13 bp of the target sequence at both ends. ORF1 and ORF2 of this L1 repeat element are thought to encode a component of the RNP particle and the reverse transcriptase, respectively. Orleans reeler was originally established by spontaneous mutation caused by L1 insertion, and this L1 sequence is considered to be potentially active for transposition in mouse genome.

Amino Acid Sequence↗
PubMed1986

Mutations induced by DNA polymerase alpha upon in vitro replication of M13mp8(+) DNA.

The forward mutation of the lacZ part of the bacteriophage M13mp8 has been used to study the fidelity of the 9S DNA polymerase alpha from calf thymus during in vitro replication of single-stranded DNA. Errors leading to a loss of alpha-complementation were identified by DNA sequencing. The overall mutation rate of the lacZ target sequence was in the range of 1:300-1:1000 which is more than one order of magnitude higher than the spontaneous mutation rate. In a mutL host the mutation rate was nearly threefold higher as compared to the wildtype host. Base substitutions comprise 86% of the errors whereas base deletions amount to 12%. The addition of a base was detected only in one mutant out of 71 sequenced ones. The frameshift mutations occurred predominantly in runs of the same base. The frequencies of individual base substitution are in the order of 2 X 10(-4)-4 X 10(-4) for most of the mismatches. Mutations involving dCTP:T and dGTP:T mismatches are observed with a lower frequency, those involving dTTP:C mismatches with a higher frequency.

Animals↗
PubMed2005

Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products.

In this paper we report that the inclusion of heat-resistant RecA protein from a thermophilic bacteria, Thermus thermophilus, and its cofactor (ATP) in PCR effectively eliminates non-specific PCR products. The effect of RecA protein, which catalyzes pairing between homologous DNA molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in PCR (i.e. priming at sites where the primer sequence is completely complementary to that of the target sequence). In addition, the RecA protein substantially reduces the primer concentration required for PCR. These experimental results have led to the realization of multiplex PCR, which involves PCR for multiple sites in the same reaction mixture. We were able to successfully perform multiplex PCR with over a dozen reactions without affecting the amplification pattern of the PCR products.

Base Pair Mismatch↗
PubMed1988

The nucleotide sequence of the LPD1 gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae: comparison between eukaryotic and prokaryotic sequences for related enzymes and identification of potential upstream control sites.

The complete nucleotide sequence of the LPD1 gene, which encodes the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes of Saccharomyces cerevisiae, has been established. The flanking region 5' to the LPD1 gene contains DNA sequences which show homology to known control sites found upstream of other yeast genes. The primary structure of the protein, determined from the DNA sequence, shows strong homology to a group of flavoproteins including Escherichia coli lipoamide dehydrogenase and pig heart lipoamide dehydrogenase. The amino acid sequence also reveals the presence of a potential targeting sequence at its N-terminus which may facilitate transport to and entry into mitochondria.

Amino Acid Sequence↗
PubMed2000

Evaluation of recA sequences for identification of Mycobacterium species.

16S rRNA sequence data have been used to provide a molecular basis for an accurate system for identification of members of the genus Mycobacterium. Previous studies have shown that Mycobacterium species demonstrate high levels (>94%) of 16S rRNA sequence similarity and that this method cannot differentiate between all species, i.e., M. gastri and M. kansasii. In the present study, we have used the recA gene as an alternative sequencing target in order to complement 16S rRNA sequence-based genetic identification. The recA genes of 30 Mycobacterium species were amplified by PCR, sequenced, and compared with the published recA sequences of M. tuberculosis, M. smegmatis, and M. leprae available from GenBank. By recA sequencing the species showed a lower degree of interspecies similarity than they did by 16S rRNA gene sequence analysis, ranging from 96.2% between M. gastri and M. kansasii to 75.7% between M. aurum and M. leprae. Exceptions to this were members of the M. tuberculosis complex, which were identical. Two strains of each of 27 species were tested, and the intraspecies similarity ranged from 98.7 to 100%. In addition, we identified new Mycobacterium species that contain a protein intron in their recA genes, similar to M. tuberculosis and M. leprae. We propose that recA gene sequencing offers a complementary method to 16S rRNA gene sequencing for the accurate identification of the Mycobacterium species.

Genes, Bacterial↗
PubMed1993

MyoD and myogenin act on the chicken myosin light-chain 1 gene as distinct transcriptional factors.

Expression of MyoD, myogenin, MRF4, and Myf-5 converts nonmuscle cells to muscle cells. In an attempt to analyze the roles of these factors, we have investigated their effects on transcription driven by the promoter of the chicken myosin alkaline light-chain (MLC1) gene. The activation by CMD1 or c-myogenin (chicken MyoD or myogenin, respectively) was dependent on the existence of a muscle-specific regulatory region located from positions -2096 to -1743. Its distal half, containing a pair of E boxes (CANNTG), had been previously characterized as an enhancer responsive to CMD1 but not to c-myogenin. In this study, we report the identification of another enhancer in the muscle-specific regulatory region which is preferentially responsive to c-myogenin. Deletion and mutation analyses indicated that this enhancer requires a single E box and its flanking sequences. Furthermore, analysis of chimeric proteins of CMD1 and c-myogenin indicated that regions outside the basic helix-loop-helix domain of c-myogenin are involved in the specificity of the enhancer. These results show that CMD1 and c-myogenin act on the MLC1 gene by recognizing different upstream DNA sequences and that direct or indirect interactions between the regions outside the basic helix-loop-helix domain and flanking sequences of E boxes are involved in the target sequence specificity.

Animals↗
PubMed2004

Sequencing of genomic DNA by combined amplification and cycle sequencing reaction.

BACKGROUND: Despite considerable advances, DNA sequencing has remained somewhat time-consuming and expensive, requiring three separate steps to generate sequencing products from a template: amplification of the target sequence; purification of the amplified product; and a sequencing reaction. Our aim was to develop a method to routinely combine PCR amplification and cycle sequencing into one single reaction, enabling direct sequencing of genomic DNA. METHODS: Combined amplification and sequencing reactions were set up with Big Dye sequencing reagents (Applied Biosystems) supplemented with variable amounts of forward and reverse primers, deoxynucleotide triphosphates (dNTPs), and input DNA. Reactions were thermal-cycled for 35 or 45 cycles. Products were analyzed by capillary electrophoresis to detect sequencing products. RESULTS: Reactions using two oligonucleotide primers at a ratio of 5:1 (500 nM primer 1 and 100 nM primer 2), 125 microM supplemental dNTPs, and 35-45 thermal cycles optimally supported combined amplification and cycle sequencing reactions. Our results suggest that these reactions are dominated by PCR during early cycles and convert to cycle sequencing in later cycles. We used this technique for a variety of sequencing applications, including the identification of germline mutations/polymorphisms in the Factor V and BRCA2 genes, sequencing of tumor DNA to identify somatic mutations in the DPC4/SMADH4 and FLT3 genes, and sequencing of 16S ribosomal DNA for bacterial speciation. CONCLUSIONS: PCR amplification and cycle sequencing can be combined into a single reaction using the conditions described. This technique allows direct sequencing of genomic DNA, decreasing the cost and labor involved in gene sequencing.

BRCA2 Protein↗
PubMed2005

Inhibition of object identity in inhibition of return: implications for encoding and retrieving inhibitory processes.

Inhibition of return (IOR) effects, in which participants detect a target in a cued box more slowly than one in an uncued box, suggest that behavior is aided by inhibition of recently attended irrelevant locations. To investigate the controversial question of whether inhibition can be applied to object identity in these tasks, in the present research we presented faces upright or inverted during cue and/or target sequences. IOR was greater when both cue and target faces were upright than when cue and/or target faces were inverted. Because the only difference between the conditions was the ease of facial recognition, this result indicates that inhibition was applied to object identity. Interestingly, inhibition of object identity affected IOR both when encoding a cue face and retrieving information about a target face. Accordingly, we propose that episodic retrieval of inhibition associated with object identity may mediate behavior in cuing tasks.

Adolescent↗