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PubMed1987

The GATATC-modification enzyme EcoRV is closely related to the GATC-recognizing methyltransferases DpnII and dam from E. coli and phage T4.

The amino acid sequence of EcoRV DNA methyltransferase which methylates the amino group of the 5'-adenine residue of the target sequence GATATC has been found to be closely related to that of three other adenine methyltransferases, DpnII, dam and damT4, the target sequence of which is GATC. Despite large differences on the DNA level, the four sequences show four blocks of homologies. One of these blocks has the sequence DVYXDPPY and is found with little modification in numerous other DNA methyltransferases. It is speculated that it could be the binding site of the methyl donor, S-adenosylmethionine. On the other hand, the identification of a DNA-binding region is more tenuous. As expected, no analogies with (dimeric) repressors and cro proteins which have the characteristic helix-turn-helix motif have been observed.

Amino Acid Sequence↗
PubMed1997

Mft52, an acid-bristle protein in the cytosol that delivers precursor proteins to yeast mitochondria.

We have identified a novel protein, Mft52, in the cytosol of yeast cells. Mft52 has a two-domain structure that includes a receptor-like carboxyl-terminal "acid-bristle" domain, which binds basic, amphipathic mitochondrial targeting sequences. Native Mft52, purified from the cytosol of yeast cells, is found as a large particle eluting in the void volume of a Superose 6 gel filtration column. Fusion proteins, consisting of mitochondrial targeting sequences fused to nonmitochondrial passenger proteins, are targeted to mitochondria in wild-type yeast cells, but defects in the gene encoding Mft52 drastically reduce the delivery of these proteins to the mitochondria. We propose that Mft52 is a subunit of a particle that is part of a system of targeting factors and molecular chaperones mediating the earliest stages of protein targeting to the mitochondria.

Amino Acid Sequence↗
PubMed1996

Effect on DNA transcription of nucleotide sequences upstream to T7 promoter.

The T7 RNA polymerase-dependent transcription was studied as a function of nucleotide sequence structures positioned upstream of the T7 promoter. Model double-stranded DNA templates were constructed for this purpose. They contained a target sequence of 485 base pairs (cDNA fragment of Venesuelian encephalomyelitis equine virus genome), T7 promoter consensus and different extra base sequences upstream of the T7 promoter. The level of the target sequence transcription was clearly determined by the extra base sequence. The presence of one extra base pair G.C ensured the most pronounced effect, transcription was increased one order of magnitude in comparison with template which has only a canonical T7 promoter sequence at the 5'-end.

Base Sequence↗
PubMed1996

Interaction of a potential vacuolar targeting receptor with amino- and carboxyl-terminal targeting determinants.

A protein of 80 kD from developing pea (Pisum sativum) cotyledons has previously been shown to exhibit characteristics of a vacuolar targeting receptor by means of its affinity for the amino-terminal vacuolar targeting sequence of proaleurain from barley (Hordeum vulgare). In this report we show that the same protein also binds to the amino-terminal targeting peptide of prosporamin from sweet potato (Ipomoea batatas) and to the carboxyl-terminal targeting determinant of pro-2S albumin from Brazil nut (Bertholletia excelsa). The receptor protein does not bind to the carboxyl-terminal propeptide (representing the targeting sequence) of barley lectin. The binding of the 80-kD protein to the sporamin determinant involves a motif (NPIR) that has been shown to be crucial for vacuolar targeting in vivo. The binding to the carboxyl-terminal targeting determinant of pro-2S albumin appears to involve the carboxyl-terminal propeptide and the adjacent five amino acids of the mature protein. The 80-kD protein does not bind to peptide sequences that have been shown to be incompetent in directing vacuolar targeting.

Amino Acid Sequence↗
PubMed1988

Sequence-specific binding and photocrosslinking of alpha and beta oligodeoxynucleotides to the major groove of DNA via triple-helix formation.

A photocrosslinking reagent (p-azidophenacyl) was covalently linked to an octathymidylate synthesized with either the natural (beta) anomer of thymidine or the synthetic (alpha) anomer. The oligothymidylate was further substituted by an acridine derivative to stabilize the hybrid formed with a complementary octadeoxyadenylate sequence via intercalation. A single-stranded 27-mer containing a (dA)8 sequence and a 27-mer duplex containing a (dA.dT)8 sequence were used as targets. Upon UV irradiation, photocrosslinking of the octathymidylate to its target sequence was observed, generating bands that migrated more slowly in denaturing gels. In the 27-mer duplex, both strands were photocrosslinked to the octathymidylate. Upon alkaline treatment of the irradiated samples, cleavage of the 27-mers was observed at specific sites. These reactions were analyzed at different salt concentrations. The location of the cleavage sites allowed us to demonstrate the following. (i) Both alpha and beta oligothymidylates can recognize a DNA double helix containing an oligo(dA).oligo(dT) sequence; the oligothymidylate binds to the major groove of DNA in a parallel orientation with respect to the adenine-containing strand of the DNA double helix. (ii) alpha oligothymidylates form helices with a complementary single-stranded oligodeoxyadenylate; the two strands have a parallel orientation independently of whether or not an intercalating agent is attached to the oligothymidylate. (iii) At low salt concentration, beta oligothymidylates form a double helix with an oligodeoxyadenylate in which, as expected, the two strands are antiparallel; at high salt concentration, a triple helix is formed in which the second oligothymidylate is oriented parallel to the adenine-containing strand. These results show that it is possible to recognize an oligopurine.oligopyrimidine sequence in a DNA double helix via local triple-helix formation and to target photochemical reactions to specific sequences in both double-stranded and single-stranded nucleic acids.

Cross-Linking Reagents↗
PubMed1995

Distinct CG and CNG DNA methyltransferases in Pisum sativum.

DNA methyltransferase activity, present in low salt extracts of nuclei from young pea shoot apices, has been fractionated into two different species by assaying with model substrates. The CG methyltransferase (an unstable enzyme believed to be of 140 kDa) methylates cytosine only in oligonucleotides with CG and Cl dinucleotide targets while an enzyme of 110 kDa (the CNG methyltransferase) methylates the cytosines in 5'-CAG-3' and 5'-CTG-3' target sequences, especially when hemimethylated, but not in 5'-CCG-3' nor in 5'-CGG-3' target sequences present in oligonucleotides.

Base Sequence↗
PubMed1994

An internal region of the peroxisomal membrane protein PMP47 is essential for sorting to peroxisomes.

Targeting sequences on peroxisomal membrane proteins have not yet been identified. We have attempted to find such a sequence within PMP47, a protein of the methylotrophic yeast, Candida boidinii. This protein of 423 amino acids shows sequence similarity with proteins in the family of mitochondrial carrier proteins. As such, it is predicted to have six membrane-spanning domains. Protease susceptibility experiments are consistent with a six-membrane-spanning model for PMP47, although the topology for the peroxisomal protein is inverted compared with the mitochondrial carrier proteins. PMP47 contains two potential peroxisomal targeting sequences (PTS1), an internal SKL (residues 320-322) and a carboxy terminal AKE (residues 421-423). Using a heterologous in vivo sorting system, we show that efficient sorting occurs in the absence of both sequences. Analysis of PMP47-dihydrofolate reductase (DHFR) fusion proteins revealed that amino acids 1-199 of PMP47, which contain the first three putative membrane spans, do not contain the necessary targeting information, whereas a fusion with amino acids 1-267, which contains five spans, is fully competent for sorting to peroxisomes. Similarly, a DHFR fusion construct containing residues 268-423 did not target to peroxisomes while residues 203-420 appeared to sort to that organelle, albeit at lower efficiency than the 1-267 construct. However, DHFR constructs containing only amino acids 185-267 or 203-267 of PMP47 were not found to be associated with peroxisomes. We conclude that amino acids 199-267 are necessary for peroxisomal targeting, although additional sequences may be required for efficient sorting to, or retention by, the organelles.

Amino Acid Sequence↗
PubMed1990

Transcriptionally active genome regions are preferred targets for retrovirus integration.

We have analyzed the transcriptional activity of cellular target sequences for Moloney murine leukemia virus integration in mouse fibroblasts. At least five of the nine random, unselected integration target sequences studied showed direct evidence for transcriptional activity by hybridization to nuclear run-on transcripts prepared from uninfected cells. At least four of the sequences contained multiple recognition sites for several restriction enzymes that cut preferentially in CpG-rich islands, indicating integration into 5' or 3' ends or flanking regions of genes. Assuming that only a minor fraction (less than 20%) of the genome is transcribed in mammalian cells, we calculated the probability that this association of retroviral integration sites with transcribed sequences is due to chance to be very low (1.6 x 10(-2]. Thus, our results strongly suggest that transcriptionally active genome regions are preferred targets for retrovirus integration.

Animals↗
PubMed1994

An oligomeric protein is imported into peroxisomes in vivo.

The mechanism of translocation of peroxisomal proteins from the cytoplasm into the matrix is largely unknown. We have been studying this problem in yeast. We show that the peroxisomal targeting sequences SKL or AKL, with or without a spacer of nine glycines (G9), are sufficient to target chloramphenicol acetyltransferase (CAT) to peroxisomes of Saccharomyces cerevisiae in vivo. The mature form of CAT is a homotrimer, and complete trimerization of CAT was found to occur within a few minutes of synthesis. In contrast, import, measured by immunoelectron microscopy and organellar fractionation, occurred over several hours. To confirm that import of preassembled CAT trimers was occurring, we co-expressed CAT-G9-AKL with CAT lacking a peroxisomal targeting sequence but containing a hemagglutinin-derived epitope tag (HA-CAT). We found that HA-CAT was not imported unless it was co-expressed with CAT-G9-AKL. Both proteins were released from the organelles under mild conditions (pH 8.5) that released other matrix proteins, indicating that import had occurred. These results strongly suggested that HA-CAT was imported as a heterotrimer with CAT-G9-AKL. The process of oligomeric import also occurs in animal cells. When HA-CAT was co-expressed with CAT-G9-AKL in CV-1 cells, HA-CAT co-localized with peroxisomes but was cytoplasmic when expressed alone. It is not clear whether the import of globular proteins into peroxisomes occurs through peroxisomal membrane pores or involves membrane internalization. Both possibilities are discussed.

Amino Acid Sequence↗
PubMed1996

Sequence variation in the 18S rRNA gene, a target for PCR-based malaria diagnosis, in Plasmodium ovale from southern Vietnam.

Field surveys of malaria were performed in southern Vietnam by using an acridine orange staining method for rapid diagnosis and a PCR-based, microtiter plate hybridization method for accurate diagnosis. A total of three patients of Plasmodium ovale infection were detected, but PCR-amplified DNA of the P. ovale isolates from two of the patients did not hybridize with the P. ovale-specific probe. Analysis of the target sequence in the 18S rRNA gene indicated that in the DNA of isolates from both patients three nucleotides in the probe region from the typical P. ovale sequence were different, with deletions of two nucleotides and the substitution of one nucleotide. These results may suggest that in addition to molecular biological methods, careful microscopic examination of stained thin blood films is still required in studies of the prevalence of different malaria species.

Animals↗
PubMedDate not supplied

Bead-based sandwich hybridization characteristics of oligonucleotide-alkaline phosphatase conjugates and their potential for quantitating target RNA sequences.

The hybridization characteristics of oligonucleotide-alkaline phosphatase conjugate probes were examined in bead-based sandwich hybridization reactions using single-stranded nucleic acid targets and oligonucleotide-polystyrene capture beads. Enzymatic activity was monitored using a chemiluminescent substrate and calibration plots of chemiluminescent signal versus conjugate concentration were used to estimate the sandwich hybridization efficiencies. Improved hybridization behavior was noted using glycerol as an additive and by increasing the length of the probe and alkyl spacer of the conjugates. The chemiluminescent assay is at least as sensitive as those employing 32P-labeled probes and can detect as little as 10-20 amol of target RNA. The linear relationship of chemiluminescent signal versus target assayed provides a method for quantitating unknown target samples. A single human immunodeficiency virus type 1 infected cell in a background of 10(6) uninfected cells is facilely detected when this enzyme-based detection assay is prefaced with a self-sustained sequence-replication amplification reaction.

Alkaline Phosphatase↗
PubMed1995

[Leu5]enkephalin-encoding sequences are targets for a specific DNA-binding factor.

A DNA-binding factor with high affinity and specificity for the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes has been characterized. The factor has the highest affinity for the [Leu5]-enkephalin-encoding sequence in the dynorphin B-encoding region of the prodynorphin gene, has relatively high affinity for other [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes, but has no apparent affinity for similar DNA sequences coding for [Met5]-enkephalin in the prodynorphin or proopiomelanocortin genes. The factor has been named [Leu5]enkephalin-encoding sequence DNA-binding factor (LEF). LEF has a nuclear localization and is composed of three subunits of about 60, 70, and 95 kDa, respectively. The highest levels were observed in rat testis, cerebellum, and spleen and were generally higher in late embryonal compared to newborn or adult animals. LEF activity was also recorded in human clonal tumor cell lines. LEF inhibited the transcription of reporter genes in artificial gene constructs where a [Leu5]enkephalin-encoding DNA fragment had been inserted between the transcription initiation site and the coding region of the reporter genes. These observations suggest that the [Leu5]enkephalin-encoding sequences in the prodynorphin and proenkephalin genes also have regulatory functions realized through interaction with a specific DNA-binding factor.

Aging↗
PubMed1988

Peptide sequences that target proteins for enhanced degradation during serum withdrawal.

Fibroblasts increase the catabolism of certain intracellular proteins in response to serum withdrawal, and these proteins contain specific peptide regions that may be required for their increased degradation. We show that the increased degradation of microinjected ribonuclease A during serum withdrawal can be blocked by co-injection of a pentapeptide corresponding to residues 7-11 of ribonuclease A, Lys-Phe-Glu-Arg-Gln. Furthermore, similar peptide sequences appear to play a widespread role in targeting proteins for enhanced degradation. Affinity-purified antibodies raised against the pentapeptide are able to precipitate 20-35% of radiolabeled cytosolic proteins from fibroblasts. Such proteins are preferentially degraded when cells are deprived of serum while nonimmunoprecipitable proteins are degraded at the same rate in the presence and absence of serum. Immunoreactive cytosolic proteins also exist in rat liver and kidney, and these proteins are depleted when protein degradation rates are enhanced due to starvation. Several types of evidence suggest that the peptides recognized in cellular proteins are similar to Lys-Phe-Glu-Arg-Gln but are not this exact sequence. Analyses of amino acid sequences for four proteins whose degradative rates are enhanced in response to serum withdrawal and for four proteins that are degraded in a serum-independent manner indicate two possible peptide motifs related to Lys-Phe-Glu-Arg-Gln that may target cellular proteins for enhanced degradation. These results, combined with previous studies (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993), suggest that these peptide regions target specific proteins to a lysosomal pathway of degradation during serum withdrawal.

Amino Acid Sequence↗
PubMed1991

The tdh and serA operons of Escherichia coli: mutational analysis of the regulatory elements of leucine-responsive genes.

The tdh promoter of Escherichia coli is induced seven- to eightfold when cells are grown in the presence of exogenous leucine. A scheme was devised to select mutants that exhibited high constitutive expression of the tdh promoter. The mutations in these strains were shown to lie within a previously identified gene (lrp) that encodes Lrp (leucine-responsive regulatory protein). By deletion analysis, the site of action of Lrp was localized to a 25-bp region between coordinates -69 and -44 of the tdh promoter. Disruption of a 12-bp presumptive target sequence found in this region of tdh resulted in constitutively derepressed expression from the tdh promoter. Similar DNA segments (consensus, TTTATTCtNaAT) were also identified in a number of other promoters, including each of the Lrp-regulated promoters whose nucleotide sequence is known. The sequence of the promoter region of serA, an Lrp-regulated gene, was determined. No Lrp consensus target sequence was present upstream of serA, suggesting that Lrp acts indirectly on the serA promoter. A previously described mutation in a leucine-responsive trans-acting factor, LivR (J. J. Anderson, S. C. Quay, and D. L. Oxender, J. Bacteriol. 126:80-90, 1976), resulted in constitutively repressed expression from the tdh promoter and constitutively induced expression from the serA promoter. The possibility that LivR and Lrp are allelic is discussed.

Alcohol Oxidoreductases↗
PubMed1997

Multivariant confirmation of sickle cell disease using a non-radioactive minisequencing reaction.

A non-radioactive solid-phase minisequencing method for confirmation of abnormal hemoglobin variants causing sickle cell disease has been developed. In this method amplified 5'-biotinylated target sequences containing normal and mutation sites are immobilized onto streptavidin-coated microplates. Detection primers corresponding to target sequences are annealed immediately adjacent to the mutation site and single-step, hapten-labeled nucleotide primer extension reactions are performed. The incorporation of the labeled nucleotide is detected through immunological reaction with an enzyme-labeled anti-hapten conjugate and a substrate. The method enables confirmation of mutations of the beta-globin gene variants (Hbs S, C, E D-Punjab, O-Arab) and the alpha-globin gene variant (Hb G-Philadelphia). The test was evaluated using characterized dried blood spot specimens (n = 100) The advantages of the procedure are easy performance and objectiveness. The non-radioactive minisequencing assay will prove helpful for genotyping in neonatal screening for hemoglobinopathies and in prenatal and pre-implantational diagnostics.

Anemia, Sickle Cell↗
PubMed1996

Identification of cis-acting sequences in the promoter of the herpes simplex virus type 1 latency-associated transcripts required for activation by nerve growth factor and sodium butyrate in PC12 cells.

In the absence of detectable viral proteins, expression of the latency-associated transcripts (LATs) is likely regulated by cellular factors during latent infection of neurons with herpes simplex virus type 1. The amounts and activation states of these factors may in turn be regulated by extracellular regulatory factors. Consistent with this hypothesis, we have recently demonstrated that LAT expression is significantly enhanced by nerve growth factor (NGF) and sodium butyrate (NaB) in neurally derived PC12 cells. With the ultimate goal of identifying trans-acting cellular factors involved in regulating LAT expression during latency, we have attempted to identify the cis-acting elements to which these putative cellular factors bind by characterizing the LAT promoter and a series of 5' promoter deletion mutants in PC12 cells following treatment with the LAT-enhancing agents NGF and NaB. Transient expression assays demonstrated that distinct cis-acting sequences mediate basal and induced LAT promoter expression. Basal activity in PC12 cells is mediated by two elements: a negative regulatory element between -435 and -270 and a positive element between -240 and -204. The positive element contains binding sites for the transactivator Sp-1, whereas the negative element bears some resemblance to known neuron-specific silencer elements. In contrast to basal expression, maximum induction of the LAT promoter by NGF and NaB requires sequences between -159 and -81. Using gel mobility shift assays, we have identified three sets of protein-DNA complexes that bind to this 78-bp region and shown by competition analysis that binding is specific. The abundance and mobility of these complexes were altered by treatment with NGF or NaB. The nucleotide sequences to which these complexes bind were fine mapped by competition analysis with oligonucleotide probes containing substitution mutations. The target sequences identified exhibit no homology to binding sites of known transcription factors. These regions were critical for complex formation in vitro and for maximum induction of the LAT promoter by NGF and NaB in transient expression assays. The protein complexes that form with target sequences likely participate in the regulation of LAT expression in response to physiological stimuli in neurons in vivo.

Animals↗
PubMed1995

MATS: a rapid and efficient method for the development of microsatellite markers from YACs.

In this report, we describe the successful application of a rapid and efficient procedure, based on subtractive hybridization and PCR amplification, for generating microsatellite-based markers directly from yeast artificial chromosomes (YACs). This strategy, termed MATS (marker addition through subtraction), exploits the fact that the only difference between a yeast host strain harboring a YAC and the host strain alone is the artificial chromosome. Given the low complexity of the yeast genome and relatively large target size presented by a YAC, only a single round of subtraction is required before amplification of the target sequences (YAC) and cloning into a plasmid vector for further analysis. Several key steps have been designed to achieve optimal subtraction and to obtain preferential amplification and recovery of the target sequences. Methods for efficient construction of small insert libraries and rapid, nonradioactive screening have also been integrated into the protocol. Using a 750-kb YAC as a target, we identified a minimum of 14 unique microsatellite containing clones, leading to the development of 12 polymorphic STSs (sequence-tagged sites). These new markers will facilitate the genetic localization of targeted locus and allow the accurate ordering by STS content mapping of a cloned contig spanning the interval. In addition to the utility of this approach in positional cloning, this strategy may provide an approach for filling gaps in the emerging genetic maps.

Base Sequence↗
PubMed1994

Noncanonical Oct-sequences are targets for mouse Oct-2B transcription factor.

We have suggested a random modification method for determining preferable binding sites of a DNA-binding protein and applied this method to the Oct-2B transcription factor. Our results indicate that the Oct-2B protein interacts with canonical oct sequence ATGC/TAAAT and degenerated sequences which contain TAAT motif in the binding site. We have determined nucleotides in the binding sites, involved in the DNA-protein interaction, and the equilibrium dissociation constants Kd for these sequences. These data show that a much greater number of potential targets for Oct proteins exist on DNA and changed our view on the gene expression regulation by this protein factor.

Animals↗