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At least 235 records · Page 13Linked to original sources
PubMed1995

Sorting of cytochrome b2 to the intermembrane space of mitochondria. Kinetic analysis of intermediates demonstrates passage through the matrix.

Precytochrome b2 is targeted to the mitochondrial intermembrane space by a dual targeting sequence comprising 80 amino acids. A kinetic analysis of intramitochondrial sorting was performed. The intermediate-size form accumulated transiently in the matrix. When import was performed in the presence of metal chelators to prevent the first processing by the matrix processing peptidase, > 40% of the imported precursor was localized in the matrix. A deletion of 13 amino acids in the intermembrane space sorting sequence caused partial inhibition of the first processing, and a transient accumulation of the precursor form in the matrix was also observed. The decrease in this matrix-localized precursor form paralleled an increase in the mature-size form in the intermembrane space. A point mutation in the mitochondrial targeting sequence (N-terminal to the sorting sequence) resulted in missorting to the matrix space. Furthermore, a chimeric protein consisting of the initial 85 residues of cytochrome b2 fused to dihydrofolate reductase was partially targeted to the matrix at 15 degrees C, but not at 25 degrees C. Together, the results presented here indicate that cytochrome b2 passes through the matrix on its sorting pathway to the intermembrane space.

Amino Acid Sequence↗
PubMed1994

Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids.

Arrays of oligonucleotides corresponding to a full set of complements of a known sequence can be made in a single series of base couplings in which each base in the complement is added in turn. Coupling is carried out on the surface of a solid support such as a glass plate, using a device which applies reagents in a defined area. The device is displaced by a fixed movement after each coupling reaction so that consecutive couplings overlap only a portion of previous ones. The shape and size of the device and the amount by which it is displaced at each step determines the length of the oligonucleotides. Certain shapes create arrays of oligonucleotides from mononucleotides up to a given length in a single series of couplings. The array is used in a hybridisation reaction to a labelled target sequence, and shows the hybridisation behaviour of every oligonucleotide in the target sequence with its complement in the array. Applications include sequence comparison to test for mutation, analysis of secondary structure, and optimisation of PCR primer and antisense oligonucleotide design.

Base Sequence↗
PubMed2024

SJPedPanel: A Pan-Cancer Gene Panel for Childhood Malignancies to Enhance Cancer Monitoring and Early Detection.

PURPOSE: The purpose of the study was to design a pan-cancer gene panel for childhood malignancies and validate it using clinically characterized patient samples. EXPERIMENTAL DESIGN: In addition to 5,275 coding exons, SJPedPanel also covers 297 introns for fusions/structural variations and 7,590 polymorphic sites for copy-number alterations. Capture uniformity and limit of detection are determined by targeted sequencing of cell lines using dilution experiment. We validate its coverage by in silico analysis of an established real-time clinical genomics (RTCG) cohort of 253 patients. We further validate its performance by targeted resequencing of 113 patient samples from the RTCG cohort. We demonstrate its power in analyzing low tumor burden specimens using morphologic remission and monitoring samples. RESULTS: Among the 485 pathogenic variants reported in RTCG cohort, SJPedPanel covered 86% of variants, including 82% of 90 rearrangements responsible for fusion oncoproteins. In our targeted resequencing cohort, 91% of 389 pathogenic variants are detected. The gene panel enabled us to detect ∼95% of variants at allele fraction (AF) 0.5%, whereas the detection rate is ∼80% at AF 0.2%. The panel detected low-frequency driver alterations from morphologic leukemia remission samples and relapse-enriched alterations from monitoring samples, demonstrating its power for cancer monitoring and early detection. CONCLUSIONS: SJPedPanel enables the cost-effective detection of clinically relevant genetic alterations including rearrangements responsible for subtype-defining fusions by targeted sequencing of ∼0.15% of human genome for childhood malignancies. It will enhance the analysis of specimens with low tumor burdens for cancer monitoring and early detection.

Humans↗
PubMed1995

Regions outside of the CAAX motif influence the specificity of prenylation of G protein gamma subunits.

A family of GTP-binding regulatory proteins (G proteins) transduces signals across the plasma membrane from a large number of receptors to a smaller number of effectors. Recent studies indicate that a series of post-translational modifications are required for their association with the plasma membrane and for their function. In the case of the G protein gamma subunits, the post-translational modifications include the prenylation of a cysteine residue within a carboxyl-terminal CAAX motif. Although prenylation has been shown to involve the addition of either a C15 farnesyl or a C20 geranylgeranyl group to proteins, the structural requirements and functional consequences of adding different types of prenyl groups to various members of the gamma subunit family have not been examined. In the present study, we have employed the baculovirus expression system to study the structural requirements for attaching different types of prenyl groups to various members of the gamma subunit family. We show that the gamma 2 subunit is modified by a C20 geranylgeranyl group, consistent with the presence of a geranylgeranylation target sequence in this protein. However, we found that the gamma 1 and mutant gamma 2(Ser-71) subunits are modified by both C15 farnesyl and C20 geranylgeranyl groups, despite the presence of an accepted farnesylation target sequence in both of these proteins. Using chimeras of the gamma 1 and gamma 2 subunits, we provide evidence indicating that structural elements upstream of the carboxyl-terminal CAAX motif play a role in the recognition of members of the gamma subunit family by the appropriate insect and mammalian prenyltransferases.

Alkyl and Aryl Transferases↗
PubMed1991

Isolation and sequence determination of cDNA encoding T-protein of the glycine cleavage system.

T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. A partial cDNA for chicken T-protein was isolated by screening a chicken liver cDNA library with oligonucleotide probes based on amino acid sequences. The clone (CT5C) contains a 675-base pair insert encoding the C-terminal half of the T-protein. Screening of a bovine liver lambda gt10 cDNA library with the insert of CT5C as a probe detected a clone, BT5A, with an insert of about 2 kilobase pairs. The 1191-base pair coding region encodes a precursor protein of 397 amino acids (Mr 42,852) comprised of a 22-residue mitochondrial targeting sequence and a 375-residue mature protein (Mr 40,534). The 33 amino acids immediately following the targeting sequence correspond exactly to those determined by sequence analysis of the amino terminus of the purified bovine T-protein. The mature protein contains several hydrophilic segments with a cluster of arginine and lysine. The T-protein cDNA probe hybridized to an mRNA species of about 2 kilobases in bovine brain, lung, heart, and liver. A probe for H-protein hybridized with two species of mRNA in these tissues and weak signals were also found in spleen. Although the enzymatic activities of T-protein and H-protein were found in these tissues where transcripts were found, activity of P-protein was detected only in liver and brain. Southern blot analysis of genomic DNA suggested that T-protein is a single copy gene.

Amino Acid Sequence↗
PubMed1995

Sequence-specific targeting of nuclear signal transduction pathways by homeodomain proteins.

Cells translate extracellular signals into specific programs of gene expression that reflect their developmental history or identity. We present evidence that one way this interpretation may be performed is by cooperative interactions between serum response factor (SRF) and certain homeodomain proteins. We show that human and Drosophila homeodomain proteins of the paired class have the ability to recruit SRF to DNA sequences not efficiently recognized by SRF on its own, thereby imparting to a linked reporter gene the potential to respond to polypeptide growth factors. This activity requires both the DNA-binding activity of the homeodomain and putative protein-protein contact residues on the exposed surfaces of homeodomain helices 1 and 2. The ability of the homeodomain to impart signal responsiveness is DNA sequence specific, and this specificity differs from the simple DNA-binding specificity of the homeodomain in vitro. The homeodomain imparts response to a spectrum of signals characteristic of the natural SRF-binding site in the c-fos gene. Response to some of these signals is dependent on the secondary recruitment of SRF-dependent ternary complex factors, and we show directly that a homeodomain can promote the recruitment of one such factor, Elk1. We infer that SRF and homeodomains interact cooperatively on DNA and that formation of SRF-homeodomain complexes permits the recruitment of signal-responsive SRF accessory proteins. The ability to route extracellular signals to specific target genes is a novel activity of the homeodomain, which may contribute to the identity function displayed by many homeodomain genes.

Animals↗
PubMed1995

Helix-stabilizing agent, CC-1065, enhances suppression of translation by an antisense oligodeoxynucleotide.

The antitumor antibiotic CC-1065 is known to bind at selected sequences in the minor groove of duplex nucleic acids and to hyperstabilize the duplexes against thermal melting. These properties suggested that CC-1065 may enhance translation inhibition by antisense oligonucleotides directed against a specific mRNA. A 585 bp mRNA transcript containing the equine infectious anemia virus (EIAV) S2 gene and a portion of the env gene was prepared. Also, a complementary 20 mer antisense oligodeoxynucleotide (5'-TGTTGGGTAATAGG-GGTTGA-3') was prepared against a target sequence in the mRNA located near the translational initiation sites of the overlapping S2 and env genes. The center of the target sequence had an expected CC-1065 recognition sequence (5'-UAUUA-3'). Translation in the presence of CC-1065 and antisense was markedly suppressed compared with that of antisense alone. Addition of a sense 20 mer strand, with or without CC-1065, had little or no effect on translation. CC-1065 and related compounds may be useful as ligands for enhancing the stability of sense-antisense duplexes and for promoting the inhibition of translation by antisense oligonucleotides.

Antibiotics, Antineoplastic↗
PubMed1994

Fluorescence energy transfer between two triple helix-forming oligonucleotides bound to duplex DNA.

An 11-mer oligopyrimidine was covalently linked via its 5'-phosphate to an acridine derivative (acridine-11-mer), and a 13-mer was covalently linked via its 3'-phosphate to an ethidium derivative (13-mer-ethidium). Each of them formed a triple helix with a 31-bp DNA fragment containing two oligopurine-oligopyrimidine sequences, 11 and 13 bp in length, separated by a variable number of base pairs. When both oligonucleotides were bound to the 31-bp DNA fragment, fluorescence energy transfer (FET) from acridine to ethidium was observed, as revealed by a quenching of acridine fluorescence and a sensitized ethidium emission. FET was temperature-dependent and occurred only when both oligonucleotides were simultaneously bound to the DNA matrix. A single base-pair change in one of the target sequences strongly reduced the energy-transfer efficiency. This method was used to discriminate between a fully complementary and a mismatched target sequence.

Base Sequence↗
PubMed1992

The polymerase chain reaction: an epidemiological tool to differentiate between two clusters of pathogenic Yersinia enterocolitica strains.

A primer set designed to amplify the enterotoxin (yst) gene of pathogenic Yersinia enterocolitica strains generated two different electrophoretic profiles of the target sequence when a collection of strains of worldwide origin was screened. Serovars O:1,3; O:2a,3; O:3; O:5,27 and O:9, known as European strains, produced a 200-bp fragment that matched the size of the target sequence. However, serovars O:4,32; O:8; O:13a,13b; O:20 and O:21, known as American strains, generated two fragments of 1.4 and 1.6 kb. The amplified products of one American strain were sequenced and the presence of the yst gene was confirmed in both fragments. Thus, the potential of the polymerase chain reaction to be used as an epidemiological tool in differentiation between the two clusters of pathogenic strains of Y. enterocolitica could be demonstrated.

Base Sequence↗
PubMed1995

COOH-terminal sequence motifs target the T cell protein tyrosine phosphatase to the ER and nucleus.

The noncatalytic domain of the human T cell protein tyrosine phosphatase (TCPTP) is alternatively spliced to generate a 45-kD form, p45TC, and a 48-kD form, p48TC (Champion-Arnaud et al., 1991; Mosinger et al., 1992). This manuscript concerns structural motifs in the noncatalytic segment of the enzyme responsible for targeting the two forms to different subcellular compartments. Endogenous and transiently expressed p48TC associates with the ER, as determined by sucrose gradient fractionation and indirect immunofluorescence, respectively. By contrast, p45TC localizes in the nucleus even though upon cell lysis it is not retained and fractionates with markers for soluble enzymes. Using fusion proteins consisting of beta-galactosidase and COOH-terminal fragments of p48TC, two motifs necessary for ER retention within a 70-residue targeting segment have been identified. These include the terminal 19 hydrophobic residues which comprise a potential membrane-spanning segment and residues 346-358 which encompass a cluster of basic amino acids that may represent another type of ER retention motif. The sequence RKRKR, which immediately precedes the splice junction, functions as a nuclear localization signal for p45TC.

Alternative Splicing↗
PubMed1992

Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA.

Carrier cattle infected with Babesia bovis are difficult to detect because of the low numbers of parasites that occur in peripheral blood. However, diagnosis of low-level infections with the parasite is important for evaluating the efficacies of vaccines and in transmission and epidemiological studies. We used the polymerase chain reaction (PCR) to amplify a portion of the apocytochrome b gene from the parasite and tested the ability of this method to detect carrier cattle. The target sequence is associated with a 7.4-kb DNA element in undigested B. bovis genomic DNA (as shown previously), and the amplified product was detected by Southern and dot blot hybridization. The assay was specific for B. bovis, since no amplification was detected with Babesia bigemina, Trypanosoma brucei, Anaplasma marginale, or leukocyte DNA. The target sequence was amplified in DNA from B. bovis Mexico, Texas, and Australia S and L strains, demonstrating the applicability of the method to strains from different geographic regions. The sensitivity of the method ranged from 1 to 10 infected erythrocytes extracted from 0.5 ml of blood. This sensitivity was about 1,000 times greater than that from the use of unamplified parasite DNA. By the PCR method, six B. bovis carrier cattle were detected 86% of the time (range, 66 to 100%) when they were tested 11 times, while with microscopic examination of thick blood smears, the same carrier cattle were detected only 36% of the time (range, 17 to 66%). The method provides a useful diagnostic tool for detecting B. bovis carrier cattle, and the sensitivity is significantly improved over that of current methods. The results also suggest that characteristics of the apocytchrome b gene may make this a valuable target DNA for PCR-based detection of other hemoparasites.

Animals↗
PubMed1990

Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes.

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif+-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10(4) cells.

Actinomycetales↗
PubMed1991

Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis.

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype.

Animals↗
PubMed1992

A rapamycin-selective 25-kDa immunophilin.

FKBP25, a previously uncharacterized 25-kDa FK506- and rapamycin-binding protein, was purified to homogeneity from calf thymus, brain, and spleen, and the sequence of a 215 amino acid (aa) 24-kDa C-terminal peptide was established. The N-terminal domain (101 aa) is unrelated to any known protein, is hydrophilic, and is predicted by circular dichroism spectroscopy to be largely alpha-helix. The C-terminal domain (114 aa) is homologous to FKBP12 and other FKBPs but has a potential nuclear targeting sequence and a unique insertion of seven amino acids in one of its loops. FKBP25 displays the rotamase activity characteristic of FKBPs; the activity is inhibited by the immunosuppressants rapamycin (Ki = 0.9 nM) and FK506 (Ki = 160 nM), but not cyclosporin A. The protein, its rapamycin selectivity, and the potential nuclear targeting sequence are discussed in terms of the structure of hFKBP12.

Amino Acid Sequence↗
PubMed1985

The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations.

The frequency and specificity of mutations produced in vitro by eucaryotic DNA polymerase-beta have been determined in a forward mutation assay using a 250-base target sequence in M13mp2 DNA. Homogeneous DNA polymerase-beta, isolated from four different sources, produces mutations at a frequency of 4-6%/single round of gap-filling DNA synthesis. DNA sequence analyses of 460 independent mutants resulting from this error-prone DNA synthesis demonstrate a wide variety of mutational events. Frameshift and base substitutions are made at approximately equal frequency and together comprise about 90% of all mutations. Two mutational "hot spots" for frameshift and base substitution mutations were observed. The characteristics of the mutations at these sites suggest that certain base substitution errors result from dislocation of template bases rather than from direct mispair formation by DNA polymerase-beta. When considering the entire target sequence, single-base frameshift mutations occur primarily in runs of identical bases, usually pyrimidines. The loss of a single base occurs 20-80 times more frequently than single-base additions and much more frequently than the loss of two or more bases. Base substitutions occur at many sites throughout the target, representing a wide spectrum of mispair formations. Averaged over a large number of phenotypically detectable sites, the base substitution error frequency is greater than one mistake for every 5000 bases polymerized. Large deletion mutations are also observed, at a frequency more than 10-fold over background, indicating that purified DNA polymerases alone are capable of producing such deletions. These data are discussed in relation to the physical and kinetic properties of the purified enzymes and with respect to the proposed role for this DNA polymerase in vivo.

Base Sequence↗
PubMed2025

Cell-type-specific response to silicon treatment in soybean leaves revealed by single-nucleus RNA sequencing and targeted gene editing.

Mineral nutrient uptake and deposition profoundly influence plant development, stress resilience, and productivity. Silicon (Si), though classified as a non-essential element, significantly influences a plant's physiology, particularly in fortifying defense responses and mitigating stress. While the genetic and molecular mechanisms of Si uptake and transport are well studied in monocots, particularly rice, their role in dicot species, such as soybean, remains unclear at the cellular and molecular levels. In this study, we utilized single-nucleus RNA sequencing (snRNA-seq) to dissect cellular responses to Si accumulation in soybean leaves. We identified distinct cellular populations, including a unique Si-induced or Si-associated cell cluster within vascular cells, suggesting a specialized mechanism of Si distribution. Si treatment notably induced the expression of defense-related genes, with a pronounced enrichment in vascular cells, underscoring their pivotal role in activating plant defense mechanisms. Moreover, Si modulated the expression of genes involved in phytoalexin biosynthesis, salicylic acid, and immune receptor signaling, suggesting transcriptional priming of genes involved in defense responses. Further investigation of Si transporters revealed precise expression of an Si efflux gene in epidermal cells in response to Si treatment. We also validated the role of efflux Si transporters using a Xenopus oocyte assay and CRISPR/Cas9 genome editing of composite soybean plant roots. This study provides critical insights into the biotic stress regulatory networks influenced by Si treatment in soybean leaves at the single-cell level, thus laying the foundation for enhancing stress tolerance through optimized mineral nutrient uptake.

Glycine max↗
PubMed1987

Efficient splicing of two yeast mitochondrial introns controlled by a nuclear-encoded maturase.

bI4 maturase encoded by the fourth intron of the yeast mitochondrial cytochrome b gene, controls the splicing of both the fourth intron of the cytochrome b gene and the fourth intron of the gene encoding subunit I of cytochrome oxidase. It has been shown previously that a cytoplasmically translated hybrid protein composed of the pre-sequence of subunit 9 of Neurospora ATPase fused to a part of the bI4 maturase can be guided to mitochondria where it could compensate maturase deficiencies. This in vivo complementation of maturase mutants can be easily estimated by restoration of respiration. This work examines the efficiency of different bI4 maturase constructions to restore respiration in different yeast maturase-deficient strains. It is shown that the N-terminal end of the bI4 maturase plays a crucial role in the maturase activity. Moreover, the 12 N-terminal amino acids of the mitochondrial outer membrane protein constitute the most efficient mitochondrial targeting sequence in this system. Surprisingly enough, it was found that the cytoplasmically translated bI4 maturase containing the 254 C-terminal amino acid coded by the intron open reading frame can complement maturase mutations without any added mitochondrial-targeting sequence.

Base Sequence↗
PubMed1995

Bacterial expression, characterization and DNA binding studies on Drosophila melanogaster c-Myb DNA-binding protein.

The Drosophila Myb homologue retains an evolutionarily conserved typical sequence of three imperfect tandem tryptophan repeat units (R1-R2-R3) of 51-53 amino acids towards its N-terminus as its presumptive DNA binding domain. Using PCR amplification and the T7 expression vector pET 11d, we have overproduced this tryptophan repeat domain of Drosophila Myb in Escherichia coli and the protein has been purified. Circular dichroic measurements indicate that the protein has a high helical component (58.6%) in its overall structure. The protein is found to recognize the same cognate target sequence TAACGG, as recognized by the vertebrate proteins. The DNA binding properties of the protein have been investigated in detail by fluorescence spectroscopy taking advantage of the large number of tryptophan residues present in the protein. The fluorescence of the native Drosophila R123 was quenched when synthetic duplex DNA oligomers were added to the protein. The oligomers containing specific Myb target sites quenched the protein fluorescence to a greater extent than the non-specific DNA. Binding constants of the protein to the targets were also length dependent for smaller oligomers. Experiments with the collisional quencher acrylamide and cysteine modification reagent indicated that the specific and non-specific target sequences interact with the protein differently. In the former case both the buried and the exposed tryptophan residues were affected by DNA binding whereas in the latter only the solvent-exposed residues were involved.

Animals↗