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PubMed2004

Crystallization of CcdB in complex with a GyrA fragment.

Plasmid addiction systems consist of a plasmid-encoded toxin-antidote pair that serves to stabilize low-copy-number plasmids in bacterial populations. CcdB, the toxin from the ccd system on the Escherichia coli F plasmid, acts as a gyrase poison. A 14 kDa fragment of gyrase, GyrA14, was found to bind to the toxin CcdB with an affinity of 1.75 x 10(-8) M. Crystals of the (GyrA14)(2) dimer in its free state belong to space group P4(3)2(1)2, with unit-cell parameters a = 86.4, c = 89.4 angstroms, and diffract to 2.4 angstroms. Crystals of the (GyrA14)(2)-(CcdB)(2) complex belong to space group P2(1)2(1)2(1), with a = 52.1, b = 83.3, c = 110.9 angstroms, and diffract to 2.8 angstroms resolution.

Bacterial Proteins↗

PubMed1990

Disruption of the gene encoding subunit VI of yeast cytochrome bc1 complex causes respiratory deficiency of cells with reduced cytochrome c levels.

A double mutant of Saccharomyces cerevisiae, in which CYCL gene is deleted and the chromosomal copy of the 17 kDa protein gene is disrupted, has been constructed. This mutant cannot grow on nonfermentable carbon sources, but normal growth can be restored by complementation of either mutation with a yeast vector containing either the wild-type 17 kDa protein gene or the CYCl gene. These results show that although the 17 kDa protein, subunit VI of yeast cytochrome bc1 complex is dispensable for yeast mitochondrial respiration in cells with the wild-type levels of cytochrome c, the 17 kDa protein is essential for respiration when the level of cytochrome c is limited, indicating that is plays a role in electron transport. This glycerol- phenotype of the double mutant can serve as the basis for further genetic studies on the function of the 17 kDa protein in yeast mitochondria and may provide insight into the physiological function of the hinge protein, the counterpart of the yeast 17 kDa protein, in beef heart mitochondria.

Blotting, Southern↗

PubMed1996

Genomic structure, chromosomal localization, start of transcription, and tissue expression of the human p40-phox, a new component of the nicotinamide adenine dinucleotide phosphate-oxidase complex.

p40-phox is a newly isolated cytosolic component of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase that copurifies with p67-phox. Although its function is not well defined, preliminary evidence indicates that it is a component of the cytosolic complex. We report the characterization of the human p40-phox gene, which is single copy and spans approximately 18 kb with 10 exons. Based on fluorescent in situ hybridization (FISH) studies and analysis of somatic hybrid cell lines, the chromosomal location of p40-phox is human chromosome 22q13.1. The start of transcription has been mapped to bp -156. The expression of p40-phox message is restricted to hematopoietic cells. In addition to identifying the mRNA transcript on Northern blot analysis in cells known to express components of the NADPH-oxidase, polymorphonuclear leukocytes, monocytes, B lymphoblastoid cell lines, and eosinophils, p40-phox is also expressed in two other cell types of white cell lineage, mast cells, and basophils. In addition, the mRNA for p40-phox is expressed in megakaryocytic cells, but not in erythroid cells.

Base Sequence↗

PubMed1992

VACUOLAR-TYPE H+-TRANSLOCATING ATPases IN PLANT ENDOMEMBRANES: SUBUNIT ORGANIZATION AND MULTIGENE FAMILIES.

Acidification of endomembrane compartments by the vacuolar-type H+-translocating ATPase (V-ATPase) is vital to the growth and development of plants. The V-ATPase purified from oat roots is a large complex of 650x10(3 )Mr that contains 10 different subunits of 70, 60, 44, 42, 36, 32, 29, 16, 13 and 12x10(3 )Mr. This set of ten polypeptides is sufficient to couple ATP hydrolysis to proton pumping after reconstitution of the ATPase into liposomes. Unlike some animal V-ATPases, the purified and reconstituted V-ATPase from oat is directly stimulated by Cl-. The peripheral complex of the ATPase includes the nucleotide-binding subunits of 70 and 60x10(3 )Mr and polypeptides of 44, 42, 36 and 29x10(3 )Mr. Six copies of the 16x10(3 )Mr proteolipid together with three other polypeptides are thought to make up the integral sector that forms the H+-conducting pathway. Release of the peripheral complex from the native membrane completely inactivates the pump; however, the peripheral subunits can be reassembled with the membrane sector to form a functional H+ pump. Comparison of V-ATPases from several plants indicates considerable variations in subunit composition. Hence, several forms of the V-ATPase may exist among, and probably within, plant species. At least four distinct cDNAs encode the 16x10(3 )Mr proteolipid subunit in oat. Multiple genes could encode different subtypes of the H+ pump that are regulated by the developmental stage and physiological function specific to the cell or tissue type.

Journal Article↗

PubMed2004

Structural basis for the interaction of Escherichia coli NusA with protein N of phage lambda.

The C terminus of transcription factor NusA from Escherichia coli comprises two repeat units, which bind during antitermination to protein N from phage lambda. To delineate the structural basis of the NusA-lambdaN interaction, we attempted to crystallize the NusA C-terminal repeats in complex with a lambdaN peptide (residues 34-47). The two NusA domains became proteolytically separated during crystallization, and crystals contained two copies of the first repeat unit in contact with a single lambdaN fragment. The NusA modules employ identical regions to contact the peptide but approach the ligand from opposite sides. In contrast to the alpha-helical conformation of the lambdaN N terminus in complex with boxB RNA, residues 34-40 of lambdaN remain extended upon interaction with NusA. Mutational analyses indicated that only one of the observed NusA-lambdaN interaction modes is biologically significant, supporting an equimolar ratio of NusA and lambdaN in antitermination complexes. Solution studies indicated that additional interactions are fostered by the second NusA repeat unit, consistent with known compensatory mutations in NusA and lambdaN. Contrary to the RNA polymerase alpha subunit, lambdaN binding does not stimulate RNA interaction of NusA. The results demonstrate that lambdaN serves as a scaffold to closely oppose NusA and the mRNA in antitermination complexes.

Amino Acid Sequence↗

PubMed1993

Visuo-spatial neglect: a new copying test to assess perceptual parsing.

A new copying test has been devised to explore different forms of perceptuo-motor impairment in visuo-spatial neglect. The stimuli are constructed so that one hypercomplex figure can be transformed into two complex figures by deletion of part of the original drawing. Task performance by five patients with left neglect after right hemisphere damage is reported. Their copies illustrate the varied spatial "reference frames" within which visual neglect can be manifest.

Aged↗

PubMed2026

Optical genome mapping enhanced by refined variant interpretation in pediatric acute lymphoblastic leukemia.

Reliable detection of structural variants (SVs) and copy number variations (CNVs) is crucial in the contemporary diagnostics of pediatric B-cell acute lymphoblastic leukemia (B-ALL). However, limitations of commonly used conventional and molecular cytogenetic methods may hinder the accurate genetic characterization of patients. Optical genome mapping (OGM) offers a reliable alternative by enabling high-resolution, genome-wide detection of CNVs and SVs. Chromosomal aberrations were screened using OGM in 51 children with B-ALL. The results were compared with those of karyotyping, fluorescence in situ hybridization (FISH), digital multiplex ligation-dependent probe amplification (digitalMLPA), and targeted RNA sequencing (RNA-seq). OGM data showed high congruency with karyotyping and FISH findings, detecting clinically relevant variants beyond G-banding results and unraveling a complex KMT2A fusion undetected by FISH. Gene fusions involved in complex ETV6::RUNX1 translocations, but not detected by RNA-seq, were confirmed using FISH. Normalization of OGM copy number values with DNA-index-improved concordance with FISH-derived copy numbers in near-tri/tetraploid cases. In the peripheral regions of OGM variants (fringe-zones), a novel evaluation strategy called 'FriZone' was applied, which significantly improved the concordance between OGM and digitalMLPA. In addition, a co-segregation analysis revealed strong associations between ETV6::RUNX1 fusion and deletions of ETV6, RAG2, and NR3C2. OGM uncovered complex rearrangements undetected by widely used methods in 15% of cases, improving genetic classification and risk stratification in 10% of the patients. The FriZone analysis and normalization by DNA-index provide a refined, more accurate approach to OGM variant interpretation, facilitating the efficient application of OGM in clinical diagnostics. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Humans↗

PubMed1995

The duplicated Saccharomyces cerevisiae gene SSM1 encodes a eucaryotic homolog of the eubacterial and archaebacterial L1 ribosomal proteins.

A previously unknown Saccharomyces cerevisiae gene, SSM1a, was isolated by screening for high-copy-number suppressors of thermosensitive mutations in the RNA14 gene, which encodes a component from the polyadenylation complex. The SSM1 a gene codes for a 217-amino-acid protein, Ssm1p, which is significantly homologous to eubacterial and archaebacterial ribosomal proteins of the L1 family. Comparison of the Ssm1p amino acid sequence with that of eucaryotic polypeptides with unknown functions reveals that Ssm1p is the prototype of a new eucaryotic protein family. Biochemical analysis shows that Ssm1p is a structural protein that forms part of the largest 60S ribosomal subunit, which does not exist in a pool of free proteins. SSM1 a is duplicated. The second gene copy, SSM1b, is functional and codes for an identical and functionally interchangeable Ssm1p protein. In wild-type cells, SSM1b transcripts accumulate to twice the level of SSM1a transcripts, suggesting that SSM1b is responsible for the majority of the Ssm1p pool. Haploid cells lacking both SSM1 genes are inviable, demonstrating that, in contrast with its Escherichia coli homolog, Ssm1p is an essential ribosomal protein. Deletion of the most expressed SSM1b gene leads to a severe decrease in the level of SSM1 transcript, associated with a reduced growth rate. Polysome profile analysis suggests that the primary defect caused by the depletion in Ssm1p is at the level of translation initiation.

5'-Nucleotidase↗

PubMed1996

IRS-Bubble PCR: An Effective Method for Representative Amplification of Human Genomic DNA Sequences from Complex Sources

A rate-limiting step in the analysis of large segments of genomic DNA is the generation of a set of representative short single-copy sequences that can be used for development of fine-structure maps. In direct response to this need, we have developed interspersed repetitive DNA sequence (IRS)-bubble PCR. IRS-bubble PCR was designed to amplify the human DNA content of somatic cell hybrids, yeast artificial chromosomes (YACs), BACs, PACs, cosmids, and lambda phage and to result in greater complexity and representation than standard inter-IRS PCR. Here, we describe the application of IRS-bubble PCR to the generation of complex clone libraries for targeted STS development and the construction of robust hybridization probes for FISH mapping, "chromosome painting," or the assembly of cosmid contigs representing the human DNA content of somatic cell hybrids or YACs.

Journal Article↗

PubMed2006

Atypical strains of Aeromonas salmonicida contain multiple copies of insertion element ISAsa4 useful as a genetic marker and a target for PCR assay.

The species Aeromonas salmonicida includes a quite complex group of pathogens that cause a variety of diseases in fishes. Best studied strains of this species are those of the subspecies salmonicida also referred to as 'typical' A. salmonicida, which cause furunculosis in salmonids. Less completely understood are bacteria assigned to other subspecies, e.g. achromogenes and masoucida, or those that cannot be assigned to a recognized subspecies. These strains are referred to collectively as 'atypical' A. salmonicida and cause diseases distinct from furunculosis, primarily affecting non-salmonids. In the course of a study to investigate the suitability of the gene product of tapA as a subunit vaccine, we discovered several atypical strains of A. salmonicida in which the tapA gene was interrupted by an insertion sequence (IS). Subsequent Southern blot analyses indicated that nearly all atypical strains (27 of 29) examined carry many copies of this IS, which we named ISAsa4. Genetic characterization of this IS element revealed it to be a member of the IS5 family, subgroup IS903. Aside from the presence of ISAsa4 in several atypical strains, the nucleotide sequence of tapA was virtually identical to that found in typical strains. This finding suggests that ISAsa4 might be a major source of genetic diversity among atypical strains which, unlike typical strains, are genetically heterogeneous. The presence of ISAsa4 in atypical strains may also help explain the host tropism of atypical strains of this bacterium. Using information on the nucleotide sequences of ISAsa4 from atypical strains of A. salmonicida, primers were designed to selectively amplify genomic DNA from most atypical strains.

Aeromonas salmonicida↗

PubMed2026

Spatial Total RNA Sequencing of Formalin-Fixed Paraffin-Embedded Tissue by spRandom-seq.

The molecular pathogenesis of infectious diseases and cancer is orchestrated by nanoscale of host and microbial RNA transcripts within the tissue microenvironment. Nevertheless, spatially resolving the comprehensive transcriptional landscape within complex clinical tissues, like formalin-fixed paraffin-embedded (FFPE) specimens, still poses a formidable challenge. Here, we present spRandom-seq, a random primer-based spatial total RNA sequencing technology designed to spatially resolve complete transcriptomes from host, bacteria, and even nanoscale viruses in FFPE tissues. Capitalizing on the random primer design, our technology not only facilitated the discovery of specific lncRNAs and alternative splicing events in mouse brain and olfactory bulb, but also delineated pronounced spatial heterogeneity in clinical FFPE sections-across distinct tumor regions in breast cancer and microbial infection sites in Klebsiella pneumoniae-infected tissues. Importantly, integrated analysis of host and viral RNAs in FFPE samples from hepatitis B virus (HBV)‑positive hepatocellular carcinoma (HCC) demonstrated that complement and coagulation pathways were specifically activated across expansive HBV‑infected tumor areas, which also exhibited an increased burden of copy number variations (CNVs). Owing to its compatibility with existing spatial transcriptomics platforms and minimal operational complexity, spRandom-seq represents a practical and scalable approach for clinical pathology applications and infection diagnostics.

Paraffin Embedding↗

PubMed1995

Structure of HIV-1 reverse transcriptase in a complex with the non-nucleoside inhibitor alpha-APA R 95845 at 2.8 A resolution.

BACKGROUND: HIV-1 reverse transcriptase (RT) is a multifunctional enzyme that copies the RNA genome of HIV-1 into DNA. It is a heterodimer composed of a 66 kDa (p66) and a 51 kDa (p51) subunit. HIV-1 RT is a crucial target for structure-based drug design, and potent inhibitors have been identified, whose efficacy, however, is limited by drug resistance. RESULTS: The crystal structure of HIV-1 RT in complex with the non-nucleoside inhibitor alpha-anilinophenyl-acetamide (alpha-APA) R95845 has been determined at 2.8 A resolution. The inhibitor binds in a hydrophobic pocket near the polymerase active site. The pocket contains five aromatic amino acid residues and the interactions of the side chains of these residues with the aromatic rings of non-nucleoside inhibitors appear to be important for inhibitor binding. Most of the amino acid residues where mutations have been correlated with high levels of resistance to non-nucleoside inhibitors of HIV-1 RT are located close to alpha-APA. The overall fold of HIV-1 RT in complex with alpha-APA is similar to that found when in complex with nevirapine, another non-nucleoside inhibitor, but there are significant conformational changes relative to an HIV-1 RT/DNA/Fab complex. CONCLUSIONS: The non-nucleoside inhibitor-binding pocket has a flexible structure whose mobility may be required for effective polymerization, and may be part of a hinge that permits relative movements of two subdomains of the p66 subunit denoted the 'palm' and 'thumb'. An understanding of the structure of the inhibitor-binding pocket, of the interactions between HIV-1 RT and alpha-APA, and of the locations of mutations that confer resistance to inhibitors provides a basis for structure-based design of chemotherapeutic agents for the treatment of AIDS.

Acetamides↗

PubMed2006

CARAT: a novel method for allelic detection of DNA copy number changes using high density oligonucleotide arrays.

BACKGROUND: DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell. RESULTS: We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100,000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods. CONCLUSION: Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.

Algorithms↗

PubMed2006

The F-box protein, Ufo1, maintains genome stability by recruiting the yeast mating switch endonuclease, Ho, for rapid proteasome degradation.

We describe a unique E3, the F-box protein, Ufo1, of yeast. Ufo1 recruits the mating switch endonuclease, Ho, to the SCF complex for ubiquitylation. In addition to the F-box and WD40 protein-protein interaction domains found in all F-box proteins, Ufo1 has a unique domain comprising multiple copies of the ubiquitin-interacting motif. Ufo1 interacts with the UbL-UbA protein, Ddi1, via its UIMs, and this is required for turnover of SCFUfo1 complexes. This is a novel function for an UbL-UbA protein. Deletion of the genomic UFO1UIMs is lethal and our data indicate that Ufo1deltaUIM acts as a dominant negative leading to inhibition of the SCF pathway of substrate degradation and to cell cycle arrest. Furthermore, we found that Ddi1 is required for the final stages of degradation of Ho endonuclease. In the absence of Ddi1, Ho does not form a complex with the 19S RP and is stabilized. Stabilization of Ho leads to perturbation of the cell cycle and to the formation of multi-budded cells. Our experiments uncover a novel role for the ubiquitin-proteasome system in maintenance of genome stability.

Cell Cycle↗

PubMed1987

A general method for cloning DNA fragments in multiple copies.

A general method for the cloning of DNA fragments in tandem arrays is presented. Synthetic directional adapters are attached to the fragment ends to establish complete control over fragment orientation during ligation. Use of these directional adapters thereby ensures the production of direct repeats, an arrangement essential for clone stability. The technique involves a protocol that is both less complex and less time-consuming than previous methods. Using this technique, a 10-min ligation has yielded a plasmid containing 20 copies of a 151-bp fragment.

Cloning, Molecular↗

PubMed2002

In vivo topography of Rap1p-DNA complex at Saccharomyces cerevisiae TEF2 UAS(RPG) during transcriptional regulation.

We have analyzed in detail the structure of RAP1-UAS(RPG) complexes in Saccharomyces cerevisiae cells using multi-hit KMnO(4), UV and micrococcal nuclease high-resolution footprinting. Three copies of the Rap1 protein are bound to the promoter simultaneously in exponentially growing cells, as shown by KMnO(4) multi-hit footprinting analysis, causing extended and diagnostic changes in the DNA structure of the region containing the UAS(RPG). Amino acid starvation does not cause loss of Rap1p from the complex; however, in vivo UV-footprinting reveals the occurrence of structural modifications of the complex. Moreover, low-resolution micrococcal nuclease digestion shows that the chromatin of the entire region is devoid of positioned nucleosomes but is susceptible to changes in accessibility to the nuclease upon amino acid starvation. The implications of these results for the mechanism of Rap1p action are discussed.

Amino Acids↗

PubMed2025

Computational Insights on the Assembly of the Dengue Virus Membrane-Capsid-RNA Complex.

Dengue virus, an arbovirus from the genus Flavivirus in the family Flaviviridae, forms a nucleocapsid structure through interactions between its genome and multiple copies of the capsid protein. Experimental studies have confirmed the interaction between the viral capsid protein and lipid droplets, indicating a protein-lipid interaction. Cryo-EM studies show that in immature viruses, the nucleocapsid is located close to the viral membrane. This study uses multiple MD simulations to explore the orientation of the capsid protein relative to the lipid membrane, focusing on how the protein's hydrophobic pocket interacts with the membrane. We also investigated the interaction between the capsid protein and RNA, considering the effects of sequence length and identity. Finally, we construct a model of the lipid-protein-RNA complex, demonstrating that the capsid protein's hydrophobic pocket interacts with the membrane, while the positively charged H4 helix interacts with the negatively charged RNA. This research may identify crucial interactions for immature virus particle formation and provide insights for future therapeutic interventions.

Dengue Virus↗

PubMed2001

cDNA cloning of the chicken branched-chain alpha-keto acid dehydrogenase complex. Chicken-specific residues of the acyltransferase affect the overall activity and the interaction with the dehydrogenase.

Branched-chain alpha-keto acid dehydrogenase complex is a macromolecule comprising three catalytic components: a dehydrogenase (E1) with alpha(2)beta(2) structure, an acyltransferase (E2) and a dihydrolipoamide dehydrogenase (E3). In the mammalian complex, the E2 component with 24 identical subunits forms a structural core, to which multiple copies of E1 and E3 bind noncovalently. We isolated cDNA clones encoding E1 alpha, E1 beta and E2 subunits from a chicken-liver cDNA library and performed nucleotide sequencing. Amino-acid sequences deduced from the nucleotide sequences revealed that chicken E1 alpha and E1 beta chains had substantially homologous sequences with the corresponding mammalian polypeptides, except for the N-terminus. Chicken E2 conserved three functional domains, a lipoyl-bearing domain, an E1/E3 binding domain and an inner-core domain, but contrasted strongly with mammalian E2 in respect of containing 11 additional residues in two interdomain linkers: nine sequential residues in one linker and two residues in the other. Replacement of many residues was also observed in the chicken linkers. When E2 activity for catalyzing the overall reaction was measured by activity reconstitution in combination with E1 and E3, chicken E2 was markedly less effective than mammalian E2. The capability of chicken E2 for binding E1 was also reduced when determined by the binding assay using sucrose density gradient centrifugation. Chicken E1 was functionally as well as structurally indistinguishable from mammalian E1. Thus the reduced catalytic activity of chicken E2 must arise from its reduced E1-binding capacity, which results from the characteristic structure of interdomain linkers in chicken E2.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗