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PubMed1995

Detection of Mycobacterium bovis in tissues by polymerase chain reaction.

A polymerase chain reaction (PCR) test was developed to detect Mycobacterium bovis in tissues. The test was based on amplification of a 248 bp segment of the insertion sequence, IS1081, present in six copies in strains of M. bovis and other members of the tuberculosis complex. The procedure involved digestion with proteinase K, lysis with sodium dodecyl sulphate, and extraction with hexadecyl tetramethyl ammonium bromide and phenol:chloroform:iso-amyl alcohol. When agarose gel electrophoresis was used for detection, the method was able to detect 1 fg of pure DNA, or 0.2 genome equivalents. It could also detect as few as 10 organisms from pure cultures and between 200-500 organisms from tissues spiked with cultured organisms. Detection by hybridization was only marginally more sensitive. The method was tested on 110 selected tissues recovered post mortem from a variety of animals. Fifty three of 58 samples diagnosed as M. bovis culture positive, including all samples containing microscopically visible acid-fast bacilli, were positive on duplicate testing by PCR. Five of 52 culture negative samples were also positive by PCR including three which contained large numbers of acid-fast organisms. Ten of the culture negative samples came from animals in a herd known to be free of bovine tuberculosis and all these were negative by PCR.

Animals↗

PubMedDate not supplied

Congenital heart defects in siblings with partial aneuploidy of chromosome 2p.

Cytogenetic anomalies are found in 13% of children with congenital heart disease, providing positional information that may aid in identifying genes governing cardiac development. We report two children born to a clinically normal female with a balanced chromosomal rearrangement, resulting from insertion of 2p24.2-p25.1 into chromosome 1. Each of her offspring had complex congenital heart disease, including left ventricular hypoplasia, and inherited a different copy number of the 2p segment. These findings suggest that proper dosage of a gene or genes in this region is important in normal cardiac development.

Aneuploidy↗

PubMed2005

Structural basis for the function of the ribosomal L7/12 stalk in factor binding and GTPase activation.

The L7/12 stalk of the large subunit of bacterial ribosomes encompasses protein L10 and multiple copies of L7/12. We present crystal structures of Thermotoga maritima L10 in complex with three L7/12 N-terminal-domain dimers, refine the structure of an archaeal L10E N-terminal domain on the 50S subunit, and identify these elements in cryo-electron-microscopic reconstructions of Escherichia coli ribosomes. The mobile C-terminal helix alpha8 of L10 carries three L7/12 dimers in T. maritima and two in E. coli, in concordance with the different length of helix alpha8 of L10 in these organisms. The stalk is organized into three elements (stalk base, L10 helix alpha8-L7/12 N-terminal-domain complex, and L7/12 C-terminal domains) linked by flexible connections. Highly mobile L7/12 C-terminal domains promote recruitment of translation factors to the ribosome and stimulate GTP hydrolysis by the ribosome bound factors through stabilization of their active GTPase conformation.

Amino Acid Sequence↗

PubMed2006

Integration of a pAL2-1 homologous mitochondrial plasmid associated with life span extension in Podospora anserina.

We isolated and characterized a novel spontaneous longevity mutant of Podospora anserina strain Wa32 carrying one of the pAL2-1 homologous mitochondrial plasmids. This mutant is at least ten fold longer-lived than the wild type, and is hence a formal suppressor of both the regular and the 'plasmid-based' senescence process. We show that the longevity trait is maternally inherited and coincides with the presence of a copy of the plasmid integrated in the 5' UTR of the mitochondrial Complex I genes nd2 and nd3. This mutation is associated with complex alterations in the respiratory chain, including a dispensable induction of the alternative oxidase. It is also associated with a stabilization of the mitochondrial chromosome and a reduction of the overall cellular level of reactive oxygen species.

5' Untranslated Regions↗

PubMed2005

Identification and characterization of purine nucleoside transporters from Crithidia fasciculata.

To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nucleoside transporter family, and the genes isolated encompassed polypeptides of 497 and 502 amino acids, respectively, each with 11 predicted membrane-spanning domains. Heterologous expression of CfNT1 cRNA in Xenopus laevis oocytes or CfNT2 in nucleoside transport-deficient Leishmania donovani demonstrated that CfNT1 is a novel high affinity adenosine transporter that also recognizes inosine, hypoxanthine, and pyrimidine nucleosides, while CfNT2 is a high affinity permease specific for inosine and guanosine. Southern blot analysis revealed that CfNT2 is present as a single copy within the C. fasciculata genome. Starvation of parasites for purines increased CfNT2 transport activity by an order of magnitude, although Northern blot analysis indicated CfNT2 transcript levels increased by <2-fold. These data imply that this metabolic adaptation can mainly be ascribed to post-transcriptional events. Conversely, Southern analysis of CfNT1 suggests that it is a member of a highly homologous multi-copy gene family, indicating that adenosine transport by C. fasciculata is more complex than previously thought.

Adenosine↗

PubMed2006

Structure of the bacteriophage phi6 nucleocapsid suggests a mechanism for sequential RNA packaging.

Bacteriophage phi6 is an enveloped dsRNA virus with a segmented genome. Phi6 specifically packages one copy of each of its three genome segments into a preassembled polymerase complex. This leads to expansion of the polymerase complex, minus and plus strand RNA synthesis, and assembly of the nucleocapsid. The phi6 in vitro assembly and packaging system is a valuable model for dsRNA virus replication. The structure of the nucleocapsid at 7.5 A resolution presented here reveals the secondary structure of the two major capsid proteins. Asymmetric P1 dimers organize as an inner T = 1 shell, and P8 trimers organize as an outer T = 13 laevo shell. The organization of the P1 molecules in the unexpanded and expanded polymerase complex suggests that the expansion is accomplished by rigid body movements of the P1 monomers. This leads to exposure of new potential RNA binding surfaces to control the sequential packaging of the genome segments.

Bacteriophage phi 6↗

PubMed2000

Cytogenetics of the chronic myeloid leukemia-derived cell line K562: karyotype clarification by multicolor fluorescence in situ hybridization, comparative genomic hybridization, and locus-specific fluorescence in situ hybridization.

The transformation of chronic myeloid leukemia (CML) from a chronic phase to an acute phase is frequently accompanied by additional chromosome changes. Extensive chromosome G-banded studies have revealed the secondary changes are nonrandom and frequently include trisomy 8, isochromosome 17q, trisomy 19, or an extra copy of the Philadelphia chromosome. In addition to these secondary chromosome changes, complex structural rearrangements often occur to form marker structures that remain unidentified by conventional G-banded analysis. The CML-derived cell line, K562, has been widely used in research since it was originally established in 1975. The K562 karyotype however, has remained incomplete, and marker structures have never been fully described. Recent advances in fluorescence in situ hybridization (FISH) technology have introduced the possibility of chromosome classification based on 24-color chromosome painting (M-FISH). In this study, we report a clarified karyotype for K562 obtained by a combination of the following molecular cytogenetic techniques: comparative genomic hybridization (CGH), FISH mapping using locus-specific probes, and M-FISH. Multicolor FISH has identified the marker structures in this cell line. The characteristic marker chromosome in K562 has been confirmed by this study to be a der(18)t(1;18). Multicolor FISH confirmed the identity of marker structures partially identified by G-banding as der(6)t(6;6),der(17)t(9;17),der(21)t(1;21),der(5)t(5;6). In addition M-FISH has revealed a deleted 20q and a complex small metacentric marker comprised of material from chromosomes 1, 6, and 20. A cryptic rearrangement was revealed between chromosomes 12 and 21 that produced a structure that looks like a normal chromosome 12 homologue by G-banding analysis. Finally, M-FISH detected regions from chromosome 13 intercalated into two acrocentric markers.

Chromosome Aberrations↗

PubMed2003

Gene cloning, purification, and stoichiometry quantification of phi29 anti-receptor gp12 with potential use as special ligand for gene delivery.

Bacterial virus phi29 is the most efficient in vitro DNA packaging system, with which up to 90% of the added DNA can be packaged into purified recombinant procapsid in vitro. The findings that phi29 virions can be assembled with the exclusive use of cloned gene products have bred a thought that phi29 has a potential to be a gene delivery vector since it is a nonpathogenic virus. gp12 of bacterial virus phi29 has been reported to be the anti-receptor that is responsible for binding the virus particle to the host cell. We cloned the gene coding gp12, overexpressed it in Escherichia coli, and purified the gene product to study the properties and functions of gp12 in virus assembly. According to SDS PloyAcrylamide Gel Electrophoresis (SDS-PAGE) analysis and N-terminal sequencing, recombinant gp12 isolated from E. coli had a molecular mass of 80 kDa, and 24 amino acids at N-terminal were cleaved after expression. The purified recombinant gp12 was incorporated into phi29 particles and converted the gp12-lacking assembly intermediates of phi29 into infectious virions in vitro. This purified protein gp12 was able to compete with infectious phi29 virions for binding to the host cell, thus inhibiting the infection by phi29. Scanning Transmission Electron Microscopy (STEM) analysis and sedimentation studies revealed that recombinant gp12 products were assembled into biologically active dimers. Analysis of the dose-response curve showed that 12 dimeric gp12 complexes were assembled onto viral particles and that each virion contained 24 copies of gp12 molecules. The results provide a basis for future research into bacteriophage-host interaction by modifying the anti-receptor protein. The ultimate goal is to re-target the bacteriophage to new host cells for the purpose of gene delivery.

Amino Acid Sequence↗

PubMed2001

Regulation and functions of myogenic regulatory factors in lower vertebrates.

The transcription factors of the MyoD family have essential functions in myogenic lineage determination and muscle differentiation. These myogenic regulatory factors (MRFs) activate muscle-specific transcription through binding to a DNA consensus sequence known as the E-box present in the promoter of numerous muscle genes. Four members, MyoD, myogenin, myf5 and MRF4/herculin/myf6, have been identified in higher vertebrates and have been shown to exhibit distinct but overlapping functions. Homologues of these four MRFs have also been isolated in a variety of lower vertebrates, including amphibians and fish. Differences have been observed, however, in both the expression patterns of MRFs during muscle development and the function of individual MRFs between lower and higher vertebrates. These differences reflect the variety of body muscle formation patterns among vertebrates. Furthermore, as a result of an additional polyploidy that occurred during the evolution of some amphibians and fish, MyoD, myogenin, myf5 and MRF4 may exist in lower vertebrates in two distinct copies that have evolved separately, acquiring specific roles and resulting in increased complexity of the myogenic regulatory network. Evidence is now accumulating that many of the co-factors (E12, Id, MEF2 and CRP proteins) that regulate MRF activity in mammals are also present in lower vertebrates. The inductive signals controlling the initial expression of MRFs within the developing somite of lower vertebrate proteins are currently being elucidated.

Animals↗

PubMed2000

Crystal structure of the complex of brugia malayi cyclophilin and cyclosporin A.

The resistance of the human parasite Brugia malayi to the antiparasitic activity of cyclosporin A (CsA) may arise from the presence of cyclophilins with relatively low affinity for the drug. The structure of the complex of B. malayi cyclophilin (BmCYP-1) and CsA, with eight independent copies in the asymmetric unit, has been determined at a resolution of 2.7 A. The low affinity of BmCYP-1 for CsA arises from incomplete preorganization of the binding site so that the formation of a hydrogen bond between His132 of BmCYP-1 and N-methylleucine 9 of CsA is associated with a shift in the backbone of approximately 1 A in this region.

Amino Acid Sequence↗

PubMedDate not supplied

A conserved DNA sequence in homoeotic genes of the Drosophila Antennapedia and bithorax complexes.

A repetitive DNA sequence has been identified in the Drosophila melanogaster genome that appears to be localized specifically within genes of the bithorax and Antennapedia complexes that are required for correct segmental development. Initially identified in cloned copies of the genes Antennapedia, Ultrabithorax and fushi tarazu, the sequence is also contained within two other DNA clones that have characteristics strongly suggesting that they derive from other homoeotic genes.

Animals↗

PubMed1990

Immunological analysis of acetyl-CoA carboxylase mass, tissue distribution and subunit composition.

Changes in the mass and subunit structure of liver acetyl-CoA carboxylase (ACC) accompany altered nutrition in vivo. Enzyme activity in different tissues and cell lines is also, in part, determined by variations in both total mass and ACC isoenzyme composition. ACC isoenzyme mass and hetero/homo-isoenzyme association were quantified by three sandwich e.l.i.s.a. assays, i.e. an avidin-based assay that measured total isoenzyme mass and two antibody-sandwich assays which measure polypeptide association. Results from the avidin-based assay reveal that the two major isoenzymes, of molecular mass 265 kDa (ACC 265) and 280 kDa (ACC 280), are present in markedly variable concentration in several rat and mouse tissues and in cell lines of rat and mouse origin. Hepatic ACC mass has been reported to be distributed between mitochondrial and cytosolic fractions and to undergo only a change in subcellular distribution without alteration in total mass on induction/repression of activity in vivo [Roman-Lopez, Shriver, Joseph & Alfred (1989) Biochem. J. 260, 927-930]. However, in the present study, immunoblotting and e.l.i.s.a. analysis reveals that, in rat liver, the mass of both isoenzymes is predominantly cytosolic in distribution, is markedly diminished on fasting and rises 6-8-fold on refeeding of a high-carbohydrate diet. These data support the results of several other investigations of hepatic ACC mass, and are consistent with known nutritionally altered changes in ACC mRNA content. By the two antibody-sandwich e.l.i.s.a. assays, isoenzyme complexes either composed of both ACC 280 and 265 or with multiple copies of ACC 265 are detectable in rat liver enzyme; their concentration varies independently of total ACC mass with the nutritional state of the rat, being lowest in fasting and highest on fasting/refeeding. E.l.i.s.a. analysis, applicable to crude tissue/cell extracts, provides a simple, sensitive and quantitative measurement of ACC mass and subunit composition. Its use may permit needed quantitative insight into the role of variable total ACC and isoenzyme mass and of alterations in ACC subunit composition that occur in vivo or in isolated cells in response to a variety of hormonal and nutritional influences.

Acetyl-CoA Carboxylase↗

PubMed1996

Gene identification in a complex chromosomal continuum by local genomic cross-referencing.

Most higher plants have complex genomes containing large quantities of repetitive DNA interspersed with low-copy-number sequences. Many of these repetitive DNAs are mobile and have homology to RNAs in various cell types. This can make it difficult to identify the genes in a long chromosomal continuum. It was decided to use genic sequence conservation and grass genome co-linearity as tools for gene identification. A bacterial artificial chromosome (BAC) clone containing sorghum genomic DNA was selected using a maize Adh1 probe. The 165 kb sorghum BAC was tested for hybridization to a set of clones representing the contiguous 280 kb of DNA flanking maize Adh1. None of the repetitive maize DNAs hybridized, but most of the low-copy-number sequences did. A low-copy-number sequence that did cross-hybridize was found to be a gene, while one that did not was found to be a low-copy-number retrotransposon that was named Reina. Regions of cross-hybridization were co-linear between the two genomes, but closer together in the smaller sorghum genome. These results indicate that local genomic cross-referencing by hybridization of orthologous clones can be an efficient and rapid technique for gene identification and studies of genome organization.

Alcohol Dehydrogenase↗

PubMed1985

Mouse DNA polymerase alpha-primase terminates and reinitiates DNA synthesis 2-14 nucleotides upstream of C2A1-2(C2-3/T2) sequences on a minute virus of mice DNA template.

The distribution of termination and initiation sites in a 5081-nucleotide minute virus of mice DNA template being copied by a highly purified mouse DNA polymerase alpha-DNA primase complex in the presence of GTP has been examined. The 3'-hydroxyl termini (17 in all) were clustered at six sites that were located 2-14 nucleotides upstream of C2A2C2, C2AC3, or C2A2T2 sequences. When either [alpha-32P]- or [gamma-32P]GTP was included in the DNA polymerase reaction mixtures, nascent DNA became radiolabeled. Analysis of the 32P-labeled material following treatment of the DNA with tobacco acid pyrophosphatase, bacterial alkaline phosphatase, or ribonuclease T1 revealed the presence of oligoribonucleotide chains averaging 5-7 nucleotides long and beginning with 5' GTP residues. Eight presumptive DNA primase initiation sites were located opposite C4 or C5 sequences 3-9 nucleotides upstream of one of the three closely related hexanucleotides C2A2C2, C2AC3, and C2A2T2. RNA-DNA junctions were found 3-10 nucleotides downstream of DNA primase initiation sites. The results indicate that hexanucleotides having the general formula C2A1-2(C2-3/T2), herein referred to as psi, are involved in promoting termination of DNA synthesis and/or de novo initiation of RNA-primed DNA chains by DNA polymerase alpha-primase.

Animals↗

PubMed2004

The functional domains of bacteriophage t4 terminase.

The packaging of double-stranded genomic DNA into some viral and all bacteriophage capsids is driven by powerful molecular motors. In bacteriophage T4, the motor consists of the portal protein assembly composed of twelve copies of gene product 20 (gp20, 61 kDa) and an oligomeric terminase complex composed of gp16 (18 kDa) and gp17 (70 kDa). The packaging motor drives the 171-kbp T4 DNA into the capsid utilizing the free energy of ATP hydrolysis. Evidence suggests that gp17 is the key component of the motor; it exhibits ATPase, nuclease, and in vitro DNA-packaging activities. The N- and C-terminal halves of gp17 were expressed and purified to homogeneity and found to have ATPase and nuclease activities, respectively. The N-terminal domain exhibited 2-3-fold higher Kcat values for gp16-stimulated ATPase than the full-length gp17. Neither of the domains, individually or together, exhibited in vitro DNA-packaging activity, suggesting that communication between the domains is essential for DNA packaging. The domains, in particular the C-terminal domain or a mixture of both the N- and C-terminal domains, inhibited in vitro DNA packaging that is catalyzed by full-length gp17. In conjunction with genetic evidence, these data suggest that the domains compete with the full-length gp17 for binding sites on the portal protein. A model for the assembly of the T4 DNA-packaging machine is presented.

Adenosine Triphosphatases↗

PubMed2006

Identification of a pH sensor in the furin propeptide that regulates enzyme activation.

The folding and activation of furin occur through two pH- and compartment-specific autoproteolytic steps. In the endoplasmic reticulum (ER), profurin folds under the guidance of its prodomain and undergoes an autoproteolytic excision at the consensus furin site Arg-Thr-Lys-Arg107/ generating an enzymatically masked furin-propeptide complex competent for transport to late secretory compartments. In the mildly acidic environment of the trans-Golgi network/endosomal system, the bound propeptide is cleaved at the internal site 69HRGVTKR75/, unmasking active furin capable of cleaving substrates in trans. Here, by using cellular, biochemical, and modeling studies, we demonstrate that the conserved His69 is a pH sensor that regulates the compartment-specific cleavages of the propeptide. In the ER, unprotonated His69 stabilizes a solvent-accessible hydrophobic pocket necessary for autoproteolytic excision at Arg107. Profurin molecules unable to form the hydrophobic pocket, and hence, the furin-propeptide complex, are restricted to the ER by a PACS-2- and COPI-dependent mechanism. Once exposed to the acidic pH of the late secretory pathway, protonated His69 disrupts the hydrophobic pocket, resulting in exposure and cleavage of the internal cleavage site at Arg75 to unmask the enzyme. Together, our data explain the pH-regulated activation of furin and how this His-dependent regulatory mechanism is a model for other proteins.

Amino Acid Sequence↗

PubMed2005

Novel SRESPHP peptide mediates specific binding to primary medullary thyroid carcinoma after systemic injection.

The efficient and specific introduction of genes into cancer cells in vivo remains a major challenge for current gene therapy modalities. Peptides possess appropriate properties to serve as tumor-targeting agents. Thus, finding new cancer-selective peptides directing gene transfer to neoplastic cells by reducing transduction of normal cells is a central goal for molecular targeting. We have previously reported identification of a peptide (HTFEPGV) that selectively binds to human medullary thyroid carcinoma (MTC)-derived TT cells in vitro and transplanted tumor xenografts in vivo, using phage display. In the present study, we have performed this approach in primary orthotopically growing murine MTCs of RET-C634R transgenic mice as a clinically relevant model for thyroid cancer by intravenous injection of a complex peptide library. Two rounds of screening on primary tumors yielded multiple copies of a phage that displays a cyclic 7-amino acid peptide, SRESPHP, with a 3000-fold increase in titer between rounds 1 and 2. The selected phage showed highly specific binding to the tumor after systemic administration, whereas binding to other organs such as lung, liver, kidney, and heart was reduced up to 90%. After tail vein injection, homing to the tumor was substantially reduced in the presence of synthetic SRESPHP peptide, indicating that tumor phage interaction strictly depends on the displayed peptide. Immunohistochemical analysis of paraffin sections from mouse tissues revealed direct binding of the SRESPHP peptide to MTC tissue. Moreover, this peptide also mediates binding to human MTC cells in vitro and in vivo, suggesting abundant expression of its cognate receptor in murine and human medullary thyroid carcinoma. Because the SRESPHP peptide is also efficiently internalized into MTC cells, it likely provides the basis for a new selective therapy of medullary thyroid carcinoma.

Amino Acid Sequence↗

PubMed2026

Using cancer profiles to identify synthetic lethal therapeutic targets and predictive biomarkers in cancer gene dependency data.

MOTIVATION: Large scale loss-of-function screens utilising CRISPR or siRNA can provide profound insights into the importance of individual genes for the survival of a cancer cell and can drive the identification of therapeutic targets and biomarkers, and the development of targeted drugs. However, the analysis of these data and the substantial bodies of metadata that relate to them, is technically challenging and typically requires substantial expertise in data science and computer coding. RESULTS: To facilitate the analysis of cancer gene dependency data by cancer biologists and clinical scientists, we have developed DepMine-a computational toolkit providing a powerful system for framing complex queries relating cancer gene dependency to the underlying genetic changes that occur in cancer cells. DepMine identifies synthetic lethal relationships between putative target genes and complex 'cancer profiles' built from user-specified combinations of mutations, copy-number variation, and expression levels, and can refine these to optimal biomarker definitions for target dependency. AVAILABILITY: The Python implementation of DepMine and associated data files can be obtained at https://github.com/UOSbioinformaticslab/depmine and is free to academics and Not-For-Profit organisations. The DepMine release referenced in this paper is archived as DOI: 10.5281/zenodo.19570601.

Humans↗