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At least 163 records · Page 9Linked to original sources
PubMed2005

Structural, functional, and genetic analysis of sorangicin inhibition of bacterial RNA polymerase.

A combined structural, functional, and genetic approach was used to investigate inhibition of bacterial RNA polymerase (RNAP) by sorangicin (Sor), a macrolide polyether antibiotic. Sor lacks chemical and structural similarity to the ansamycin rifampicin (Rif), an RNAP inhibitor widely used to treat tuberculosis. Nevertheless, structural analysis revealed Sor binds in the same RNAP beta subunit pocket as Rif, with almost complete overlap of RNAP binding determinants, and functional analysis revealed that both antibiotics inhibit transcription by directly blocking the path of the elongating transcript at a length of 2-3 nucleotides. Genetic analysis indicates that Rif binding is extremely sensitive to mutations expected to change the shape of the antibiotic binding pocket, while Sor is not. We suggest that conformational flexibility of Sor, in contrast to the rigid conformation of Rif, allows Sor to adapt to changes in the binding pocket. This has important implications for drug design against rapidly mutating targets.

Aminoglycosides↗
PubMed2006

The missense genetic polymorphisms of human CYP2A13: functional significance in carcinogen activation and identification of a null allelic variant.

Cytochrome P450 2A13 (CYP2A13), an enzyme predominantly expressed in human respiratory tissues, is highly efficient for the metabolic activation of two suspected human lung carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and aflatoxin B1 (AFB1). Functional genetic polymorphisms of CYP2A13 may therefore be an important factor in human susceptibility to related lung cancers. Among the reported CYP2A13 polymorphisms with missense variations, only CYP2A13*2 variant (containing either a single or double variation of R25Q and R257C) was studied for its NNK-metabolizing activity. The present study demonstrated that there was no remarkable difference in AFB1- and NNK-induced toxicity between the Flp-In Chinese Hamster Ovary (CHO) cells stably expressing wild-type CYP2A13 and the cells expressing the individual polymorphic variants R25Q, D158E, R257C, R25Q/R257C, V323L, F453Y, and R494C. In contrast, cells transfected with R101Q variant complementary DNA (cDNA), same as the vector control cells, showed no significant death even at highest concentrations of AFB1 (10microM) and NNK (200microM). This result correlated with the lack of CYP2A13 protein in the R101Q-CHO cells, although the genomic integration of transfected R101Q cDNA and the expression of R101Q messenger RNA were clearly demonstrated in these stable transfectants. Consistent with the possibility that the variation might reduce the protein stability, R101Q variant protein expressed in insect cells showed a loss of P450 peak and coumarin 7-hydroxylase activity as well as an increased susceptibility to limited protein digestion. Thus, the R101Q polymorphic change results in a null allelic variant of CYP2A13. Our results should be useful in designing and interpreting molecular epidemiological studies related to CYP2A13 genetic polymorphisms.

Aflatoxin B1↗
PubMed1996

The Bacillus subtilis genes for ribonucleotide reductase are similar to the genes for the second class I NrdE/NrdF enzymes of Enterobacteriaceae.

We have cloned and sequenced the nrd (nucleotide reductase) locus of Bacillus subtilis. The locus seems to be organized in an operon comprising four ORFs. The first three encode polypeptides highly similar to the product of the coding sequences characterizing the nrdEF operons of Enterobacteriaceae. The sequencing of the conditional lethal mutation ts-A13, localized in the nrdE cistron, and the lethality of insertional mutations targeted in the internal region of nrdE and nrdF, demonstrated the essential role of this locus. The fourth ORF, ymaB, part of the putative operon, which is not similar to any known protein, is also essential. The regulation of expression of the operon, monitored by lacZ transcriptional fusions, is similar to the regulation of the functionally relevant nrdAB operon of Escherichia coli. The operon was induced by thymidine starvation and its expression was directly or indirectly affected by RecA function. Genetic and functional analysis strongly indicates that in B. subtilis the class I ribonucleotide reductase encoded by this nrd operon is evolutionarily distant from the homologous class I enzyme of Enterobacteria.

Amino Acid Sequence↗
PubMed1980

Fine structure in the thermal denaturation of DNA: high temperature-resolution spectrophotometric studies.

Fine structures which appear in the optical melting profile of DNA are examined from both the experimental and theoretical aspects. After a brief historical survey of the DNA melting experiments during the pre-fine-structure era in Section II, the high temperature-resolution experimental techniques which are essential to the investigation of fine structure are described in Section III. Then, the current status of the high-resolution study is reviewed first by a phenomenological description of the melting profile (Section IV) and then of the refolding profile (Section V), where a general idea about the cooperatively melting region and several factors affecting it is given. Sections VI and VII are devoted to the review of current theoretical works. Several well-established theoretical frameworks which correlate the base sequence with the melting phenomena are examined in terms of their rigorousness and usefulness. The molecular thermodynamic parameters concerning the DNA melting which have been evaluated by several research groups are compared and discussed. Finally, in Section VIII, current ideas on the correlation between the fine structure and genetic functions and genetic maps are reviewed. Some future problems relating to the fine structure are also discussed.

Animals↗
PubMed1995

The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity.

The genetic and functional basis of phosphoribosylpyrophosphate synthetase (PRS) superactivity associated with purine nucleotide inhibitor-resistance was studied in six families with this X chromosome-linked purine metabolic and neurodevelopmental disorder. Cloning and sequencing of PRS1 and PRS2 cDNAs, derived from fibroblast total RNA of affected male patients by reverse transcription and PCR amplification, demonstrated that each PRS1 cDNA contained a distinctive single base substitution predicting a corresponding amino acid substitution in the PRS1 isoform. Overall, the array of substitutions encompassed a substantial portion of the translated sequence of PRS1 cDNA. Plasmid-mediated expression of variant PRS1 cDNAs in Escherichia coli BL21 (DE3/pLysS) yielded recombinant mutant PRS1s, which, in each case, displayed a pattern and magnitude of purine nucleoside diphosphate inhibitor-resistance comparable to that found in cells of the respective patient. Kinetic analysis of recombinant mutant PRS1s showed that widely dispersed point mutations in the X chromosome-linked PRPS1 gene encoding the PRS1 isoform result in alteration of the allosteric mechanisms regulating both enzyme inhibition by purine nucleotides and activation by inorganic phosphate. The functional consequences of these mutations provide a tenable basis for the enhanced production of phosphoribosylpyrophosphate, purine nucleotides, and uric acid that are the biochemical hallmarks of PRS superactivity.

Base Sequence↗
PubMed2005

Identification of zebrafish insertional mutants with defects in visual system development and function.

Genetic analysis in zebrafish has been instrumental in identifying genes necessary for visual system development and function. Recently, a large-scale retroviral insertional mutagenesis screen, in which 315 different genes were mutated, that resulted in obvious phenotypic defects by 5 days postfertilization was completed. That the disrupted gene has been identified in each of these mutants provides unique resource through which the formation, function, or physiology of individual organ systems can be studied. To that end, a screen for visual system mutants was performed on 250 of the mutants in this collection, examining each of them histologically for morphological defects in the eye and behaviorally for overall visual system function. Forty loci whose disruption resulted in defects in eye development and/or visual function were identified. The mutants have been divided into the following phenotypic classes that show defects in: (1) morphogenesis, (2) growth and central retinal development, (3) the peripheral marginal zone, (4) retinal lamination, (5) the photoreceptor cell layer, (6) the retinal pigment epithelium, (7) the lens, (8) retinal containment, and (9) behavior. The affected genes in these mutants highlight a diverse set of proteins necessary for the development, maintenance, and function of the vertebrate visual system.

Animals↗
PubMed2003

Genetic and functional differences between multipotent neural and pluripotent embryonic stem cells.

Stem cells (SCs) are functionally defined by their abilities to self-renew and generate differentiated cells. Although much effort has been focused on defining the common characteristics among various types of SCs, the genetic and functional differences between multipotent and pluripotent SCs have garnered less attention. We report a direct genetic and functional comparison of molecularly defined and clonally related populations of neural SCs (NSCs) and embryonic SCs (ESCs), using the Sox2 promoter for isolation of purified populations by fluorescence-activated cell sorting. A stringent expression profile comparison of promoter-defined NSCs and ESCs revealed a striking dissimilarity, and subsequent chimera analyses confirmed the fundamental differences in cellular potency between these populations. This direct comparison elucidates the molecular basis for the functional differences in pluripotent ESCs and multipotent NSCs.

Animals↗
PubMed2003

Arabidopsis phosphatidylglycerophosphate synthase 1 is essential for chloroplast differentiation, but is dispensable for mitochondrial function.

Genetic dissection of the lipid bilayer composition provides essential in vivo evidence for the role of individual lipid species in membrane function. To understand the in vivo role of the anionic phospholipid, phosphatidylglycerol, the loss-of-function mutation was identified and characterized in the Arabidopsis thaliana gene coding for phosphatidylglycerophosphate synthase 1, PGP1. This mutation resulted in pigment-deficient plants of the xantha type in which the biogenesis of thylakoid membranes was severely compromised. The PGP1 gene coded for a precursor polypeptide that was targeted in vivo to both plastids and mitochondria. The activity of the plastidial PGP1 isoform was essential for the biosynthesis of phosphatidylglycerol in chloroplasts, whereas the mitochondrial PGP1 isoform was redundant for the accumulation of phosphatidylglycerol and its derivative cardiolipin in plant mitochondrial membranes. Together with findings in cyanobacteria, these data demonstrated that anionic phospholipids play an important, evolutionarily conserved role in the biogenesis and function of the photosynthetic machinery. In addition, mutant analysis suggested that in higher plants, mitochondria, unlike plastids, could import phosphatidylglycerol from the endoplasmic reticulum.

Arabidopsis↗
PubMed2000

The N-terminal region of DNA polymerase delta catalytic subunit is necessary for holoenzyme function.

Genetic and biochemical studies have shown that DNA polymerase delta (Poldelta) is the major replicative Pol in the eukaryotic cell. Its functional form is the holoenzyme composed of Poldelta, proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C). In this paper, we describe an N-terminal truncated form of DNA polymerase delta (DeltaN Poldelta) from calf thymus. The DeltaN Poldelta was stimulated as the full-length Poldelta by PCNA in a RF-C-independent Poldelta assay. However, when tested for holoenzyme function in a RF-C-dependent Poldelta assay in the presence of RF-C, ATP and replication protein A (RP-A), the DeltaN Poldelta behaved differently. First, the DeltaN Poldelta lacked holoenzyme functions to a great extent. Second, product size analysis and kinetic experiments showed that the holoenzyme containing DeltaN Poldelta was much less efficient and synthesized DNA at a much slower rate than the holoenzyme containing full-length Poldelta. The present study provides the first evidence that the N-terminal part of the large subunit of Poldelta is involved in holo-enzyme function.

Animals↗
PubMed2001

Fibulin-1 binds the amino-terminal head of beta-amyloid precursor protein and modulates its physiological function.

Genetic studies have implicated amyloid precursor protein (APP) in the pathogenesis of Alzheimer's disease. While accumulating lines of evidence indicate that APP has various functions in cells, little is known about the proteins that modulate its biological activity. Toward this end, we employed a two-hybrid system to identify potential interacting factors. We now report that fibulin-1, which contains repetitive Ca(2+)-binding EGF-like elements, binds to APP at its amino-terminal growth factor-like domain, the region that is responsible for its neurotrophic activities. Fibulin-1 expression in the brain is confined to neurons, and is not expressed significantly by astrocytes or microglia. Direct binding of fibulin-1 to the secreted form of APP (sAPP) was demonstrated with a pull-down assay using fragments of both fibulin-1 fused with glutathione-S transferase and sAPP, produced in bacteria and yeast, respectively. The fibulin-1/sAPP heteromer was shown to form in the conditioned medium of transfected COS-7 cells. Furthermore, fibulin-1 blocks sAPP-mediated proliferation of primary cultured rat neural stem cells. These results suggest that fibulin-1 may play a significant role in modulating the neurotrophic activities of APP.

Amino Acid Sequence↗
PubMed1999

p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function.

Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. In this study, we examined the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.

Animals↗
PubMed2001

Effects of mercury contamination on the culturable heterotrophic, functional and genetic diversity of the bacterial community in soil.

This study investigates the effect of mercury contamination on the culturable heterotrophic, functional and genetic diversity of the bacterial community in soil. The changes in diversity were monitored in soil microcosms, enriched with 25 &mgr;g Hg(II) g(-1) soil, over a period of 3 months. The culturable heterotrophic diversity was investigated by colony morphology and colony appearance on solid LB medium. Functional diversity was analysed as sole carbon utilisation patterns in ECOplates. Genetic diversity was measured as bands on denaturing gradient gel electrophoresis (DGGE) gels obtained by purification of total soil DNA and amplification of bacterial 16S rDNA fragments by polymerase chain reaction. Concentrations of bioavailable and total mercury were measured throughout the experiment. The effect on the culturable heterotrophic and genetic diversity was very similar, showing an immediate decrease after mercury addition but then slowly increasing throughout the entire experimental period. Pre-exposure levels were not reached within the time span of this investigation. The DGGE band pattern indicated that a shift in the community structure was responsible for recovered diversity. When analysed by Shannon-Weaver indices, functional diversity was found to increase almost immediately after mercury addition and to remain at a level higher than the control soil for the rest of the experiment. The fraction of culturable heterotrophic bacteria increased from 1% to 10% of the total bacterial number as a result of mercury addition, and the mercury-resistant population increased to represent the entire heterotrophic population.

Journal Article↗
PubMed1977

Rh sytem. Genetics and function.

Little is known about the exact genetic control of the Rh system. The Rh antigen is a large protein molecule carrying many antigenic determinants. The structural genes controlling its production comprise a gene complex that has probably evolved by unequal crossover and mutation at the duplicated points. No convincing evidence for crossing-over within the gene complex has been found since the discovery of Rh. Studies of Rhnull families provide evidence for unlinked control genes that influence the ability of the structural Rh genes to function. It was hoped that the Rhnull bloods would provide information to establish the function of the Rh antigen, because the antigen appears to be necessary to maintain the integrity of the red cell membrane. However, the abnormalities of the Rhnull cells (increased Na+K+ pumps) have not been tied directly to their lack of Rh antigens.

Alleles↗
PubMed2005

Cardiac troponin I mutations in Australian families with hypertrophic cardiomyopathy: clinical, genetic and functional consequences.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder caused by mutations in sarcomeric proteins. Cardiac troponin I (cTnI) is a key switch molecule in the sarcomere. Mutations in cTnI have been identified in <1% of genotyped HCM families. METHODS: To study the prevalence, clinical significance and functional consequences of cTnI mutations, genetic testing was performed in 120 consecutive Australian families with HCM referred to a tertiary referral centre, and results correlated with clinical phenotype. Each cTnI mutation identified was tested in a mammalian two-hybrid system to evaluate the functional effects of these mutations on troponin complex interactions. RESULTS: Disease-causing missense mutations were identified in four families (3.3%). Two mutations were located at the same codon in exon 7 (R162G, R162P), and two in exon 8 (L198P, R204H). All four mutations change amino acid residues which are highly conserved and were not found in normal populations. Follow-up family screening has identified a total of seven clinically affected members in these four families, with a further four members who carry the gene mutation but have no clinical evidence of disease. Age at clinical presentation was variable (range 15-68 years) and the mean septal wall thickness was 19.3 +/- 4.6 mm (range 7-33 mm) in clinically affected individuals, including children. In all four families, at least one member had a sudden cardiac death event, including previous cardiac arrest, indicating a more malignant form of HCM. All four mutations disrupted functional interactions with troponin C and T and this may account for the increased severity of disease in these families. CONCLUSIONS: Gene mutations in cTnI occur in Australian families with HCM with a prevalence higher than previously reported and may be associated with a clinically more malignant course, reflecting significant disruptions to troponin complex interactions.

Adolescent↗
PubMed1987

Influences of conjugal genetic transfer functions of colicin V plasmids on adhesion of Escherichia coli to murine intestinal tissue.

Cells of strains of Escherichia coli K-12 291 bearing one of three colicin V plasmids, pF54, pH247, or pF70, were tested in comparison with cells of strain 291 for their ability to adhere to murine intestinal tissue in vitro. The plasmids were either repressed or derepressed in conjugal genetic transfer functions. The strains bearing pF54 and pH247 repressed in transfer functions (pColVF54 luminal diameter r and pColVH247 luminal diameter r) adhered in higher numbers to the murine tissue than did the host strain lacking the plasmid or the strains containing the plasmids with active transfer functions (pColVF54drd and pColVH247drd). The number of cells (CFU) of strain 291(pColVF54 luminal diameter r) adherent to the tissue was related directly to the time of incubation (up to 30 min) and to the number of cells (CFU) to which the tissues were exposed. As indicated by tests for sensitivity to F factor (F)-pilus-specific bacteriophages, the cells of strains bearing the plasmids derepressed for conjugal functions had F pili on their surfaces, while such structures were missing from cells of the parental strain (291) and the strains containing the plasmids in repressed form. This finding was supported by transmission electron microscopy of cells of strain 291, 291(pColVF54 luminal diameter r), and 291(pColVF54drd). F pili could be seen on cells of the latter strain but not on those of the parental strain or the strain bearing pColVF54 luminal diameter r. Pili other than F pili were not seen on cells of the strains bearing pF54 in either form. Strains 291(pColVF70drd) and 291(pColVF70 luminal diameter r) adhered to the tissues in numbers comparable to those of strain 291. Nevertheless, these findings are further evidence that for certain colicin V plasmids (A. M. Nilius and D. C. Savage, Infect. Immun. 43:947-953, 1984), conjugal genetic transfer functions influence properties that may be important in the pathogenesis of invasive E. coli strains.

Animals↗
PubMed2004

Genetic and functional characteristics of the human in vivo LRP1/A2MR receptor suggested as a risk marker for Alzheimer's disease and other complex (degenerative) diseases.

LDL receptor-related protein/alpha2-macroglobulin receptor (LRP1/A2MR) a multiligand receptor is considered as not only being a possible risk factor of neurodegenerative diseases like Alzheimer's disease but also as determining the progression of other complex diseases like atherosclerosis and cancer. Although a large number of in vitro studies have highlighted its functional importance, as yet not enough is known about the clinical importance of the genetic background of LRP1 in human diseases. The aim of this ex vivo/in vivo study of 448 subjects was to present data on genetic LRP1 variants of healthy European Caucasians from Central Germany. Genotype-dependent LRP1 expression was analyzed in a representative subgroup (gene expression: n = 127, protein expression: n = 44). These data were evaluated in comparison to other published clinical LRP1 studies. For 15 functionally interesting genetic variants the genotype and allele distributions of the German Caucasians were presented in relation to their in vivo LRP1 gene and protein expression. A direct influence of the LRP1 promoter polymorphism c.1-25C>G on the human in vivo LRP1 expression level was demonstrated. In an analysis of 48 further studies genomic and functional results were evaluated. The analysis especially on Alzheimers's disease partly highlighted contradictory results, but suggested that ethnic as well as genomic characteristics determine LRP1 expression and must be considered in clinical investigations on human LRP1.

Adult↗
PubMed1985

Pattern of genetic influence on pulmonary function.

Whether genetic factors influence small airway function and lung volume was examined in 20 adolescent (mean age, 16.2 +/- SD 1.1 years) monozygotic twin pairs, 11 adolescent (mean age, 16.7 +/- 0.7 years) dizygotic twin pairs, and 20 adult (mean age, 38.7 +/- 9.3 years) monozygotic twin pairs. Within-pair variances of adolescent dizygotic twins for height, FVC, FEV1/FVC, V50, V25, V50/FVC, FRC, and delta N2 were significantly larger than those of adolescent monozygotic twins, indicating that these pulmonary function variables are influenced predominantly by genetic factors in adolescence. Within-pair variances of adult monozygotic twins were similar to those of adolescent monozygotic twins in terms of V50 and V25, indicating that these pulmonary function variables are influenced predominantly by genetic factors in adulthood. These results indicate that small airway dynamics, in terms of V50 and V25, are influenced in larger part by genetic factors in adolescence as well as adulthood, whereas lung volumes in terms of FRC and FVC are controlled by genetics only in adolescence.

Adolescent↗