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At least 829 records · Page 46Linked to original sources
PubMed2000

The growth of the dermomyotome and formation of early myotome lineages in thoracolumbar somites of chicken embryos.

Myotome formation in the epaxial and hypaxial domains of thoraco-lumbar somites was analyzed using fluorescent vital dye labeling of dermomyotome cells and cell-fate assessment by confocal microscopy. Muscle precursor cells for the epaxial and hypaxial myotomes are predominantly located in the dorsomedial and ventrolateral dermomyotome lips, respectively, and expansion of the dermomyotome is greatest along its mediolateral axis coincident with the dorsalward and ventralward growth directions of the epaxial and hypaxial myotomes. Measurements of the dermomyotome at different stages of development shows that myotome growth begins earlier in the epaxial than in the hypaxial domain, but that after an initial lag phase, both progress at the same rate. A combination of dye injection and/or antibody labeling of early and late-expressed muscle contractile proteins confirms the myotome mediolateral growth directions, and shows that the myotome thickness increases in a superficial (near dermis) to deep (near sclerotome) growth direction. These findings also provide a basis for predicting the following gene expression sequence program for the earliest muscle precursor lineages in mouse embryos: Pax-3 (stem cells), myf-5 (myoblast cells) and myoD (myocytes). The movements and mitotic activity of early muscle precursor cells lead to the conclusion that patterning and growth in the myotome specifically, and in the epaxial and hypaxial domains of the body generally, are governed by morphogenetic cell movements.

Animals↗
PubMed2004

Genetic analysis of violacein biosynthesis by Chromobacterium violaceum.

Chromobacterium violaceum presents a distinctive phenotypic characteristic, the production of a deep violet pigment named violacein. Although the physiological function of this pigment is not well understood, the sequencing of the genome of this bacterium has given some insight into the mechanisms and control of violacein production. It was found that erythrose-4-phosphate (E4P), a precursor to aromatic amino acid biosynthesis, is produced by the non-oxidative portion of the hexose monophosphate pathway, since it lacks 6-phosphogluconate dehydrogenase. All genes leading from E4P plus phosphoenolpyruvate to tryptophan are present in the genome. Nevertheless, these genes are not organized in an operon, as in E. coli, indicating that other mechanisms are involved in expression. The sequencing data also indicated the presence and organization of an operon for violacein biosynthesis. Three of the four gene products of this operon presented similarity with nucleotide-dependent monooxygenases and one with a limiting enzyme polyketide synthase. As previously suggested, genes encoding proteins involved in quorum sensing control by N-hexanoyl-homoserine-lactone, an autoinducer signal molecule, are present in the bacterial genome. These data should help guide strategies to increase violacein biosynthesis, a potentially useful molecule.

Carboxylic Ester Hydrolases↗
PubMed2005

Inheritance and genetic mapping of tuber eye depth in cultivated diploid potatoes.

Tuber eye depth of the potato (Solanum tuberosum L.) is an important trait for the processing quality and appearance of potatoes. In the present study, we used a cultivated diploid potato family (12601) of 107 plants to dissect the mode of inheritance and to map the gene(s) controlling the trait. The family segregated for both eye depth (deep vs shallow) and tuber shape (round vs long) traits. The deep eye (Eyd) phenotype was found to be associated with round tubers (Ro) in most progeny clones. Further evaluation of this population with molecular markers including simple sequence repeats, amplified fragment length polymorphism, and sequence-characterized amplified regions revealed that the primary locus for eye depth is located on chromosome 10. This map location was confirmed by evaluating a second diploid family (12586). The results of this study led to the following conclusions: (1) there is a major locus controlling the eye depth trait; (2) deep eye (Eyd) is dominant to shallow (eyd); (3) the Eyd/eyd locus is located on chromosome 10; and (4) the Eyd/eyd locus is closely linked with the major locus for tuber shape (Ro/ro), at a distance of about 4 cM.

Agriculture↗
PubMed1994

Early development of the somatotopic map and barrel patterning in rat somatosensory cortex.

Several lines of evidence implicate a crucial role for thalamic afferents from the ventroposterior nucleus (VP) in the development of barrels and their characteristic pattern in the primary somatosensory cortex (S1) of rodents. We sought to determine the stage in development when VP thalamocortical afferents are first distributed in a periphery-related pattern and the sequence of events that culminate in a mature pattern. Using acetylcholinesterase (AChE) histochemistry, an early marker for VP thalamocortical afferents, and the anterograde axon tracer DiI, we show that VP thalamocortical afferents become distributed into a periphery-related pattern earlier than was previously reported, including their parcellation into a barrel-related pattern that mirrors the distribution of sensory hairs on the face. The earliest periphery-related patterning observed is transiently present in the deep cortical layers prior to the emergence of layer 4, the layer in which barrels later develop. AChE histochemistry reveals a clear sequence of maturation of the barrel pattern in the distribution of VP afferents: An initially patternless distribution of AChE-reactive afferents is followed by their distribution in a nascent trigeminal representation, from which rows subsequently emerge; barrel-related clusters of afferents then emerge from the rows. This process begins before birth, and the transition from row-related to barrel-related distributions of VP afferents is evident during the first postnatal day (P0). This demonstration of a periphery-related pattern in developing rat S1 precedes by about 2 days that revealed by any other marker reported to delineate barrels. These findings confirm that VP thalamocortical afferents are the first barrel component to have a periphery-related pattern and support the hypothesis that thalamocortical afferents provide to immature S1 the patterning information that initiates the formation of barrels and their characteristic array. Furthermore because these findings show an earlier onset for barrel formation than was previously realized, they necessitate a reevaluation of conclusions drawn from experiments examining developmental plasticity in barrel patterning.

Acetylcholinesterase↗
PubMed2006

Sequences from 14 mitochondrial genes provide a well-supported phylogeny of the Charadriiform birds congruent with the nuclear RAG-1 tree.

Because of the difficulties of constructing a robust phylogeny for Charadriiform birds using morphological characters, recent studies have turned to DNA sequences to resolve the systematic uncertainties of family-level relationships in this group. However, trees constructed using nuclear genes or the mitochondrial Cytochrome b gene suggest deep-level relationships of shorebirds that differ from previous studies based on morphology or DNA-DNA hybridization distances. To test phylogenetic hypotheses based on nuclear genes (RAG-1, myoglobin intron-2) and single mitochondrial genes (Cytochrome b), approximately 13,000 bp of mitochondrial sequence was collected for one exemplar species of 17 families of Charadriiformes plus potential outgroups. Maximum likelihood and Bayesian analyses show that trees constructed from long mitochondrial sequences are congruent with the nuclear gene topologies [Chardrii (Lari, Scolopaci)]. Unlike short mitochondrial sequences (such as Cytochrome b alone), longer sequences yield a well-supported phylogeny for shorebirds across various taxonomic levels. Examination of substitution patterns among mitochondrial genes reveals specific genes (especially ND5, ND4, ND2, and COI) that are better suited for phylogenetic analyses among shorebird families because of their relatively homogeneous nucleotide composition among lineages, slower accumulation of substitutions at third codon positions, and phylogenetic utility in both closely and distantly related lineages. For systematic studies of birds in which family and generic levels are examined simultaneously, we recommend the use of both nuclear and mitochondrial sequences as the best strategy to recover relationships that most likely reflect the phylogenetic history of these lineages.

Animals↗
PubMed2002

Solution structure of protein SRP19 of Archaeoglobus fulgidus signal recognition particle.

Protein SRP19 is an essential RNA-binding component of the signal recognition particle (SRP) in Archaea and Eucarya. A three-dimensional solution structure of the 104 residue SRP19 from the hyperthermophilic archaeon Archaeoglobus fulgidus, designated as Af19, was determined by NMR spectroscopy. Af19 contains three beta-strands, two alpha-helical regions, arranged in a betaalphabetabetaalpha topology, a 3(10) helix, and a disordered C-terminal tail. This fold is similar to the betaalphabetabetaalphabeta RNP motif present in numerous other RNA-binding proteins, which engage their cognate RNAs using conserved sequence motifs present within beta-strands 1 and 3. Mutagenesis studies of human SRP19, however, reveal the major contact sites with SRP RNA reside within loops 1, 3, and 4. These contacts were verified by the crystal structure of human SRP19 complexed to SRP RNA helix 6 reported subsequent to the submission of the manuscript. The crystal structure also reveals that, unlike canonical RNP motifs, SRP19 does not engage specific RNA bases through conserved sequence motifs present within beta-strands 1 and 3. Instead, SRP19 uses residues both within and flanking beta-strand 1 to stabilize the complex through direct and indirect contacts to the phosphate backbone of the tetraloop, leaving the bases of the tetraloop exposed. This, coupled with the fact that SRP19 appears relatively rigid and undergoes only minor changes in structure upon RNA binding, may underlie the molecular basis by which SRP19 functions to initiate SRP assembly.

Amino Acid Sequence↗
PubMed2002

Prevalence and microdiversity of Alteromonas macleodii-like microorganisms in different oceanic regions.

The presence, prevalence and variability of microorganisms related to the species Alteromonas macleodii, a well known culturable gamma-Proteobacterium, has been studied in different seawater samples from diverse geographical locations, in both the Northern and Southern hemispheres, and tested with two molecular techniques (rRNA hybridization and gene cloning and sequencing). Results show that A. macleodii-like microorganisms are present in high proportions in North Atlantic and, especially, Mediterranean waters, being higher at deep samples and particle-associated fractions, in agreement with previous findings. In contrast, Southern samples (all from very cold areas near Antarctica) presented no significant hybridization signals. The analysis of the ribosomal ITS (16S-23S internal transcribed spacers) revealed that A. macleodii-like microorganisms from Mediterranean, North Atlantic, Caribbean and Red Sea waters differed in both size and sequence, mostly depending on their geographical origin, with Mediterranean and North Atlantic clones clustering into two main groups whereas Caribbean and Red Sea clones appeared separated.

Alteromonas↗
PubMed2003

An Escherichia coli MG1655 lipopolysaccharide deep-rough core mutant grows and survives in mouse cecal mucus but fails to colonize the mouse large intestine.

The ability of E. coli strains to colonize the mouse large intestine has been correlated with their ability to grow in cecal and colonic mucus. In the present study, an E. coli MG1655 strain was mutagenized with a mini-Tn5 Km (kanamycin) transposon, and mutants were tested for the ability to grow on agar plates with mouse cecal mucus as the sole source of carbon and nitrogen. One mutant, designated MD42 (for mucus defective), grew poorly on cecal-mucus agar plates but grew well on Luria agar plates and on glucose minimal-agar plates. Sequencing revealed that the insertion in MD42 was in the waaQ gene, which is involved in lipopolysaccharide (LPS) core biosynthesis. Like "deep-rough" E. coli mutants, MD42 was hypersensitive to sodium dodecyl sulfate (SDS), bile salts, and the hydrophobic antibiotic novobiocin. Furthermore, its LPS core oligosaccharide was truncated, like that of a deep-rough mutant. MD42 initially grew in the large intestines of streptomycin-treated mice but then failed to colonize (<10(2) CFU per g of feces), whereas its parent colonized at levels between 10(7) and 10(8) CFU per g of feces. When mouse cecal mucosal sections were hybridized with an E. coli-specific rRNA probe, MD42 was observed in cecal mucus as clumps 24 h postfeeding, whereas its parent was present almost exclusively as single cells, suggesting that clumping may play a role in preventing MD42 colonization. Surprisingly, MD42 grew nearly as well as its parent during growth in undiluted, highly viscous cecal mucus isolated directly from the mouse cecum and, like its parent, survived well after reaching stationary phase, suggesting that there are no antimicrobials in mucus that prevent MD42 colonization. After mini-mariner transposon mutagenesis, an SDS-resistant suppressor mutant of MD42 was isolated. The mini-mariner insertion was shown to be in the bipA gene, a known regulator of E. coli surface components. When grown in Luria broth, the LPS core of the suppressor mutant remained truncated; however, the LPS core was not truncated when the suppressor mutant was grown in the presence of SDS. Moreover, when the suppressor mutant was grown in the presence of SDS and fed to mice, it colonized the mouse large intestine. Collectively, the data presented here suggest that BipA may play a role in E. coli MG1655 LPS core biosynthesis and that because MD42 forms clumps in intestinal mucus, it is unable to colonize the mouse large intestine.

Animals↗
PubMed2006

Mitochondrial genomics identifies major haplogroups in Aboriginal Australians.

We classified diversity in eight new complete mitochondrial genome sequences and 41 partial sequences from living Aboriginal Australians into five haplogroups. Haplogroup AuB belongs to global lineage M, and AuA, AuC, AuD, and AuE to N. Within N, we recognize subdivisions, assigning AuA to haplogroup S, AuD to haplogroup O, AuC to P4, and AuE to P8. On available evidence, (S)AuA and (M)AuB are widespread in Australia. (P4)AuC is found in the Riverine region of western New South Wales, and was identified by others in northern Australia. (O)AuD and (P8)AuE were clearly identified only from central Australia. Our eight Australian full mt genome sequences, combined with 20 others (Ingman and Gyllensten 2003 Genome Res. 13:1600-1606) and compared with full mt genome sequences from regions to the north that include Papua New Guinea, Malaya, and Andaman and Nicobar Islands, show that ancestral connections between regions are deep and limited to clustering at the level of the N and M macrohaplogroups. The Australian-specific distribution of the five haplogroups identified indicates genetic isolation over a long period. Ancestral connections within Australia are deeper than those reflected by known linguistic or culturally based affinities. Applying a coalescence analysis to a gene tree for the coding regions of the eight genomic sequences, we made estimates of time depth that support a continuity of presence for the descendants of a founding population already established by 40,000 years ago.

DNA, Mitochondrial↗
PubMed2004

A small bacterial RNA regulates a putative ABC transporter.

A small noncoding bacterial ribonucleic acid of 62-64 nucleotides, RydC, was identified in the genomes of Escherichia coli, Salmonella, and Shigella. In vivo, RydC binds to the RNA-binding protein Hfq, and it is unstable when Hfq is absent. Mobility assays reveal that complex formation between RydC and Hfq is specific, with an apparent binding constant of approximately 300 nm. Sequence alignments combined with structural probing demonstrate that RydC folds as a pseudoknot. Hfq binds the loops crossing the deep and shallow grooves of the pseudoknotted RNA and reorganizes its overall conformation. An interaction with a polycistronic mRNA, yejABEF, which encodes a putative ABC transporter, was detected by affinity purification of immobilized RNA-Hfq complexes. In vivo, the yejABEF operon is expressed on minimal medium. Remarkably, its expression is reduced when RydC is absent, and the operon is degraded when RydC expression is stimulated. This observation correlates with the growth defects associated with a stimulation of its expression in vivo, generating a thermosensitive phenotype that affects growth on minimal media supplemented with glycerol, maltose, or ribose. We conclude that RydC regulates the yejABEF-encoded ABC permease at the mRNA level. This small RNA may contribute to optimal adaptation of some Enterobacteria to environmental conditions.

ATP-Binding Cassette Transporters↗
PubMed1986

Development of cell and fiber lamination in the mouse superior colliculus.

The emergence of laminar organization in the superior colliculus was investigated in the mouse with several anatomical methods, including tritiated-thymidine autoradiography, Golgi impregnation, and general stains for cell bodies and for fibers. The sequence of neurogenesis, cell migration, and early morphological differentiation of neurons was shown to exhibit a discontinuity between the lower and upper divisions (i.e., between the deep and intermediate "gray" and "white" layers and the superficial "gray" and "white" layers). These events proceed in an inside-out order within the lower division, but the same events within the upper division commence in advance of the completion of this progression. Thus, peak generation times for layers of the lower division proceed from (embryonic day) E11 to E13 and for the upper division from E12 to E13. Cell migration, as monitored with tritiated-thymidine labelling, reflects closely the pattern of cytogenesis. This is most clearly evident on E15 when a population of E11-labelled cells is divided into superficial and deep layers (the strata superficiale and profundum--SS and SP) by the interposition of E13-labelled cells at an intermediate level (stratum intermedium--SI). A contingent of the latter cells continue their migration and join their predecessors within the SS on E17, a time point when cell migrations are largely complete. Paralleling this sequence of arrival of neurons and the formation of three primary layers, both the time course of accumulation of fiber fascicles and the early morphological differentiation of neurons in the interval from E13 to E17 tends to proceed from SP to SS and from SS to SI. Thus, the transverse fiber system and large multipolar neurons of SP develop in advance of the longitudinal fiber system and vertically oriented neurons of SS, which in turn develop precociously with respect to the longitudinal fibers and medium-sized multipolar neurons of SI. In contrast, later events of differentiation that underly a major radial growth and an architectonic sublamination of the primary strata proceed in a simpler inside-out sequence from E17 to (postnatal day) P6. The major morphogenetic events underlying the establishment of statification in the colliculus appear to involve the operation of relatively independent programs of assembly for the two basic subdivisions. It is probable that selective cell-cell interactions contribute to the delivery of concurrently generated neurons to different laminae as well as to the deployment of axons in a manner that respects laminar boundaries.

Animals↗
PubMed2006

An unusual phylogeography in the bushcricket Ephippiger ephippiger from Southern France.

Pleistocene glaciations have played a major role in species divergence. The bushcricket Ephippiger ephippiger shows unusual patterns of intraspecific variation in multiple traits across Southern Europe. This is centred in Southern France, and evidence implies that it results from secondary contact after differentiation in Pleistocene refugia. However, the possible time scales involved, locations of the refugia and patterns of expansion remain obscure. This study sequenced the COII (507 BP) and cyt b (428 BP) mitochondrial genes to examine the intraspecific phylogeography of Western European samples of E. ephippiger. A minimum evolution tree revealed little resolution between described subspecies of E. ephippiger. Strikingly, populations from the Pyrenees and Mediterranean coastal region contained a complex genetic structure corresponding to major river valleys, independent of the traditional taxonomy. Samples of the subspecies E. e. vitium formed a distinct clade, perhaps supporting their taxonomic status. However, other forms (cruciger and cunii) were not genetically distinct, which is surprising given differences in their morphology and behaviour. The extent of the genetic divergence between Pyreneen valleys is unexpectedly deep, with average Tamura-Nei distances of around 14% (net distances of 11%) separating the main clades of coding COII sequences. Cyt b showed a similar pattern, but was confounded by some non-coding probable pseudogenes. If a conventional insect molecular clock is applied, these cryptic clades must pre-date the Pleistocene, and hypotheses for their history are discussed. However, mtDNA divergence in Ephippiger is not evolving in a clock-like manner, because a likelihood ratio test rejects clock assumptions for the COII sequences.

Animals↗
PubMed1997

Solution structure of the calicheamicin gamma 1I-DNA complex.

Calicheamicin gamma 1I is an enediyne antibiotic possessing antitumour activity associated with its ability to bind and following activation, affect double-strand cleavage at oligopyrimidine-oligopurine tracts on DNA. Footprinting and chemical modification studies have identified the (T-C-C-T).(A-G-G-A) sequence as a preferred calicheamicin gamma 1I binding site and established the importance of the 5'-guanine residue as critical for high affinity binding. The sequence specificity of intermolecular recognition has been identified with the aryltetrasaccharide component of the drug together with an important contribution from the iodine atom on the thiobenzoate ring to the affinity of complex formation. Calicheamicin gamma 1I binds to the minor groove of the DNA duplex and in the process positions the enediyne ring to abstract hydrogen atoms from partner strands leading to double-strand cleavage. We report on the solution structure of the calicheamicin gamma 1I-DNA hairpin duplex complex containing a central (T-C-C-T).(A-G-G-A) segment based on a combined analysis of NMR and molecular dynamics calculations including intensity refinement in a water box. The refined solution structures of the complex provide a molecular explanation of the sequence specificity of binding and cleavage by this member of the enediyne family of antitumor antibiotics. Calicheamicin gamma 1I binds to the DNA minor groove with its aryltetrasaccharide segment in an extended conformation spanning the (T-C-C-T).(A-G-G-A) segment of the duplex. Further, the thio sugar B molecule and the thiobenzoate ring C molecule are inserted in an edgewise manner deep into the minor groove with their faces sandwiched between the walls of the groove. A range of intermolecular hydrophobic and hydrogen-bonding interactions account for the sequence specific recognition in the complex. These include critical intermolecular contacts between the iodine and sulfur atoms of the thiobenzoate ring of the drug with the exposed exocyclic amino protons of the 5' and 3'-guanine bases, respectively, of the A-G-G-A segment on the DNA. The bound aryltetrasaccharide in turn positions the enediyne ring deep in the minor groove such that the pro-radical carbon centers of the enediyne are proximal to their anticipated proton abstraction sites. Specifically, the pro-radical C-3 and C-6 atoms are aligned opposite the abstractable H-5' (pro-S) and H-4' protons on partner strands across the minor groove, respectively, in the complex. The DNA duplex is right-handed with Watson-Crick base-pairing in the complex. The helix exhibits a B-DNA type minor groove width at the aryltetrasaccharide binding-site while there is widening of the groove at the adjacent enediyne binding-site in the complex. The DNA helix exhibits localized perturbations at the binding-site as reflected in imino proton complexation shifts and specific altered sugar pucker geometrics associated with complex formation. Sequence-specific binding of calicheamicin gamma 1I to the (T-C-C-T).(A-G-G-A) containing DNA hairpin duplex is favored by the complementarity of the fit through hydrophobic and hydrogen-bonding interactions between the drug and the floor and walls of the minor groove of a minimally perturbed DNA helix.

Aminoglycosides↗
PubMed1994

Sequence analysis of the third dnaK homolog gene in Synechococcus sp. PCC7942.

The nucleotide sequence of the third dnaK homolog gene from Synechococcus sp. PCC7942 has been determined. Deduced amino acid sequences of dnaK3 and of previously reported dnaK1 and dnaK2 were compared with Hsp70s from various species. Among the three DnaK homologs, DnaK2 shows close relationship to chloroplast proteins and DnaK3 also has some similarities to them. On the other hand, DnaK1 has few chloroplast-specific motifs and forms a deep branch of the prokaryotic cluster in the phylogenetic tree.

Amino Acid Sequence↗
PubMed1988

Carcinoma of the urinary bladder: MR imaging with a double surface coil.

Twenty-four patients with carcinoma of the urinary bladder were examined with MR imaging by using both a conventional body coil and a newly designed wraparound double surface coil. All patients had T1- and two patients had T2-weighted pulse sequences with both coils. Because of the long examination times, T2-weighted sequences in all other patients were obtained by using the double surface coil only. Nine patients underwent radical cystectomy, while the other patients had deep transurethral tumor resection and a clinical follow-up of at least 1.5 years. The results of imaging with the surface coil showed a twofold improvement of spatial resolution compared with the images obtained with the body coil. The RF field homogeneity was excellent, and the field of view was sufficiently large to achieve a complete study of the pelvis and the lower abdomen in one sequence. For the T1-weighted images, the higher spatial resolution of the double surface coil resulted in a significant (p less than .10) improvement in tumor staging. The accuracies of double-surface-coil and body-coil imaging were 79% and 54%, respectively. Double-surface-coil imaging appears to provide better MR images of the urinary bladder than body-coil imaging does, resulting in more accurate staging of tumors on T1-weighted pulse sequences.

Carcinoma, Transitional Cell↗
PubMed2001

Decoupling of molecular and morphological evolution in deep lineages of a meiobenthic harpacticoid copepod.

Molecular and biochemical genetic analyses have revealed that many marine invertebrate taxa, including some well-studied and presumably cosmopolitan species, are actually complexes of sibling species. When morphological differences are slight and estimated divergence times are old, data suggest either unusually high rates of sequence evolution or long-term morphological stasis. Here, five gene regions (mitochondrial cytochrome oxidase subunit I and large-subunit ribosomal 16S rDNA and nuclear ITS1, 5.8S rDNA, and ITS2) were analyzed in four geographic samples of the meiobenthic harpacticoid copepod Cletocamptus deitersi. Molecular sequences revealed four extremely differentiated molecular lineages with unalignable nuclear intergenic spacers and mitochondrial uncorrected divergences reaching 25% (cytochrome oxidase) and 36% (16S rDNA). These levels of divergence are greater than those reported previously for congeneric species in diverse invertebrate taxa, including crustaceans. The nominally intraspecific divergence matches or exceeds the corresponding divergence from a known congener (Cletocamptus helobius). A molecular clock applied to the cytochrome oxidase subunit I data suggests that these lineages split in the Miocene, consistent with the fossil record of a North American Cletocamptus from the same period. Morphological differences among the major lineages are subtle but congruent with the patterns of genetic differentiation. Our conclusion, based on concordant patterns of variation in two mitochondrial and three nuclear gene regions, as well as morphological observations, is that C. deitersi in North America is composed of at least four separate species by the genealogical concordance, phylogenetic, and morphological-species criteria. Alternative explanations for the deep phylogenetic nodes and apparent morphological stasis, including high rates of sequence evolution, balancing selection, and genetic signatures of historical events, are considered unlikely.

Animals↗
PubMed2004

Insights in metabolism and toxin production from the complete genome sequence of Clostridium tetani.

The decryption of prokaryotic genome sequences progresses rapidly and provides the scientific community with an enormous amount of information. Clostridial genome sequencing projects have been finished only recently, starting with the genome of the solvent-producing Clostridium acetobutylicum in 2001. A lot of attention has been devoted to the genomes of pathogenic clostridia. In 2002, the genome sequence of C. perfringens, the causative agent of gas gangrene, has been released. Currently in the finishing stage and prior to publication are the genomes of the foodborne botulism-causing C. botulinum and of C. difficile, the causative agent of a wide spectrum of clinical manifestations such as antibiotic-associated diarrhea. Our team sequenced the genome of neuropathogenic C. tetani, a Gram-positive spore-forming bacterium predominantly found in the soil. In deep wound infections it occasionally causes spastic paralysis in humans and vertebrate animals, known as tetanus disease, by the secretion of potent neurotoxin, designated tetanus toxin. The toxin blocks the release of neurotransmitters from presynaptic membranes of interneurons of the spinal cord and the brainstem, thus preventing muscle relaxation. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid, a formaldehyde-treated tetanus toxin, but nevertheless, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The World Health Organization has stated that neonatal tetanus is the second leading cause of death from vaccine preventable diseases among children worldwide. This minireview focuses on an analysis of the genome sequence of C. tetani E88, a vaccine production strain, which is a toxigenic non-sporulating variant of strain Massachusetts. The genome consists of a 2,799,250 bp chromosome encoding 2618 open reading frames. The tetanus toxin is encoded on a 74,082 kb plasmid, containing 61 genes. Additional virulence-related factors as well as an insight into the metabolic strategy of C. tetani with regard to its pathogenic phenotype will be presented. The information from other clostridial genomes by means of comparative analysis will also be explored.

Journal Article↗
PubMed1994

Molecular analysis of enrichment cultures of marine ammonia oxidisers.

Marine ammonia oxidising bacteria were enriched by incubation of sea water, amended with ammonium sulphate, and subsequent subculture in liquid inorganic medium. PCR primers were designed to be specific for rDNA sequences from ammonia oxidisers belonging to the beta-sub-group of the proteobacteria. These primers were then used to amplify rRNA genes from ammonia oxidiser enrichment cultures containing heterotrophs. PCR products were recovered from all cultures in which complete ammonia oxidation occurred. Subsequent rDNA sequence analysis indicated the presence of three new lineages within the clade defined by sequences of cultured beta-sub-group ammonia oxidisers. Two of the new lineages showed moderate similarity to sequences from pure cultures of ammonia oxidisers previously isolated from marine and brackish environments. The third lineage (AEM-3) was deep branching and occupied an intermediate position between clades defined by Nitrosomonas or Nitrosospira, which were isolated from soil or sewage. The phylogenetic analysis suggests that, in enrichment cultures, the primers are specific for members of the target group, the beta-proteobacteria ammonia oxidisers. The results also indicate the presence of previously unknown ammonia oxidisers in marine samples. The approach enabled analysis of ammonia oxidiser enrichments at an early stage and without the requirement for isolation of pure cultures, significantly reducing the time required and facilitating quantitative assessment of relatedness of strains.

Ammonia↗