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John M Burke

Publications and source records attributed to John M Burke.

At least 19 recordsLinked to original sources
PubMed2006

EST databases as a source for molecular markers: lessons from Helianthus.

Expressed sequence tag (EST) databases represent a potentially valuable resource for the development of molecular markers for use in evolutionary studies. Because EST-derived markers come from transcribed regions of the genome, they are likely to be conserved across a broader taxonomic range than are other sorts of markers. This paper describes a case study in which the publicly available cultivated sunflower (Helianthus annuus) EST database was used to develop simple sequence repeat (SSR) markers for use in the genetic analysis of a rare sunflower species, Helianthus verticillatus, as well as the more widespread Helianthus angustifolius. EST-derived SSRs were found to be more than 3 times as transferable across species as compared with anonymous SSRs (73% vs. 21%, respectively). Moreover, EST-SSRs whose primers were located within protein-coding sequence were more readily transferable than those derived from untranslated regions, and the former loci were no less variable than the latter. The utility of existing EST databases as a means for facilitating population genetic analyses in plants was further explored by cross-referencing publicly available EST resources against available lists of rare or invasive flowering plant taxa. This survey revealed that more than one-third of all plant-derived EST collections of sufficient size could conceivably serve as a source of EST-SSRs for the analysis of rare, endangered, or invasive plant species worldwide.

Asteraceae↗
PubMed2006

Three conserved guanosines approach the reaction site in native and minimal hammerhead ribozymes.

Native hammerhead ribozymes contain RNA domains that enable high catalytic activity under physiological conditions, where minimal hammerheads show little activity. However, little is known about potential differences in native versus minimal ribozyme folding. Here, we present results of photocross-linking analysis of native and minimal hammerheads containing photoreactive nucleobases 6-thioguanosine, 2,6-diaminopurine, 4-thiouridine, and pyrrolocytidine, introduced at specific sites within the catalytic core. Under conditions where catalytic activity is observed, the two substrate nucleobases spanning the cleavage site approach and stack upon G8 and G12 of the native hammerhead, two conserved nucleobases that show similar behavior in minimal constructs, have been implicated in general acid-base catalysis, and are >15 A from the cleavage site in the crystal structures. Pyrrolocytidine at cleavage site position 17 forms an efficient crosslink to G12, and the crosslinked RNA retains catalytic activity. Multiple cross-linked species point to a structural rearrangement within the U-turn, positioning residue G5 in the vicinity of cleavage site position 1.1. Intriguing crosslinks were triggered by nucleotide analogues at positions distal to the crosslinked residues; for example, 6-thioguanosine at position 5 induced a crosslink between G12 and C17, suggesting an intimate functional communication among these three nucleobases. Together, these results support a model in which the native hammerhead folds to an active structure similar to that of the minimal ribozyme, and significantly different from the crystallographic structures.

Base Sequence↗
PubMed2006

Chloroplast DNA variation confirms a single origin of domesticated sunflower (Helianthus annuus L.).

Although sunflower was long thought to be the product of a single domestication in what is now the east-central United States, recent archaeological and genetic evidence have suggested the possibility of an independent origin of domestication, perhaps in Mexico. We therefore used hypervariable chloroplast simple-sequence repeat markers to search for evidence of a possible Mexican origin of domestication. This work resulted in the identification of 45 chloroplast haplotypes from 26 populations across the range of wild sunflower as well as 3 haplotypes from 15 domesticated lines, representing both primitive and improved cultivars. The 3 domesticated haplotypes were characterized by 1 primary haplotype (found at a frequency of 6.7% in the wild) as well as 2 rare haplotypes, which are most likely the products of mutation or introgression. One of these rare haplotypes was not observed in the wild, bringing the total number of haplotypes identified to 46. A principal coordinate analysis revealed the presence of 3 major haplotype clusters, one of which contained the primary domesticated haplotype, the 2 rare domesticated variants, as well as haplotypes found across much of the range of wild sunflower. The Mexican haplotypes, on the other hand, fell well outside of this cluster. Although our data do not provide insight into the specific location of sunflower domestication, the relative rarity of the primary domesticated haplotype in the wild, combined with the dissimilarity between this haplotype and those found in the Mexican populations surveyed, provides further evidence that the extant domesticated sunflowers are the product of a single domestication event somewhere outside of Mexico.

Cluster Analysis↗
PubMed2006

Cation-specific structural accommodation within a catalytic RNA.

Metal ions facilitate the folding of the hairpin ribozyme but do not participate directly in catalysis. The metal complex cobalt(III) hexaammine supports folding and activity of the ribozyme and also mediates specific internucleotide photocrosslinks, several of which retain catalytic ability. These crosslinks imply that the active core structure organized by [Co(NH3)6]3+ is different from that organized by Mg2+ and that revealed in the crystal structure [Rupert, P. B., and Ferre-D'Amare, A. R. (2001) Nature 410, 780-786] (1). Residues U+2 and C+3 of the substrate, in particular, adopt different conformations in [Co(NH3)6]3+. U+2 is bulged out of loop A and stacked on residue G36, whereas the nucleotide at position +3 is stacked on G8, a nucleobase crucial for catalysis. Cleavage kinetics performed with +2 variants and a C+3 U variant correlate with the crosslinking observations. Variants that decreased cleavage rates in magnesium up to 70-fold showed only subtle decreases or even increases in observed rates when assayed in [Co(NH3)6]3+. Here, we propose a model of the [Co(NH3)6]3+-mediated catalytic core generated by MC-SYM that is consistent with these data.

Base Sequence↗
PubMed2006

Physicochemical modelling of cell signalling pathways.

Physicochemical modelling of signal transduction links fundamental chemical and physical principles, prior knowledge about regulatory pathways, and experimental data of various types to create powerful tools for formalizing and extending traditional molecular and cellular biology.

Algorithms↗
PubMed2006

Letting the gene out of the bottle: the population genetics of genetically modified crops.

Genetically modified (GM) plants are rapidly becoming a common feature of modern agriculture. This transition to engineered crops has been driven by a variety of potential benefits, both economic and ecological. The increase in the use of GM crops has, however, been accompanied by growing concerns regarding their potential impact on the environment. Here, we focus on the escape of transgenes from cultivation via crop x wild hybridization. We begin by reviewing the literature on natural hybridization, with particular reference to gene flow between crop plants and their wild relatives. We further show that natural selection, and not the overall rate of gene flow, is the most important factor governing the spread of favorable alleles. Hence, much of this review focuses on the likely effects of transgenes once they escape. Finally, we consider strategies for transgene containment.

Crops, Agricultural↗
PubMed2005

Patterns of nucleotide diversity in wild and cultivated sunflower.

Interest in the level and organization of nucleotide diversity in domesticated plant lineages has recently been motivated by the potential for using association-based mapping techniques as a means for identifying the genes underlying complex traits. To date, however, such data have been available only for a relatively small number of well-characterized plant taxa. Here we provide the first detailed description of patterns of nucleotide polymorphism in wild and cultivated sunflower (Helianthus annuus), using sequence data from nine nuclear genes. The results of this study indicate that wild sunflower harbors at least as much nucleotide diversity as has been reported in other wild plant taxa, with randomly selected sequence pairs being expected to differ at 1 of every 70 bp. In contrast, cultivated sunflower has retained only 40-50% of the diversity present in the wild. Consistent with this dramatic reduction in polymorphism, a phylogenetic analysis of our data revealed that the cultivars form a monophyletic clade, adding to the growing body of evidence that sunflower is the product of a single domestication. Eight of the nine loci surveyed appeared to be evolving primarily under purifying selection, while the remaining locus may have been the subject of positive selection. Linkage disequilibrium (LD) decayed very rapidly in the self-incompatible wild sunflower, with the expected LD falling to negligible levels within 200 bp. The cultivars, on the other hand, exhibited somewhat higher levels of LD, with nonrandom associations persisting up to approximately 1100 bp. Taken together, these results suggest that association-based approaches will provide a high degree of resolution for the mapping of functional variation in sunflower.

Base Pairing↗
PubMed2005

Genetic consequences of selection during the evolution of cultivated sunflower.

We mapped quantitative trait loci (QTL) controlling differences in seed oil content and composition between cultivated and wild sunflower and used the results, along with those of a previous study of domestication-related QTL, to guide a genome-wide analysis of genetic variation for evidence of past selection. The effects of the seed oil QTL were almost exclusively in the expected direction with respect to the parental phenotypes. A major, oil-related QTL cluster mapped near a cluster of domestication-related QTL on linkage group six (LG06), the majority of which have previously been shown to have effects that are inconsistent with the parental phenotypes. To test the hypothesis that this region was the target of a past selective sweep, perhaps resulting in the fixation of the antagonistic domestication-related QTL, we analyzed simple sequence repeat (SSR) diversity from 102 markers dispersed throughout the sunflower genome. Our results indicate that LG06 was most likely the target of multiple selective sweeps during the postdomestication era. Strong directional selection in concert with genetic hitchhiking therefore offers a possible explanation for the occurrence of numerous domestication-related QTL with apparently maladaptive phenotypic effects.

Biological Evolution↗
PubMed2005

Model for general acid-base catalysis by the hammerhead ribozyme: pH-activity relationships of G8 and G12 variants at the putative active site.

We have used nucleobase substitution and kinetic analysis to test the hypothesis that hammerhead catalysis occurs by a general acid-base mechanism, in which nucleobases are directly involved in deprotonation of the attacking 2'-hydroxyl group and protonation of the 5'-oxygen that serves as the leaving group in the cleavage reaction. We demonstrate that simultaneous substitution of two important nucleobases, G8 and G12, with 2,6-diaminopurine shifts the pH optimum of the cleavage reaction from greater than 9.5 to approximately 6.8 in two different hammerhead constructs. Controls involving substitution with other nucleobases and combinations of nucleobases at G5, G8, and/or G12 do not show this behavior. The observed changes in the pH-rate behavior are consistent with a mechanism in which N1 protonation-deprotonation events of guanine or 2,6-diaminopurine at positions 8 and 12 are essential for catalysis. Further support for the participation of G8 and G12 comes from photochemical cross-linking experiments, which show that G8 and G12 can stack upon the two substrate nucleobases at the reactive linkage, G(or U)1.1 and C17 (Heckman, J. E., Lambert, D., and Burke, J. M. (2005) Photocrosslinking detects a compact active structure of the hammerhead ribozyme, Biochemistry 44, 4148-4156). Together, these results support a model in which the hammerhead undergoes a transient conformational change into a catalytically active structure, in which stacking of G8 and G12 upon the nucleobases spanning the cleavage site provides an appropriate architecture for general acid-base catalysis. The hammerhead and hairpin ribozymes may share similarities in the organization of their active sites and their catalytic mechanism.

Binding Sites↗
PubMed2005

Photocrosslinking detects a compact, active structure of the hammerhead ribozyme.

The hammerhead ribozyme has been intensively studied for approximately 15 years, but its cleavage mechanism is not yet understood. Crystal structures reveal a Y-shaped molecule in which the cleavage site is not ideally aligned for an S(N)2 reaction and no RNA functional groups are positioned appropriately to perform the roles of acid and base or other functions in the catalysis. If the ribozyme folds to a more compact structure in the transition state, it probably does so only transiently. We have used photocrosslinking as a tool to trap hammerhead ribozyme-substrate complexes in various stages of folding. Results suggest that the two substrate residues flanking the cleavage site approach and stack upon two guanosines (G8 and G12) in domain 2, moving 10-15 A closer to domain 2 than they appear in the crystal structure. Most crosslinks obtained with the nucleotide analogues positioned in the ribozyme core are catalytically inactive; however, one cobalt(III) hexaammine-dependent crosslink of an unmodified ribozyme retains catalytic activity and confirms the close stacking of cleavage site residue C17 with nucleotide G8 in domain 2. These findings suggest that residues involved in the chemistry of hammerhead catalysis are likely located in that region containing G8 and G12.

Animals↗
PubMed2005

Chloroplast SSR polymorphisms in the Compositae and the mode of organellar inheritance in Helianthus annuus.

Because organellar genomes are often uniparentally inherited, chloroplast (cp) and mitochondrial (mt) DNA polymorphisms have become the markers of choice for investigating evolutionary issues such as sex-biased dispersal and the directionality of introgression. To the extent that organellar inheritance is strictly maternal, it has also been suggested that the insertion of transgenes into either the chloroplast or mitochondrial genomes would reduce the likelihood of gene escape via pollen flow from crop fields into wild plant populations. In this paper we describe the adaptation of chloroplast simple sequence repeats (cpSSRs) for use in the Compositae. This work resulted in the identification of 12 loci that are variable across the family, seven of which were further shown to be highly polymorphic within sunflower (Helianthus annuus). We then used these markers, along with a novel mtDNA restriction fragment length polymorphism (RFLP), to investigate the mode of organellar inheritance in a series of experimental crosses designed to mimic the initial stages of crop-wild hybridization in sunflower. Although we cannot rule out the possibility of extremely rare paternal transmission, our results provide the best evidence to date of strict maternal organellar inheritance in sunflower, suggesting that organellar gene containment may be a viable strategy in sunflower. Moreover, the portability of these markers suggests that they will provide a ready source of cpDNA polymorphisms for use in evolutionary studies across the Compositae.

Asteraceae↗
PubMed2005

Parallel genotypic adaptation: when evolution repeats itself.

Until recently, parallel genotypic adaptation was considered unlikely because phenotypic differences were thought to be controlled by many genes. There is increasing evidence, however, that phenotypic variation sometimes has a simple genetic basis and that parallel adaptation at the genotypic level may be more frequent than previously believed. Here, we review evidence for parallel genotypic adaptation derived from a survey of the experimental evolution, phylogenetic, and quantitative genetic literature. The most convincing evidence of parallel genotypic adaptation comes from artificial selection experiments involving microbial populations. In some experiments, up to half of the nucleotide substitutions found in independent lineages under uniform selection are the same. Phylogenetic studies provide a means for studying parallel genotypic adaptation in non-experimental systems, but conclusive evidence may be difficult to obtain because homoplasy can arise for other reasons. Nonetheless, phylogenetic approaches have provided evidence of parallel genotypic adaptation across all taxonomic levels, not just microbes. Quantitative genetic approaches also suggest parallel genotypic evolution across both closely and distantly related taxa, but it is important to note that this approach cannot distinguish between parallel changes at homologous loci versus convergent changes at closely linked non-homologous loci. The finding that parallel genotypic adaptation appears to be frequent and occurs at all taxonomic levels has important implications for phylogenetic and evolutionary studies. With respect to phylogenetic analyses, parallel genotypic changes, if common, may result in faulty estimates of phylogenetic relationships. From an evolutionary perspective, the occurrence of parallel genotypic adaptation provides increasing support for determinism in evolution and may provide a partial explanation for how species with low levels of gene flow are held together.

Adaptation, Biological↗
PubMed2005

Inhibition of viral replication by ribozyme: mutational analysis of the site and mechanism of antiviral activity.

A controlled mutational study was used to determine the site and mechanism of the antiviral action of ribozymes that inhibit Sindbis virus replication. A hairpin ribozyme targeting G575 of the Sindbis virus genomic RNA was designed and cloned into a minimized alphavirus amplicon vector. Cells that were stably transfected with this construct expressed low levels of a constitutive transcript containing the ribozyme plus recognition sequences for Sindbis RNA replicase. Upon infection, the ribozyme transcript was amplified to high levels by the viral replicase, resulting in decreased viral production from infected ribozyme-expressing cells. Mutations were then introduced into the viral RNA target sequence to interfere with ribozyme binding, and compensatory changes were generated in the ribozyme recognition sequence. Single mutations in the virus or ribozyme decreased the efficacy of the ribozyme's inhibition of viral replication, and compensatory mutations restored it. To confirm that ribozyme-catalyzed RNA cleavage was actually needed for inhibition, we performed tests with a cell line expressing an inactivated ribozyme and with a virus containing a single nucleotide target mutation that allowed the ribozyme to bind but blocked cleavage at the recognition site. The results show that most of the antiviral activity of ribozymes is due to ribozyme-catalyzed cleavage at the targeted RNA sequence, but some additional inhibition seems to occur through an antisense mechanism.

Animals↗
PubMed2005

Extensive chromosomal repatterning and the evolution of sterility barriers in hybrid sunflower species.

New species may arise via hybridization and without a change in ploidy. This process, termed homoploid hybrid speciation, is theoretically difficult because it requires the development of reproductive barriers in sympatry or parapatry. Theory suggests that isolation may arise through rapid karyotypic evolution and/or ecological divergence of hybrid neospecies. Here, we investigate the role of karyotypic change in homoploid hybrid speciation by generating detailed genetic linkage maps for three hybrid sunflower species, Helianthus anomalus, H. deserticola, and H. paradoxus, and comparing these maps to those previously generated for the parental species, H. annuus and H. petiolaris. We also conduct a quantitative trait locus (QTL) analysis of pollen fertility in a BC2 population between the parental species and assess levels of pollen and seed fertility in all cross-combinations of the hybrid and parental species. The three hybrid species are massively divergent from their parental species in karyotype; gene order differences were observed for between 9 and 11 linkage groups (of 17 total), depending on the comparison. About one-third of the karyoypic differences arose through the sorting of chromosomal rearrangements that differentiate the parental species, but the remainder appear to have arisen de novo (six breakages/six fusions in H. anomalus, four breakages/three fusions in H. deserticola, and five breakages/five fusions in H. paradoxus). QTL analyses indicate that the karyotypic differences contribute to reproductive isolation. Nine of 11 pollen viability QTL occur on rearranged chromosomes and all but one map close to a rearrangement breakpoint. Finally, pollen and seed fertility estimates for F1's between the hybrid and parental species fall below 11%, which is sufficient for evolutionary independence of the hybrid neospecies.

Chromosome Mapping↗
PubMed2004

Contrasting genetic structure of adults and progeny in a Louisiana iris hybrid population.

Studies of natural hybridization have suggested that it may be a creative stimulus for adaptive evolution and speciation. An important step in this process is the establishment of fit recombinant genotypes that are buffered from subsequent recombination with unlike genotypes. We used molecular markers and a two-generation sampling strategy to infer the extent of recombination in a Louisiana iris hybrid zone consisting predominantly of Iris fulva-type floral phenotypes. Genotypic diversity was fairly high, indicating that sexual reproduction is frequent relative to clonal reproduction. However, we observed strong spatial genetic structure even after controlling for clonality, which implies a low level of pollen and seed dispersal. We therefore used cluster analysis to explore the hypothesis that the fulva-type hybrids are an admixture of groups between which there has been limited recombination. Our results indicate that several such groups are present in the population and are strongly localized spatially. This spatial pattern is not attributable strictly to a lack of mating opportunities between dissimilar genotypes for two reasons: (1) relatedness of flowering pairs was uncorrelated with the degree of overlap in flowering, and (2) paternity analysis shows that pollen movement among the outcross fraction occurred over large distances, with roughly half of all paternity attributed to pollen flow from outside the population. We also found evidence of strong inbreeding depression, indicated by contrasting estimates of the rate of self-fertilization and the average inbreeding coefficient of fulva-type hybrids. We conclude that groups of similar hybrid genotypes can be buffered from recombination at small spatial scales relative to pollen flow, and selection against certain recombinant genotypes may be as important as or more important than clonal reproduction and inbreeding.

Cluster Analysis↗
PubMed2004

Modifications and deletions of helices within the hairpin ribozyme-substrate complex: an active ribozyme lacking helix 1.

Within the hairpin ribozyme, structural elements required for formation of the active tertiary structure are localized in two independently folding domains, each consisting of an internal loop flanked by helical elements. Here, we present results of a systematic examination of the relationship between the structure of the helical elements and the ability of the RNA to form the catalytically active tertiary structure. Deletions and mutational analyses indicate that helix 1 (H1) in domain A can be entirely eliminated, while segments of helices 2, 3, and 4 can also be deleted. From these results, we derive a new active minimal ribozyme that contains three helical elements, an internal loop, and a terminal loop. A three-dimensional model of this truncated ribozyme was generated using MC-SYM, and confirms that the catalytic core of the minimized construct can adopt a tertiary structure that is very similar to that of the nontruncated version. A new strategy is described to study the functional importance of various residues and chemical groups and to identify specific interdomain interactions. This approach uses two physically separated and truncated domains derived from the minimal motif.

Nucleic Acid Conformation↗
PubMed2004

Comparative mapping and rapid karyotypic evolution in the genus helianthus.

Comparative genetic linkage maps provide a powerful tool for the study of karyotypic evolution. We constructed a joint SSR/RAPD genetic linkage map of the Helianthus petiolaris genome and used it, along with an integrated SSR genetic linkage map derived from four independent H. annuus mapping populations, to examine the evolution of genome structure between these two annual sunflower species. The results of this work indicate the presence of 27 colinear segments resulting from a minimum of eight translocations and three inversions. These 11 rearrangements are more than previously suspected on the basis of either cytological or genetic map-based analyses. Taken together, these rearrangements required a minimum of 20 chromosomal breakages/fusions. On the basis of estimates of the time since divergence of these two species (750,000-1,000,000 years), this translates into an estimated rate of 5.5-7.3 chromosomal rearrangements per million years of evolution, the highest rate reported for any taxonomic group to date.

Biological Evolution↗