Publications by authors named "Phillip San Miguel"

13 Publications

  • Page 1 of 1

Characterization of the Gray Whale Eschrichtius robustus Genome and a Genotyping Array Based on Single-Nucleotide Polymorphisms in Candidate Genes.

Biol Bull 2017 06 1;232(3):186-197. Epub 2017 Sep 1.

Genetic and genomic approaches have much to offer in terms of ecology, evolution, and conservation. To better understand the biology of the gray whale Eschrichtius robustus (Lilljeborg, 1861), we sequenced the genome and produced an assembly that contains ∼95% of the genes known to be highly conserved among eukaryotes. From this assembly, we annotated 22,711 genes and identified 2,057,254 single-nucleotide polymorphisms (SNPs). Using this assembly, we generated a curated list of candidate genes potentially subject to strong natural selection, including genes associated with osmoregulation, oxygen binding and delivery, and other aspects of marine life. From these candidate genes, we queried 92 autosomal protein-coding markers with a panel of 96 SNPs that also included 2 sexing and 2 mitochondrial markers. Genotyping error rates, calculated across loci and across 69 intentional replicate samples, were low (0.021%), and observed heterozygosity was 0.33 averaged over all autosomal markers. This level of variability provides substantial discriminatory power across loci (mean probability of identity of 1.6 × 10 and mean probability of exclusion >0.999 with neither parent known), indicating that these markers provide a powerful means to assess parentage and relatedness in gray whales. We found 29 unique multilocus genotypes represented among our 36 biopsies (indicating that we inadvertently sampled 7 whales twice). In total, we compiled an individual data set of 28 western gray whales (WGSs) and 1 presumptive eastern gray whale (EGW). The lone EGW we sampled was no more or less related to the WGWs than expected by chance alone. The gray whale genomes reported here will enable comparative studies of natural selection in cetaceans, and the SNP markers should be highly informative for future studies of gray whale evolution, population structure, demography, and relatedness.
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http://dx.doi.org/10.1086/693483DOI Listing
June 2017

An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation.

Nucleic Acids Res 2016 09 13;44(16):7605-17. Epub 2016 May 13.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA

Coordinated regulation of gene expression that involves activation of lineage specific genes and repression of pluripotency genes drives differentiation of embryonic stem cells (ESC). For complete repression of pluripotency genes during ESC differentiation, chromatin at their enhancers is silenced by the activity of the Lsd1-Mi2/NuRD complex. The mechanism/s that regulate DNA methylation at these enhancers are largely unknown. Here, we investigated the affect of the Lsd1-Mi2/NuRD complex on the dynamic regulatory switch that induces the local interaction of histone tails with the Dnmt3 ATRX-DNMT3-DNMT3L (ADD) domain, thus promoting DNA methylation at the enhancers of a subset of pluripotency genes. This is supported by previous structural studies showing a specific interaction between Dnmt3-ADD domain with H3K4 unmethylated histone tails that is disrupted by histone H3K4 methylation and histone acetylation. Our data suggest that Dnmt3a activity is triggered by Lsd1-Mi2/NuRD-mediated histone deacetylation and demethylation at these pluripotency gene enhancers when they are inactivated during mouse ESC differentiation. Using Dnmt3 knockout ESCs and the inhibitors of Lsd1 and p300 histone modifying enzymes during differentiation of E14Tg2A and ZHBTc4 ESCs, our study systematically reveals this mechanism and establishes that Dnmt3a is both reader and effector of the epigenetic state at these target sites.
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http://dx.doi.org/10.1093/nar/gkw426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027477PMC
September 2016

A Search for Parent-of-Origin Effects on Honey Bee Gene Expression.

G3 (Bethesda) 2015 Jun 5;5(8):1657-62. Epub 2015 Jun 5.

Department of Entomology, Purdue University, West Lafayette, Indiana 47907

Parent-specific gene expression (PSGE) is little known outside of mammals and plants. PSGE occurs when the expression level of a gene depends on whether an allele was inherited from the mother or the father. Kin selection theory predicts that there should be extensive PSGE in social insects because social insect parents can gain inclusive fitness benefits by silencing parental alleles in female offspring. We searched for evidence of PSGE in honey bees using transcriptomes from reciprocal crosses between European and Africanized strains. We found 46 transcripts with significant parent-of-origin effects on gene expression, many of which overexpressed the maternal allele. Interestingly, we also found a large proportion of genes showing a bias toward maternal alleles in only one of the reciprocal crosses. These results indicate that PSGE may occur in social insects. The nonreciprocal effects could be largely driven by hybrid incompatibility between these strains. Future work will help to determine if these are indeed parent-of-origin effects that can modulate inclusive fitness benefits.
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http://dx.doi.org/10.1534/g3.115.017814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528322PMC
June 2015

A snapshot of the hepatic transcriptome: ad libitum alcohol intake suppresses expression of cholesterol synthesis genes in alcohol-preferring (P) rats.

PLoS One 2014 26;9(12):e110501. Epub 2014 Dec 26.

Purdue University Interdisciplinary Life Sciences Graduate Program, Purdue University, West Lafayette, IN, United States of America; Department of Animal Science, Purdue University, West Lafayette, IN, United States of America; Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, United States of America.

Research is uncovering the genetic and biochemical effects of consuming large quantities of alcohol. One prime example is the J- or U-shaped relationship between the levels of alcohol consumption and the risk of atherosclerotic cardiovascular disease. Moderate alcohol consumption in humans (about 30 g ethanol/d) is associated with reduced risk of coronary heart disease, while abstinence and heavier alcohol intake is linked to increased risk. However, the hepatic consequences of moderate alcohol drinking are largely unknown. Previous data from alcohol-preferring (P) rats showed that chronic consumption does not produce significant hepatic steatosis in this well-established model. Therefore, free-choice alcohol drinking in P rats may mimic low risk or nonhazardous drinking in humans, and chronic exposure in P animals can illuminate the molecular underpinnings of free-choice drinking in the liver. To address this gap, we captured the global, steady-state liver transcriptome following a 23 week free-choice, moderate alcohol consumption regimen (∼ 7.43 g ethanol/kg/day) in inbred alcohol-preferring (iP10a) rats. Chronic consumption led to down-regulation of nine genes in the cholesterol biosynthesis pathway, including HMG-CoA reductase, the rate-limiting step for cholesterol synthesis. These findings corroborate our phenotypic analyses, which indicate that this paradigm produced animals whose hepatic triglyceride levels, cholesterol levels and liver histology were indistinguishable from controls. These findings explain, at least in part, the J- or U-shaped relationship between cardiovascular risk and alcohol intake, and provide outstanding candidates for future studies aimed at understanding the mechanisms that underlie the salutary cardiovascular benefits of chronic low risk and nonhazardous alcohol intake.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0110501PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277277PMC
August 2015

Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing.

mBio 2014 Jul 8;5(4):e01442-14. Epub 2014 Jul 8.

Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA

We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3' transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5' ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are complex, with internal promoters and terminators generating multiple transcription units and allowing differential gene expression within these operons. We discovered extensive antisense transcription that results from more than 500 operons, which fully overlap or extensively overlap adjacent divergent or convergent operons. The genomic regions corresponding to these antisense transcripts are highly conserved in E. coli (including Shigella species), although it remains to be proven whether or not they are functional. Our observations of features unearthed by single-nucleotide transcriptome mapping suggest that deeper layers of transcriptional regulation in bacteria are likely to be revealed in the future.
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http://dx.doi.org/10.1128/mBio.01442-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161252PMC
July 2014

RNA-seq analysis identifies potential modulators of gravity response in spores of Ceratopteris (Parkeriaceae): evidence for modulation by calcium pumps and apyrase activity.

Am J Bot 2013 Jan 9;100(1):161-74. Epub 2012 Oct 9.

The University of Texas at Austin, 1 University Station A6700, Austin, Texas 78712, USA.

Premise Of The Study: Gravity regulates the magnitude and direction of a trans-cell calcium current in germinating spores of Ceratopteris richardii. Blocking this current with nifedipine blocks the spore's downward polarity alignment, a polarization that is fixed by gravity ∼10 h after light induces the spores to germinate. RNA-seq analysis at 10 h was used to identify genes potentially important for the gravity response. The data set will be valuable for other developmental and phylogenetic studies.

Methods: De novo Newbler assembly of 958 527 reads from Roche 454 sequencing was executed. The sequences were identified and analyzed using in silico methods. The roles of endomembrane Ca(2+)-ATPase pumps and apyrases in the gravity response were further tested using pharmacological agents.

Key Results: Transcripts related to calcium signaling and ethylene biosynthesis were identified as notable constituents of the transcriptome. Inhibiting the activity of endomembrane Ca(2+)-ATPase pumps with 2,5-di-(t-butyl)-1,4-hydroquinone diminished the trans-cell current, but increased the orientation of the polar axis to gravity. The effects of applied nucleotides and purinoceptor antagonists gave novel evidence implicating extracellular nucleotides as regulators of the gravity response in these fern spores.

Conclusions: In addition to revealing general features of the transcriptome of germinating spores, the results highlight a number of calcium-responsive and light-receptive transcripts. Pharmacologic assays indicate endomembrane Ca(2+)-ATPases and extracellular nucleotides may play regulatory roles in the gravity response of Ceratopteris spores.
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http://dx.doi.org/10.3732/ajb.1200292DOI Listing
January 2013

Developmental changes in the metabolic network of snapdragon flowers.

PLoS One 2012 11;7(7):e40381. Epub 2012 Jul 11.

Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, United States of America.

Evolutionary and reproductive success of angiosperms, the most diverse group of land plants, relies on visual and olfactory cues for pollinator attraction. Previous work has focused on elucidating the developmental regulation of pathways leading to the formation of pollinator-attracting secondary metabolites such as scent compounds and flower pigments. However, to date little is known about how flowers control their entire metabolic network to achieve the highly regulated production of metabolites attracting pollinators. Integrative analysis of transcripts and metabolites in snapdragon sepals and petals over flower development performed in this study revealed a profound developmental remodeling of gene expression and metabolite profiles in petals, but not in sepals. Genes up-regulated during petal development were enriched in functions related to secondary metabolism, fatty acid catabolism, and amino acid transport, whereas down-regulated genes were enriched in processes involved in cell growth, cell wall formation, and fatty acid biosynthesis. The levels of transcripts and metabolites in pathways leading to scent formation were coordinately up-regulated during petal development, implying transcriptional induction of metabolic pathways preceding scent formation. Developmental gene expression patterns in the pathways involved in scent production were different from those of glycolysis and the pentose phosphate pathway, highlighting distinct developmental regulation of secondary metabolism and primary metabolic pathways feeding into it.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040381PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3394800PMC
March 2013

Orthologous comparisons of the Hd1 region across genera reveal Hd1 gene lability within diploid Oryza species and disruptions to microsynteny in Sorghum.

Mol Biol Evol 2010 Nov 3;27(11):2487-506. Epub 2010 Jun 3.

Department of Agronomy, Purdue University.

Heading date is one of the most important quantitative traits responsible for the domestication of rice. We compared a 155-kb reference segment of the Oryza sativa ssp. japonica cv. Nipponbare genome surrounding Hd1, a major heading date gene in rice, with orthologous regions from nine diploid Oryza species that diverged over a relatively short time frame (∼16 My) to study sequence evolution around a domestication locus. The orthologous Hd1 region from Sorghum bicolor was included to compare and contrast the evolution in a more distant relative of rice. Consistent with other observations at the adh1/adh2, monoculm1, and sh2/a1 loci in grass species, we found high gene colinearity in the Hd1 region amidst size differences that were lineage specific and long terminal repeat retrotransposon driven. Unexpectedly, the Hd1 gene was deleted in O. glaberrima, whereas the O. rufipogon and O. punctata copies had degenerative mutations, suggesting that other heading date loci might compensate for the loss or nonfunctionality of Hd1 in these species. Compared with the japonica Hd1 region, the orthologous region in sorghum exhibited micro-rearrangements including gene translocations, seven additional genes, and a gene triplication and truncation event predating the divergence from Oryza.
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http://dx.doi.org/10.1093/molbev/msq133DOI Listing
November 2010

Detailed analysis of a contiguous 22-Mb region of the maize genome.

PLoS Genet 2009 Nov 20;5(11):e1000728. Epub 2009 Nov 20.

Arizona Genomics Institute, School of Plant Sciences and Department of Ecology, BIO5 Institute for Collaborative Research, University of Arizona, Tucson, Arizona, United States of America.

Most of our understanding of plant genome structure and evolution has come from the careful annotation of small (e.g., 100 kb) sequenced genomic regions or from automated annotation of complete genome sequences. Here, we sequenced and carefully annotated a contiguous 22 Mb region of maize chromosome 4 using an improved pseudomolecule for annotation. The sequence segment was comprehensively ordered, oriented, and confirmed using the maize optical map. Nearly 84% of the sequence is composed of transposable elements (TEs) that are mostly nested within each other, of which most families are low-copy. We identified 544 gene models using multiple levels of evidence, as well as five miRNA genes. Gene fragments, many captured by TEs, are prevalent within this region. Elimination of gene redundancy from a tetraploid maize ancestor that originated a few million years ago is responsible in this region for most disruptions of synteny with sorghum and rice. Consistent with other sub-genomic analyses in maize, small RNA mapping showed that many small RNAs match TEs and that most TEs match small RNAs. These results, performed on approximately 1% of the maize genome, demonstrate the feasibility of refining the B73 RefGen_v1 genome assembly by incorporating optical map, high-resolution genetic map, and comparative genomic data sets. Such improvements, along with those of gene and repeat annotation, will serve to promote future functional genomic and phylogenomic research in maize and other grasses.
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http://dx.doi.org/10.1371/journal.pgen.1000728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773423PMC
November 2009

Comparative physical mapping between Oryza sativa (AA genome type) and O. punctata (BB genome type).

Genetics 2007 May 4;176(1):379-90. Epub 2007 Mar 4.

Arizona Genomics Institute, University of Arizona, Tucson, Arizona 85721, USA.

A comparative physical map of the AA genome (Oryza sativa) and the BB genome (O. punctata) was constructed by aligning a physical map of O. punctata, deduced from 63,942 BAC end sequences (BESs) and 34,224 fingerprints, onto the O. sativa genome sequence. The level of conservation of each chromosome between the two species was determined by calculating a ratio of BES alignments. The alignment result suggests more divergence of intergenic and repeat regions in comparison to gene-rich regions. Further, this characteristic enabled localization of heterochromatic and euchromatic regions for each chromosome of both species. The alignment identified 16 locations containing expansions, contractions, inversions, and transpositions. By aligning 40% of the punctata BES on the map, 87% of the punctata FPC map covered 98% of the O. sativa genome sequence. The genome size of O. punctata was estimated to be 8% larger than that of O. sativa with individual chromosome differences of 1.5-16.5%. The sum of expansions and contractions observed in regions >500 kb were similar, suggesting that most of the contractions/expansions contributing to the genome size difference between the two species are small, thus preserving the macro-collinearity between these species, which diverged approximately 2 million years ago.
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http://dx.doi.org/10.1534/genetics.106.068783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1893071PMC
May 2007

Integration of hybridization-based markers (overgos) into physical maps for comparative and evolutionary explorations in the genus Oryza and in Sorghum.

BMC Genomics 2006 Aug 8;7:199. Epub 2006 Aug 8.

Department of Agronomy, Purdue University, West Lafayette, Indiana 47907, USA.

Background: With the completion of the genome sequence for rice (Oryza sativa L.), the focus of rice genomics research has shifted to the comparison of the rice genome with genomes of other species for gene cloning, breeding, and evolutionary studies. The genus Oryza includes 23 species that shared a common ancestor 8-10 million years ago making this an ideal model for investigations into the processes underlying domestication, as many of the Oryza species are still undergoing domestication. This study integrates high-throughput, hybridization-based markers with BAC end sequence and fingerprint data to construct physical maps of rice chromosome 1 orthologues in two wild Oryza species. Similar studies were undertaken in Sorghum bicolor, a species which diverged from cultivated rice 40-50 million years ago.

Results: Overgo markers, in conjunction with fingerprint and BAC end sequence data, were used to build sequence-ready BAC contigs for two wild Oryza species. The markers drove contig merges to construct physical maps syntenic to rice chromosome 1 in the wild species and provided evidence for at least one rearrangement on chromosome 1 of the O. sativa versus Oryza officinalis comparative map. When rice overgos were aligned to available S. bicolor sequence, 29% of the overgos aligned with three or fewer mismatches; of these, 41% gave positive hybridization signals. Overgo hybridization patterns supported colinearity of loci in regions of sorghum chromosome 3 and rice chromosome 1 and suggested that a possible genomic inversion occurred in this syntenic region in one of the two genomes after the divergence of S. bicolor and O. sativa.

Conclusion: The results of this study emphasize the importance of identifying conserved sequences in the reference sequence when designing overgo probes in order for those probes to hybridize successfully in distantly related species. As interspecific markers, overgos can be used successfully to construct physical maps in species which diverged less than 8 million years ago, and can be used in a more limited fashion to examine colinearity among species which diverged as much as 40 million years ago. Additionally, overgos are able to provide evidence of genomic rearrangements in comparative physical mapping studies.
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http://dx.doi.org/10.1186/1471-2164-7-199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1590032PMC
August 2006

Analysis of expressed sequence tags from Gibberella zeae (anamorph Fusarium graminearum).

Fungal Genet Biol 2003 Mar;38(2):187-97

Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA.

Gibberella zeae is a broad host range pathogen that infects many crop plants, including wheat and barley, and causes head blight and rot diseases throughout the world. To better understand fungal development and pathogenicity, we have generated 7996 ESTs from three cDNA libraries. Two libraries were generated from carbon-(C-) and nitrogen- (N-) starved mycelia and one library was generated from cultures of maturing perithecia (P). In other fungal pathogens, starvation conditions have been shown to act as cues to induce infection-related gene expression. To assign putative function to cDNAs, sequences were initially assembled using StackPack. The estimated total number of genes identified from the three EST databases was 2110: 1088 contigs and 1022 singleton sequences. These 2110 sequences were compared to a yeast protein sequence reference set and to the GenBank nonredundant database using BLASTX. Based on presumptive gene function identified by this process, we found that the two starved cultures had similar, but not identical, patterns of gene expression, whereas the developmental cultures were distinct in their pattern of expression. Of the three libraries, the perithecium library had the greatest percentage (46%) of ESTS falling into the "unclassified" category. Homologues of some known fungal virulence or pathogenicity factors were found primarily in the N- and C-libraries. Comparisons also were made with ESTs from the related fungi, Neurospora crassa and Magnaporthe grisea and the genomic sequence of N. crassa.
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http://dx.doi.org/10.1016/s1087-1845(02)00529-7DOI Listing
March 2003

Putative subunits of the maize origin of replication recognition complex ZmORC1-ZmORC5.

Nucleic Acids Res 2003 Jan;31(2):619-28

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.

The finding in animal species of complexes homologous to the products of six Saccharomyces cerevisiae genes, origin of replication recognition complex (ORC), has suggested that ORC-related mechanisms have been conserved in all eukaryotes. In plants, however, the only cloned putative homologs of ORC subunits are the Arabidopsis ORC2 and the rice ORC1. Homologs of other subunits of plant origin have not been cloned and characterized. A striking observation was the absence from the Arabidopsis genome of an obvious candidate gene-homolog of ORC4. This fact raised compelling questions of whether plants, in general, and Arabidopsis, in particular, may have lost the ORC4 gene, whether ORC-homologous subunits function within a complex in plants, whether an ORC complex may form and function without an ORC4 subunit, whether a functional (but not sequence) protein homolog may have taken up the role of ORC4 in Arabidopsis, and whether lack of ORC4 is a plant feature, in general. Here, we report the first cloned and molecularly characterized five genes coding for the maize putative homologs of ORC subunits ZmORC1, ZmORC2, ZmORC3, ZmORC4 and ZmORC5. Their expression profiles in tissues with different cell-dividing activities are compatible with a role in DNA replication. Based on the potential of ORC-homologous maize proteins to bind each other in yeast, we propose a model for their possible assembly within a maize ORC. The isolation and molecular characterization of an ORC4-homologous gene from maize argues that, in its evolution, Arabidopsis may have lost the homologous ORC4 gene.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC140504PMC
http://dx.doi.org/10.1093/nar/gkg138DOI Listing
January 2003