Cell Biology & Molecular Genetics Research Works

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Alternative divalent cations (Zn2+, Co2+, and Mn2+) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity
(Springer Nature, 2015-05-03) Achuthan, Vasudevan; DeStefano, Jeffrey J
Fidelity of DNA polymerases can be influenced by cation co-factors. Physiologically, Mg2+ is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; however, alternative cations including Mn2+, Co2+, and Zn2+ can also support catalysis. Although Zn2+ supports DNA synthesis, it inhibits HIV RT by significantly modifying RT catalysis. Zn2+ is currently being investigated as a component of novel treatment options against HIV and we wanted to investigate the fidelity of RT with Zn2+. We used PCR-based and plasmid-based alpha complementation assays as well as steady-state misinsertion and misincorporation assays to examine the fidelity of RT with Mn2+, Co2+, and Zn2+. The fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn2+ when compared to Mg2+ at cation conditions optimized for nucleotide catalysis. Consistent with this, RT extended primers with mismatched 3′ nucleotides poorly and inserted incorrect nucleotides less efficiently using Zn2+ than Mg2+. In agreement with previous literature, we observed that Mn2+ and Co2+ dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM). However, surprisingly, the fidelity of HIV RT with Mn2+ and Co2+ remained similar to Mg2+ at lower concentrations that are optimal for catalysis. This study shows that Zn2+, at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.
The Alveolate Perkinsus marinus: Biological Insights from EST Gene Discovery
(Springer Nature, 2010-04-07) Joseph, Sandeep J; Fernández-Robledo, José A; Gardner, Malcolm J; El-Sayed, Najib M; Kuo, Chih-Horng; Schott, Eric J; Wang, Haiming; Kissinger, Jessica C; Vasta, Gerardo R
Perkinsus marinus, a protozoan parasite of the eastern oyster Crassostrea virginica, has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of the United States. It is classified as a member of the Perkinsozoa, a recently established phylum considered close to the ancestor of ciliates, dinoflagellates, and apicomplexans, and a key taxon for understanding unique adaptations (e.g. parasitism) within the Alveolata. Despite intense parasite pressure, no disease-resistant oysters have been identified and no effective therapies have been developed to date. To gain insight into the biological basis of the parasite's virulence and pathogenesis mechanisms, and to identify genes encoding potential targets for intervention, we generated >31,000 5' expressed sequence tags (ESTs) derived from four trophozoite libraries generated from two P. marinus strains. Trimming and clustering of the sequence tags yielded 7,863 unique sequences, some of which carry a spliced leader. Similarity searches revealed that 55% of these had hits in protein sequence databases, of which 1,729 had their best hit with proteins from the chromalveolates (E-value ≤ 1e-5). Some sequences are similar to those proven to be targets for effective intervention in other protozoan parasites, and include not only proteases, antioxidant enzymes, and heat shock proteins, but also those associated with relict plastids, such as acetyl-CoA carboxylase and methyl erythrithol phosphate pathway components, and those involved in glycan assembly, protein folding/secretion, and parasite-host interactions. Our transcriptome analysis of P. marinus, the first for any member of the Perkinsozoa, contributes new insight into its biology and taxonomic position. It provides a very informative, albeit preliminary, glimpse into the expression of genes encoding functionally relevant proteins as potential targets for chemotherapy, and evidence for the presence of a relict plastid. Further, although P. marinus sequences display significant similarity to those from both apicomplexans and dinoflagellates, the presence of trans-spliced transcripts confirms the previously established affinities with the latter. The EST analysis reported herein, together with the recently completed sequence of the P. marinus genome and the development of transfection methodology, should result in improved intervention strategies against dermo disease.
Antimicrobial and Antivirulence Impacts of Phenolics on Salmonella Enterica Serovar Typhimurium
(MDPI, 2020-10-03) Alvarado-Martinez, Zabdiel; Bravo, Paulina; Kennedy, Nana-Frekua; Krishna, Mayur; Hussain, Syed; Young, Alana C.; Biswas, Debabrata
Salmonella enterica serovar Typhimurium (ST) remains a major infectious agent in the USA, with an increasing antibiotic resistance pattern, which requires the development of novel antimicrobials capable of controlling ST. Polyphenolic compounds found in plant extracts are strong candidates as alternative antimicrobials, particularly phenolic acids such as gallic acid (GA), protocatechuic acid (PA) and vanillic acid (VA). This study evaluates the effectiveness of these compounds in inhibiting ST growth while determining changes to the outer membrane through fluorescent dye uptake and scanning electron microscopy (SEM), in addition to measuring alterations to virulence genes with qRT-PCR. Results showed antimicrobial potential for all compounds, significantly inhibiting the detectable growth of ST. Fluorescent spectrophotometry and microscopy detected an increase in relative fluorescent intensity (RFI) and red-colored bacteria over time, suggesting membrane permeabilization. SEM revealed severe morphological defects at the polar ends of bacteria treated with GA and PA, while VA-treated bacteria were found to be mid-division. Relative gene expression showed significant downregulation in master regulator hilA and invH after GA and PA treatments, while fliC was upregulated in VA. Results suggest that GA, PA and VA have antimicrobial potential that warrants further research into their mechanism of action and the interactions that lead to ST death.
Benchmarking AlphaFold for protein complex modeling reveals accuracy determinants
(Wiley, 2022-07-13) Yin, Rui; Yeng, Brandon F.; Varshney, Amitabh; Pierce, Brian G.
High-resolution experimental structural determination of protein–protein interactions has led to valuable mechanistic insights, yet due to the massive number of interactions and experimental limitations there is a need for computational methods that can accurately model their structures. Here we explore the use of the recently developed deep learning method, AlphaFold, to predict structures of protein complexes from sequence. With a benchmark of 152 diverse heterodimeric protein complexes, multiple implementations and parameters of AlphaFold were tested for accuracy. Remarkably, many cases (43%) had near-native models (medium or high critical assessment of predicted interactions accuracy) generated as top-ranked predictions by AlphaFold, greatly surpassing the performance of unbound protein–protein docking (9% success rate for near-native top-ranked models), however AlphaFold modeling of antibody–antigen complexes within our set was unsuccessful. We identified sequence and structural features associated with lack of AlphaFold success, and we also investigated the impact of multiple sequence alignment input. Benchmarking of a multimer-optimized version of AlphaFold (AlphaFold-Multimer) with a set of recently released antibody–antigen structures confirmed a low rate of success for antibody–antigen complexes (11% success), and we found that T cell receptor–antigen complexes are likewise not accurately modeled by that algorithm, showing that adaptive immune recognition poses a challenge for the current AlphaFold algorithm and model. Overall, our study demonstrates that end-to-end deep learning can accurately model many transient protein complexes, and highlights areas of improvement for future developments to reliably model any protein–protein interaction of interest.
Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase
(Springer Nature, 2009-12-14) Poliakov, Eugenia; Gentleman, Susan; Chander, Preethi; Cunningham, Francis X Jr; Grigorenko, Bella L; Nemuhin, Alexander V; Redmond, T Michael
β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a β-carotene-accumulating E. coli system. Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our ab initio model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326. Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.
Breaking the conformational ensemble barrier: Ensemble structure modeling challenges in CASP15
(Wiley, 2023-10-23) Kryshtafovych, Andriy; Montelione, Gaetano T.; Rigden, Daniel J.; Mesdaghi, Shahram; Karaca, Ezgi; Moult, John
or the first time, the 2022 CASP (Critical Assessment of Structure Prediction) community experiment included a section on computing multiple conformations for protein and RNA structures. There was full or partial success in reproducing the ensembles for four of the nine targets, an encouraging result. For protein structures, enhanced sampling with variations of the AlphaFold2 deep learning method was by far the most effective approach. One substantial conformational change caused by a single mutation across a complex interface was accurately reproduced. In two other assembly modeling cases, methods succeeded in sampling conformations near to the experimental ones even though environmental factors were not included in the calculations. An experimentally derived flexibility ensemble allowed a single accurate RNA structure model to be identified. Difficulties included how to handle sparse or low-resolution experimental data and the current lack of effective methods for modeling RNA/protein complexes. However, these and other obstacles appear addressable.
Carbohydrase Systems of Saccharophagus degradans Degrading Marine Complex Polysaccharides
(MDPI, 2011-04-21) Hutcheson, Steven W.; Zhang, Haitao; Suvorov, Maxim
Saccharophagus degradans 2–40 is a γ-subgroup proteobacterium capable of using many of the complex polysaccharides found in the marine environment for growth. To utilize these complex polysaccharides, this bacterium produces a plethora of carbohydrases dedicated to the processing of a carbohydrate class. Aiding in the identification of the contributing genes and enzymes is the known genome sequence for this bacterium. This review catalogs the genes and enzymes of the S. degradans genome that are likely to function in the systems for the utilization of agar, alginate, α- and β-glucans, chitin, mannans, pectins, and xylans and discusses the cell biology and genetics of each system as it functions to transfer carbon back to the bacterium.
A Case to Study: Fostering Scientific Literacy in Non-Majors
(2012) Schalk, Kelly; Benson, Spencer
Preparing a person for life beyond a classroom has been a priority of the science education reform movement. Researchers have proposed the use of Socioscientific Issues (SSI) as a way of helping learners become scientifically literate members of society. This investigation examined the effects of a SSI based curriculum on students’ ability to interpret scientific information and changes in their epistemological beliefs. Qualitative data from multiple sources were analyzed. Students’ interpretative skills of science as well as their beliefs about the nature of knowledge, simplicity of knowledge, and perceptions of their ability to learn science improved.
Collaborative learning in an undergraduate life sciences living-learning program: case studies at multiple planes of analysis.
(2017) Jardine, Hannah; Levin, Daniel; Quimby, B. Bryn; Cooke, Todd
The authors report on a living-learning program (LLP) designed to transform life sciences education. One goal of the LLP is to engage students in collaborative learning. Little research describes interactions and experiences within an LLP that encourage collaborative learning. This qualitative ethnographic study explores the following questions: What are some of the ways in which collaborative learning occurs in an LLP? and What factors influence how, when, and to what extent collaborative learning occurs in an LLP? The authors aim to identify ways to promote collaborative learning in an LLP and provide insight for others wishing to construct LLPs with similar goals.
Comparative genomic analysis reveals evidence of two novel Vibrio species closely related to V. cholerae
(2010-05-27) Haley, Bradd J; Grim, Christopher J; Hasan, Nur A; Choi, Seon-Young; Chun, Jongsik; Brettin, Thomas S; Bruce, David C; Challacombe, Jean F; Detter, J Chris; Han, Cliff S; Huq, Anwar; Colwell, Rita R
Background: In recent years genome sequencing has been used to characterize new bacterial species, a method of analysis available as a result of improved methodology and reduced cost. Included in a constantly expanding list of Vibrio species are several that have been reclassified as novel members of the Vibrionaceae. The description of two putative new Vibrio species, Vibrio sp. RC341 and Vibrio sp. RC586 for which we propose the names V. metecus and V. parilis, respectively, previously characterized as non-toxigenic environmental variants of V. cholerae is presented in this study. Results: Based on results of whole-genome average nucleotide identity (ANI), average amino acid identity (AAI), rpoB similarity, MLSA, and phylogenetic analysis, the new species are concluded to be phylogenetically closely related to V. cholerae and V. mimicus. Vibrio sp. RC341 and Vibrio sp. RC586 demonstrate features characteristic of V. cholerae and V. mimicus, respectively, on differential and selective media, but their genomes show a 12 to 15% divergence (88 to 85% ANI and 92 to 91% AAI) compared to the sequences of V. cholerae and V. mimicus genomes (ANI <95% and AAI <96% indicative of separate species). Vibrio sp. RC341 and Vibrio sp. RC586 share 2104 ORFs (59%) and 2058 ORFs (56%) with the published core genome of V. cholerae and 2956 (82%) and 3048 ORFs (84%) with V. mimicus MB-451, respectively. The novel species share 2926 ORFs with each other (81% Vibrio sp. RC341 and 81% Vibrio sp. RC586). Virulenceassociated factors and genomic islands of V. cholerae and V. mimicus, including VSP-I and II, were found in these environmental Vibrio spp. Conclusions: Results of this analysis demonstrate these two environmental vibrios, previously characterized as variant V. cholerae strains, are new species which have evolved from ancestral lineages of the V. cholerae and V. mimicus clade. The presence of conserved integration loci for genomic islands as well as evidence of horizontal gene transfer between these two new species, V. cholerae, and V. mimicus suggests genomic islands and virulence factors are transferred between these species.
Comprehensive analysis of alternative splicing in rice and comparative analyses with Arabidopsis
(Springer Nature, 2006-12-28) Campbell, Matthew A; Haas, Brian J; Hamilton, John P; Mount, Stephen M; Buell, C Robin
Recently, genomic sequencing efforts were finished for Oryza sativa (cultivated rice) and Arabidopsis thaliana (Arabidopsis). Additionally, these two plant species have extensive cDNA and expressed sequence tag (EST) libraries. We employed the Program to Assemble Spliced Alignments (PASA) to identify and analyze alternatively spliced isoforms in both species. A comprehensive analysis of alternative splicing was performed in rice that started with >1.1 million publicly available spliced ESTs and over 30,000 full length cDNAs in conjunction with the newly enhanced PASA software. A parallel analysis was performed with Arabidopsis to compare and ascertain potential differences between monocots and dicots. Alternative splicing is a widespread phenomenon (observed in greater than 30% of the loci with transcript support) and we have described nine alternative splicing variations. While alternative splicing has the potential to create many RNA isoforms from a single locus, the majority of loci generate only two or three isoforms and transcript support indicates that these isoforms are generally not rare events. For the alternate donor (AD) and acceptor (AA) classes, the distance between the splice sites for the majority of events was found to be less than 50 basepairs (bp). In both species, the most frequent distance between AA is 3 bp, consistent with reports in mammalian systems. Conversely, the most frequent distance between AD is 4 bp in both plant species, as previously observed in mouse. Most alternative splicing variations are localized to the protein coding sequence and are predicted to significantly alter the coding sequence. Alternative splicing is widespread in both rice and Arabidopsis and these species share many common features. Interestingly, alternative splicing may play a role beyond creating novel combinations of transcripts that expand the proteome. Many isoforms will presumably have negative consequences for protein structure and function, suggesting that their biological role involves post-transcriptional regulation of gene expression.
A computational survey of candidate exonic splicing enhancer motifs in the model plant Arabidopsis thaliana
(Springer Nature, 2007-05-21) Pertea, Mihaela; Mount, Stephen M; Salzberg, Steven L
Algorithmic approaches to splice site prediction have relied mainly on the consensus patterns found at the boundaries between protein coding and non-coding regions. However exonic splicing enhancers have been shown to enhance the utilization of nearby splice sites. We have developed a new computational technique to identify significantly conserved motifs involved in splice site regulation. First, 84 putative exonic splicing enhancer hexamers are identified in Arabidopsis thaliana. Then a Gibbs sampling program called ELPH was used to locate conserved motifs represented by these hexamers in exonic regions near splice sites in confirmed genes. Oligomers containing 35 of these motifs have been shown experimentally to induce significant inclusion of A. thaliana exons. Second, integration of our regulatory motifs into two different splice site recognition programs significantly improved the ability of the software to correctly predict splice sites in a large database of confirmed genes. We have released GeneSplicerESE, the improved splice site recognition code, as open source software. Our results show that the use of the ESE motifs consistently improves splice site prediction accuracy.
Conservation in first introns is positively associated with the number of exons within genes and the presence of regulatory epigenetic signals
(Springer Nature, 2014-06-26) Park, Seung Gu; Hannenhalli, Sridhar; Choi, Sun Shim
Genomes of higher eukaryotes have surprisingly long first introns and in some cases, the first introns have been shown to have higher conservation relative to other introns. However, the functional relevance of conserved regions in the first introns is poorly understood. Leveraging the recent ENCODE data, here we assess potential regulatory roles of conserved regions in the first intron of human genes. We first show that relative to other downstream introns, the first introns are enriched for blocks of highly conserved sequences. We also found that the first introns are enriched for several chromatin marks indicative of active regulatory regions and this enrichment of regulatory marks is correlated with enrichment of conserved blocks in the first intron; the enrichments of conservation and regulatory marks in first intron are not entirely explained by a general, albeit variable, bias for certain marks toward the 5’ end of introns. Interestingly, conservation as well as proportions of active regulatory chromatin marks in the first intron of a gene correlates positively with the numbers of exons in the gene but the correlation is significantly weakened in second introns and negligible beyond the second intron. The first intron conservation is also positively correlated with the gene’s expression level in several human tissues. Finally, a gene-wise analysis shows significant enrichments of active chromatin marks in conserved regions of first introns, relative to the conserved regions in other introns of the same gene. Taken together, our analyses strongly suggest that first introns are enriched for active transcriptional regulatory signals under purifying selection.
Critical assessment of methods of protein structure prediction (CASP)—Round XV
(Wiley, 2023-11-02) Kryshtafovych, Andriy; Schwede, Torsten; Topf, Maya; Fidelis, Krzysztof; Moult, John
Computing protein structure from amino acid sequence information has been a long-standing grand challenge. Critical assessment of structure prediction (CASP) conducts community experiments aimed at advancing solutions to this and related problems. Experiments are conducted every 2 years. The 2020 experiment (CASP14) saw major progress, with the second generation of deep learning methods delivering accuracy comparable with experiment for many single proteins. There is an expectation that these methods will have much wider application in computational structural biology. Here we summarize results from the most recent experiment, CASP15, in 2022, with an emphasis on new deep learning-driven progress. Other papers in this special issue of proteins provide more detailed analysis. For single protein structures, the AlphaFold2 deep learning method is still superior to other approaches, but there are two points of note. First, although AlphaFold2 was the core of all the most successful methods, there was a wide variety of implementation and combination with other methods. Second, using the standard AlphaFold2 protocol and default parameters only produces the highest quality result for about two thirds of the targets, and more extensive sampling is required for the others. The major advance in this CASP is the enormous increase in the accuracy of computed protein complexes, achieved by the use of deep learning methods, although overall these do not fully match the performance for single proteins. Here too, AlphaFold2 based method perform best, and again more extensive sampling than the defaults is often required. Also of note are the encouraging early results on the use of deep learning to compute ensembles of macromolecular structures. Critically for the usability of computed structures, for both single proteins and protein complexes, deep learning derived estimates of both local and global accuracy are of high quality, however the estimates in interface regions are slightly less reliable. CASP15 also included computation of RNA structures for the first time. Here, the classical approaches produced better agreement with experiment than the new deep learning ones, and accuracy is limited. Also, for the first time, CASP included the computation of protein–ligand complexes, an area of special interest for drug design. Here too, classical methods were still superior to deep learning ones. Many new approaches were discussed at the CASP conference, and it is clear methods will continue to advance.
Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche
(Springer Nature, 2022-03-04) Patel, Akshay; Wu, Yicong; Han, Xiaofei; Su, Yijun; Maugel, Tim; Shroff, Hari; Roy, Sougata
Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Niche-specific AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.
Defects in sarcolemma repair and skeletal muscle function after injury in a mouse model of Niemann-Pick type A/B disease
(Springer Nature, 2019-01-05) Michailowsky, V.; Li, H.; Mittra, B.; Iyer, S. R.; Mazála, D. A. G.; Corrotte, M.; Wang, Y.; Chin, E. R.; Lovering, R. M.; Andrews, N. W.
Niemann-Pick disease type A (NPDA), a disease caused by mutations in acid sphingomyelinase (ASM), involves severe neurodegeneration and early death. Intracellular lipid accumulation and plasma membrane alterations are implicated in the pathology. ASM is also linked to the mechanism of plasma membrane repair, so we investigated the impact of ASM deficiency in skeletal muscle, a tissue that undergoes frequent cycles of injury and repair in vivo. Utilizing the NPDA/B mouse model ASM−/− and wild type (WT) littermates, we performed excitation-contraction coupling/Ca2+ mobilization and sarcolemma injury/repair assays with isolated flexor digitorum brevis fibers, proteomic analyses with quadriceps femoris, flexor digitorum brevis, and tibialis posterior muscle and in vivo tests of the contractile force (maximal isometric torque) of the quadriceps femoris muscle before and after eccentric contraction-induced muscle injury. ASM−/− flexor digitorum brevis fibers showed impaired excitation-contraction coupling compared to WT, a defect expressed as reduced tetanic [Ca2+]i in response to electrical stimulation and early failure in sustaining [Ca2+]i during repeated tetanic contractions. When injured mechanically by needle passage, ASM−/− flexor digitorum brevis fibers showed susceptibility to injury similar to WT, but a reduced ability to reseal the sarcolemma. Proteomic analyses revealed changes in a small group of skeletal muscle proteins as a consequence of ASM deficiency, with downregulation of calsequestrin occurring in the three different muscles analyzed. In vivo, the loss in maximal isometric torque of WT quadriceps femoris was similar immediately after and 2 min after injury. The loss in ASM−/− mice immediately after injury was similar to WT, but was markedly larger at 2 min after injury. Skeletal muscle fibers from ASM−/− mice have an impairment in intracellular Ca2+ handling that results in reduced Ca2+ mobilization and a more rapid decline in peak Ca2+ transients during repeated contraction-relaxation cycles. Isolated fibers show reduced ability to repair damage to the sarcolemma, and this is associated with an exaggerated deficit in force during recovery from an in vivo eccentric contraction-induced muscle injury. Our findings uncover the possibility that skeletal muscle functional defects may play a role in the pathology of NPDA/B disease.
Derepression of Cancer/Testis Antigens in cancer is associated with distinct patterns of DNA Hypomethylation
(Springer Nature, 2013-03-22) Kim, Robert; Kulkarni, Prakash; Hannenhalli, Sridhar
The Cancer/Testis Antigens (CTAs) are a heterogeneous group of proteins whose expression is typically restricted to the testis. However, they are aberrantly expressed in most cancers that have been examined to date. Broadly speaking, the CTAs can be divided into two groups: the CTX antigens that are encoded by the X-linked genes and the non-X CT antigens that are encoded by the autosomes. Unlike the non-X CTAs, the CTX antigens form clusters of closely related gene families and their expression is frequently associated with advanced disease with poorer prognosis. Regardless however, the mechanism(s) underlying their selective derepression and stage-specific expression in cancer remain poorly understood, although promoter DNA demethylation is believed to be the major driver. Here, we report a systematic analysis of DNA methylation profiling data from various tissue types to elucidate the mechanism underlying the derepression of the CTAs in cancer. We analyzed the methylation profiles of 501 samples including sperm, several cancer types, and their corresponding normal somatic tissue types. We found strong evidence for specific DNA hypomethylation of CTA promoters in the testis and cancer cells but not in their normal somatic counterparts. We also found that hypomethylation was clustered on the genome into domains that coincided with nuclear lamina-associated domains (LADs) and that these regions appeared to be insulated by CTCF sites. Interestingly, we did not observe any significant differences in the hypomethylation pattern between the CTAs without CpG islands and the CTAs with CpG islands in the proximal promoter. Our results corroborate that widespread DNA hypomethylation appears to be the driver in the derepression of CTA expression in cancer and furthermore, demonstrate that these hypomethylated domains are associated with the nuclear lamina-associated domains (LADS). Taken together, our results suggest that wide-spread methylation changes in cancer are linked to derepression of germ-line-specific genes that is orchestrated by the three dimensional organization of the cancer genome.
Dermestes maculatus: an intermediate-germ beetle model system for evo-devo
(Springer Nature, 2015-10-16) Xiang, Jie; Forrest, Iain S.; Pick, Leslie
Understanding how genes change during evolution to direct the development of diverse body plans is a major goal of the evo-devo field. Achieving this will require the establishment of new model systems that represent key points in phylogeny. These new model systems must be amenable to laboratory culture, and molecular and functional approaches should be feasible. To date, studies of insects have been best represented by the model system Drosophila melanogaster. Given the enormous diversity represented by insect taxa, comparative studies within this clade will provide a wealth of information about the evolutionary potential and trajectories of alternative developmental strategies. Here we established the beetle Dermestes maculatus, a member of the speciose clade Coleoptera, as a new insect model system. We have maintained a continuously breeding culture in the lab and documented Dermestes maculatus embryogenesis using nuclear and phalloidin staining. Anterior segments are specified during the blastoderm stage before gastrulation, and posterior segments are added sequentially during germ band elongation. We isolated and studied the expression and function of the pair-rule segmentation gene paired in Dermestes maculatus. In this species, paired is expressed in stripes during both blastoderm and germ band stages: four primary stripes arise prior to gastrulation, confirming an intermediate-germ mode of development for this species. As in other insects, these primary stripes then split into secondary stripes. To study gene function, we established both embryonic and parental RNAi. Knockdown of Dmac-paired with either method resulted in pair-rule-like segmentation defects, including loss of Engrailed expression in alternate stripes. These studies establish basic approaches necessary to use Dermestes maculatus as a model system. Methods are now available for use of this intermediate-germ insect for future studies of the evolution of regulatory networks controlling insect segmentation, as well as of other processes in development and homeostasis. Consistent with the role of paired in long-germ Drosophila and shorter-germ Tribolium, paired functions as a pair-rule segmentation gene in Dermestes maculatus. Thus, paired retains pair-rule function in insects with different modes of segment addition.
Detection of lineage-specific evolutionary changes among primate species
(2011-07-04) Pertea, Mihaela; Pertea, Geo M; Salzberg, Steven L
Background: Comparison of the human genome with other primates offers the opportunity to detect evolutionary events that created the diverse phenotypes among the primate species. Because the primate genomes are highly similar to one another, methods developed for analysis of more divergent species do not always detect signs of evolutionary selection. Results: We have developed a new method, called DivE, specifically designed to find regions that have evolved either more or less rapidly than expected, for any clade within a set of very closely related species. Unlike some previous methods, DivE does not rely on rates of synonymous and nonsynonymous substitution, which enables it to detect evolutionary events in noncoding regions. We demonstrate using simulated data that DivE compares favorably to alternative methods, and we then apply DivE to the ENCODE regions in 14 primate species. We identify thousands of regions in these primates, ranging from 50 to >10000 bp in length, that appear to have experienced either constrained or accelerated rates of evolution. In particular, we detected 4942 regions that have potentially undergone positive selection in one or more primate species. Most of these regions occur outside of protein-coding genes, although we identified 20 proteins that have experienced positive selection. Conclusions: DivE provides an easy-to-use method to predict both positive and negative selection in noncoding DNA, that is particularly well-suited to detecting lineage-specific selection in large genomes.
Distinct double flower varieties in Camellia japonica exhibit both expansion and contraction of C-class gene expression
(Springer Nature, 2014-10-25) Sun, Yingkun; Fan, Zhengqi; Li, Xinlei; Liu, Zhongchi; Li, Jiyuan; Yin, Hengfu
Double flower domestication is of great value in ornamental plants and presents an excellent system to study the mechanism of morphological alterations by human selection. The classic ABC model provides a genetic framework underlying the control of floral organ identity and organogenesis from which key regulators have been identified and evaluated in many plant species. Recent molecular studies have underscored the importance of C-class homeotic genes, whose functional attenuation contributed to the floral diversity in various species. Cultivated Camellia japonica L. possesses several types of double flowers, however the molecular mechanism underlying their floral morphological diversification remains unclear. In this study, we cloned the C-class orthologous gene CjAG in C. japonica. We analyzed the expression patterns of CjAG in wild C. japonica, and performed ectopic expression in Arabidopsis. These results revealed that CjAG shared conserved C-class function that controls stamen and carpel development. Further we analyzed the expression pattern of CjAG in two different C. japonica double-flower varieties, `Shibaxueshi’ and `Jinpanlizhi’, and showed that expression of CjAG was highly contracted in `Shibaxueshi’ but expanded in inner petals of `Jinpanlizhi’. Moreover, detailed expression analyses of B- and C-class genes have uncovered differential patterns of B-class genes in the inner organs of `Jinpanlizhi’. These results demonstrated that the contraction and expansion of CjAG expression were associated with the formation of different types of double flowers. Our studies have manifested two different trajectories of double flower domestication regarding the C-class gene expression in C. japonica.