RESUMEN
UNLABELLED: Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc(2)155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCE: The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.
Asunto(s)
Familia de Multigenes , Micobacteriófagos/clasificación , Micobacteriófagos/genética , Mycobacterium smegmatis/virología , ARN de Transferencia/genética , ARN Viral , Composición de Base , Secuencia de Bases , Codón , Secuencia Conservada , Orden Génico , Tamaño del Genoma , Genoma Viral , Secuencias Invertidas Repetidas , Lisogenia/genética , Micobacteriófagos/ultraestructura , Sistemas de Lectura Abierta , Filogenia , ARN de Transferencia/química , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de Secuencia , Virión/genética , Virión/ultraestructura , Ensamble de Virus/genéticaRESUMEN
The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.
Asunto(s)
Actinobacteria/virología , Bacteriófagos/genética , Variación Genética , Genoma Viral , Bacteriófagos/clasificación , Bacteriófagos/aislamiento & purificación , Composición de Base , ADN Viral/genética , Genes Virales , Genómica , Filogenia , Proteínas Virales de Fusión/genéticaRESUMEN
Cluster BE1 Streptomyces bacteriophages belong to the Siphoviridae, with genome sizes over 130 kbp, and they contain direct terminal repeats of approximately 11 kbp. Eight newly isolated closely related cluster BE1 phages contain 43 to 48 tRNAs, one transfer-messenger RNA (tmRNA), and 216 to 236 predicted open reading frames (ORFs), but few of their genes are shared with other phages, including those infecting Streptomyces species.
RESUMEN
Drosophila melanogaster heterochromatin protein 2 (HP2) interacts with heterochromatin protein 1 (HP1). In polytene chromosomes, HP2 and HP1 colocalize at the chromocenter, telomeres, and the small fourth chromosome. We show here that HP2 is present in the arms as well as the centromeric regions of mitotic chromosomes. We also demonstrate that Su(var)2-HP2 exhibits a dosage-dependent modification of variegation of a yellow reporter transgene, indicating a structural role in heterochromatin formation. We have isolated and characterized 14 new mutations in the Su(var)2-HP2 gene. Using wm4h, many (but not all) mutant alleles show dominant Su(var) activity. Su(var)2-HP2 mutant larvae show a wide variety of mitotic abnormalities, but not the telomere fusion seen in larvae deficient for HP1. The Su(var)2-HP2 gene codes for two isoforms: HP2-L (approximately 365 kDa) and HP2-S (approximately 175 kDa), lacking exons 5 and 6. In general, mutations that affect only the larger isoform result in more pronounced defects than do mutations common to both isoforms. This suggests that an imbalance between large and small isoforms is particularly deleterious. These results indicate a role for HP2 in the structural organization of chromosomes and in heterochromatin-induced gene silencing and show that the larger isoform plays a critical role in these processes.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Silenciador del Gen , Alelos , Animales , Proteínas Cromosómicas no Histona/química , Cruzamientos Genéticos , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Dosificación de Gen , Marcadores Genéticos , Proteínas Fluorescentes Verdes/metabolismo , Indoles , Larva , Mitosis , Peso Molecular , Mutación , Técnicas de Amplificación de Ácido Nucleico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análisis de Secuencia de ADNRESUMEN
The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (â¼5.2 Mb) is the smallest chromosome in Drosophila melanogaster, but it is substantially larger (>18.7 Mb) in D. ananassae To identify the major contributors to the expansion of the F element and to assess their impact, we improved the genome sequence and annotated the genes in a 1.4-Mb region of the D. ananassae F element, and a 1.7-Mb region from the D element for comparison. We find that transposons (particularly LTR and LINE retrotransposons) are major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F-element genes exhibit distinct characteristics compared to D-element genes (e.g., larger coding spans, larger introns, more coding exons, and lower codon bias), but these differences are exaggerated in D. ananassae Compared to D. melanogaster, the codon bias observed in D. ananassae F-element genes can primarily be attributed to mutational biases instead of selection. The 5' ends of F-element genes in both species are enriched in dimethylation of lysine 4 on histone 3 (H3K4me2), while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F-element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves our understanding of how transposons can affect genome size and how genes can function within highly repetitive domains.
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Cromosomas/genética , Drosophila/genética , Retroelementos/genética , Animales , Composición de Base/genética , Secuencia de Bases , Codón/genética , Femenino , Perfilación de la Expresión Génica , Genes de Insecto , Histonas/metabolismo , Procesamiento Proteico-Postraduccional/genética , Wolbachia/genéticaRESUMEN
The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.
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Proteínas de Drosophila/genética , Drosophila/genética , Evolución Molecular , Genoma , Genómica , Animales , Codón , Biología Computacional , Elementos Transponibles de ADN , Drosophila melanogaster/genética , Exones , Reordenamiento Génico , Heterocromatina , Intrones , Anotación de Secuencia Molecular , Cromosomas Politénicos , Secuencias Repetitivas de Ácidos Nucleicos , Selección Genética , Especificidad de la EspecieRESUMEN
UNLABELLED: Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students' interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. IMPORTANCE: Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.
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Bacterias/virología , Bacteriófagos/genética , Genómica/educación , Microbiología/educación , Adulto , Femenino , Humanos , Masculino , Estudiantes , Adulto JovenRESUMEN
There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge. We have developed a bioinformatics project that provides a course-based research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. An alumni survey revealed that time spent on a research project is also a significant factor in the value former students assign to the experience one or more years later. We conclude: 1) implementation of a bioinformatics project within the biology curriculum provides a mechanism for successfully engaging large numbers of students in undergraduate research; 2) benefits to students are achievable at a wide variety of academic institutions; and 3) successful implementation of course-based research experiences requires significant investment of instructional time for students to gain full benefit.
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Biología/educación , Curriculum , Investigación/educación , Actitud , Conducta Cooperativa , Recolección de Datos , Docentes , Genoma , Genómica/educación , Humanos , Conocimiento , Aprendizaje , Anotación de Secuencia Molecular , Evaluación de Programas y Proyectos de Salud , Investigadores , Autoinforme , Encuestas y Cuestionarios , Factores de TiempoRESUMEN
Five newly isolated mycobacteriophages--Angelica, CrimD, Adephagia, Anaya, and Pixie--have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them--with the exception of TM4--form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species.
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Evolución Molecular , Micobacteriófagos/genética , Sitios de Ligazón Microbiológica , Secuencia de Bases , Mapeo Cromosómico , Análisis por Conglomerados , Secuencia Conservada/genética , Eliminación de Gen , Genoma Viral/genética , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Familia de Multigenes/genética , Mutación/genética , Micobacteriófagos/crecimiento & desarrollo , Micobacteriófagos/aislamiento & purificación , Micobacteriófagos/ultraestructura , Análisis de Secuencia de ADN , Temperatura , Proteínas Virales/genética , Virión/genética , Virión/ultraestructura , Integración Viral/genética , Replicación Viral/fisiologíaRESUMEN
The distal arm of the fourth ("dot") chromosome of Drosophila melanogaster is unusual in that it exhibits an amalgamation of heterochromatic properties (e.g., dense packaging, late replication) and euchromatic properties (e.g., gene density similar to euchromatic domains, replication during polytenization). To examine the evolution of this unusual domain, we undertook a comparative study by generating high-quality sequence data and manually curating gene models for the dot chromosome of D. virilis (Tucson strain 15010-1051.88). Our analysis shows that the dot chromosomes of D. melanogaster and D. virilis have higher repeat density, larger gene size, lower codon bias, and a higher rate of gene rearrangement compared to a reference euchromatic domain. Analysis of eight "wanderer" genes (present in a euchromatic chromosome arm in one species and on the dot chromosome in the other) shows that their characteristics are similar to other genes in the same domain, which suggests that these characteristics are features of the domain and are not required for these genes to function. Comparison of this strain of D. virilis with the strain sequenced by the Drosophila 12 Genomes Consortium (Tucson strain 15010-1051.87) indicates that most genes on the dot are under weak purifying selection. Collectively, despite the heterochromatin-like properties of this domain, genes on the dot evolve to maintain function while being responsive to changes in their local environment.
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Cromosomas de Insectos/genética , Drosophila/genética , Evolución Molecular , Genoma de los Insectos/genética , Animales , Mapeo Cromosómico , Drosophila/clasificación , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Eucromatina/genética , Genes de Insecto/genética , Heterocromatina/genética , Mutación INDEL/genética , Sistemas de Lectura Abierta/genética , Especificidad de la Especie , Sintenía , Secuencias Repetidas en Tándem/genéticaRESUMEN
Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 million bases of draft genomic sequence from several species of Drosophila and have produced hundreds of gene models using evidence-based manual annotation. Students appreciate their ability to make a contribution to ongoing research, and report increased independence and a more active learning approach after participation in GEP projects. They show knowledge gains on pre- and postcourse quizzes about genes and genomes and in bioinformatic analysis. Participating faculty also report professional gains, increased access to genomics-related technology, and an overall positive experience. We have found that using a genomics research project as the core of a laboratory course is rewarding for both faculty and students.
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Investigación Genética , Genómica/educación , Laboratorios , Universidades , Animales , Docentes , Estudiantes/psicologíaRESUMEN
The fourth chromosome of Drosophila melanogaster has a number of unique properties that make it a convenient model for the study of chromatin structure. Only 4.2 Mb overall, the 1.2 Mb distal arm of chromosome 4 seen in polytene chromosomes combines characteristics of heterochromatin and euchromatin. This domain has a repeat density of ~35%, comparable to some pericentric chromosome regions, while maintaining a gene density similar to that of the other euchromatic chromosome arms. Studies of position-effect variegation have revealed that heterochromatic and euchromatic domains are interspersed on chromosome 4, and both cytological and biochemical studies have demonstrated that chromosome 4 is associated with heterochromatic marks, such as heterochromatin protein 1 and histone 3 lysine 9 methylation. Chromosome 4 is also marked by POF (painting-of-fourth), a chromosome 4-specific chromosomal protein, and utilizes a dedicated histone methyltransferase, EGG. Studies of chromosome 4 have helped to shape our understanding of heterochromatin domains and their establishment and maintenance. In this review, we provide a synthesis of the work to date and an outlook to the future.
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Cromosomas/metabolismo , Drosophila melanogaster/metabolismo , Animales , Proteínas de Drosophila/metabolismo , Eucromatina/metabolismo , Heterocromatina/metabolismo , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Chromosome four of Drosophila melanogaster, known as the dot chromosome, is largely heterochromatic, as shown by immunofluorescent staining with antibodies to heterochromatin protein 1 (HP1) and histone H3K9me. In contrast, the absence of HP1 and H3K9me from the dot chromosome in D. virilis suggests that this region is euchromatic. D. virilis diverged from D. melanogaster 40 to 60 million years ago. RESULTS: Here we describe finished sequencing and analysis of 11 fosmids hybridizing to the dot chromosome of D. virilis (372,650 base-pairs) and seven fosmids from major euchromatic chromosome arms (273,110 base-pairs). Most genes from the dot chromosome of D. melanogaster remain on the dot chromosome in D. virilis, but many inversions have occurred. The dot chromosomes of both species are similar to the major chromosome arms in gene density and coding density, but the dot chromosome genes of both species have larger introns. The D. virilis dot chromosome fosmids have a high repeat density (22.8%), similar to homologous regions of D. melanogaster (26.5%). There are, however, major differences in the representation of repetitive elements. Remnants of DNA transposons make up only 6.3% of the D. virilis dot chromosome fosmids, but 18.4% of the homologous regions from D. melanogaster; DINE-1 and 1360 elements are particularly enriched in D. melanogaster. Euchromatic domains on the major chromosomes in both species have very few DNA transposons (less than 0.4 %). CONCLUSION: Combining these results with recent findings about RNAi, we suggest that specific repetitive elements, as well as density, play a role in determining higher-order chromatin packaging.
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Mapeo Cromosómico , Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Drosophila/genética , Heterocromatina/genética , Animales , ADN/genética , Proteínas de Drosophila/genética , Etiquetas de Secuencia Expresada , Genoma , Hibridación in Situ , Modelos Genéticos , Modelos Estadísticos , Interferencia de ARN , Secuencias Repetitivas de Ácidos Nucleicos , Retroelementos/genética , Estadísticas no ParamétricasRESUMEN
Heterochromatin protein 1 (HP1), first discovered in Drosophila melanogaster, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. We report here characterization of an HP1-interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT-hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing.