RESUMEN
The body reserves of adult Lepidoptera are accumulated during larval development. In the Glanville fritillary butterfly, larger body size increases female fecundity, but in males fast larval development and early eclosion, rather than large body size, increase mating success and hence fitness. Larval growth rate is highly heritable, but genetic variation associated with larval development is largely unknown. By comparing the Glanville fritillary population living in the Åland Islands in northern Europe with a population in Nantaizi in China, within the source of the post-glacial range expansion, we identified candidate genes with reduced variation in Åland, potentially affected by selection under cooler climatic conditions than in Nantaizi. We conducted an association study of larval growth traits by genotyping the extremes of phenotypic trait distributions for 23 SNPs in 10 genes. Three genes in clip-domain serine protease family were associated with larval growth rate, development time and pupal weight. Additive effects of two SNPs in the prophenoloxidase-activating proteinase-3 (ProPO3) gene, related to melanization, showed elevated growth rate in high temperature but reduced growth rate in moderate temperature. The allelic effects of the vitellin-degrading protease precursor gene on development time were opposite in the two sexes, one genotype being associated with long development time and heavy larvae in females but short development time in males. Sexually antagonistic selection is here evident in spite of sexual size dimorphism.
Asunto(s)
Alelos , Mariposas Diurnas/crecimiento & desarrollo , Larva/crecimiento & desarrollo , Serina Proteasas/genética , Temperatura , Animales , Mariposas Diurnas/genética , Femenino , Masculino , TranscriptomaRESUMEN
Allozyme variation at the phosphoglucose isomerase (PGI) locus in the Glanville fritillary butterfly (Melitaea cinxia) is associated with variation in flight metabolic rate, dispersal rate, fecundity and local population growth rate. To map allozyme to DNA variation and to survey putative functional variation in genomic DNA, we cloned the coding sequence of Pgi and identified nonsynonymous variable sites that determine the most common allozyme alleles. We show that these single-nucleotide polymorphisms (SNPs) exhibit significant excess of heterozygotes in field-collected population samples as well as in laboratory crosses. This is in contrast to previous results for the same species in which other allozymes and SNPs were in Hardy-Weinberg equilibrium or exhibited an excess of homozygotes. Our results suggest that viability selection favours Pgi heterozygotes. Although this is consistent with direct overdominance at Pgi, we cannot exclude the possibility that heterozygote advantage is caused by the presence of one or more deleterious alleles at linked loci.
Asunto(s)
Mariposas Diurnas/fisiología , Glucosa-6-Fosfato Isomerasa/genética , Polimorfismo de Nucleótido Simple/genética , Animales , Mariposas Diurnas/genética , Mariposas Diurnas/metabolismo , Femenino , Variación Genética , Genotipo , Masculino , FenotipoRESUMEN
Important general insights into the mechanism of pre-mRNA splicing have emerged from studies of the U12-dependent spliceosome. Here, photochemical cross-linking analyses during U12-dependent spliceosome assembly have surprisingly revealed that an upstream 5' exon region is required for establishing two essential catalytic core interactions, U12/U6atac helix Ib and U6atac/5' splice site contacts, but not for U5/5' exon interactions or partial unwinding of U4atac/U6atac. A novel intermediate, representing an alternative pathway for catalytic core formation, is a ternary snRNA complex containing U4atac/U6atac stem II and U12/U6atac helix Ia that forms even without U6atac replacing U11 at the 5' splice site. A powerful oligonucleotide displacement method suggests that the blocked complexes analyzed to deduce the interdependence of these multiple RNA exchanges are authentic intermediates in U12-dependent spliceosome assembly.
Asunto(s)
Dominio Catalítico/genética , ARN Mensajero/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/genética , Exones/genética , Conformación de Ácido Nucleico , Oligonucleótidos/genética , ARN Mensajero/química , ARN Mensajero/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismoRESUMEN
We have investigated the formation of prespliceosomal complex A in HeLa nuclear extracts on a splicing substrate containing an AT-AC (U12-type) intron from the P120 gene. Using an RNase H protection assay and specific blocking oligonucleotides, we find that recognition of the 5' splice-site (5'ss) and branchpoint sequence (BPS) elements by U11 and U12 snRNPs, respectively, displays strong cooperativity, requiring both sites in the pre-mRNA substrate for efficient complex formation. Deletion analysis indicates that beside the 5'ss and BPS, no additional elements in the pre-mRNA are necessary for A-complex formation, although 5' exon sequences provide stimulation. Cross-linking studies with pre-mRNAs containing the 5'ss or BPS alone indicate that recognition of the BPS by the U12 snRNP is stimulated at least 20- to 30-fold by the binding of the U11 snRNP to the 5'ss in the same pre-mRNA molecule, whereas recognition of the 5'ss by U11 is stimulated approximately fivefold by the U12/BPS interaction. These results argue that intron recognition in the U12-dependent splicing pathway is carried out by a single U11/U12 di-snRNP complex, suggesting greater rigidity in the intron recognition process than in the major spliceosome.
Asunto(s)
Empalme del ARN/fisiología , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Adenoviridae/genética , Northern Blotting , Relación Dosis-Respuesta a Droga , Evolución Molecular , Ficusina/metabolismo , Células HeLa , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Intrones , Modelos Genéticos , Oligonucleótidos/metabolismo , Ribonucleasa H/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteínas/metabolismo , Factores de TiempoRESUMEN
Empty procapsids of the segmented dsRNA virus phi 6, produced in Escherichia coli from a cloned L genome segment, package plus-strand phi 6 ssRNA genomic segments, synthesize minus strands, and transcribe the newly formed dsRNA templates. Procapsids can be restricted to minus-strand synthesis by high concentrations of CaCl2 or low concentrations of nucleotides, enabling us to separate the viral minus-strand (replication) and plus-strand (transcription) RNA-dependent RNA polymerase activities in vitro. Reaction conditions for minus-strand synthesis were optimized. Plus-strand synthesis by procapsids could be activated by binding of purine nucleoside triphosphates to a low-affinity NTP-binding site. The second 5'-terminal nucleotide of the phi 6 plus-sense ssRNA L genomic segment is important for determining the level of transcription of that segment and the generation of infectious procapsids.
Asunto(s)
Bacteriófago phi 6/fisiología , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Bicatenario/biosíntesis , ARN Viral/biosíntesis , Replicación Viral , Bacteriófago phi 6/genética , Sitios de Unión , Escherichia coli , Genoma Viral , Nucleótidos de Purina/metabolismo , ARN Viral/aislamiento & purificación , Transcripción Genética , Uridina Trifosfato/metabolismoRESUMEN
Bacteriophage phi6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (L, M, S). These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4, and P7. Empty preformed procapsids are able to package the plus-sense single-stranded RNA (ssRNA) of each genome segment, to synthesize the corresponding minus strands ("replication") to form dsRNA segments, and to continue to the plus strand synthesis ("transcription") in which the dsRNA segments are used as templates in production of plus-sense ssRNA. In this study, we have investigated the requirements for the switch-on of minus and plus strand syntheses. We show that there exists an inverse relationship between regulation of the ssRNA packaging and minus strand synthesis. The packaging of single-stranded l, which has previously been shown to be packaged as the last, is the necessary signal for the onset of the minus strand synthesis. The absolute requirement for plus strand synthesis is minus strand synthesis of l, but in addition, the minus strand synthesis of m and the packaging of s segment are needed for efficient plus strand synthesis. Furthermore, the second nucleotide at the 5'-end of each segment regulates the extent of the transcription.
Asunto(s)
Bacteriófago phi 6/genética , ARN Bicatenario/biosíntesis , ARN Viral/biosíntesis , Bacteriófago phi 6/crecimiento & desarrollo , Secuencia de Bases , Cápside/metabolismo , Genoma Viral , Modelos Genéticos , Datos de Secuencia Molecular , ARN Polimerasa Dependiente del ARN/metabolismo , Transcripción Genética , Ensamble de Virus , Replicación ViralRESUMEN
Bacteriophage phi 6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (l, m, s). These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4 and P7. Expression of the cDNA copy of the L segment in Escherichia coli leads to the formation of empty procapsid particles. These particles are able to package the plus-sense single-stranded RNA (ssRNA)s of each genome segment in vitro. We have used this in vitro system for a detailed study of phi 6 RNA packaging. The reaction conditions for RNA packaging were optimized using a RNase protection assay. The RNA packaging reaction is dependent on divalent cations (either Mg2+ or Mn2+) and requires a nucleoside triphosphate (NTP) as an energy source. Any one of the rNTPs, dNTPs or ddNTPs can support the RNA packaging. Purine nucleotides support packaging better than pyrimidine nucleotides, GTP being preferred to ATP. The plus-sense ssRNA of each the three genome segments can be packaged independently into the procapsid. However, when two or three segments are packaged simultaneously, regulatory effects modulating the packaging efficiency can be detected between the segments. The packaging of the s and m segments is more efficient when they are packaged alone, compared to a situation in which they are packaged with the other segments. In contrast, the packaging of the l segment is very inefficient alone, but is enhanced when packaged together with the m segment. We propose that each segment has a preferred high-affinity binding site in the procapsid particle and packaging of the m segment creates the high-affinity binding site for the l segment. If any of the segments is missing from the packaging reaction the other segments can occupy its binding site.
Asunto(s)
Bacteriófago phi 6/genética , ARN Bicatenario/genética , ARN Viral/genética , Unión Competitiva , Cinética , NucleótidosRESUMEN
Bacteriophage phi 6 has a genome consisting of three segments of double-stranded RNA designated L, M, and S. Each virion contains one of each genomic segment. Empty procapsids can package plus-strand transcripts of the genomic segments if the 5' regions are intact. Minus-strand synthesis takes place if all three segments are packaged and if the 3' end of the segment is intact. The 3' ends of the segments contain four hairpin structures within a region of high sequence conservation. We now show that removal of parts of this region leads to progressive but limited loss of ability to support minus-strand synthesis. The defective 3' ends can be corrected by heterologous recombination with the termini of other segments. Segments that have small deletions in the conserved region and that support apparently normal minus-strand synthesis are highly recombinogenic.
Asunto(s)
Bacteriófago phi 6/genética , Conformación de Ácido Nucleico , ARN Bicatenario/química , ARN Viral/química , Transcripción Genética , Secuencia de Bases , ADN Viral , Datos de Secuencia Molecular , ARN Bicatenario/biosíntesis , ARN Viral/biosíntesis , ARN Viral/metabolismoRESUMEN
Bacteriophage phi 6 has a genome of three segments of double-stranded RNA enclosed in a procapsid composed of four different proteins. The preformed procapsid is capable of packaging plus-strand transcripts of the genomic segments in an in vitro reaction. The packaging-specific sequences on the RNA molecules are located near the 5' ends. In this study we show that the packaging sequences are different for each of the three segments and that they are of about 250 nucleotides in length. Although these sequences are consistent with some secondary structure, there is no clear structural similarity between the packaging regions of the three segments.
Asunto(s)
Bacteriófago phi 6/crecimiento & desarrollo , Bacteriófago phi 6/genética , ARN Mensajero/metabolismo , ARN Viral/genética , Secuencia de Bases , Análisis Mutacional de ADN , Genoma Viral , Datos de Secuencia Molecular , Eliminación de SecuenciaRESUMEN
Bacteriophage phi 6 has a genome of three segments of double-stranded RNA, designated L, M, and S. A 1.2-kbp kanamycin resistance gene was inserted into segment M but was shown to be genetically unstable because of a high recombination rate between segment M and the 3' ends of segments S and L. The high rate of recombination is due to complementary homopolymer tracts bounding the kan gene. Removal of one arm of this potential hairpin stabilizes the insertion. The insertion of a 241- or 427-bp lacZ' gene into segment M leads to a stable Lac+ phage. The insertion of the same genes bounded by complementary homopolymer arms leads to recombinational instability. A stable derivative of this phage was shown to have lost one of the homopolymer arms. Several other conditions foster recombination. The truncation of a genomic segment at the 3' end prevents replication, but such a damaged molecule can be rescued by recombination. Similarly, insertion of the entire 3-kb lacZ gene prevents normal formation of virus, but the viral genes can be rescued by recombination. It appears that conditions leading to the retardation or absence of replication of a particular genomic segment facilitate recombinational rescue.
Asunto(s)
Bacteriófago phi 6/genética , Pseudomonas/genética , ARN Viral/metabolismo , Recombinación Genética , Bacteriófago T7/enzimología , Secuencia de Bases , Clonación Molecular , ADN , ADN Viral/genética , ADN Viral/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Electroforesis en Gel de Agar , Modelos Genéticos , Modelos Estructurales , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , ARN Bicatenario/genética , ARN Bicatenario/aislamiento & purificación , ARN Viral/genética , ARN Viral/aislamiento & purificación , Mapeo Restrictivo , Transcripción Genética , Ensayo de Placa ViralRESUMEN
Bacteriophage phi 6 has a segmented genome consisting of three pieces of double-stranded RNA (dsRNA). The viral procapsid is the structure that packages plus strands, synthesizes the complementary negative strands to form dsRNA, and then transcribes dsRNA to form plus-strand message. The minus-strand synthesis of a particular genomic segment is dependent on prior packaging of the other segments. The 5' end of the plus strand is necessary and sufficient for packaging, while the normal 3' end is necessary for synthesis of the negative strand. We have now investigated the ability of truncated RNA segments which lack the normal 3' end of the molecules to stimulate the synthesis of minus strands of the other segments. Fragments missing the normal 3' ends were able to stimulate the minus-strand synthesis of intact heterologous segments. Minus-strand synthesis of one intact segment could be stimulated by the presence of two truncated nonreplicating segments. The 5' fragments of each single-stranded genomic segment can compete with homologous full-length single-stranded genomic segments in minus-strand synthesis reactions, suggesting that there is a specific binding site in the procapsid for each segment.
Asunto(s)
Bacteriófagos/genética , Cápside/genética , Escherichia coli/genética , Genoma Viral , Pseudomonas/genética , ARN Bicatenario/genética , Transcripción Genética , Proteínas del Núcleo Viral/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Mapeo RestrictivoRESUMEN
The genome of bacteriophage phi 6 contains three segments of double-stranded RNA. Procapsid structures whose formation was directed by cDNA copies of the large genomic segment are capable of packaging the three viral message sense RNAs in the presence of ATP. Addition of UTP, CTP, and GTP results in the synthesis of minus strands to form double-stranded RNA. In this report, we show that procapsids are capable of taking up any of the three plus-strand single-stranded RNA segments independently of the others. In manganese-containing buffers, synthesis of the corresponding minus strand takes place. In magnesium-containing buffers, individual message sense viral RNA segments were packaged, but minus-strand replication did not take place unless all three viral single-stranded RNA segments were packaged. Since the conditions of packaging in magnesium buffer more closely resemble those in vivo, these results indicated that there is no specific order or dependence in packaging and that replication is regulated so that it does not begin until all segments are in place.
Asunto(s)
Cápside/metabolismo , Fagos ARN/crecimiento & desarrollo , ARN Bicatenario/metabolismo , Virión/crecimiento & desarrollo , Replicación Viral , Magnesio/farmacología , Manganeso/farmacología , Fagos ARN/efectos de los fármacos , Virión/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
We have determined the nucleotide sequence of the late region (11 kbp) of the lipid-containing bacteriophage PRD1. Gene localization was carried out by complementing nonsense phage mutants with genomic clones containing specific reading frames. The localization was confirmed by sequencing the N-termini of isolated gene products as well as sequencing the N-termini of tryptic fragments of the phage membrane-associated proteins. This, with the previously obtained sequence of the early regions, allowed us to organize most of the phage genes in the phage genome.