RESUMEN
The complete absence of eyes in the medaka fish mutation eyeless is the result of defective optic vesicle evagination. We show that the eyeless mutation is caused by an intronic insertion in the Rx3 homeobox gene resulting in a transcriptional repression of the locus that is rescued by injection of plasmid DNA containing the wild-type locus. Functional analysis reveals that Six3- and Pax6- dependent retina determination does not require Rx3. However, gain- and loss-of-function phenotypes show that Rx3 is indispensable to initiate optic vesicle evagination and to control vesicle proliferation, by that regulating organ size. Thus, Rx3 acts at a key position coupling the determination with subsequent morphogenesis and differentiation of the developing eye.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Ojo/crecimiento & desarrollo , Proteínas de Peces , Oryzias/crecimiento & desarrollo , Oryzias/genética , Retina/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/genética , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Proteínas Represoras , Proteínas de Dominio T Box/genética , Temperatura , Proteína Homeobox SIX3RESUMEN
Notch encodes a transmembrane protein that functions in intercellular signaling. Although there is one Notch gene in Drosophila, vertebrates have three or more with overlapping patterns of embryonic expression. We cloned the entire 7575-bp coding region of an amphioxus Notch gene (AmphiNotch), encoding 2524 amino acids, and obtained the exon/intron organization from a genomic cosmid clone. Southern blot and PCR data indicate that AmphiNotch is the only Notch gene in amphioxus. AmphiNotch, like Drosophila Notch and vertebrate Notch1 and Notch2, has 36 EGF repeats, 3 Notch/lin-12 repeats, a transmembrane region, and 6 ankyrin repeats. Phylogenetic analysis places it at the base of all the vertebrate genes, suggesting it is similar to the ancestral gene from which the vertebrate Notch family genes evolved. AmphiNotch is expressed in all three embryonic germ layers in spatiotemporal patterns strikingly similar to those of all the vertebrate homologs combined. In the developing nerve cord, AmphiNotch is first expressed in the posteriormost part of the neural plate, then it becomes more broadly expressed and later is localized dorsally in the anteriormost part of the nerve cord corresponding to the diencephalon. In late embryos and larvae, AmphiNotch is also expressed in parts of the pharyngeal endoderm, in the anterior gut diverticulum, and, like AmphiPax2/5/8, in the rudiment of Hatschek's kidney. A comparison with Notch1 and Pax5 and Pax8 expression in the embryonic mouse kidney helps support homology of the amphioxus and vertebrate kidneys. AmphiNotch is also an early marker for presumptive mesoderm, transcripts first being detectable at the gastrula stage in a ring of mesendoderm just inside the blastopore and subsequently in the posterior mesoderm, notochord, and somites. As in sea urchins and vertebrates, these domains of AmphiNotch expression overlap with those of several Wnt genes and brachyury. These relationships suggest that amphioxus shares with other deuterostomes a common mechanism for patterning along the anterior/posterior axis involving a posterior signaling center in which the Notch and Wnt pathways and brachyury interact.
Asunto(s)
Cordados no Vertebrados/embriología , Cordados no Vertebrados/genética , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Tipificación del Cuerpo/genética , Cordados no Vertebrados/crecimiento & desarrollo , Clonación Molecular , Secuencia Conservada , Cartilla de ADN/genética , ADN Complementario/genética , Evolución Molecular , Exones , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Intrones , Riñón/crecimiento & desarrollo , Ratones , Datos de Secuencia Molecular , Filogenia , Receptores Notch , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad de la Especie , VertebradosRESUMEN
A prerequisite to understanding the evolution of the human X chromosome is the analysis of synteny of X-linked genes in different species. We have focused on the spermine synthase gene in human Xp22. 1. We show that whereas the human gene spans a genomic region of 54 kb, the Fugu rubripes gene is encompassed in a 4.7-kb region. However, we could not find conserved synteny between this region of human Xp22 and the equivalent F. rubripes region. A cosmid clone containing the F. rubripes gene does not contain other X-linked genes. Instead we identified homologs of human genes that are autosomally localized: the ryanodine receptor type I (RYRI), which is implicated in malignant hyperthermia and central core disease, and the HE6 gene. Comparison of the F. rubripes, Tetraodon fluviatilis, mouse, human, and Danio rerio 5'UTRs of spermine synthase highlights conserved sequences potentially involved in regulation. Interestingly, pseudogenes of this gene that are present in the human and mouse genomes seem to be absent in the compact F. rubripes genome. Analysis of a D. rerio PAC clone containing spermine synthase shows an intermediate genomic size in this fish. Sequence analysis of this PAC clone did not reveal other known genes: neither the RYRI gene, nor the HE6 gene, nor other human Xp22 genes were identified.
Asunto(s)
Peces/genética , Espermina Sintasa/genética , Pez Cebra/genética , Empalme Alternativo , Animales , Exones , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Cromosoma XRESUMEN
To gain an insight into vertebrate genome evolution, we have analysed the organization of an approximately 40-kb genomic clone of an amphioxus (Branchiostoma floridae) cosmid library. Amphioxus is considered as being the last non-vertebrate relative to vertebrates. Sequencing and analysis of the above clone using three different exon prediction programs (Grail, GenScan, Mzef) have led to the identification of a gene of the aldo-keto reductase family as well as further exons that gave a significant database match to known genes.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Cordados no Vertebrados/genética , Aldehído Reductasa , Aldo-Ceto Reductasas , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cósmidos/genética , Datos de Secuencia Molecular , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de SecuenciaRESUMEN
The use of genomic libraries maintained in arrayed format is becoming a more and more popular tool for the analysis of molecular evolution and comparative molecular development. Being able to use already existing reference libraries considerably reduces the work load, and if results are made publicly available, it will facilitate in silica experiments in the future. Here we describe the construction and preliminary characterization of six cosmid libraries of different chordate species, Ciona intestinalis (Hemichordate), Branchiostoma floridae (Cephalochordate), Lampetra fluviatilis (Cyclostoma), Xiphophorus maculatus, and Danio rerio (Osteichthyes) in Lawrist7 and Fugu rubripes in Lawrist4.
Asunto(s)
Cordados no Vertebrados/genética , Biblioteca Genómica , Animales , Ciona intestinalis/genética , Clonación Molecular , Cósmidos , Ciprinodontiformes/genética , ADN/genética , Peces Venenosos/genética , Genoma , Lampreas/genética , Hibridación de Ácido Nucleico , Pez Cebra/genéticaRESUMEN
We describe a novel approach for the identification and mapping of polymorphic markers. Amplicons are generated by ligation of double-stranded adaptor molecules to genomic DNA cleaved with a restriction enzyme. Using primers that extend beyond the restriction site, reduced-complexity subsets of fragments are generated by PCR. Differences in the composition of complex probes generated from DNA of different strains are revealed through hybridization against high-density filter grids of large-insert genomic clones. Genetic mapping of genomic clones is achieved by hybridizing complex probes derived from backcross animals against the polymorphic clones. The mouse was chosen as a model system to test the feasibility of this technique because of the general availability of backcross resources and genomic libraries. Nevertheless, we would expect the method to be of particular use to generate markers for species that have not yet been extensively studied, because a substantial number of easy-to-use markers can be recruited in a relatively short period of time.
Asunto(s)
Mapeo Cromosómico/métodos , Polimorfismo Genético , Animales , Clonación Molecular/métodos , Cruzamientos Genéticos , Marcadores Genéticos , Biblioteca Genómica , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Ratones Endogámicos , Muridae/genética , Reacción en Cadena de la PolimerasaRESUMEN
We present a gridded genomic library of the honey-bee (Apis mellifera) for comparative and basic genetic study of the honeybee genome. The library will be established as a "Reference Library" system, and clones as well as data will be shared with the entire scientific community. This will accelerate the molecular level of honeybee genetics, combining the efforts of different laboratories. Because of male haploidy and the high rate of recombination, the honeybee is becoming a model organism for genomic studies of naturally occurring traits and behavioral genetics. The library consists of about 110,000 clones spotted at high density onto four filter membranes, representing 22 genome equivalents. Preliminary analysis using single-copy sequences revealed a positive clone number of the same order. The techniques for library generation and preliminary analysis as well as library access are described.
Asunto(s)
Abejas/genética , Biblioteca Genómica , Himenópteros/genética , Animales , Clonación Molecular/métodos , Bases de Datos Factuales , Hibridación de Ácido NucleicoRESUMEN
We have used telomeric DNA to break two acrocentric derivatives of the human Y chromosome into mini-chromosomes that are small enough to be size- fractionated by pulsed-field gel electrophoresis. One of the mini-chromosomes is about 7 Mb in size and sequence-tagged site analysis of this molecule suggests that it corresponds to a simple truncation of the short arm of the Y chromosome. Five of the mini-chromosomes are derived from the long arm, are all rearranged by more than a simple truncation, and range in size from 4.0 Mb to 9 Mb. We have studied the mitotic stabilities of these mini-chromosomes and shown that they are stably maintained by cells proliferating in culture for about 100 cell divisions.
Asunto(s)
Aberraciones Cromosómicas , Telómero , Cromosoma Y , Animales , Células CHO , Carboxiliasas/biosíntesis , Carboxiliasas/genética , División Celular , Células Cultivadas , Mapeo Cromosómico , Cricetinae , Genes Fúngicos , Humanos , Hibridación Fluorescente in Situ , Masculino , Mitosis , Plásmidos , Saccharomyces cerevisiae/genética , Lugares Marcados de Secuencia , TransfecciónRESUMEN
We have used telomeric DNA to break the human Y chromosome within the centromeric array of alphoid satellite DNA and have created two derivative chromosomes; one consists of the short arm and 140 kb of alphoid DNA, the other consists of the long arm and 480 kb of alphoid DNA. Both segregate accurately at mitosis. It is known that there is no large scale sequence duplication around the alphoid DNA and so the simplest interpretation of our results is that the sequence responsible for accurate segregation is the alphoid DNA itself. Although the long arm acrocentric derivative segregates accurately it lags with respect to the other chromosomes in about 10% of anaphase cells and thus additional sequences may be required for orderly segregation. The short arm acrocentric chromosome is probably no larger than 12 Mb in size and thus our results also demonstrate that chromosomes of this size are capable of accurate segregation.
Asunto(s)
Centrómero/genética , Clonación Molecular , ADN/genética , Cromosoma Y , Células Cultivadas , Humanos , Mapeo Restrictivo , Telómero/genéticaRESUMEN
Using DNA from Drosophila hydei KUN-DH-33 cells we describe an efficient method for selective and representative cloning of complex mixtures of satellite DNAs from eukaryotic genomes. Effective separation of satellite DNA from the bulk of all other sequences it obtained by fractionation of high molecular weight DNA by PFGE after treating it with '6 bp' restriction enzymes. Since extended clusters of tandemly arranged, so called simple sequence, repeats are inert to cleavage by most '6 bp' restriction enzymes the DNA fraction recovered from the gel region > 50 kb is mainly a mixture of satellites. Efficient and representative cloning of this DNA is performed by sonication to an average size of 50-500 bp and ligation of the blunt ended DNA fragments into the Bluescript vector pBS.