RESUMO
The advent of human round spermatid microinjection (ROSI) into oocytes as a treatment for severe male infertility raises the question of whether spermatids have undergone all of the maturation processes necessary for normal development. It is particularly important to know whether spermatids have undergone correct genomic imprinting, which results in the parent-of-origin-specific expression of only one allele of a gene. We assessed the imprinting status of three maternally and three paternally expressed genes in interspecific hybrid embryos generated by injecting Mus castaneus spermatids into Mus musculus oocytes. We used the single nucleotide primer extension (SNuPE) assay to measure the relative expression of maternal and paternal alleles on the basis of sequence polymorphisms in the transcripts. Expression of imprinted genes in mouse embryos derived by ROSI did not differ from controls, indicating that paternal genes have undergone proper imprinting by the round spermatid stage.
Assuntos
Impressão Genômica , Espermátides/fisiologia , Animais , Feminino , Humanos , Inseminação Artificial , Masculino , Camundongos , Interações Espermatozoide-ÓvuloRESUMO
The centromere protein B (CENP-B) is a centromeric DNA/binding protein. It recognizes a 17-bp sequence motif called the CENP-B box, which is found in the centromeric region of most chromosomes. It binds DNA through its amino terminus and dimerizes through its carboxy terminus. CENP-B protein has been proposed to perform a vital role in organizing chromatin structures at centromeres. However, other evidence does not agree with this view. For example, CENP-B is found at inactive centromeres on stable dicentric chromosomes, and also mitotically stable chromosomes lacking alpha-satellite DNA have been reported. To address the biological function of CENP-B, we generated mouse null mutants of CENP-B by homologous recombination. Mice lacking CENP-B were viable and fertile, indicating that mice without CENP-B undergo normal somatic and germline development. Thus, both mitosis and meiosis are able to proceed normally in the absence of CENP-B.
Assuntos
Autoantígenos , Proteínas Cromossômicas não Histona/fisiologia , Proteínas de Ligação a DNA/fisiologia , Animais , Linhagem Celular , Proteína B de Centrômero , Proteínas Cromossômicas não Histona/química , Cruzamentos Genéticos , Fertilidade/genética , Viabilidade Fetal/genética , Genótipo , Histocitoquímica , Imuno-Histoquímica , Tamanho da Ninhada de Vivíparos , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , RNA Mensageiro/metabolismo , Contagem de Espermatozoides , Testículo/citologiaRESUMO
The plutonium (plu) gene product controls DNA replication early in Drosophila development. plu mutant females lay unfertilized eggs that have undergone extensive DNA synthesis. In fertilized embryos from plu mutant mothers, S-phase is uncoupled from mitosis. The gene is expressed only in ovaries and embryos, null alleles are strict maternal effect mutations, and the phenotype of inappropriate DNA replication is the consequence of loss-of-gene function. plu therefore negatively regulates S-phase at a time in early development when commitment to S-phase does not depend on cyclic transcription. plu encodes a protein with two ankyrin-like repeats, a domain for protein-protein interaction. plu is immediately adjacent to, but distinct from, the PCNA gene.
Assuntos
Anquirinas/genética , Replicação do DNA , Proteínas de Ligação a DNA , Proteínas de Drosophila , Drosophila/genética , Hormônios de Inseto/genética , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Drosophila/embriologia , Feminino , Humanos , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Mutations in the Drosophila maternal genes plutonium (plu) and pan gu (png) have the striking phenotype that DNA replication initiates in unfertilized eggs. Fertilized eggs from plu or png mutant mothers also have a mutant phenotype; DNA replication is uncoupled from nuclear division, resulting in giant, polyploid nuclei. Analysis of multiple alleles of these genes indicates that their wild-type function is required to maintain repression of DNA replication until fertilization. The phenotype of two png alleles suggests that this gene also may play a direct role in coupling S phase and mitosis during the early cleavage divisions. We describe genetic interactions among png, plu, and the previously identified gene gnu that demonstrate these three genes regulate the same process.
Assuntos
Replicação do DNA , Drosophila melanogaster/genética , Fertilização , Alelos , Animais , Ciclo Celular , Fase de Clivagem do Zigoto , Drosophila melanogaster/embriologia , Genes , Genes Reguladores , Teste de Complementação Genética , Mitose , MutaçãoRESUMO
We used human oncogene DNA to transform the nontumorigenic, revertant, human osteosarcoma cell line HOS TE-85 clone 5 (ATCC CRL 1543) to tumorigenicity in athymic nude mice with latency periods as short as 3 weeks. These cells were also transformed by genetic markers in genomic DNA samples. Because of their low rate of spontaneous tumor formation and the simplicity of culturing them, HOS cells provide a human cell alternative to NIH 3T3 murine fibroblasts for oncogene transfection studies.
