No abstract

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.

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.

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.

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.Until recently, DNA transfection studies aimed at identifying human oncogenes (genetic elements capable of inducing malignant transformation) were limited to the use of NIH 3T3 cells in culture, This assay involves the development of morphologically altered foci in cell monolayers on tissue culture dishes. Very often the genes from human tumors that had focus-forming activity were found to be members'of the ras family of oncogenes. However, the vast majority of the DNA samples from human tumors do not have focusforming activity on NIH 3T3 cells. One explanation for this observation could be that the many genetic elements responsible for tumor formation in humans cannot effect morphological transformation of rodent cells. shown that this approach, direct tumor formation by transfected NIH 3T3 cells, can lead to the detection of new tumorigenic genetic loci of human origin. However, the NIH 3T3-transforming gene assay system suffers from a relatively high rate of spontaneous transformation, and we observed that tumors arising 12 or more weeks after cells were injected into mice were likely to be attributable to spontaneous transformation, rather than to the acquisition of specific human DNA sequences.