Location of enhancers is essential for the imprinting of H19 and Igf2 genes

Genomic imprinting is the process in mammals by which gamete-specific epigenetic modifications establish the differential expression of the two alleles of a gene. The tightly linked H19 and Igf2 genes are expressed in tissues of endodermal and mesodermal origin, with H19 expressed from the maternal chromosome and Igf2 expressed from the paternal chromosome. A model has been proposed to explain the reciprocal imprinting of these genes; in this model, expression of the genes is governed by competition between their promoters for a common set of enhancers. An extra set of enhancers might be predicted to relieve the competition, thereby eliminating imprinting. Here we tested this prediction by generating mice with a duplication of the endoderm-specific enhancers. The normally silent Igf2 gene on the maternal chromosome was expressed in liver, consistent with relief from competition. We then generated a maternal chromosome containing a single set of enhancers located equidistant from 1gf2 and H19; the direction of the imprint was reversed. Thus, the location of the enhancers determines the outcome of competition in liver, and the strength of the H19 promoter is not sufficient to silence Igf2.     

Loss of H19 imprinting in esophageal cancer

Recent articles have reported that loss of imprinting (LOI) of the endogenous gene H19 was frequently found in lung cancer and chorio-carcinoma, common adulthood cancers. Consequently, we examined the status of genomic imprinting of H19 in 29 esophageal and 48 colorectal cancer specimens, and studied its relation to the expression of H19. Of 12 esophageal cancer specimens heterozygous for the RsaI polymorphism, 6 (50%) exhibited LOI of H19, but none of the 18 colorectal cancer specimens heterozygous for the RsaI polymorphism exhibited LOI of H19. The present study suggests that LOI of H19 may play an important role in the pathogenesis of esophageal cancer. Moreover, H19 expression was frequently abundant in both cancers, and all six esophageal cancers carried LOI with overexpressed H19. Therefore, this overexpression of H19 seems to be an important phenomenon for the development of esophageal and colorectal cancer cells.     

Imprinting of Igf2 and H19 from a 130 kb YAC transgene

A stringent test for imprint control elements is to examine their function at ectopic loci in transgenic experiments. Igf2 and H19 are part of a larger imprinting region and as a first step, we examined these reciprocally imprinted genes in transgenic experiments using a 130 kb YAC clone. After paternal inheritance, H19 was appropriately repressed and Igf2 was expressed, irrespective of copy number or genetic background. After maternal inheritance H19 was consistently expressed, albeit with some variability. The levels of H19 expression per copy of the transgene inversely correlated with Igf2 (-lacZ) expression in cis. The consistent imprinting of H19 from this YAC contrasts with the previously described imprinting of mini-H19 transgenes, which only occurs at multi-copy loci, is inconsistent, and is prone to genetic background effects. We propose a novel model in which silencing of the H19 gene is the default state and its activation after maternal inheritance is the key mechanistic event for imprinting in this region. In addition, in situ analysis of the Igf2-lacZ reporter indicates that additional mesoderm-specific enhancers are present within the YAC clone. No obvious phenotype was detected from the excess gene dosage of H19.     

The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.     

The Drosophila zygotic lethal gene shuttle craft is required maternally for proper embryonic development

The Drosophila gene shuttle craft (stc) is expressed zygotically in the embryonic central nervous system (CNS) where it is required to maintain the proper morphology of motoneuronal axon nerve routes following their migration from the ventral cord. Here, we report that a prominent maternal source of STC protein is also present throughout both oogenesis and embryogenesis. To determine whether this maternal component is required in the ovary and/or embryo, we used the Drosophila autosomal dominant female sterile technique to generate germ-line clones that lacked the stc maternal function. Our results demonstrate that a maternally derived source of STC protein is required during embryogenesis but not oogenesis. In contrast to the zygotic phenotype, the primary defect in embryos derived from stc germ-line clones affects segmentation by causing disruptions and deletions in distinct thoracic (T1-T3) and abdominal (A4-A8) segments. These localized defects are responsible for additional phenotypes observed later in development which include gaps in the ventral nerve cord and deletions of denticle belts in the cuticle. An additional phenotype occurring in all other neuromeric segments consists of the misguided migration of motoneuronal axons as they project out of the ventral nerve cord. Thus, the stc zygotic function is required later in development and cannot correct the segmentation and subsequent CNS abnormalities associated with loss of its earlier acting maternally derived activity.     

The yeast SEN1 gene is required for the processing of diverse RNA classes

A single base change in the helicase superfamily 1 domain of the yeast Saccharomyces cerevisiae SEN1 gene results in a heat-sensitive mutation that alters the cellular abundance of many RNA species. We compared the relative amounts of RNAs between cells that are wild-type and mutant after temperature-shift. In the mutant several RNAs were found to either decrease or increase in abundance. The affected RNAs include tRNAs, rRNAs and small nuclear and nucleolar RNAs. Many of the affected RNAs have been positively identified and include end-matured precursor tRNAs and the small nuclear and nucleolar RNAs U5 and snR40 and snR45. Several small nucleolar RNAs co-immunoprecipitate with Sen1 but differentially associate with the wild-type and mutant protein. Its inactivation also impairs precursor rRNA maturation, resulting in increased accumulation of 35S and 6S precursor rRNAs and reduced levels of 20S, 23S and 27S rRNA processing intermediates. Thus, Sen1 is required for the biosynthesis of various functionally distinct classes of nuclear RNAs. We propose that Sen1 is an RNA helicase acting on a wide range of RNA classes. Its effects on the targeted RNAs in turn enable ribonuclease activity.     

The mouse RecA-like gene Dmc1 is required for homologous chromosome synapsis during meiosis

The mouse Dmc1 gene is an E. coli RecA homolog that is specifically expressed in meiosis. The DMC1 protein was detected in leptotene-to-zygotene spermatocytes, when homolog pairing likely initiates. Targeted gene disruption in the male mouse showed an arrest of meiosis of germ cells at the early zygotene stage, followed by apoptosis. In female mice lacking the Dmc1 gene, normal differentiation of oogenesis was aborted in embryos, and germ cells disappeared in the adult ovary. Meiotic chromosome analysis of Dmc1-deficient mouse spermatocytes revealed random spread of univalent axial elements without correct pairing between homologs. In rare cases, however, we observed complex pairing among nonhomologs. Thus, the mouse Dmc1 gene is required for homologous synapsis of chromosomes in meiosis.     

Loss of H19 imprinting and up-regulation of H19 and SNRPN in a case with malignant mixed Müllerian tumor of the uterus

An unusual clinical presentation of a patient with neuronal intestinal dysplasia is presented. A 46-year-old male noted a palpable mass in the right lower quadrant of his abdomen for two months. A computed axial tomographic scan showed a thickened wall of the cecum with a tumor-like appearance. The excised specimen consisted of a mass caused by the thickened, edematous wall of the dilated cecum and appendix. The wall of the cecum and appendix measured up to 2.5 and 0.8 cm, respectively, in thickness. Microscopic studies showed extensive hyperplasia and hypertrophy of the ganglia and nerve plexuses and hypertrophy of the muscularis propria, consistent with neuronal intestinal dysplasia.     

The STUD gene is required for male-specific cytokinesis after telophase II of meiosis in Arabidopsis thaliana

During male meiosis in wild-type Arabidopsis the pollen mother cell (PMC) undergoes two meiotic nuclear divisions in the absence of cell division. Only after telophase II is a wall formed which partitions the PMC into four microspores. Each microspore undergoes two subsequent mitotic divisions to produce one vegetative cell and two sperm cells in the mature pollen grain. In this paper we describe the isolation and the phenotypic characterization of mutations in the STUD (STD) gene, which is specifically required for male-specific cytokinesis after telophase II of meiosis. Although the male meiotic nuclear divisions are normal in std mutant plants, no walls are formed resulting in a tetranucleate microspore. Despite the absence of cell division in the PMC, postmeiotic development in the coenocytic microspore proceeds relatively normally, resulting in the formation of large pollen grains which contain four vegetative nuclei and up to eight sperm cells. Interestingly, these enlarged pollen grains which contain multiple vegetative nuclei and extra sperm cells behave as single male gametophytes, producing only single pollen tubes and resulting in partial male fertility in std mutant plants. Characterization of the process of pollen development and pollen function in std mutants thus reveals two different types of developmental regulation. Each of the four nuclei found in a std microspore following meiosis is capable of independently undergoing the complete mitotic cell division (including cytokinesis) which the single nucleus of a wild-type microspore would normally undertake. The ability of the four meiotic products to independently continue through mitosis does not depend on their division into separate cells, but is controlled by some subcellular component found within the coenocytic microspore. By contrast, the mature std pollen grain functions as a unit and produces only a single pollen tube despite the presence of multiple nuclei within the vegetative cell, suggesting that this process is controlled at the cellular level independently of the extra subcellular components.     

The GURKE gene is required for normal organization of the apical region in the Arabidopsis embryo

Dicot plant embryos undergo a transition from radial to bilateral symmetry. In Arabidopsis, this change reflects patterning within the apical region, resulting in the formation of the cotyledon and shoot meristem primordia. Mutations in the GURKE gene give seedlings with highly reduced or no cotyledons. Both strong and weak gurke alleles confer this phenotypic variability although strong alleles often eliminate the entire apex and sometimes also part of the hypocotyl. The root and the root meristem as well as the radial pattern of concentric tissue layers are essentially normal. The mutant seedling phenotype can be traced back to the triangular/early-heart stage of embryogenesis when abnormal cell divisions occur within the apical region such that no or only rudimentary cotyledon primordia are established. The postembryonic development of gurke seedlings was examined in culture. In weak alleles, apical growth gave rise to abnormal leaves and stem-like structures and, eventually, abnormal flowers. In strong alleles, the apical region often failed to grow but occasionally produced fused leaf-like structures with no dorso-ventral polarity and a totally unorganized vascular system while no stems developed. The observations suggest that the GURKE gene is involved primarily in the organization of the apical region in the embryo and may also play a role during postembryonic development.     

DAD1 is required for the function and the structural integrity of the oligosaccharyltransferase complex

Asparagine-linked glycosylation is a highly conserved protein modification reaction that occurs in all eukaryotic organisms. The oligosaccharyltransferase (OST), which has its active site exposed on the luminal face of the endoplasmic reticulum (ER), catalyzes the transfer of preassembled high mannose oligosaccharides onto certain asparagine residues of nascent polypeptides. The mammalian OST complex was initially thought to be composed of three transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Most recently, a small integral membrane protein of 12 kDa called DAD1 has been identified as an additional member of the mammalian OST complex. A point mutation in the DAD1 gene is responsible for the temperature-sensitive phenotype of a baby hamster kidney-derived cell line (tsBN7) that undergoes apoptosis at the non-permissive temperature. Furthermore, the mutant protein DAD1 is not detectable in tsBN7 cells 6 h after shifting the cells to the non-permissive temperature. This temperature-sensitive cell line offered unique opportunities to study the effects caused by the loss of one OST subunit on the other three subunits and also on N-linked glycosylation. Western blot analysis of cell lysates showed that after 6 h at the non-permissive temperature, steady-state levels of the ribophorins were reduced by about 50%, and OST48 was barely detectable. On the other hand, steady-state levels of other components of the rough ER, such as the alpha-subunits of the TRAP (translocon-associated membrane protein) and the Sec61 complex, which are components of the translocation apparatus, are not affected by the instability of the OST subunits. Furthermore, N-glycosylation of the ribophorins was seriously affected 6 h after shifting the cells to the non-permissive temperature, and after 12 h they were synthesized only in the non-glycosylated form. As may be expected, this defect in the OST complex at the non-permissive temperature caused also the underglycosylation of a secretory glycoprotein. We concluded that degradation of DAD1 at the non-permissive temperature not only affects the stability of OST48 and the ribophorins but also results in the functional inactivation of the OST complex.     

The Schizosaccharomyces pombe cdc14 gene is required for septum formation and can also inhibit nuclear division

A conditional heat-sensitive mutation in the cdc14 gene of the fission yeast Schizosaccharomyces pombe results in failure to form a septum. Cells become highly elongated and multinucleate as growth and nuclear division continue in the absence of cell division. This article describes the cloning of the cdc14 gene and the identification of its product, a protein of 240 amino acids, p28cdc14. A null allele of the cdc14 gene shows that the gene is essential for septum formation and completion of the cell-division cycle. Overexpression of the gene product, p28cdc14, causes cell-cycle arrest in late G2 before mitosis. Cells leaking past the block activate p34cdc2 kinase and show condensed chromosomes, but the normal rearrangements of the microtubules and microfilaments that are associated with the transition from interphase to mitosis do not occur. Overexpression of p28cdc14 in mutants, in which the timing of mitosis is altered, suggests that these effects may be mediated upstream of the mitotic inhibitor wee1. These data are consistent with the idea that p28cdc14 may play a role in both the initiation of mitosis and septum formation and, by doing so, be part of the mechanism that coordinates these two cell-cycle events.     

The imprinting box of the mouse Igf2r gene

Genomic imprinting is a phenomenon characterized by parent-of-origin-specific expression. The imprint is a mark established during germ-cell development to distinguish between the paternal and maternal copies of the imprinted genes. This imprint is maintained throughout embryo development and erased in the embryonic gonads to set the stage for a new imprint. DNA methylation is essential in this process as shown by the presence of differentially methylated regions (DMRs) in all imprinted genes and by the loss of imprinting in mice that are deficient in DNA methylation or upon deletion of DMRs. Here we show that a DMR in the imprinted Igf2r gene (which encodes the receptor for insulin-like growth factor type-2) that has been shown to be necessary for imprinting includes a 113-base-pair sequence that constitutes a methylation imprinting box. We identify two new cis-acting elements in this box that bind specific proteins: a de novo methylation signal and an allele-discrimination signal. We propose that this regulatory system, which we show to be involved in the establishment of differential methylation in the Igf2r DMR, represents a critical element in the imprinting process.     

Chromatin modification of imprinted H19 gene in mammalian spermatozoa

To date, most functional analysis studies have focused on the effects of treatment contingencies on specific targeted aberrant and alternative responses. In the current investigation, the main and collateral effects of the assessment and treatment of attention-maintained self-injury were assessed. Specifically, we evaluated the effects of noncontingent and contingent social attention on four categories of behavior: self-injury, a novel mand, preexisting prosocial responses (e.g., babbling and reaching out), and other aberrant responses (i.e., aggression and destruction). Results suggested that self-injury, prosocial responses, and other aberrant behaviors were within the same functional response class. Possible impact of these results when selecting mands for functional communication training is discussed.     

The Caenorhabditis elegans spe-5 gene is required for morphogenesis of a sperm-specific organelle and is associated with an inherent cold-sensitive phenotype

The nonrandom segregation of organelles to the appropriate compartment during asymmetric cellular division is observed in many developing systems. Caenorhabditis elegans spermatogenesis is an excellent system to address this issue genetically. The proper progression of spermatogenesis requires specialized intracellular organelles, the fibrous body-membranous organelle complexes (FB-MOs). The FB-MOs play a critical role in cytoplasmic partitioning during the asymmetric cellular division associated with sperm meiosis II. Here we show that spe-5 mutants contain defective, vacuolated FB-MOs and usually arrest spermatogenesis at the spermatocyte stage. Occasionally, spe-5 mutants containing defective FB-MOs will form spermatids that are capable of differentiating into functional spermatozoa. These spe-5 spermatids exhibit an incomplete penetrance for tubulin mis-segregation during the second meiotic division. In addition to morphological and FB-MO segregation defects, all six spe-5 mutants are cold-sensitive, exhibiting a more penetrant sterile phenotype at 16 degrees than 25 degrees. This cold sensitivity could be an inherent property of FB-MO morphogenesis.