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At least 109 records · Page 6Linked to original sources
PubMed2006

Zygote arrest 1 gene in pig, cattle and human: evidence of different transcript variants in male and female germ cells.

BACKGROUND: Zygote arrest 1 (ZAR1) is one of the few known oocyte-specific maternal-effect genes essential for the beginning of embryo development discovered in mice. This gene is evolutionary conserved in vertebrates and ZAR1 protein is characterized by the presence of atypical plant homeobox zing finger domain, suggesting its role in transcription regulation. This work was aimed at the study of this gene, which could be one of the key regulators of successful preimplantation development of domestic animals, in pig and cattle, as compared with human. METHODS: Screenings of somatic cell hybrid panels and in silico research were performed to characterize ZAR1 chromosome localization and sequences. Rapid amplification of cDNA ends was used to obtain full-length cDNAs. Spatio-temporal mRNA expression patterns were studied using Northern blot, reverse transcription coupled to polymerase chain reaction and in situ hybridization. RESULTS: We demonstrated that ZAR1 is a single copy gene, positioned on chromosome 8 in pig and 6 in cattle, and several variants of correspondent cDNA were cloned from oocytes. Sequence analysis of ZAR1 cDNAs evidenced numerous short inverted repeats within the coding sequences and putative Pumilio-binding and embryo-deadenylation elements within the 3'-untranslated regions, indicating the potential regulation ways. We showed that ZAR1 expressed exclusively in oocytes in pig ovary, persisted during first cleavages in embryos developed in vivo and declined sharply in morulae and blastocysts. ZAR1 mRNA was also detected in testis, and, at lower level, in hypothalamus and pituitary in both species. For the first time, ZAR1 was localized in testicular germ cells, notably in round spermatids. In addition, in pig, cattle and human only shorter ZAR1 transcript variants resulting from alternative splicing were found in testis as compared to oocyte. CONCLUSION: Our data suggest that in addition to its role in early embryo development highlighted by expression pattern of full-length transcript in oocytes and early embryos, ZAR1 could also be implicated in the regulation of meiosis and post meiotic differentiation of male and female germ cells through expression of shorter splicing variants. Species conservation of ZAR1 expression and regulation underlines the central role of this gene in early reproductive processes.

3' Untranslated Regions↗
PubMed1997

Embryo research: the challenge for public policy.

Complete moral consensus on the status of the human embryo is neither feasible nor necessary for the formulation of ethically acceptable public policy for human embryo research. Significant consensus on permissible human embryo research can rest upon diverse but overlapping moral traditions. Thus, human embryo research policy should do more than reflect mere abstract assertions about the moral status of human embryos. Rather, the moral underpinnings of human embryo research should be derived from a range of values, including the facilitation of human procreation, the advancement of applied scientific knowledge, the reduction of human suffering, and the protection of vulnerable persons from coercion and exploitation.

Abortion, Induced↗
PubMed2001

Gene expression studies on human primordial germ cells and preimplantation embryos.

Research on the regulation of gene expression in human germ cells and preimplantation embryos is restricted due to the scarcity of samples and the requirement for highly sensitive molecular techniques to investigate the few cells available. To overcome these difficulties, we have developed a reliable procedure capable of generating amplified cDNA preparations from single cells. Using this procedure, we prepared cDNA from primordial germ cells (PGCs) isolated from the gonads of fetuses at 10 weeks gestation and from preimplantation embryos at the 1-cell, 4-cell, 8-cell and blastocyst stages. Our cDNA preparations allow us to investigate the expression profile of an almost unlimited number of different genes in the same sample preparation. This is of great advantage for studies of a panel of genes in a particular family or functional group, or with related mechanisms of regulation, e.g., developmental genes, oncogenes, cell cycle-control genes and imprinted genes. We have used these cDNA preparations in conjunction with differential display to identify genes specifically expressed in PGCs and preimplantation embryos in a sex- and developmental stage-specific manner. Genes specifically expressed in PGCs, oocytes and embryos were further analysed for their expression in embryonal carcinoma cells and in their differentiated derivatives following treatment by retinoic acid. Our strategy will disclose genes essential for gametogenesis and embryonic development which may only be expressed at certain stages of their development. The germ cell- and embryo-specific cDNA molecules, cDNA libraries and microarrays are a valuable resource for other researchers in this field.

Blastocyst↗
PubMed2003

Amplification of representative cDNA pools from single human oocytes and pronucleate embryos.

In the human embryo, gene expression studies have been hindered by the scarcity of material and the fact that in vitro fertilisation (IVF) embryos available for research are usually of poor quality and are, therefore, not representative of normal development. This has led most authors to study individual human embryos, using conventional RT-PCR strategies, which permit analysis of only a few genes. Variability in the expression of genes between individual embryos is characteristic of these studies. In this study, a global RT-PCR strategy has been used, allowing the analysis of an almost infinite number of genes from a single embryo. We have used oocytes, which failed to fertilise and representative pronucleate embryos donated from cycles in which the patient conceived, to investigate possible variability in transcript abundance between individual embryos. We have screened oocytes and embryos for a panel of genes including beta-actin (expressed in 24/28 oocytes, 6/6 pronuclear embryos), the integrins beta1 (17/28 oocytes, 6/6 pronuclear embryos) and beta5 (8/28 oocytes, 5/6 pronuclear embryos), and the apoptotic regulators BCL-2 (20/28 oocytes, 2/6 pronuclear embryos) and BAX (21/28 oocytes, 5/6 pronuclear embryos). The expression of the pro-apoptotic regulator BAX increased in human oocytes following prolonged periods of culture. Overall, patterns of gene transcript presence showed variation between embryos and this was independent of either zona removal or lysis conditions. Pronucleate embryos showed less variation, however, even sibling embryos from the patient did not express an identical subset of genes.

DNA, Complementary↗
PubMed1998

Analysis of a coded panel of licensed vaccines by polymerase chain reaction-based reverse transcriptase assays: a collaborative study [seecomments].

BACKGROUND: A recent publication reporting the presence of low levels of reverse transcriptase (RT) activity in certain vaccines for human use necessitated that regulatory agencies address the issue of whether this RT activity presented a risk to humans. Detection of low levels of RT activity corresponding to fewer than ten virions became possible with the development of highly-sensitive polymerase chain reaction (PCR)-based RT (PBRT) assays. Variations of the PBRT assay were developed in three laboratories. These assays were reported as being at least one million-fold more sensitive than conventional RT assays. OBJECTIVE: To ascertain the sensitivity and reliability of PBRT assays in different laboratories and to determine which vaccine samples possessed RT activity. STUDY DESIGN: Coded panels of licensed vaccines together with positive and negative controls was assembled at the Center for Biologics Evaluation and Research (CBER) of the Food and Drug Administration (FDA) and distributed to five cooperating laboratories as well as to our laboratory at CBER. Each laboratory carried out their version of the PBRT assay and submitted the results to the coordinator at CBER. RESULTS: Results of the PBRT analyses carried out in the six laboratories are presented. Five of the six laboratories reported results that were highly consistent. RT activity was detected in live attenuated vaccines that were prepared in chick embryo cells (mumps, measles and yellow fever), but very low or undetectable RT activity was found in vaccines produced in mammalian cells (rabies and rubella). Influenza vaccines from several manufacturers included in the panel displayed the most variability, with different products of this inactivated vaccine having differing amounts of RT activity. CONCLUSIONS: Only vaccines produced in chick embryo cells had significant RT activity. Because RT activity was present in the allantoic fluid of uninfected chick embryos and culture medium from chick embryo fibroblasts, the RT activity arises from the cell substrate used for vaccine production. The PBRT assays were reliably able to detect the low levels of RT activity in chicken-derived vaccines.

Animals↗
PubMed1987

Isolation of cross-reactive, subtype-specific monoclonal antibodies against influenza virus HA1 and HA2 hemagglutinin subunits.

The large (HA1) and small (HA2) subunits of influenza virus A/Vict/3/75 hemagglutinin were purified in denatured form by preparative electrophoresis. Both polypeptides were used to immunize mice from which monoclonal antibodies were obtained. These antibodies reacted not only with the corresponding hemagglutinin subunit but also with purified virions. When tested by radioimmunoassay against a panel of human viruses, most anti-HA1 and -HA2 antibodies behaved as subtype-specific, whereas anti-HA antibodies, raised against purified virus, were more restricted. The anti-subunit antibodies were negative in hemagglutination-inhibition and neutralization tests. The interest of these antibodies as reagents for research and diagnosis is discussed.

Animals↗
PubMed2002

Complete mutation analysis panel of the 39 human HOX genes.

BACKGROUND: The HOX gene family consists of highly conserved transcription factors that specify the identity of the body segments along the anteroposterior axis of the embryo. Because the phenotypes of mice with targeted disruptions of Hox genes resemble some patterns of human malformations, mutations in HOX genes have been expected to be associated with a significant number of human malformations. Thus far, however, mutations have been documented in only three of the 39 human HOX genes (HOXD13, HOXA13, and HOXA11) partly because current knowledge on the complete coding sequence and genome structure is limited to only 20 of the 39 human HOX genes. METHODS: Taking advantage of the human and mouse draft genome sequences, we attempted to characterize the remaining 19 human HOX genes by bioinformatic analysis including phylogenetic footprinting, the probabilistic prediction method, and comparison of genomic sequences with the complete set of the human anonymous cDNA sequences. RESULTS: We were able to determine the full coding sequences of 19 HOX genes and their genome structure and successfully designed a complete set of PCR primers to amplify the entire coding region of each of the 39 HOX genes from genomic DNA. CONCLUSIONS: Our results indicate the usefulness of bioinformatic analysis of the draft genome sequences for clinically oriented research projects. It is hoped that the mutation panel provided here will serve as a launchpad for a new discourse on the genetic basis of human malformations.

Animals↗
PubMed1983

Monoclonal antibody which recognizes a common antigenic determinant on intermediate filament proteins, actin, and myosin.

A monoclonal antibody is described which reacts with the intermediate filament proteins vimentin, desmin, keratins, actin, and myosin. This is the first report of an epitope common to intermediate filament proteins and myosin. X1, the wide-spectrum monoclonal antibody in question, was isolated in the course of screening monoclonal antibodies to chicken thymocytes. Cross-reactivities were investigated by immunofluorescence on various types of cultured cells and sectioned tissues, ELISA with a panel of purified antigens, immunoprecipitation, immunodot tests, and immunoblotting.

Actins↗
PubMed1998

Genomic structure of the gene for the SH2 and pleckstrin homology domain-containing protein GRB10 and evaluation of its role in Hirschsprung disease.

Hirschsprung disease (HSCR), or congenital aganglionic megacolon, is the most frequent cause of congenital bowel obstruction. Germline mutations in the RET receptor tyrosine kinase have been shown to cause HSCR. Mice that carry null alleles for RET or for its ligand, glial cell line-derived neurotrophic factor (GDNF), both exhibit complete intestinal aganglionosis and renal defects. Recently, the Src homology 2 (SH2) domain-containing protein Grb10 has been shown to interact with RET in vitro and in vivo, early in development. We have confirmed the map location of GRB10 on human chromosome 7, isolated human BACs containing the gene, elucidated its genomic structure, isolated a highly polymorphic microsatellite marker adjacent to exon 14 and scanned the gene for mutations in a large panel of HSCR patients. No evidence of linkage was detected in HSCR kindreds and no mutations were found in patients. These data suggest that while GRB10 may be important for signal transduction in developing embryos, it does not play an obvious role in HSCR.

Blood Proteins↗