Independent regulation of H-2K and H-2D gene expression in murine teratocarcinoma somatic cell hybrids.

Cells of two teratocarcinoma stem cell lines (PCC4 azaguanine [aza] 1 and F9 5-bromodeoxyuridine [BrdU]) were fused with normal mouse spleen cells and mouse thymoma-derived cells (BW 5147), respectively. Hybrid clones were tested for the expression of molecules coded by the H-2K and -2D genes both by absorption analysis of conventional H-2 sera and by indirect antibody-binding radioimmunoassay with monoclonal antibodies. Somatic cell hybrids between PCC4 aza 1 and spleen cells morphologically resemble teratocarcinoma stem cells and do not express H-2 antigens. However, after differentiation in vitro, one of these hybrid clones expresses the H-2K and -2D gene products of both parental cell lines, one close expresses H-2-D- but not H-2K-coded antigenic determinants, and one clone remains H-2 negative. Somatic cell hybrids between F9 BrdU and BW 5147 resemble fibroblasts. Analysis of a series of hybrid clones revealed some clones that express both the H-2K- and H-2D-coded antigenic specificities of both parental alleles, some that express H-2D gene products strongly and the H-2K gene products very weakly, and some that express H-2D- but not H-2K-coded molecules. These results imply independent regulation of expression of the H-2K and -2D genes. The H-2D gene products appear to be preferentially expressed if the hybrid cells are capable of expressing H-2. The results suggest complex regulatory mechanisms that are H-2K and H-2D specific.

Complement-mediated antiserum cytotoxic reactions to human chromosome 7 coded antigen(s): immunoselection of rearranged human chromosome 7 in human-mouse somatic cell hybrids.

Immunoselection via complement-dependent lysis of human-mouse somatic cell hybrids containing chromosome 7, with antisera reactive to cell surface antigen(s) coded for by chromosome 7, has resulted in growth of somatic cell hybrids containing rearranged human chromosome 7s. Investigation of these hybrids has localized the gene(s) coding for the relevant cell surface antigen(s) to the short arm of human chromosome 7. The simian virus 40 integration site and the gene coding for human beta-glucuronidase appear to be localized to the long arm of chromosome 7 in this hybrid clone.

Inability of mouse blastomere nuclei transferred to enucleated zygotes to support development in vitro.

More than 90 percent of enucleated one-cell mouse embryos receiving pronuclei from other one-cell embryos successfully develop to the blastocyst stage in vitro. In this investigation, nuclei from successive preimplantation cleavage stages were introduced into enucleated one-cell embryos and the embryos were tested for development in vitro. Although two-cell nuclei supported development to the morula or blastocyst stage, four-cell, eight-cell, and inner cell mass cell nuclei did not. The inability of cell nuclei from these stages to support development reflects rapid loss of totipotency of the transferred nucleus and is not the result of simultaneous transfer of membrane or cytoplasm.

Nuclear transplantation in the mouse embryo by microsurgery and cell fusion.

Nuclear transplantation in the mouse embryo was achieved by using a method that combines microsurgical removal of the zygote pronuclei with the introduction of a donor nucleus by a virus-mediated cell fusion technique. Survival of embryos was greater than 90 per cent in tests of this procedure. The embryos developed to term at a frequency not significantly different from that of nonmanipulated control embryos. Because nuclei and cytoplasm from genetically distinct inbred mouse strains can be efficiently interchanged, this procedure may be useful in characterizing possible cytoplasmic contributions to the embryonic and adult phenotype.

Raf, a trans-acting locus, regulates the alpha-fetoprotein gene in a cell-autonomous manner.

Genetic analysis provides an approach for identifying regulatory loci that govern the expression of specific genes within the context of the entire organism. Such analyses have defined two unlinked regulatory loci, termed raf and Rif, that modulate the levels of alpha-fetoprotein in liver. Of primary importance for the isolation and characterization of the raf product is to determine whether it is produced by the hepatocyte or whether it is produced by a different cell type. By means of analysis of alpha-fetoprotein expression in livers of embryo aggregation chimeras derived from mice of different raf genotypes it was possible to conclude that the product of the raf locus is expressed as a hepatocyte autonomous function that acts in trans to regulate the level of alpha-fetoprotein messenger RNA.

Tumor location detected with radioactively labeled monoclonal antibody and external scintigraphy.

Murine teratocarcinomas were located in mice by external gamma-ray scintigraphy with an iodine-125-labeled monoclonal antibody specific to the tumors. The specificity of the method was increased by subtracting the radiation produced by an iodine-125-labeled indifferent monoclonal antibody of the same immunoglobulin class as the tumor-specific antibody.

Metastatic hibernomas in transgenic mice expressing an alpha-amylase-SV40 T antigen hybrid gene.

Mice transgenic for a hybrid gene containing the liver promoter of the mouse amylase gene (Amy-1a) fused to the SV40 tumor antigen coding region unexpected developed malignant brown adipose tissue tumors (malignant hibernomas). Expression of the alpha-amylase gene had previously been thought to be confined to the liver parotid, and pancreas; however, analysis of white and brown adipose tissue from nontransgenic mice revealed expression of the endogenous Amy-1a gene in these tissues. Gene constructs driven by the Amy-1a liver promoter thus provide a means of targeting gene expression to the adipocyte cell lineage in transgenic mice. Moreover the high frequency of metastases in the liver, lungs, spleen, heart, and adrenals of these mice provides an experimental system in which to study the development of disseminated malignancy.

Unintended changes in cognition, mood, and behavior arising from cell-based interventions for neurological conditions: ethical challenges.

The prospect of using cell-based interventions (CBIs) to treat neurological conditions raises several important ethical and policy questions. In this target article, we focus on issues related to the unique constellation of traits that characterize CBIs targeted at the central nervous system. In particular, there is at least a theoretical prospect that these cells will alter the recipients' cognition, mood, and behavior-brain functions that are central to our concept of the self. The potential for such changes, although perhaps remote, is cause for concern and careful ethical analysis. Both to enable better informed consent in the future and as an end in itself, we argue that early human trials of CBIs for neurological conditions must monitor subjects for changes in cognition, mood, and behavior; further, we recommend concrete steps for that monitoring. Such steps will help better characterize the potential risks and benefits of CBIs as they are tested and potentially used for treatment.

Expression and regulation of the pituitary- and placenta-specific human glycoprotein hormone alpha-subunit gene is restricted to the pituitary in transgenic mice.

Expression of the glycoprotein hormone alpha subunit occurs in both the pituitary and placenta in humans. However, this study found that expression of this subunit is restricted to the pituitary in mice. An interspecies analysis of human alpha-subunit gene regulation was undertaken, using the transgenic-mouse approach. In mice transgenic for a genomic clone containing the complete human alpha-subunit gene and several kilobases of 5'- and 3'-flanking sequences, cell-type-specific expression and hormonal regulation of the human alpha-subunit transgene occurred in the mouse pituitary, whereas no expression of the transgene was detectable in the mouse placenta. These findings provide strong evidence that a common trans-acting factor(s) regulates glycoprotein hormone alpha-subunit gene expression in the human and mouse pituitaries; however, this factor(s) or a unique factor(s), though functional in the human placenta, is either nonfunctional or absent in the mouse placenta.

A DNA element that regulates expression of an endogenous retrovirus during F9 cell differentiation is E1A dependent.

The retinoic acid-induced differentiation of F9 cells into parietal endoderm-like cells activates transcription of the endogenous mouse retrovirus, the intracisternal A-particle (IAP). To investigate the elements that control IAP gene differentiation-specific expression, we used methylation interference, Southwestern (DNA-protein), and transient-transfection assays and identified the IAP-proximal enhancer (IPE) element that directs differentiation-specific expression. We find that the IPE is inactive in undifferentiated F9 cells and active in differentiated parietal endoderm-like PYS-2 cells. Three proteins of 40, 60, and 68 kDa bind to the sequence GAGTAGAC located between nucleotides -53 and -47 within the IPE. The 40- and 68-kDa proteins from both the undifferentiated and differentiated cells exhibit similar DNA-binding activities. However, the 60-kDa protein from differentiated cells has greater binding activity than that from undifferentiated cells, suggesting a role for this protein in F9 differentiation-specific expression of the IAP gene. The IAP gene is negatively regulated by the adenovirus E1A proteins, and the E1A sequence responsible for repression is located at the N terminus, between amino acids 2 and 67. The DNA sequence that is the target of E1A repression also maps to the IPE element. Colocalization of the differentiation-specific and E1A-sensitive elements to the same protein-binding site within the IPE suggests that the E1A-like activity functions in F9 cells to repress IAP gene expression. Activation of the IAP gene may result when the E1A-like activity is lost or inactivated during F9 cell differentiation, followed by binding of the 60-kDa positive regulatory protein to the enhancer element.

Immunohistochemical localization of the mouse stage-specific embryonic antigen 1 in human tissues and tumors.

Normal human tissues and various human tumors were surveyed by immunohistochemical techniques for expression of the stage-specific embryonic antigen 1 (SSEA-1). The antibody reacted with many normal and neoplastic human tissues. In most instances, equivalent human and mouse tissues expressed SSEA-1; however, different tissue localization patterns were sometimes seen between these two species. Most SSEA-1-positive tumors originate from tissues that normally expressed this antigen; however, some breast and ovarian tumors are SSEA-1 positive, and these organs are SSEA-1 negative. SSEA-1-positive tumors were composed of both immunoreactive and nonreactive tumor cells. These data show that SSEA-1, initially defined as a mouse embryonic antigen, represents a heterogenetic antigen present in many normal human tissues. It is retained on many but not all neoplastic cells originating in these normal tissues and also appears on the surface of some tumor cells developing in SSEA-1-negative tissues.

Expression of a carbohydrate differentiation antigen, stage-specific embryonic antigen 1, in human colonic adenocarcinoma.

The expression of the carbohydrate structure defined by monoclonal antibody to murine stage-specific embryonic antigen 1 (SSEA-1) was examined, using immunofluorescence, in formalin-fixed, paraffin-embedded sections of normal fetal and adult human colon and human colonic adenocarcinoma. SSEA-1 was expressed in all human colonic adenocarcinoma tissues examined, although in some cases the staining was heterogeneous. In normal human colonic mucosa, under the conditions used, faint staining was seen in the lower crypts and in only 26% of the crypts examined. When human fetal colon was tested, SSEA-1 was expressed in much larger amounts and in over 50% of all crypts. Transitional mucosa, immediately adjacent to human colonic adenocarcinomas, was also tested, and in this case, increased SSEA-1 expression was seen not only in the lower crypts but also in the upper crypts and surface epithelium. These results show that the increased expression of SSEA-1 in human colonic adenocarcinoma is an oncodevelopmental marker for this cancer. In addition, the results suggest that increased expression of SSEA-1 may be a preneoplastic change in human colon.

Cell line derived from a metastasis of a human testicular germ cell tumor.

A cell line, designated 833K-E, has been established from a metastasis of a human testicular germ cell tumor that consisted of four histological types of tumor cells. The 833K-E cells have morphological and ultrastructural characteristics of epithelial cells and a hyperdiploid karyotype indicative of their human male origin. The cells grow in agar cultures and produce in nude mice tumors which have the hstological features of embryonal carcinoma without differentiated elements. Many of the cells express a stage-specific mouse embryonic antigen, and low levels of the major histocompatibility antigens and beta 2-microglobulin also were detected on a large percentage of the cells. A lymphoblastoid cell line (833K-LC) established from the same tumor specimen expresses major histocompatibility antigens and beta 2-microglobulin but does not express the embryonic antigen.

Epigenetic Control of Early Mouse Development.

Although the genes sequentially transcribed in the mammalian embryo prior to implantation have been identified, understanding of the molecular processes ensuring this transcription is still in development. The genomes of the sperm and egg are hypermethylated, hence transcriptionally silent. Their union, in the prepared environment of the egg, initiates their epigenetic genomic reprogramming into a totipotent zygote, in which the genome gradually becomes transcriptionally activated. During gametogenesis, sex-specific processes result in sperm and eggs with disparate epigenomes, both of which require drastic reprogramming to establish the totipotent genome of the zygote and the pluripotent inner cell mass of the blastocyst. Herein, we describe the factors, DNA and histone modifications, activation and repression of retrotransposons, and cytoplasmic localizations, known to influence the activation of the mammalian genome at the initiation of new life.

Characterization of a monoclonal antibody that binds equally to all apolipoprotein and lipoprotein forms of human plasma apolipoprotein B. I. Specificity and binding studies.

A stable mouse hybridoma cell line has been developed that produces monoclonal antibody to human plasma apolipoprotein B. This antibody was proven to be specific for apolipoprotein B immunoblotting and an enzyme immunoassay using apolipoprotein B and other apolipoproteins. The antibody bound with comparable affinities to soluble apolipoprotein B, chylomicrons, very-low-density (VLDL) and low-density lipoproteins (LDL). Coupled to agarose, this antibody allowed complete removal of apolipoprotein B-containing lipoproteins from normolipidemic, hypertriglyceridemic and hypercholesterolemic plasma. Desialyzation and deglycosylation had no effect on its binding to LDL. The described antibody had no effect on the receptor-mediated binding of radiolabeled LDL to the human hepatoma cells (HepG2) in culture. Analysis of 25 different samples of human plasma indicated identical expression of the corresponding epitope in these individuals. The described monoclonal antibody, most likely, binds to a rather stable domain of apolipoprotein B that is not altered by the interaction with lipids or polymorphism of the apolipoprotein B. We propose that this antibody be called 'Pan B' antibody.

Mechanistic and developmental aspects of genetic imprinting in mammals.

Genetic imprinting in mammals allows the recognition and differential expression of maternal and paternal alleles of certain genes. Recent results from a number of laboratories indicate that, at least for some genes, gametic imprints, which must exist in order to mark chromosomes or genes as having been transmitted via sperm or ovum, are not by themselves sufficient to determine allele expression. Other postfertilization events are required, and these events are subject to both tissue-specific and developmental stage-specific regulation. Changes in imprinted gene methylation during preimplantation and fetal life indicate that the establishment of additional allele-specific modifications is likely to contribute to imprinted regulation. Disruptions in imprinting processes, loss of imprints, and loss of nonimprinted alleles through uniparental disomy are likely to contribute to a variety of developmental abnormalities and pathological conditions in both mice and humans.

Relevance of genomic imprinting to human diseases.

The existence of functional differences between parts of the maternal and paternal genome has been demonstrated. The control of gene expression through genomic imprinting plays a significant role in normal developmental processes in mammals and is also instrumental in several human pathological conditions.

Imprinting.

Imprinting provides a fascinating mechanism of control of gene expression so that the maternal and paternal alleles of some genes are unequally expressed. Imprinting is most likely established during gametogenesis by a mechanism not completely clear, though DNA methylation probably plays a certain role. Expression of imprinted gene significantly affects mammalian development so that only the maternal or only the paternal diploid genomes cannot support normal development. Since imprinting results in functional hemizygocity, mutation of the expressed allele can have the drastic consequences of a null mutation. For this reason identification of imprinted genes and further understanding of the imprinting mechanism represent an important change for human medical genetics.

Protein parameters of differential gene activation during development and tumorigenesis.

Various models of normal and abnormal developmental systems were addressed to get an insight into molecular parameters of cell differentiation at the level of protein gene products. Electrophoretic analysis of heterogeneous protein mixtures permitted qualitative analysis of developing systems, particularly during organogenesis in mammals, as well as of neoplastic growth in the animal and plant kingdoms. From our earlier findings indicating that the definite protein patterns characteristic of adult organs are acquired long after the adult morphological and histological characteristics of these tissues have developed, it has been repeatedly proven that quantitative changes in whole proteins is not a dependable indicator of cell differentiation.

Construction of primary and subtracted cDNA libraries from early embryos.

By modifying current cDNA cloning and electroporation methods, large and representative murine cDNA libraries were synthesized from 10 to 100 ng mRNA isolated from unfertilized egg and preimplantation mouse embryos. High cloning efficiency is essential for complete representation of genes expressed in egg and preimplantation embryos and for the isolation of stage-specific genes using subtractive hybridization. Because the mouse embryo contains no more than 50 pg of poly(A)+ mRNA at any stage of preimplantation development, approximately 5000-10,000 embryos are required to obtain enough mRNA to synthesize libraries using current methods. To obtain a representative library that also includes rare transcripts, the size of the library should be at least 10(6) clones. The average percent conversion of mRNA to single-stranded cDNA was 20-40%, so that a cloning efficiency of nearly 2 x 10(8) cfu/microgram cDNA is required for such a cDNA library. No previous methods have provided directional cloning of cDNA into plasmids with these high efficiencies. The advent of electroporation methods for the introduction of nucleic acids into bacteria has made possible the use of standard plasmid vectors for high-efficiency cDNA cloning. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and T3 RNA polymerase promoter sequences can be used to generate sense and anti-sense transcripts for subtractive hybridization and riboprobe synthesis. The cDNA libraries we derived using this methodology are a reusable and abundant source of genetic information about the control of preimplantation development. Specialized subtractive cDNA libraries enriched for genes expressed exclusively at a predetermined time in development give access to genes expressed in a stage-specific manner. The ability to construct new cDNA libraries from limited amounts of starting material ensures the provision of new and important resources for the identification and study of novel genes or gene families, and it is an important new tool for understanding the molecular control of mammalian development.