Brenda Kahan

Autonomous gene expression on the human inactive X chromosome.

Local derepression of the hpt locus on the human inactive X chromosome obtained in human female fibroblast x mouse L cell somatic cell hybrids was not correlated with the presence or absence of any specific human chromosome in the hybrids. Loss of the human active X, in particular, did not result in observable derepression of genes on the inactive X. Introduction of an active X, via a second hybridization of human cells having an active X with hybrid cells containing a locally derepressed X chromosome, did not restore repression of the derepressed hpt allele. The rate of hpt locus derepression in hybrid cells was estimated to be 10−6 per inactive X chromosome per cell generation.

Ovarian localization by embryonal teratocarcinoma cells derived from female germ gells.

Embryonal carcinoma cells derived from several different spontaneous ovarian teratocarcinomas of strain LT mice form tumors that are located exclusively, in many cases, in the ovaries of female mice. Embryonal cells previously unselected for site specificity localize in the ovaries regardless of route of entry of the cells, and produce very few tumors in males following intraperitoneal injections. The ovary tumors have been verified as originating from the injected cells by chromosomal and drug resistance markers, as well as by general in vitro growth characteristics. Cell-cell adhesion studies suggest specificity at the level of tumor cell-ovary organ cell interaction.

Elimination of tumorigenic stem cells from differentiated progeny and selection of definitive endoderm reveals a Pdx1+ foregut endoderm stem cell lineage

Embryonic stem cell (ESC) derivatives offer promise for generating clinically useful tissues for transplantation, yet the specter of producing tumors in patients remains a significant concern. We have developed a simple method that eliminates the tumorigenic potential from differentiated ESC cultures of murine and human origin while purifying lineage-restricted, definitive endoderm-committed cells. A three-stage scheme utilizing magnetic bead sorting and specific antibodies to remove undifferentiated ESCs and extraembryonic endoderm cells, followed by positive selection of definitive endoderm cells on the basis of epithelial cell adhesion molecule (EpCAM) expression, was used to isolate a population of EpCAM(+)SSEA1(-)SSEA3(-) cells. Sorted cells do not form teratomas after transplantation into immunodeficient mice, but display gene and protein expression profiles indicative of definitive endoderm cells. Sorted cells could be subsequently expanded in vitro and further differentiated to express key pancreas specification proteins. In vivo transplantation of sorted cells resulted in small, benign tissues that uniformly express PDX1. These studies describe a straightforward method without genetic manipulation that eliminates the risk of teratoma formation from ESC differentiated derivatives. Significantly, enriched populations isolated by this method appear to be lineage-restricted definitive endoderm cells with limited proliferation capacity.

Adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase immunoprecipitation reactions in human-mouse and human-hamster cell hybrids

SummaryMale New Zealand White rabbits were immunized with human adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which were purified about 2000-fold and 800-fold, respectively, from erythrocytes by DEAE-cellulose chromatography, ammonium sulfate precipitation and preparative polyacrylamide gel electrophoresis. Specific immunoprecipitations of APRT and HGPRT were achieved with the antisera that were obtained and by using polyethylene glycol as a substitute for goat anti-(rabbit) gamma globulin. The activities of the human forms of these enzymes, whether from red blood cells or from cultured cells, were almost completely eliminated under the conditions of immunoprecipitation used. Little or no redution of APRT and HGPRT activities from mouse and Chinese hamster cells was observed. This discriminatory capacity of the antisera was successfully used for the identification of human APRT and HGPRT in human-mouse and human-hamster cell hybrids using the immunoprecipitation reaction.

Epitope mapping of monoclonal antibodies toEscherichia coli ribosomal protein S3

The antigenic structure ofEscherichia coli ribosomal protein S3 has been investigated by use of monoclonal antibodies. Six S3-specific monoclonal antibodies secreted by mouse hybridomas have been identified by immunoblotting of two-dimensional ribosomal protein separation gels. By using a competitive enzyme-linked immunosorbent assay, we have divided these monoclonal antibodies into three mutual inhibition groups, members of which are directed to three distinct regions of the S3 molecule. The independence of these monoclonal antibody-defined regions was confirmed by the failure of pairs of monoclonal antibodies from two inhibition groups to block the binding of biotinylated monoclonal antibodies of the third group. To determine the regions recognized by these monoclonal antibodies, chemically cleaved S3 peptides were fractionated by gel filtration and reverse-phase high-performance liquid chromatography. The fractionated peptides were coated on plates and examined for specific interaction with monoclonal antibody by enzyme immunoassay. In this manner, two epitopes have been mapped at the ends of the S3 molecule: one, in the last 22 residues, is recognized by three monoclonal antibodies; and the second, in the first 21 residues, is defined by two monoclonal antibodies. The third S3 epitope, recognized by a single monoclonal antibody, has been localized in a central segment of about 90 residues by gel electrophoresis and immunoblotting. These epitope-mapped monoclonal antibodies are valuable probes for studying S3 structurein situ.

Epitopes of Escherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S131–22; three monoclonal antibodies bound to S131–40; the binding sites of three other antibodies have been located in S1323–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.

The Stability of X-Chromosome Inactivation: Studies with Mouse-Human Cell Hybrids and Mouse Teratocarcinomas

The control scheme for the expression of X chromosomes in female mammals can be considered to consist of two parts (Lyon 1972, 1974). The first of these involves the initiation of inac-tivation of one X chromosome (Kratzer and Gartler 1978; Monk 1978; Chapman, West, and Adler 1978). This initial choice may be either random, as it is in eutherian embryonic somatic tissues, or preferential, as occurs in extraembryonic components of some species (Takagi 1978; West, Papaioannou, Frels, and Chapman 1978) and in somatic tissues of marsupial mammals (Cooper, Johnston, Murtagh, Sharman, VandeBerg, and Poole 1975). This report will focus on the second part of the problem of X-chromosome differentiation: how genetic repression of the inactive X is maintained and propagated. It will consider specifically some of the insights into the regulatory mechanisms involved in the maintenance of repression that may be gained from experiments designed to derepress genes on the inactive X.

Detecting immunocomplex formation in sucrose gradients by enzyme immunoassay: Application in determining epitope accessibility on ribosomes

A sensitive method using enzyme immunoassay and sucrose gradient to analyze immunocomplexes of biological particles has been developed. The sensitivity and application of this method were demonstrated by that the in situ accessibility of ribosomal protein epitopes could be easily determined. We used sucrose gradients to separate the ribosome-bound and the free antibodies and traced the antibodies in the gradients by an enzyme-linked immunosorbent assay. Epitopes exposed in situ are bound by specific antibodies, which in turn are detected in sucrose gradients migrating with ribosomes. This method of detecting antibody migration is more sensitive than the conventional means of using A260nm to monitor the antibody-mediated dimerization of ribosomes. Furthermore, an epitope defined by a biotin-labeled monoclonal antibody can be analyzed in the presence of other unlabeled antibodies. Thus, the relationship of different accessible epitopes in situ can be readily examined. Versatility and sensitivity of this method should make it useful in analyzing a variety of immunocomplex systems.

Embryonic Stem Cells as a Source of Pancreatic Precursors and Islet Cells in Vitro

cells or β cells from an abundant, renewable, and readily accessible source for transplantation would probably render current transplantation therapies obsolete. Although this ultimate goal is on the distant horizon, recent progress, representing the first step, has been made in identifying pancreatic precursor cells and differentiated islet cells generated from both mouse and human embryonic stem (ES) cells. The next hurdle, achieving enrichment of these cell types from ES cell cultures and isolating purified populations for functional testing, may be a more challenging step. It is becoming clear that a better understanding of the sequential genetic and epigenetic signals occurring during normal mouse and human development will be necessary. Particularly relevant is the need to understand the nature and identity of true embryonic pancreatic precursor cells and islet progenitor cells, and to identify conditions that allow their efficient, large-scale isolation. An ES cell-based in vitro differentiation system can facilitate these goals by providing a straightforward means to select and purify progenitor cells, and to investigate conditions that promote their expansion and differentiation ex vivo. Specifically, a human ES cell-based in vitro model system would be invaluable for studying human islet development and for providing cells for transplantation. The Clinical Problem