One gene and one pseudogene for the cytokeratin endo A.

Abstract

The recombinant cDNA RecXVI, which hybridizes to the endo A mRNA, detects two specific bands on a Southern blot of EcoRI-digested genomic mouse DNA. We have screened a mouse genomic library with this cDNA and isolated these two sequences. Endo A is encoded by a 7.5-kilobase gene and by a 1.6-kilobase pseudogene devoid of introns. A repetitive sequence belonging to the B2 family is located in the third intron of the gene. We have observed that transcription of B2 sequences and of the endo A gene are mutually exclusive.The first detectable morphological differentiation of mouse embryonic cells takes place during blastocyst formation, in the course of which two different types of cells, the trophectoderm and the inner cell mass cells, become differentiated (1). This differentiation is accompanied by modifications in the pattern of protein synthesis, revealing a difference in the regulation of gene expression (2). Among these differences, some intermediate filament proteins are synthesized in the trophectoderm but not in inner cell mass cells (3, 4). Analysis of the onset of synthesis of these proteins might provide an insight into molecular events directly related to the cell commitment program.We describe here the isolation and characterization of the genes encoding endo A (5), also referred to as cytokeratin A (6). Endo A appears during blastocyst formation and has been identified in trophoblast but not in inner cell mass cells (7). It is therefore a valuable marker of the modifications in gene expression during the first binary choice made by embryonic cells. Later in development, endo A is still expressed in a tissue-specific manner; it is expressed in visceral and parietal endoderm and in epithelial cells derived from mesoderm and endoderm (i.e., in liver and kidney) but not in fibroblasts, myoblast, neural tissues, or keratinocytes (8). In vitro, endo A is found in trophoblastoma cell line TDM-1, but not in embryonal carcinoma cells.A cDNA clone, RecXVI, was isolated (9) from a cDNA library prepared from TDM-1. This cDNA hybridizes to a specific 18S mRNA encoding endo A. We have used this cDNA to isolate the endo A genes to analyze the regulation signals involved in the expression of this protein during the first embryonic differentiation. We describe here the structure of the genes and discuss the possible regulatory role of a repetitive sequence that is included in one of them.MATERIALS AND METHODS Cells. Teratocarcinoma cell lines F9 and PCC3 (10) were cultured under standard conditions. TDM-1 is a trophoblastoma cell line (10).Isolation of Mouse Genomic Clones. A genomic library of BALB/c mouse DNA restricted with EcoRI and cloned into phage X Charon 4A was plated to a density of 15,000 phages per 10-cm plate and screened as described (11) using the cDNA RecXVI.Electron Microscopy of Heteroduplexes. Heteroduplexes were prepared by renaturing the DNAs in 50% formamide/0.58 M NaCl/50 mM Pipes, pH 6.8/1 mM EDTA at 640C for 1 hr. After fixation with glyoxal for 2 hr at 12'C, a solution was prepared f...

Identification of the gene coding for the Endo B murine cytokeratin and its methylated, stable inactive state in mouse nonepithelial cells.

“…The presence of processed pseudogenes for Endo A (Vasseur et al 1985) and Endo B (see below) and the evi dence for expression of only a single Endo B gene (Trevor and Oshima 1985) in both mice and humans (Kulesh and Oshima 1988) suggest that many of the hybridizing bands found in primate DNA may represent pseudogenes.…”

Mid-gestational lethality in mice lacking keratin 8.

“…It contains two arms of homology to the inK8 gene {Endo A al), a 1.8-kb XbaI fragment and a 1.2-kb SmaI fragment, respectively (Vasseur et al 1985). The neo r gene (pMClneopolA, Stratagene, La Jolla, CA) replaces part of the first exon, including the ATG translation initiation codon (Semat et al 1988).…”

“…Five micrograms of XbaI-digested DNA was loaded onto a 0.65% agarose gel and transferred to a Zetaprobe membrane {Bio-Rad Laboratories, Cambridge, MA). The filter was hybridized with the 2.5-kb EcoRI pseudogene fragment (Endo A a2) (Vasseur et al 1985), which is homologous to the exonic sequences of the inK8 gene, or with the XhoI-SalI fragment from the pMClneopA plasmid. In some cases, genomic DNA cut with XhoI and HindlII was hybridized with a XhoI-XbaI fragment located 5' of the inK8 first exon (5' probe), or with the EA-R6 EcoRI fragment of the mK8 eDNA (Semat et al 1988).…”

JunB does not inhibit the induction of c‐Jun during the retinoic acid induced differentiation of F9 cells

“…The c d u n probe was a 1,625 bp fragment of the RSVc-J u n vector (Angel et al, 1988) derived by digestion with HindIII and NotI. For detection of mK8 RNA, the 295 bp SmaI fragment of the Endo A a1 gene (Vasseur et al, 1985) was used. The laminin B1 probe was the 705 bp XbaI and EcoRI fragment of the cDNA fragment cloned in pPE386 (Barlow et al, 1984).…”