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Transcription of the c-jun gene is up-regulated by either retinoic acid (RA) or adenovirus E1A during the differentiation of F9 cells. We show here that RA and E1A induce phosphorylation of the E1A-associated 300 kDa protein (p300) during the differentiation of F9 cells. The region of E1A that is required for interaction with cellular protein p300 overlaps with the region of E1A required for E1A to induce expression of the c-jun gene. Treatment of F9 cells with RA or infection of the cells by adenovirus led to a decrease in the electrophoretic mobility of p300. Phosphatase treatment of p300 from RA-treated or adenovirus-infected F9 cells reversed the changes in migration of p300, indicating that RA- and E1A-mediated changes in the mobility of p300 were due to phosphorylation. We also found factors, designated DRF1 and DRF2, that bound specifically to a sequence element that is necessary and sufficient for RA- and E1A-mediated up-regulation of the c-jun gene. The mobility of DRF complexes was changed by E1A or RA and the complexes were supershifted by addition of a polyclonal p300 antiserum. Moreover, overexpression of p300 resulted in an increase in the level of DRF1 complex. p300 fused to the DNA binding domain of the E2 protein of papilloma virus stimulated E2-dependent reporter activity in response to RA or E1A in F9 cells. Our results suggest that p300 is part of the DRF complexes, that it is differentially phosphorylated in undifferentiated versus differentiated cells and that it is likely involved in regulating transcription of the c-jun gene during F9 cell differentiation.

Melanomas are most frequently infiltrated by actively proliferating T-lymphocytes (1). Some of these T-cells are cytolytic and recognize peptide antigens derived from melanoma-specific antigens (2). However, with the noteworthy exception of rare immune-mediated, sponaneous regressions of melanomas (3), or in the particular case of the halo nevus phenomenon in which normal melanocytes are killed by CD8(+)-specific T-cells (4), the ongoing melanocyte-specific T-cell responses are most frequently incapable of controlling the growth of the tumor, resulting in the malignant melanocytic tumors escaping an otherwise specific immune T-cell response. The understanding of the mechanisms that underlie the switch of efficient to inefficient (and vice versa) T-cell responses is thus of primary importance in conceiving specific immunotherapies of melanomas.

Ag fragments derived from the cytosol are transported into the endoplasmic reticulum (ER) lumen, where they bind to nascent MHC class I molecules. However, it is not known whether only high affinity peptides enter the ER, or whether ER proteases must trim longer precursor peptides down to optimal size. To evaluate the feasibility of proteolytic fine trimming in vitro, soluble Kd and Kb were preincubated with peptides that bind to Kd or Kb and the mixture was exposed to three different proteases. Class I protected allele-specific peptides against proteolysis, whereas the other peptides were degraded to the amino acid level. When a Kd/S11E (SYIPSAEYILE) complex was immunoprecipitated after incubation with carboxypeptidase, both S11E and the optimal sized S9I (SYIPSAEYI) were found to be specifically bound to Kd. However, only S91 was recovered if S11E, Kd and carboxypeptidase were mixed at the same time, and there was no fine-trimming of bound S11E if high protease concentrations and short proteolysis times were used, which suggests that trimming occurs only in the unbound state. The amount of peptide that immunoprecipitated with Kd increased after carboxypeptidase treatment of Kd/S11E, implying that the peptide affinity had increased. Kd also protected S9I against proteolysis by a lysed microsome preparation, demonstrating that class I could also protect high affinity peptides in vivo. These results suggest that class I participates in the selection of high affinity peptides in the ER, by sampling transported unbound peptides are degraded by ER proteases or efflux back to the cytosol.

The development of entomology and medical entomology in France is discussed in the context of the prevalence of Lamarckian ideas concerning heredity and evolution. Lamarckian ideas have greatly influenced research carried out at the Institut Pasteur by Emile Roubaud and more generally in Felix Mesnil's laboratory, as well as research in general entomology at the Museum national d'histoire naturelle. By contrast, it did not influence research and teaching at the Faculté de médecine of Paris or that of physicians more generally including those in overseas Instituts Pasteur, which clearly kept away from theoretical discussion concerning the origin of variations and adaptation in insects of medical interest.

Isolation of cDNA clones encoding the beta-chain of the human T cell surface glycoprotein CD8 revealed the presence of five distinct forms of cDNA resulting from alternative splicing. In the process of analysis of the gene organization, we found that there exist two recently duplicated genes for CD8 beta. These genes, designated CD8 beta 1 and beta 2, consist of nine and seven exons, respectively. The organization of CD8 beta 1 and beta 2 genes is almost identical except in their 3'-ends. There are nine nucleotide differences between the coding regions of the CD8 beta 1 and beta 2 genes in spite of the extremely high similarity of these genes which extends over the entire genes including introns. Pulse field gel analysis demonstrated that CD8 beta 1 and beta 2 genes are located more than 1.5 Mb apart. It was found that the CD8 beta 1 gene is approximately 25 kb upstream from the CD8 alpha gene in the same transcriptional orientation on chromosome 2. Although both CD8 beta 1 and beta 2 genes appear functional from the nucleotide sequence, the five distinct forms of CD8 beta cDNAs and corresponding mRNAs found in thymus, PBL, and leukemic cell line HPB-ALL are all derived by alternative splicing from CD8 beta 1 transcripts.

The class I Ag encoded in the Qa/T1a regions of the murine MHC are much less polymorphic, and usually have a more restricted tissue distribution than the classical histocompatibility class I Ag, encoded by genes located in the H-2K, D, and L loci. The isolation of a quasi-ubiquitously expressed, poorly polymorphic class I gene of the T1a region of the H-2d mouse MHC, namely gene 37 (or T18d), has been recently reported. We describe the nucleotide sequence of a closely related gene, T10c gene, the counterpart of the gene 37 in the large duplicated parts of T1a region of the BALB/c (H-2d) MHC. The T10c gene structure and sequence are very similar to those of gene 37, but T10c gene is most likely a pseudogene. In A/J mouse strain, there appears to be a single gene related to 37, which is also found expressed in a variety of tissues. We show that this gene is likely to be a chimeric one derived from T10c for its 3' part, and from a gene closely related to gene 37 for its 5' part, which potentially encodes for an unusual class I molecule composed of the first two domains. Finally, Southern blot analysis of a number of wild mice and related animals suggests that a gene closely related to the present T10c gene may be the ancestor of this subfamily of class I genes characterized by the presence of an unusual second domain.

A cDNA encoding subunits 8 and 6 of mitochondrial ATPase of the cephalochordate lancelet (Branchiostoma lanceolatum), a direct ancestor of vertebrates, has been cloned and sequenced. A unique transcript encodes the 8 and 6 subunits. In the course of isolation of the 5' end of the ATPase 8 subunit gene, we also determined the sequence of the 5' adjacent tRNA-Lys gene. The anticodon used by mitochondrial tRNA-Lys is TTT.

Objective This epidemiologic study was undertaken after a cluster of five cases of rare forms of cancer (bone sarcoma, non-Hodgkin's lymphoma) had been observed among biomedical research workers at the Pasteur Institute in Paris to ascertain whether their disease was connected with exposure during this research. Methods A mortality study included 3765 people who worked at the Pasteur Institute between 1971 and 1986 and were followed until the end of 1987. Within this cohort a nested case-referent study included 23 cases of cancer [non-Hodgkin's lymphoma (6), multiple myeloma (1), leukemia (3), pancreatic cancer (7), bone cancer (3), brain tumor (3)], and four referents per case, matched for gender and year of birth. Results Total mortality from cancer was less than expected, the standardized mortality ratio (SMR) being 72 for the men and 82 for the women. Among the women the proportion of pancreatic cancer cases was larger than expected [SMR 490, 95% confidence interval (95%CI) 158--1144], as was the number of brain cancer cases (SMR 239, 95% CI 48--696). Among the men, mortality from bone cancer was greater than expected (SMR 553, 95% CI 62--2006). In the nested case-referent study, more cases than referents had worked in the areas of molecular biology [odds ratio (OR) 7.1, 95% CI 1.5--33] and microbial genetics (OR 6.7, 95% CI 1.3--35). These cases especially included non-Hodgkin's lymphoma and bone cancer. Associated with this finding was the fact that more cases had used certain chemicals, including ethidium bromide, acrylamide, methylnitronitrosoguanidine and ethylmethanesulfonate, and radioactive compounds (essentially 32phosphorus). Conclusion As the products used are potent genotoxicants, the present findings suggest that work in biomedical research might well involve an increased risk of certain types of cancer; this conclusion should be balanced by the fact that two of the five index cases were included in the mortality study and four in the nested case-referent study.

In primary rodent cells transformed by the E1A region of the highly oncogenic adenovirus type 12, repression of transcription mediated by the far upstream TATA-like element was observed only in conjunction with either possible juxtaposition of a CAA repeated element in the presence of E1A and was dependent upon the relative arrangement of both the TATA-like and CAA repeated motifs in both homologous and heterologous promoter constructs. A gel shift competition study demonstrated that the TATA-binding protein (TBP) or a TBP-like protein can bind to both the upstream TATA-like sequence and the regular TATA box on the H-2Kb basal promoter. Moreover, employing immunoselection and cyclic amplification and selection of targets (CASTing) methods with nuclear extracts derived from Ad12-E1A transformants, we have identified a high affinity binding site in the H-2Kb class I promoter for E1A-associated DNA-binding proteins. The sequences of the binding sites were identified and were found to contain both the upstream TATA-like motif and the CAA repeated motifs. Our results suggest that the TATA-like sequence in the far upstream region of the H-2Kb gene is one of the elements that is required for Ad12-E1A-mediated negative repression.

MRL mice homozygous for the lpr/lpr gene develop a massive lymphadenopathy caused by the accumulation of CD4-CD8-, Thy-1-positive T cells that express B220. This phenotypically unusual T cell population coexists with normal, B220- T cells in lpr/lpr animals. To investigate the origin and differentiation pathway of B220+ T cells, the expression of a panel of developmentally regulated cell surface markers including TCR, CD4, CD8, Thy-1, and B220 was examined. Thymocytes and peripheral T lymphocytes from lpr/lpr mice were analyzed by four-color flow cytometry. The results showed that both B220+ and B220- thymocytes contained all of CD4-CD8-, CD4+CD8+, and CD4 or CD8 single positive T cell subpopulation in the lpr thymus. Expression of the V beta 11 TCR, measured by flow cytometry and reverse polymerase chain reaction, was demonstrated in lpr thymus. However, the number of T cells expressing V beta 11 was greatly reduced in both the B220+ and B220- T cell populations in lymph node, spleen, and liver. Taken together, the data provide evidence for maturation and selection of a distinct population of B220+ T cells in the thymus of MRL lpr/lpr mice.

Ia specificities were studied on germinal cells from C3H and 129 mice by using an AT.H anti-AT.L antiserum. By absorption experiments and immunofluorescence studies on sections of testes from mice of various ages, we were able to detect Ia specificities on cells of the germinal line from primary spermatocyte to spermatozoa. Ia specificities were not detectable on gonocytes or spermatogonia.

The high-molecular-weight glycopeptides characteristic of early embryonic cells were isolated from F9 embryonal carcinoma cells grown in vitro and also from the cells grown in vivo as subcutaneous tumors. The two preparations had similar carbohydrate compositions. The major components were galactose and N-acetylglucosamine (molar ratio 1:0.86) in the glycan isolated from the cultured cells. In addition, small amounts of fucose, N-acetylgalactosamine and mannose were present. The glycan from the in vitro grown cells was found to have a molecular weight of more than 10,000 by gel filtration after mild alkaline treatment or hydrazinolysis. The structural characteristics of the core portion of the glycan were studied by using the radioactively labeled glycopeptide from the in vitro grown cells. Methylation analysis provided the following informations. 1) The glycan was highly branched at galactosyl residues. 2) Large numbers of galactosyl residues were also present at non-reducing termini. 3) Monosubstitution of galactose occurred at C-3. 4) Glucosamine residues were mainly monosubstituted. That the disaccharide GlcNAc-Gal was the major structural unit of the glycan was suggested by the isolation of the deacetylated disaccharide after alkaline thiophenol cleavage followed by acid hydrolysis. Furthermore, methylation analysis of the glycan from the in vivo grown tumors indicated that monosubstitution of glucosamine occurred at C-4 and that disubstitution of galactose occurred at least mainly at C-3 and C-6. We propose that the basic structural unit of the core portion is 4GlcNAc 1 leads to 3Gal, and that the galactosyl residue serves as a branching point at C-6. Thus, the structural unit of the core portion of the large glycan appears to be the same as that of lactosaminoglycans found in adult cells.

Several cell lines (either of embryonal carcinoma or of differentiated cells derived from teratomas) have been established in vitro from transplantable testicular teratomas. Primitive cell lines, propagated in vitro as embryonal carcinoma have retained the ability of the original tumor to differentiate in vivo or in vitro into must embryonic cell types. Features of this model system for study of early embryogenesis are described. Emphasis is placed on the description of the cell surface antigens of several cell lines. Syngeneic antisera raised against two primitive lines (F9 and PCC4) and against a differentiated one (Endo) have allowed the detection of three groups of cell surface antigens, present on teratoma cells, tumor cells and embryonic cells. The F9 antigen appears to be specific to be specific of the very early steps of egg development (morula and blastocyst). After egg implantation, it keeps expressed on the cells of the male germ line. The PCC4 antigen has a similar cell type distribution but appears to be more specific of multipotential cells. The Endo antigen is essentially specific of endodermal derivatives. The F9 antigen is probably specified by the wild type allele (+ tl2) of the tl2 gene at the T-Locus of the mouse, a gene which plays some critical role in early development. The molecular structure of this antigen, as determined from immunoprecipitates is very similar to that of H-2 antigens. In addition, a cross-reacting material is found in Man, with a tissue distribution identical to that found in the mouse.

400 MHz NMR spectrum was recorded for the glycoprotein -bound large carbohydrates (embryoglycan) isolated from F9 embryonal carcinoma cells. Two intense signals at 4.13 ppm and 4.69 ppm were assigned to be H-4 of galactosyl residues substituted at C-3 and H-1 of G1cNAc beta 1----3, respectively. The result is consistent with the proposal that the fundamental building unit of the large glycan is G1cNAc beta 1----3Ga1 beta. Furthermore, the spectral data confirmed a conclusion obtained by glycosidase digestion that fucosyl residues are linked mostly to N-acetylglucosamine rather than galactose.

The recent isolation, by recombinant DNA techniques, of cloned probes for mouse and human class I major transplantation antigens has initiated the molecular analysis of the corresponding genes. Mouse genes belong to a relatively large multigene family, whose members share extensive structural homologies. Sequence analyses suggest that some genes could have a mosaic structure. This feature might help us to understand one of the distinctive traits of these antigens: their large antigenic polymorphism.

Once molecular probes detecting mouse H-2 (Kvist et al., 1981) and human HLA (Ploegh et al., 1980; Sood et al., 1981) class I sequences had been isolated, it was quickly realized that the mouse class I H-2 multigene family was far more complex than initially anticipated (Cami et al., 1981; Steinmetz et al., 1981a). A considerable amount of cloning work performed in several laboratories has led to the isolation of almost the entire H-2 locus from both BALB/c (Steinmetz et al., 1982) and B10 mice (Weiss et al., 1984). These studies have established that there exists of the order of 26 to 36 class I H-2 genes, depending on the mouse strain. It has become usual to divide these genes into two major groups predominantly on the basis of serological data. The first group includes those genes which map to the K and D loci. They encode the quasi-ubiquitous and highly polymorphic class -I major transplantation antigens, which serve as restriction elements in the recognition of target cells by cytotoxic T lymphocytes (Zinckernagel and Doherty, 1979; see also Kourilsky et al., 1987). The other group of genes (23 to 29 depending on the mouse strain) lie in the Qa-Tla region and includes the genes encoding the serologically detected Qa and Tla differentiation cell surface antigens. The latter are much less polymorphic than. the class I molecules encoded in the K and D regions, and display a limited tissue distribution, being expressed mostly in B and/or T cells. Accordingly, it was believed for some time that the Qa-Tla region encoded only non-ubiquitous cell surface antigens.

Endo-beta-N-acetylglucosaminidase H released four major oligosaccharides from high-mannose glycopeptides prepared from embryonal carcinoma cells. The oligosacchaides were indistinguishable from (Man)9GlcNAc, (Man)8GlcNAc, (Man)7GlcNAc, and (Man)6GlcNAc isolated from fibroblasts. This result suggests that the biosynthetic pathway of asparagine-linked oligosaccharides in early embryonic cells is controlled as in adult cells, at least to the initial stage of processing of the nascent oligosaccharide transferred from lipid-linked intermediate.

A rabbit antiserum raised against papain-solubilized H-2 antigens has been used to investigate the eventual expression of H-2 antigens and related molecules on embryonal carcinoma cells and on other types of mouse cells. No material reacting with this serum could be detected on cells carrying the F9 antigen. It is concluded that no H-2 antigen or cross-reacting material is expressed on these cell types.