Serum-inducible expression of transfected human c-myc genes.

Activation of the c-myc proto-oncogene is implicated in the initiation or progression of many vertebrate cancers. In nontransformed cells, the expression of c-myc is induced by growth factors. Studies have indicated that such induction is effected by multiple mechanisms.

Normal expression of a rearranged and mutated c-myc oncogene after transfection into fibroblasts.

Expression of the c-myc oncogene is deregulated in a variety of malignancies. Rearrangement and mutation of the c-myc locus is a characteristic feature of human Burkitt's lymphoma. Whether deregulation is solely a result of mutation of c-myc or whether it is influenced by the transformed B cell context has not been determined.

Diversity in T-cell receptor gamma gene usage in intestinal epithelium.

The intraepithelial cells of the murine small intestine include a significant number of CD3+ T cells that use T-cell receptor gamma genes rather than T-cell receptor beta genes. As with other sites of T-cell receptor gamma expression, combinatorial diversity is limited, but there is junctional diversity, and this, together with the specific variable region gamma gene segments used, distinguishes gamma gene expression in the gut epithelium from that in cells derived from the dermal epithelium. The restriction of productive gamma gene expression largely to one V-J-C (V, variable; J, joining; C, constant) gene combination may result from nonproductive joining of other V-J combinations and from productively rearranged genes rendered nonfunctional by incorrect splicing.

Developmental control of lymphokine gene expression in fetal thymocytes during T-cell ontogeny.

We have used the technique of in situ hybridization to investigate the expression of lymphokine genes by immature thymocytes during intrathymic development. In 13-day fetal thymocytes a population of cells constitutively produces low levels of interleukin 2 (IL-2) and interleukin 4 (IL-4) mRNAs. A second phase of lymphokine gene expression occurs in the majority of 15-day thymocytes, and a population of cells constitutively produces both IL-2 and IL-4 mRNAs.

Molecular analysis of T cell receptor gamma gene expression in allo-activated splenic T cells of adult mice.

Northern analysis, hybridization in situ and cDNA sequence analysis have been used to demonstrate that the induction of T cell gamma-gene expression is a general occurrence when primary splenic T cells of adult mice are cultured in short-term mixed lymphocyte reactions (MLR). Splenic T cells from nine strains of mice examined in eleven different MLR all showed significant induction of gamma-RNA, even when the primary T cell response was to only a three amino acid mismatch in a major histocompatibility complex class I antigen. In MLR examined in detail, the expression is highly enriched for in CD3+ "double-negative" T cells (lacking both CD4 and CD8 expression).

Use of a chemically modified T7 DNA polymerase for manual and automated sequencing of supercoiled DNA.

Procedures are presented for reliable and accurate nucleotide sequence analysis using as template supercoiled DNA prepared by a modified rapid boiling minipreparation protocol. This method yields DNA templates suitable for sequencing within 1 h of bacterial harvest. We describe optimal reaction conditions for supercoiled miniprep DNA sequencing using a modified T7 DNA polymerase (Sequenase) in dideoxynucleotide chain termination reactions.

T-cell receptor gene rearrangement in primary tumors: effect of genetic background and inducing agent.

The status of T-cell receptor beta and gamma genes has been assessed in a series of primary tumors induced by a chemical carcinogen or by gamma-irradiation using two inbred strains of mice. It appears that these well-characterized regimens of carcinogenesis yield T-cell tumors showing gene rearrangements consistent with a clonal origin of the tumors. Individual rearranged bands seem to represent orthodox, intralocus recombination events.

Transcripts of functionally rearranged gamma genes in primary T cells of adult immunocompetent mice.

The T-cell specific, rearranging gamma-chain genes bear striking resemblance to T-cell receptor and immunoglobulin genes, but the role of gamma remains unknown. A central problem is to understand the conditions under which gamma RNA is expressed in cells. The transcription of gamma is abundant in T cells of fetal thymi, but is negligible in peripheral T cells of adults, suggesting that gamma is involved in development of the T-cell repertoire.

A frameshift at a mutational hotspot in the polyoma virus early region generates two new proteins that define T-antigen functional domains.

A frameshift mutation, arising from the deletion of any one of nine consecutive cytidines in the region of Py DNA encoding both the midregion of large T-Ag and the C-terminal region of middle T-Ag, yields unstable flat cell revertants that synthesize two novel viral proteins in which shuffling of the different domains of the Py T-Ags has occurred. The first protein (37 kd) is a hybrid containing the N-terminus of large T-Ag and the hydrophobic C-terminus of middle T-Ag. The latter domain is responsible for membrane association, even in the 37 kd hybrid protein.

Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells.

The technique of DNA transfer by electroporation was investigated in an effort to evaluate its utility for the identification of developmentally controlled regulatory sequences. Transient and stable gene expression was detected in a variety of lymphoid cell lines subjected to electroporation. No correlation existed between the levels of chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol 3-O-acetyltransferase, EC 2.

Unusual organization and diversity of T-cell receptor alpha-chain genes.

T lymphocytes recognize cell-bound antigens in the molecular context of the self major histocompatibility complex (MHC) gene products through the surface T-cell receptor(s). The minimal component of the T-cell receptor is a heterodimer composed of alpha and beta subunits, each of relative molecular mass (Mr) approximately 45,000 (refs 1-3). Recently, complementary DNA clones encoding these subunits have been isolated and characterized along with that of a third subunit of unknown function, termed gamma (refs 4-9).

Structure, organization, and somatic rearrangement of T cell gamma genes.

We present the initial characterization of a novel family of genes that rearrange in T cells, but do not encode either of the defined (alpha/beta) subunits of the clone-specific heterodimer of the T cell receptor. The family comprises at least three variable (V) gene segments, three constant (C) gene segments, and three junction (J) gene segments. In a cloned cytolytic T lymphocyte, 2C, one of each of these fragments has productively rearranged to yield an expressed VJC transcription unit, which shows no evidence for somatic mutation.

Activation of a translocated c-myc gene: role of structural alterations in the upstream region.

The translocated c-myc gene in AW-Ramos, a Burkitt lymphoma cell line carrying the 8;14 translocation, is expressed at 2- to 5-fold higher levels than c-myc in lymphoblastoid cell lines. The translocation event has joined c-myc to the IgM switch region. As a consequence, a recently identified immunoglobulin transcriptional enhancer element is not linked to the translocated c-myc gene.

A third rearranged and expressed gene in a clone of cytotoxic T lymphocytes.

In addition to the two previously identified genes rearranged and expressed in a cytotoxic T-lymphocyte clone, we have identified a third gene that is also rearranged and expressed in the same clone. This new gene shows clonal diversity, codes for a polypeptide chain that contains immunoglobulin-like variable and constant domains, carries potential N-glycosylation sites and is a particularly attractive candidate for the gene that encodes the alpha-subunit of the heterodimeric antigen receptor of this T-cell clone.

Complete primary structure of a heterodimeric T-cell receptor deduced from cDNA sequences.

Two related, but distinct, cDNA clones have been isolated and sequenced from a functional murine cytotoxic T-lymphocyte clone. The genes corresponding to these cDNA are expressed and rearranged specifically in T cells and both have similarities to immunoglobulin variable and constant region genes. It is concluded that these genes code for the two subunits of the heterodimeric antigen receptor on the surface of the T cell; its complete deduced primary structure is presented.

Activation of a translocated human c-myc gene by an enhancer in the immunoglobulin heavy-chain locus.

A tissue-specific transcriptional enhancer element that is associated with the human immunoglobulin heavy-chain locus is defined. In a non-Hodgkin's lymphoma that contains a translocated c-myc gene this enhancer is retained on the 14q+ chromosome and occurs within sequences shown to activate previously cryptic promoters of the c-myc gene.

Activation of the c-myc gene by translocation: a model for translational control.

We have shown that the human cellular oncogene c-myc is composed of three exons and is transcribed from two initiation sites separated by 175-base-pair DNA in HeLa cells. For both resulting mRNA species, exon 1 composes the 5' untranslated region and the initiator methionine is located 16 base pairs down-stream from the 5' splice acceptor of exon 2. In a non-Hodgkin lymphoma, Manca, harboring a t(8; 14) translocation, c-myc gene is broken within intron 1, and its exons 2 and 3 are translocated to a site between the heavy chain joining region cluster and C mu-coding DNA segment of the immunoglobulin heavy chain locus.

Loss of polyoma virus infectivity as a result of a single amino acid change in a region of polyoma virus large T-antigen which has extensive amino acid homology with simian virus 40 large T-antigen.

The polyoma virus (Py) transformed cell line 7axB, selected by in vivo passage of an in vitro transformed cell, contains an integrated tandem array of 2.4 genomes and produces the large, middle, and small Py T-antigen species, with molecular weights of 100,000, 55,000, and 22,000, respectively (Hayday et al., J.

Structural and biological analysis of integrated polyoma virus DNA and its adjacent host sequences cloned from transformed rat cells.

EcoRI fragments containing integrated viral and adjacent host sequences were cloned from two polyoma virus-transformed cell lines (7axT and 7axB) which each contain a single insert of polyoma virus DNA. Cloned DNA fragments which contained a complete coding capacity for the polyoma virus middle and small T-antigens were capable of transforming rat cells in vitro. Analysis of the flanking sequences indicated that rat DNA had been reorganized or deleted at the sites of polyoma virus integration, but none of the hallmarks of retroviral integration, such as the duplication of host DNA, were apparent.

Active viral genes in transformed cells lie close to the nuclear cage.

Nuclear DNA is looped by attachment to a matrix or cage. Using nine different lines transformed by polyoma or avian sarcoma virus, we have mapped viral sequences integrated within these loops. In all lines that contain high concentrations of viral transcripts and express the transformed phenotype, the integrated viral genes lie close to the points of attachment to the cage.

Detection in situ of foreign DNA in eukaryotic cells.

A simple technique is described that allows mixed populations of eukaryotic cells to be screened for clones containing multiple copies of a particular DNA. Essentially, eukaryotic cells are transferred to either nitrocellulose of Whatman 541 filters, and their DNA is immobilised in situ. Exposure of the filters to a 32P-labeled DNA "probe" results in detectable hybridisation only at the positions of clones containing multiple copies of the DNA.