Biomedical subjects
Publications and source records attributed to A Hayday.
The sequences of productive T-cell gamma/delta receptor transcripts were compared in different murine fetal tissues. Differences from tissue to tissue suggest that the sequence repertoires are at least in part the products of selection, presumably through interaction of T cells bearing the gamma delta receptor with fetal self-ligands.
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The inflammatory response in the lungs of mice infected with an influenza A virus consists largely of macrophages and CD3+ T cells. Most T lymphocytes recovered before day 7 after infection express mRNA for the T cell receptor alpha/beta (TCR-alpha/beta), while TCR-gamma/delta mRNA+ cells are found at much higher frequency over the next 7 d. The predominant surface phenotype for the TCR-gamma/delta mRNA+ population is CD3+4-8-TCR-alpha/beta-. Some lymphocytes expressing all the known V gamma genes are found in the inflammatory exudate, but V gamma 2+/V gamma 1+ and V gamma 4+ T cells are present at highest frequency. The response is staged, with maximal numbers of V gamma 4+ cells occurring on day 10 after infection, while the predominant phenotype on day 13 is V gamma 2/V gamma 1+. The emerging peak in numbers of V gamma 4+ lymphocytes is paralleled by increasing numbers of macrophages expressing hsp mRNA. The later maxima found for the V gamma 2+/V gamma 1+ T cells is consistent with the possibility that at least some of these lymphocytes are responding to the hsp+ cells and are functioning to resolve the inflammatory process.
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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. A translocated and mutated allele of c-myc was stably transfected into fibroblasts. The rearranged allele was expressed indistinguishably from a normal c-myc gene: it had serum-regulated expression, was transcribed with normal promoter preference, and was strongly attenuated. Thus mutations by themselves are insufficient to deregulate c-myc transcription.
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.
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. To study regulation of c-myc expression, a transfection system has been developed in which introduced c-myc genes exhibit serum-responsive activity. The responsiveness assayed is not mediated by increased transcription initiation. Rather, it is effected at a point(s) between transcription and stabilization of the RNA.
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). A cDNA sequence analysis, constituting the first such analysis of any size of gamma-gene transcripts from circulating, peripheral cells of adult mice, revealed transcription to be frequently of productively rearranged genes. These genes display extensive junctional diversity.
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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. We demonstrate that under these conditions, the sequencing data obtained with miniprep DNA is indistinguishable from that obtained with CsCl purified supercoiled DNA or from that obtained using single stranded DNA templates. We further show that the supercoiled DNA sequencing reactions can be analyzed on a commercially available automated DNA sequencing system that detects 32P labeled DNA during its electrophoretic separation. Taken together, these developments represent a significant improvement in the process of nucleotide sequence analysis.
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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. The second protein (43 kd), which contains the N-terminal 75% of middle T-Ag, has an associated protein kinase activity and forms a complex with c-src, but cannot induce a transformed phenotype.
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. Integration of polyoma DNA induces outlying cellular sequences to become closely associated with the cage. The strength of this correlation between gene activity and proximity to the cage was examined using sub-clones of one avian sarcoma virus transformant. Proviral sequences are closely associated with the cage in this transformant, much less so in two untransformed 'flat revertants' which contain no detectable viral transcripts but regain their close association with the cage in two retransformed derivatives.
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. There was no obvious similarity of DNA sequences in the four virus-host joins. In one case the virus-host junction sequence predicted the virus-host fusion protein which was detected in the transformed cell line. DNA homologous to the flanking sequences of three out of four of the joins was present in single copy in untransformed cells. One copy of the flanking host sequences existed in an unaltered form in the two transformed cell lines, indicating that a haploid copy of the viral transforming sequences is sufficient to maintain transformation. The flanking sequences from one cell line were further used as a probe to isolate a target site (unoccupied site) for polyoma virus integration from uninfected cellular DNA. The restriction map of this DNA was in agreement with that of the flanking sequences, but the sequence of the unoccupied site indicated that viral integration did not involve a simple recombination event between viral and cellular sequences. Instead, sequence rearrangements or alterations occurred immediately adjacent to the viral insert, possibly as a consequence of the integration of viral DNA.
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. Using Whatman 541 paper, a portion of the cells, evenly distributed throughout the mixed population is retained on the culture dish, and can be propagated further for subsequent cell cloning. The technique has allowed rapid distinction of clones of transformed rat cells that contain a single or only a few copies per cell of polyoma viral DNA from clones maintaining multiple copies. The technique has also been used to distinguish between clones of mouse L-cells containing multiple and only a few copies of 0X174 DNA. In this manner the technique allows rapid detection of cells amplifying a particular species of DNA. Finally, the method can be used to detect cells assimilating many copies of a foreign DNA, even in the absence of a co-transfected selectable marker.
We have analyzed the state, arrangement, and expression of polyoma viral DNA sequences in a number of in vitro-transformed Fischer rat cells before and after growth in vivo as tumour cells. When the in vitro lines used to induce the tumors contained only a single insert of viral sequences and did not produce either a full-size 100,000-dalton (100K) large T-antigen or free viral genomes, no differences in the above-mentioned properties were observed. By contrast, in vitro cell lines containing multiple inserts of viral sequences, a functional 100K large T-antigen, and free viral genome induced tumor cells which displayed a reduced number of inserts of viral sequences and which did not produce either a functional 100K large T-antigen or free viral genomes. All of the in vitro lines and their tumor cell derivatives expressed the polyoma virus 55K middle and 22K small T-antigen species. Possible mechanisms for the selection in vivo against cells containing a functional 100K large T-antigen and consequently free viral genomes are discussed.