Geoffrey R. Kitchingman

Transmembrane orientation of glycoproteins encoded by the v-fms oncogene

The retroviral oncogene v-fms encodes a glycoprotein whose transport to the plasma membrane is required for transformation. Tryptic digestion of microsomes from transformed cells yielded membrane-protected amino-terminal fragments 40 kd smaller than intact molecules. These fragments were glycosylated, and they included v-fms-coded epitopes expressed at the cell surface. Deletion of the predicted membrane-spanning peptide generated polypeptides that were completely sequestered within microsomes. The mutant glycoproteins acquired more asparagine-linked oligosaccharide chains than did wild-type molecules, lacked kinase activity in vitro, were not transported to the cell surface, and had no transforming activity. Thus, the membrane-spanning segment in the middle of the glycoprotein interrupts translocation of nascent chains into the endoplasmic reticulum, ultimately orienting the amino-terminal domain outside the cell and the carboxy-terminal kinase domain in the cytoplasm. These topological features are similar to those of several growth factor receptors, suggesting that v-fms transforms cells through modified receptor-mediated signals.

Sequence of the DNA-binding protein of a human subgroup E adenovirus (type 4): comparisons with subgroup A (type 12), subgroup B (type 7), and subgroup C (type 5).

The nucleotide sequence of the gene for the single-stranded DNA-binding protein of adenovirus type 4 (Ad4) has been determined. The gene codes for a protein of 512 amino acids. Comparison of the amino acid sequence with those previously determined for Ad5, Ad12, and Ad7 allowed identification of regions that are conserved between the four serotypes. These include stretches of 9, 9, and 12 amino acids in the carboxy-terminal domain of the protein; these sequences are similar to those identified in the single-stranded DNA-binding proteins of procaryotes as being important for interaction of the protein with single-stranded DNA. A conserved region of four amino acids in the amino-terminal domain is identical in sequence to a region of the SV40 large T antigen that has recently been implicated in the nuclear localization of the protein. Other conserved amino acids that may be important for the three-dimensional structure of the protein have also been identified. The overall homology between the DBPs of the four serotypes is 17.2% in the amino-terminal domain, 47.8% in the carboxy-terminal domain. Two-way comparisons between the DBPs of the four serotypes indicates that the DBP of Ad4 is most closely related to that of Ad7.

Isolation of activated ras transforming genes from two patients with Hodgkin's disease

Despite impressive advances in the clinical management of Hodgkins disease, little is known about its cellular origin or the mechanism(s) of Hodgkinogenesis. Recent findings that certain human cellular oncogenes can cause malignant transformation suggest that aberrant activation of these genes may play a role in carcinogenesis. To determine if such genes are operative in Hodgkins cells, we isolated DNA from splenic nodules of three patients with nodular sclerosis Hodgkins disease and tested its ability to transform mouse NIH 3T3 cells, the standard assay for oncogene-mediated malignant transformation. Transformed cells containing human DNA were obtained from two patients. DNA from these primary transformants yielded secondary transformants of NIH 3T3 fibroblasts; one also transformed normal mouse bone marrow macrophages, a cell type probably related to the Hodgkins cell. When analyzed by Southern blot methods for homology with closed oncogene probes, the transforming genes from both patients had homology with N-ras. The homology and size of the restriction fragments were similar to those of transforming genes isolated from patients with acute nonlymphocytic leukemias. The presence of the same activated oncogene in tumor tissue from two different patients suggests that it may play an important role in Hodgkinogenesis.

Restriction mapping and molecular cloning of adenovirus type 4 (subgroup E) DNA

The locations of thirty restriction endonuclease cleavage sites were determined on the genome of adenovirus type 4 (Ad4), the sole member of the subgroup E adenovirions. The restriction endonucleases Bg/II, EcoRI, HindIII, HpaI, KpnI, SalI, and XbaI cut Ad4 DNA 10, 3, 2, 3, 5, 5 and 3 times, respectively. Orientation of the linear Ad4 map with respect to left and right molecular ends was accomplished by taking advantage of the limited sequence homology between Ad2 and Ad4. Ten non-overlapping fragments of Ad4 DNA representing 98% of the genome, map units 1.6 to 99.6, have been cloned into the plasmid vector pKC7.

Cytotoxic T cells and immunotherapy

Abstract Promising immunotherapies for viral infections and malignancies reflect the successful, rapid translation of laboratory findings into clinical practice. Fletcher et al. [1] present imaging studies of Epstein-Barr virus (EBV)-associated lymphomas before and after immunotherapy. Here, we briefly review the scientific bases of such novel therapies, which have evolved from advances in understanding of immune effector cells, of the cytokines that drive immune responses, and of the mechanisms underlying cell death.