Human immune interferon gene is located on chromosome 12.

A cDNA clone for human immune interferon (IFN-gamma) gene sequences, plasmid p69, was used to chromosomally map the IFN-gamma gene by detecting human IFN-gamma gene sequences in DNA isolated from human-rodent somatic cell hybrids. We were able to map the IFN-gamma gene by correlating the human chromosomes present in these hybrids with the human specific 8.8 and 2.0 kilobase pair fragments produced by EcoRI digestion of genomic DNA. Southern blot analysis of 37 hybrid cell lines indicated that the gene for IFN-gamma was on human chromosome 12. A hybrid containing a portion of chromosome 12 localized the IFN-gamma gene to the p1205 leads to qter region.

Human c-Ki-ras2 proto-oncogene on chromosome 12.

A human colonic adenocarcinoma transforming gene, recently identified as a cellular homolog of the Kirsten sarcoma gene (v-ras), was used to assign the human cellular Kirsten ras2 gene to chromosome 12 by the Southern hybridization method. A single 640 base-pair Eco RI--Hind III fragment of the transforming gene, isolated by DNA transfection and molecular cloning, can detect a single Eco RI fragment (2.9 kilobase pairs) of DNA from phenotypically normal cells. The data suggest a constant chromosomal location of c-Ki-ras2.

Clustering of leukocyte and fibroblast interferon genes of human chromosome 9.

At least ten leukocyte interferon genes and the single known fibroblast interferon gene have been localized on the pter leads to q12 region of human chromosome 9. Gene mapping was accomplished by blot hybridization of cloned interferon complementary DNA to DNA from human-mouse cell hybrids with a translocation involving human chromosome 9. Supporting evidence suggests these genes are clustered.

Restriction fragment length polymorphism studies show consistent loss of chromosome 3p alleles in small cell lung cancer patients' tumors.

Previous karyotypic analysis of human small cell lung cancer cell lines has demonstrated a consistent deletion of a portion of the short arm of chromosome 3(p14-23). DNA prepared from tumors and normal tissues obtained from 24 small cell lung cancer and two extrapulmonary small cell cancer patients was hybridized to four probes that detect restriction fragment length polymorphisms within chromosome region 3p14-21. Of the 25 patients who were heterozygous for at least one marker in this region in the DNA from normal tissue, 23 (92%) showed an unequivocal loss of heterozygosity in the DNA from their tumor tissue. From these studies we conclude that loss of alleles from the short arm of chromosome 3 is a consistent finding in unselected small cell lung cancer patients' tumor DNA.

Regional localization of two human cellular Kirsten ras genes on chromosomes 6 and 12.

Human cellular Kirsten ras1 and ras2 genes were localized to chromosomes 6p23 ----q12 and 12p12 .05----pter, respectively, using human-rodent cell hybrids. Thus, the short arms of human chromosomes 11 (encoding lactate dehydrogenase-A and the proto-oncogene c-Ha- ras1 ) and 12 (encoding lactate dehydrogenase B and c-Ki- ras2 ) share at least two pairs of genes that probably evolved from common ancestral genes.

Altered structure and expression of the human retinoblastoma susceptibility gene in small cell lung cancer.

Karyotypic and molecular genetic evidence has indicated that deletion or rearrangement of both chromosomes 3 and 13 may be important in the pathology of human small cell lung cancer (SCLC). The retinoblastoma susceptibility gene, RB, on chromosome 13 band q14, has previously been shown to be altered in SCLC [J. W. Harbour et al., Science (Wash. DC), 241: 353-357, 1988; J. Yokota et al., Oncogene, 3: 471-475, 1988]. Our studies of 26 SCLC tumor and normal DNA samples indicate that 6 of 6 patients whose normal cell DNA was heterozygous for an RB restriction fragment length polymorphism have lost one of the two alleles in their tumor DNA. Consistent with other studies, we find 2 of 26 tumors with homozygous deletions within the RB gene. Of 13 SCLC cell lines examined, only 3 expressed greater than trace amounts of RB mRNA. RB protein was detected in 2 of 14 SCLC cell lines examined, unlike the results of Yokota et al. (Oncogene, 3: 471-475, 1988) which showed no RB protein in any of the 9 cell lines they examined. Only unphosphorylated RB protein was detected in SCLC cell line H209, suggesting that the RB protein may be inactivated by a novel mechanism in this cell line. These data suggest that inactivation of the RB gene is a frequent if not universal event in SCLC.

Use of the single strand conformation polymorphism technique and PCR to detect p53 gene mutations in small cell lung cancer.

Recent studies have suggested that the p53 oncoprotein might function normally as a tumor suppressor. Mutations in highly conserved regions of the p53 gene have been observed in numerous types of tumors and tumor cell lines. To detect in a more sensitive manner p53 gene mutations in small cell lung cancer (SCLC) we utilized the single strand conformation polymorphism (SSCP) technique of Orita et al., (1989). Using PCR primers for the most highly conserved regions of the p53 gene, including exons 4-9, we have identified p53 mutations in 5 of 9 small cell lung cancer (SCLC) tumor DNA samples and in 1 SCLC cell line. None of the mutations seen in tumor DNA samples were present in normal DNA from the same patients, indicating that mutation of the p53 gene in these tumors was a somatic event. Of the six mutations observed, two were found in exon 7, three were found in the region encompassing exons 8 and 9, and one was found in the region encompassing exons 5 and 6. Nucleotide sequencing of one of the exon 7 mutations and one of the exon 8-9 mutations indicated that each was a C to T transition. In SCLC-6 the mutation resulted in substitution of serine for proline at amino acid 278 and in SCLC-4 substitution of tryptophan for arginine at amino acid 248, both nonconservative amino acid substitutions. Both of these changes are in regions of the p53 gene where mutations have been observed in other tumors. Two additional mutations were observed in SCLC cell lines using conventional PCR techniques. One of these is a mutation which results in altered splicing of the p53 pre-mRNA.

Activation of the colony-stimulating factor 1 receptor leads to the rapid tyrosine phosphorylation of GTPase-activating protein and activation of cellular p21ras.

We have previously reported that platelet-derived growth factor (PDGF) induced tyrosine phosphorylation of GTPase-activating protein (GAP) in intact quiescent fibroblasts under conditions in which insulin and basic fibroblast growth factor (bFGF) were ineffective (Molloy et al., 1988). In the present study, we have provided evidence that colony-stimulating factor 1 (CSF-1) is capable of inducing tyrosine phosphorylation of GAP and its associated cellular proteins, p62 and p190, in NIH3T3 cells overexpressing the human CSF-1 receptor (CSF-1R). However, the extent of GAP tyrosine phosphorylation induced by CSF-1 was approximately 10% of that induced by PDGF-BB in the NIH3T3 fibroblasts. Despite this significant difference, both PDGF-BB and CSF-1 increased the activation of p21ras, the extent of which correlated well with the mitogenic response induced by each growth factor in these cells. Taken together, our findings provide evidence for a possible role of tyrosine phosphorylation of GAP and GAP-associated phosphoproteins in regulating transduction of CSF-1-induced mitogenic signals through p21ras activation.

Neutrophil collagenase (MMP-8) is expressed during early development in neural crest cells as well as in adult melanoma cells.

Matrix metalloproteinase-8 (MMP-8) is a neutral metalloproteinase of the fibrillar collagenase family that also includes MMP-1 and MMP-13. In contrast to the other collagenases, MMP-8 has a very limited tissue distribution, thought to be restricted to neutrophils and chondrocytes. In a previous study, we observed MMP-8 expression in human melanoma cells. This observation led us to assess in more detail the expression of MMP-8 in normal and malignant melanocytic cells. We found that MMP-8 was expressed by 11 out of 12 human melanoma cell lines tested and all 10 primary melanomas we examined, but was not expressed by four primary neonatal melanocyte strains. Since melanocytes arise from highly motile neural crest cells, we examined the hypothesis that MMP-8 might be expressed by neural crest cells. RT-PCR analysis of post-implantation mouse embryos indicated the presence of MMP-8 transcripts at E9.5. In situ hybridization and immunohistochemistry of mouse embryos between E9.5-E14.5 demonstrated MMP-8 expression in the surface ectoderm, neural crest cells and chondrocytes. MMP-8 was also detected in neural crest cell migration located in the circumference of the neural tube, branchial arches and the notochord. In addition, MMP-8 expression was observed between the somites, in circumscriptive areas of the developing brain, heart, and eye, and in the interdigital zones of the limbs. In summary, we found MMP-8 to be the first fibrillar collagenase expressed during development. In contrast to its restricted tissue expression post-partum, MMP-8 was present in multiple embryonic tissues, including neural crest cells. The production of MMP-8 by migrating neural crest cells may contribute to their ability to degrade fibrillar collagen matrices while in transit.

Decline of signal transduction by phospholipase C gamma 1 in IMR 90 human diploid fibroblasts at high population doubling levels.

During cellular senescence in vitro, the cells do not respond mitogenically to serum growth factors at high population doubling levels. Phospholipase C activity in low PDL IMR 90 cells showed a 4.7-fold stimulation in response to 10% serum compared to 3.3-fold in high PDL cells when measured in whole cell extracts. Immunoaffinity purified tyrosine phosphorylated protein fraction showed a greater increase (5.2-fold) in phospholipase C activity in low PDL than high PDL cells (2.1-fold) in response to serum. Serum stimulated PLC gamma 1 activity was diminished in high PDL cells. Immunokinase assay of PLC gamma 1 immunoprecipitates from serum stimulated IMR 90 fibroblasts suggested that diminished enzymatic activity in high PDL cells is not due to less receptor coupled tyrosine phosphorylated PLC gamma 1 enzyme. Serum stimulated [3H]thymidine incorporation into DNA declined in parallel with the activity of PLC gamma 1, suggesting that its activation might play significant roles in this in vitro model for cellular senescence.

The kinase insert domain of colony stimulating factor-1 receptor is dispensable for CSF-1 induced phosphatidylcholine hydrolysis.

Mouse NIH 3T3 fibroblasts transfected with human colony stimulating factor-1 receptor produced diacylglycerol in response to CSF1 and this correlated with elevated phosphatidylcholine hydrolyzing activity measured in an in vitro assay. Treatment of cells with the isoflavone derivative genistein attenuated PC hydrolysis in vitro suggesting a role for CSF1R tyrosine kinase activity. A CSF1R mutant lacking 67 amino acids of the kinase insert domain, which may affect the association of receptor with certain substrates, stimulated PC hydrolysis in response to CSF1. Coupling to PC hydrolysis is likely a general property of CSF1R and the kinase insert domain is dispensable for this activity.

Site-directed deletion mutagenesis using phagemid vectors and genetic selection.

Oligonucleotide-directed mutagenesis was used along with the dut and ung genetic selection method of Kunkel to introduce large site-specific deletions into cDNAs cloned into phagemid vectors. We find that large deletions can be achieved with an efficiency equal to that of single point mutations, with a very low frequency of aberrent clones. To facilitate screening of clones, E. coli strain DH5 alpha was used as the recipient host cell to genetically select for deletion mutants. Comparisons were made to deletion mutagenesis without genetic selection, and to reactions utilizing two oligonucleotide primers simultaneously. The low frequency of deletion mutants observed without genetic selection renders random screening for deletion mutant clones cumbersome. The results provide representative expectations and a useful guide for those contemplating the construction of deletion mutants.

A human c-src gene resides on the proximal long arm of chromosome 20 (cen----q131).

A molecular clone of v-src, the oncogene of Rous sarcoma virus, was used to detect and regionally localize a human c-src proto-oncogene on chromosome #20. The human c-src gene, detected as either a 28-kbp EcoRI DNA fragment or as a 15.4-kbp BglII DNA fragment, was localized to 20 cen----q131 by filter hybridization analysis of DNAs from human-rodent somatic cell hybrids. The results indicate that c-src is on the same chromosome arm as aberrations associated with myeloproliferative disease, although the possible involvement of c-src in these aberrations is unknown.

Gene transfer and gene mapping in mammalian cells in culture.

The ability to transfer mammalian genes parasexually has opened new possibilities for gene mapping and fine structure mapping and offers great potential for contributing to several aspects of mammalian biology, including gene expression and genetic engineering. The DNA transferred has ranged from whole genomes to single genes and smaller segments of DNA. The transfer of whole genomes by cell fusion forms cell hybrids, which has promoted the extensive mapping of human and mouse genes. Transfer, by cell fusion, of rearranged chromosomes has contributed significantly to determining close linkage and the assignment of genes to specific chromosomal regions. Transfer of single chromosomes has been achieved utilizing microcells fused to recipient cells. Metaphase chromosomes have been isolated and used to transfer single-to-multigenic DNA segments. DNA-mediated gene transfer, simulating bacterial transformation, has achieved transfer of single-copy genes. By utilizing DNA cleaved with restriction endonucleases, gene transfer is being empolyed as a bioassay for the purification of genes. Gene mapping and the fate of transferred genes can be examined now at the molecular level using sequence-specific probles. Recently, single genes have been cloned into eucaryotic and procaryotic vectors for transfer into mammalian cells. Moreover, recombinant libraries in which entire mammalian genomes are represented collectively are a rich new source of transferable genes. Methodology for transferring mammalian genetic information and applications for mapping mammalian genes is presented and prospects for the future discussed.

Coronavirus 229E susceptibility in man-mouse hybrids is located on human chromosome 15.

Human coronavirus 229E, n enveloped, RNA-containing virus, causes respiratory illness in man and is serologically related to murine coronavirus JHM, which causes acute and chronic demyelination in rodents. 229E displays a species-specific host range restriction whose genetic basis was studied in human-mouse hybrids. 229E replicated in human WI-38 cells but not in three mouse cell lines tested (RAG, LM/TK-, and A9). Human coronavirus sensitivity (HCVS) was expressed as a dominant phenotype in hybrids, indicating that mouse cells do not actively suppress 229E replication. HCVS segregated concordantly with the human chromosome 15 enzyme markers mannose phosphate isomerase (MPI) and the muscle form of pyruvate kinase (PKM2), and analysis of hybrids containing an X/15 translocation [t(X;15)(p11;q11)] localized HCVS to the q11 leads to qter region of chromosome 15. HCVS might code for a specific surface receptor, allowing 229E to be absorbed to and received within the host cell.

Migration inhibition factor study in Histoplasma capsulatum.

Mice sublethally infected with viable Histoplasma capsulatum or immunized with merthiolate-killed yeast phase cells showed decreased mortality on subsequent challenge infection as compared to controls. Migration inhibition (MI) assays using peritoneal and spleen cells from immunized but unchallenged mice showed no parallel correlation with percent mortality. MI assay indices fluctuated without concomitant changes in resistance to challenge injection with live yeast phase cells. Viable vaccines induced greater resistance to challenge infection than killed cells, although both were comparable in sensitizing ability as measured by MI assay techniques with this mouse model.