The AluQuant Human DNA Quantitation System has been developed for human-specific quantitation of forensic samples. This system uses probes specific to repetitive genetic elements allowing quantitation without target amplification. Target immobilization is unnecessary with employment of solution hybridization. The AluQuant Human DNA Quantitation System uses a series of enzymatic reactions to produce a luminescent signal proportional to the quantity of human DNA present. This report demonstrates a range of quantitation from 0.1-50 ng of human DNA. Signal from non-human DNAs tested was insignificant and addition of non-human DNAs into a human sample did not alter quantitation. Lastly, the system was unaffected by degradation of sample through sonication. The AluQuant Human DNA Quantitation System is a simple and sensitive method for quantitating the concentration of human DNA in forensic samples.

The accurate analysis of STR fragments that have been amplified by PCR is dependent on using a narrow range of DNA concentration in the amplification reaction. For casework samples, determining DNA concentrations can be further complicated by contamination with microbial, fungal, and occasionally animal DNA. The current available human-specific DNA quantitation kits use hybridization of a repetitive sequence probe to sample DNA attached to membranes. While these systems provide the necessary sensitivity, they have a limited dynamic range frequently compared by eye with known standards. In addition, these techniques require the manipulation of membranes and suffer the inherent problems of filter hybridization.

Denaturing polyacrylamide gel electrophoretic analysis of amplified polymorphic short tandem repeat (STR) loci using fluorescent markers is a mainstay of forensic and paternity testing. To reduce the drawback of preparing gels or using expensive precast gels, we have developed a simple and rapid method to reuse gels between 2 and 8 times over a period of several days. Following the initial electrophoresis and scan, the original samples are removed from the gel by a 1-1.5-h reverse-electrophoresis step. This step heats the gel for the next set of samples and can be performed several days after the initial electrophoresis. Sample bands remain sharp on subsequent runs, but edge effects (frowning of the outside lanes) become progressively worse and ultimately limit gel reuse. Well distortions and separation of the gel from the plates become problems if the gel is used more than twice. However, degassing the gel solution and bonding the gel to both plates eliminate these problems. Precast gels also can be used multiple times. Using this technique, we have successfully analyzed samples amplified with a nine-locus multiplex system and characterized the separated products using a fluorescent scanner and software.

Protein kinases and phosphatases play an important role in a variety of cellular functions. Thus, it is of interest to develop an assay system that can be used to quantify the activity of individual enzymes specifically in a crude cellular extract, is simple to perform, and is amenable to automation. Here we report on the development of a protein kinase assay that addresses these points and circumvents the pitfalls of existing methodologies. The assay is based on the high affinity and strong binding of streptavidin toward biotin-linked peptide substrates. The biotinylated peptide substrate is phosphorylated by the cognate protein kinase using [γ-32P]ATP under optimal enzyme condition, and the phosphorylated peptide product is then captured by a streptavidin-linked disk. After removal of free [γ-32P]ATP, the 32P incorporated into the peptide substrate can be used as an expression of enzyme activity. In contrast to the commonly used phosphocellulose method, only the phospho-, biotinylated peptide (and not other phosphorylated proteins present in the extract) will bind to the disks, thus giving a true estimate of enzymatic activity. In addition to specificity, this assay does not require the peptide substrate to contain basic amino acids or to be modified by the addition of basic amino acid residues as required for the phosphocellulose method which may result in altered specificity of the substrate. Finally, the binding of the modified peptide substrate to the disks is of high affinity, rapid, and, once formed, unaffected by a wide extreme of pH, temperature, ionic and nonionic detergents, organic solvents, and other denaturing agents.

Journal Article The Gene for Aromatase (P450arom) in the Chicken Is Located on the Long Arm of Chromosome 1 Get access A. Tereba, A. Tereba 2Department of Virology and Molecular Biology, St. Jude's Children's Research HospitalMemphis, Tennesse Address reprint requests to Dr. Jean D. wilson, Department of Internal Medicine, University of TExas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75235-8857. Search for other works by this author on: Oxford Academic PubMed Google Scholar M. J. McPhaul, M. J. McPhaul 1From the Department of Internal Medicine, The University of Texas Southwestern Medical CenterDallas Search for other works by this author on: Oxford Academic PubMed Google Scholar J. D. Wilson J. D. Wilson 1From the Department of Internal Medicine, The University of Texas Southwestern Medical CenterDallas Search for other works by this author on: Oxford Academic PubMed Google Scholar Journal of Heredity, Volume 82, Issue 1, January 1991, Pages 80–81, https://doi.org/10.1093/jhered/82.1.80 Published: 01 January 1991

An I-125-labeled partial cDNA for the chicken aromatase P450 was used for in situ hybridization to chromosomes from primary chicken embryo fibroblast cultures. The results indicate that the gene that encodes aromatase is located on the long arm of chromosome 1 at approximately position 0.16.

Amplification of one of three growth-stimulating myc genes is a common method by which many tumor types gain a proliferative advantage. In metastatic human neuroblastoma, the amplification of the N-myc locus, located on chromosome 2, is a dominant feature of this usually fatal pediatric cancer. Of the many models proposed to explain this amplification, all incorporate as the initial step either disproportionate overreplication of the chromosomal site or recombination across a loop structure. The original locus is retained within the chromosome in the overreplication models but is excised in the recombination models. To test these models, we have used somatic cell hybrids to separate and analyze the chromosomes 2 from a neuroblastoma cell line containing in vivo amplified N-myc. Our results demonstrate that N-myc is excised from one of the chromosomes, suggesting that deletion is a requisite part of gene amplification in a naturally occurring system.

Cytogenetic analyses have documented the consistent deletion of part of the short arm of chromosome 1 in neuroblastoma cells suggesting the presence of a suppressor gene in this chromosomal region. To determine the smallest region of deletion overlap at the molecular level on independently derived tumors and to define the location of the breakpoints more precisely, Southern analyses were performed on a somatic cell hybrid panel containing the normal and altered chromosomes 1 from seven neuroblastoma lines. By this method we were able to analyze a panel of 20 cloned sequences and two isozymes to determine the location of the breakpoints. Our findings indicate that the proximal breakpoints of chromosome 1 deletions range over a distance of more than 50 cM with the most distal deletion breakpoint occurring between MYCL1 and DIS57. In addition, using restriction fragment length polymorphisms, it was determined that in at least three of the five cell lines in which MYCL1 was deleted from a chromosome I, the gene was translocated to another chromosome thus retaining the diploid complement. We propose that the neuroblastoma susceptibility gene is located distal to MYCL1 and that there is another gene which is linked to MYCL1 that may be involved in this neoplasm.

Tumor cells from 70% of neuroblastoma patients contain a deletion of part of the short arm of chromosome 1, indicating that this chromosomal region includes a gene involved in tumor formation. To more precisely evaluate the boundaries and mechanisms involved in generating these deletions, we have examined four neuroblastoma cell lines using a combination of somatic cell hybridization, isozyme analysis, and nucleic acid hybridization employing both standard and restriction fragment length polymorphic probes. The data suggest that the truncation of chromosome 1 in these neuroblastomas was most likely due to a complex translocation and deletion mechanism rather than a simple unbalanced translocation or terminal or interstitial deletion. This conclusion is supported by the frequent removal of MYCL from the altered chromosome 1 to another chromosome. Furthermore, the data suggest that the frequency of breakpoints previously assigned by karyotypic analysis to bands other than 1p32 in neuroblastomas may be overestimated. Finally, this study identified a breakpoint at 1p32 that was localized between the genes JUN and MYCL for one neuroblastoma thus establishing the order of these genes as centromere, JUN, MYCL, telomere. We conclude that the observed breakpoints within chromosome 1p in human neuroblastoma are not as variable as previously described and suggest the results of this study provide evidence for the involvement of specific DNA sequences within 1p32 in the generation of neuroblastoma.

Through the use of drug-adapted tissue culture cells, correlations have been observed between the level of specific enzymes and drug resistance. Drug resistance, however, may be due to multiple factors. To test whether the activity of daunorubicin reductase or NADPH diaphorase independently influences in vitro daunorubicin-induced cytotoxicity, we developed somatic cell hybrid clones to partially isolate these factors. This was accomplished by fusing daunorubicin-resistant myeloblast cells obtained from a patient with monosomy 7 leukemia to a daunorubicin-sensitive Chinese hamster cell line. The in vitro cytotoxicity of daunorubicin was compared in hybrid clones having variable enzyme activities; the concentrations of daunorubicin that inhibited the growth of clones by 50% did not differ by more than 2-fold, whereas daunorubicin reductase activities and NADPH diaphorase isozyme activities differed by more than 100- and 15-fold, respectively. These large differences in enzymatic activity were obtained in part by the suppression of specific hamster genes, indicating a regulatory control mechanism for xenobiotic enzymes. Our findings suggest that in this system substantial intercellular variation in the activity of these xenobiotic enzymes does not independently influence cellular resistance to daunorubicin.

We recently derived a series of transformed cell lines by transfecting mouse bone marrow cells highly enriched for macrophage progenitors with a newly described human gene, R-myc, which has homology to the c-myc oncogene. In this report, we show that these lines share some features characteristic of cells of the mononuclear phagocyte lineage. Specifically, all cell lines had macrophage- or monocytelike morphology, contained nonspecific esterase, were phagocytic for latex beads, secreted lysozyme, bore the Mac-1 antigen, and contained a minority of cells with Fc receptors. However, only a single monocytelike clone had appreciable numbers of cells which bore complement receptor 1, and none were phagocytic for antibody or complement-coated particles, or constitutively secreted Interleukin-1. All these cell lines secreted a growth factor capable of supporting the in vitro proliferation of bone marrow macrophages. Radioimmunoassay and receptor binding studies indicate that this factor is colony stimulating factor 1.

Publisher Summary This chapter discusses the concept of protooncogenes and describes the studies by which these genes have been located in a variety of species, with a marked emphasis on man. The chapter summarizes the chromosomal location of 22 protooncogenes in chicken, mouse, and human cells in tabulated form. It also lists the diseases associated with the homologous retrovirus oncogene and associations with naturally occurring tumors. The chicken protooncogenes were the first examined, mainly for determining the chromosomal association between endogenous retrovirus loci and protooncogenes. This examination, along with Southern blot analyses of diverse species, has revealed no correlation, and it is assumed that these protooncogenes are important growth and/or regulatory genes that infrequently act as nonspecific targets for exogenous retrovirus genomes. The localization of human protooncogenes has progressed at breakneck speed since the discovery that chicken c -myc was involved in avian leukosis virus-induced bursal lymphomas. The localization of c- myc and c- abl to the breakpoints of very consistent translocations in Burkitt's lymphoma and chronic myelogenous leukemia, respectively, support the importance of pursuing the localization of protooncogenes by linking specific genes with the disease state of human malignancies.

We have examined the transcriptional expression of the cellular homologues of several retrovirus-associated oncogenes, myc, rel, rasH, myb, src, and erb, in uncultured childhood leukemia and normal hematopoietic cells, as a first step in determining their normal function and possible association with human neoplasia. Cellular myc-specific RNA was detected in all 30 samples of hematopoietic tissue examined, including 18 leukemias of both the lymphoid and myeloid series, three lymphomas, five normal leukocytes, and four cell lines. Although the level of expression varied over a 25-fold range, no general pattern based on cell type or disease state was evident. In addition, in all cell types examined, a single-molecular-weight myc-specific RNA species was observed. Transcriptional expression of the rel and rasH genes showed a similar lack of specificity, with the rasH gene being expressed at a low uniform level in all cell types examined. myb expression was marginally detectable in most samples, although the myeloid leukemia cells possessed approximately 4-fold higher levels. The expression of src was relatively low in most samples, with markedly elevated levels in a few diverse leukemia samples. erb expression was undetectable in all but two acute myelogenous leukemia samples. Analysis of one patient who had high levels of myc, erb, and src expression before therapy revealed a dramatic reduction in erb and src expression but not myc expression while the patient was in remission. These results indicate that primary human leukemia cells, as well as normal leukocytes, do express the cell homologues to several retrovirus-associated oncogenes, that some leukemia cells express high levels of several oncogenes, and that some of these genes are differentially expressed in specific subpopulations of cells.