Amplification of DNA sequences from chromosome 19q13.1 in human pancreatic cell lines.

Conventional cytogenetics and comparative genomic hybridization (CGH) were utilized to identify recurrent chromosomal imbalances in 12 pancreatic adenocarcinoma cell lines. Multiple deletions and gains were observed in all cell lines. Losses affecting chromosomes or chromosome arms 9p, 13, 18q, 8p, 4, and 10p and gains involving chromosome arms or bands 19q13.1, 20q, 5p, 7p, 11q, 3q25-qter, 8q24, and 10q were commonly observed. Interestingly, 19 distinct sites of high-level amplification were found by CGH. Recurrent sites involved 19q13.1 (6 cases), 5p (3 cases), and 12p and 16p (2 cases). Amplification of KRAS2 was demonstrated in 2 cell lines and that of ERBB2 in another. To define the occurrence of chromosome 19 amplification further, two-dimensional analysis of NotI genomic restriction digests and fluorescence in situ hybridization using probes from band 19q13.1 were utilized. High-level amplification of overlapping sets of chromosome 19 NotI fragments was exhibited in 3 cell lines of which 2 showed amplification of both OZF and AKT2 genes and 1 that of AKT2 alone. In these 3 cell lines, amplification of chromosome 19 sequences was associated with the presence of a homogeneously staining region. Our results provide evidence of heterogeneity in the extent of chromosome 19 amplification and suggest the existence of yet unknown amplified genes that may play a role in pancreatic carcinogenesis.

Two-dimensional separation and cloning of chromosome 1 NotI-EcoRV-derived genomic fragments.

The two-dimensional (2-D) separation of genomic digests has provided the means to analyze over 2000 unique restriction fragments simultaneously in a single gel, for genetic variation as well as for genomic alterations in cancer. By utilizing different combinations of restriction enzymes or different electrophoretic conditions, the number of analyzable fragments in multiple 2-D patterns can be augmented. We have previously shown the feasibility of distinguishing between spot intensities representing fragments from one allele and from two alleles and have implemented approaches for the cloning of fragments of interest in 2-D gels. In this study, the 2-D separation and cloning of chromosome 1 NotI-EcoRV-derived genomic fragments was performed. Three hundred forty-six NotI fragments in whole genomic preparations were assigned to chromosome 1. To verify the reliability of the assignment, two of the NotI fragments attributed to chromosome 1 were cloned and sequenced. The fragments that contained CpG islands were mapped by FISH to 1p35-p36.1 and to 1p13.3-p21, respectively. Our study indicates the feasibility of analyzing 2-D separations of whole genomic digests for the detection of alterations in specific chromosomes. The large number of restriction fragments attributed to chromosome 1 provides the means to screen 2-D patterns for chromosome 1 deletions and amplifications with a high marker density.

Generation of five high-complexity painting probe libraries from flow-sorted mouse chromosomes.

Mouse metaphase chromosomes were purified by flow sorting from the murine fibroblast cell line Mus spretus clone 5A. We sorted chromosomes that fell into five individual peaks based on the Hoechst 33258/chromomycin A3 DNA histogram: three peaks corresponding to the least amount of DNA and two peaks representing chromosomes with the most DNA content. This is the first example of the successful application of bivariate flow karyotyping to murine chromosome sorting. We then applied primer-directed in vitro DNA amplification using the polymerase chain reaction (PCR) to generate and label larger amounts of chromosome-specific DNA. In situ hybridization showed specific binding of the PCR products to mouse chromosomes Y, 19, 18, 3, and X as well as chromosomes 1 and 2. The combination of chromosome sorting from the M. spretus cell line and PCR proved to be highly valuable for generation of pools of DNA fragments that exhibit specific binding to mouse chromosomes and can be used to identify and delineate mouse metaphase chromosomes.

Molecular mapping of mouse chromosomes 4 and 6: use of a flow-sorted Robertsonian chromosome.

The development of dense genetic maps of mammalian chromosomes is facilitated when chromosome-specific libraries are used as a source of genetic markers. To saturate the genetic maps of mouse chromosomes 4 and 6, we have made use of fluorescent-activated chromosome sorting to purify a 4:6 Robertsonian chromosome from a cell line harboring the Rb(4:6)2Bnr translocation. After staining with chromomycin A3 and Hoechst 33528, this chromosome was separated from the other mouse chromosomes. DNA was isolated from the fraction containing the Robertsonian chromosome and subcloned into the insertion vector lambda gt10, generating a library with 4.6 x 10(5) independent phage. A total of 19 single-copy sequences were used to type the progeny of a C57BL/6J x Mus spretus backcross that had previously been typed for loci on chromosomes 4 and 6. Approximately 70% of the clones in the library mapped to either chromosome 4 or 6 as assessed by genetic mapping and by use of a somatic cell hybrid panel. Simple sequence repeats have also been isolated from this library. Further characterization of these microsatellites should accelerate efforts to map mouse chromosomes 4 and 6 using PCR. In addition, flow sorting of Robertsonian chromosomes suggests a general approach for making chromosome-specific libraries in mouse.

Use of somatic cell hybrids for quantitation of mutagenesis: reduction in background mutants by fluorescence-activated cell sorting (FACS).

Environmentally induced mutations, especially those involving large scale genetic damage such as deletions and chromosome loss, are of central importance in the production of human genetic disease and cancer. We have developed a methodology, the AL assay, that permits detection of such extensive genetic changes which often escape detection in other systems in which they are lethal. The AL assay employs a human-Chinese hamster ovary cell hybrid that retains a single human chromosome, number 11. A set of specific cell surface antigens result from genes located on opposite arms of this chromosome. Exposure to mutagens produces mutants which form colonies in the presence of complement and specific antiserum that kill nonmutant cells. The frequency and pattern of marker loss provides a measure of single gene mutation, large and small deletion, and loss of the entire chromosome 11. We have employed the indirect fluorescein conjugated isothiocyanate (FITC) technique and fluorescence-activated cell sorting (FACS) to remove spontaneous mutants from the initial population. The 100-fold reduction in background thus far achieved should allow accurate analysis of mutation by ionizing radiation at doses of less than 10 rad.

Preparation of chromosome suspensions for flow cytometry.

Certain variables in the preparation of chromosome suspensions for flow cytometric analysis have been investigated. The optimal conditions have been determined. The results of this series of experiments have been incorporated to yield a preparation protocol that gives chromosome profiles with a low amount of small particle debris and few chromosome clumps. The method reduces variability that results from sample preparation. Chromosomes are optimally isolated in a hypotonic solution buffered to pH 8.0. MgSO4 and dithiothreitol added to the buffer reduce the number of clumps and small fluorescent particles. The presence of MgSO4 also stabilizes the chromosomes and precludes the need for other stabilizing agents such as propidium iodide. When the swelling buffer developed in this investigation is used, unstained chromosomes are stable for at least 1 wk if stored at 4 degrees C. The preparation procedure can be used with the DNA stains, propidium iodide, Hoechst 33258, and mithramycin. Preliminary experiments show that this procedure can also be used for bivariate analysis of human and Chinese hamster chromosomes. The importance of this improvement for studies on chromosome damage caused by irradiation or mutagens is discussed.

Community-acquired bacteremia in the elderly: analysis of one hundred consecutive episodes.

A retrospective analysis was made of the records of 100 consecutive geriatric patients with community-acquired bacteremia, admitted to a suburban hospital. The most frequently identified tissue sources for these bacteremias were the urinary tract (34 percent), biliary tract (20 percent), and lungs (13 percent). In 11 percent of the patients, the tissue focus was not established. E. coli, Klebsiella species and Streptococcus pneumoniae were the most common organisms isolated, and they contributed to 73 percent of the bacteremias. Of the 100 patients, 26 succumbed to the infection. Clinical manifestations unique to the geriatric patient are described.

Chromatographic analysis of hydrocarbons in marine sediments and seawater.

The low concentration of hydrocarbons anticipated in pollution baseline studies necessitates the development of analytical techniques sensitive at the sub-microgram per kilogram concentration level. The method of analysis developed in this laboratory involves dynamic headspace sampling for volatile hydrocarbon components of the sample, followed by coupled-column liquid chromatography for the non-volatile components. These techniques require minimal sample handling, reducing the risk of sample component loss and/or sample contamination. Volatile sample components are separated from the matrix in a closed system and concentrated on a TENAX-GC packed pre-column, free from large amounts of solvent and ready for GC/GC-MS analysis. Non-volatile compounds, such as the benzpyrenes, may be extracted from large volumes of water and concentrated on a Bondapak C18 packed pre-column for coupled-column liquid chromatographic separation and analysis. Results of the application of these techniques to the analysis of samples from sites of known low level hydrocarbon contamination are presented and discussed.