Mitchell S. Turker

Preparation of multivesicular liposomes

A novel type of liposome, named here multivesicular liposomes, was prepared by evaporation of organic solvents from chloroform-ether spherules suspended in water. Within each spherule were numerous water droplets that contained solutes to be trapped in liposomes upon solvent evaporation. Liposome preparations of different average diameters were made, varying from 29 +/- 10 microns to 5.6 +/- 1.7 microns. The liposomes were morphologically characterized by light microscopy and transmission electron microscopy. Materials successfully trapped within the liposomes ranged in molecular size from glucose to nucleic acids. Extremely high percentages of encapsulation (up to 89%) were achieved.

Formation of methylation patterns in the mammalian genome.

Cytosine methylation in mammals is an epigenetic modification required for viability of the developing embryo. It has been suggested that DNA methylation plays important roles in X-chromosome inactivation, imprinting, protection of the genome from invasive DNA sequences, and compartmentalization of the genome into active and condensed regions. Despite the significance of DNA methylation in mammalian cells, the mechanisms used to establish methylation patterns during development are not understood. This review will summarize the current state of knowledge about potential roles for cis- and trans-acting factors in the formation of methylation patterns in the mammalian genome.

Genetic alterations on chromosome 17 distinguish different types of epithelial ovarian tumors

Epithelial tumors of the ovary are the most common ovarian tumors of adult women. They exist in several different histological patterns and exhibit varying degrees of aggressiveness. Molecular genetic studies in epithelial ovarian cancer have shown that loss of heterozygosity (LOH) for regions of chromosome 17 is a common event, probably reflecting the inactivation of one or more tumor suppressor genes present on this chromosome. We examined 87 sporadic epithelial ovarian tumors of different grade and histological type at 16 loci on this chromosome and found that 35% of them showed LOH for chromosome 17. Of these, 84% showed LOH for all informative markers, suggesting that loss of the entire chromosome 17 homologue may have occurred. Interestingly, chromosome 17 loss was observed frequently in serous tumors (49%), was less common in endometrioid tumors (15%), and was rare in mucinous tumors (4%) (P = .01 and P = .0002, respectively). Our findings support the concept that the histological subtypes of epithelial ovarian cancer may be the result of different molecular genetic events.

Molecular Genetic Changes in Human Epithelial Ovarian Malignancies

The frequent finding of loss of heterozygosity (LOH) for a specific chromosomal marker in tumor DNA compared to normal DNA suggests the presence of a closely linked tumor-suppressor gene. Using Southern blot analysis, 34 primary ovarian epithelial tumors were examined for the presence of tumor-specific allelic losses, using six probes for chromosomes 6q, 11p, 13q, 16q, and 17p. A high incidence of LOH was observed on 11p, 13q, and 17p. LOH for 17p was present in 3 of 4 (75%) informative benign ovarian tumors, 1 of 5 (20%) borderline tumors, and 16 of 24 (67%) invasive ovarian cancers. Allelic loss with the H-ras1 probe on 11p was present in 10 of 19 (53%) invasive tumors but was not identified in 6 benign or borderline tumors. LOH on 13q was present in 18 of 31 (58%) informative cases including 8 of 10 (80%) Stage 1 tumors. This preliminary study suggests that loss of tumor-suppressor genes on chromosomes 13q and 17p may be early events in ovarian tumorigenesis and that changes on chromosome 11p are later events.

Hypermethylation at a chromosome 17 "hot spot" is a common event in ovarian cancer.

Alterations of normal DNA methylation patterns have been reported in various types of human tumors. These alterations are represented by genome wide hypomethylation and by region specific hypermethylation. One commonly hypermethylated region is 17p13.3 (D17S5), the putative site of a tumor suppressor gene. In this study we report that hypermethylation at this locus occurs frequently (33%) in ovarian tumors. We reported previously that loss of chromosome 17 is a common event in serous epithelial ovarian tumors. A correlation of the methylation event and chromosome 17 loss suggests that hypermethylation at D17S5 precedes chromosome 17 loss.

Molecular evidence for the induction of large interstitial deletions on mouse chromosome 8 by ionizing radiation

The P19H22 mouse embryonal carcinoma cell line is characterized by a hemizygous deficiency for the chromosome 8 encoded aprt (adenine phosphoribosyltransferase) gene and heterozygosity for many chromosome 8 loci. We have previously demonstrated that this cell line is suitable for mutational studies because it is permissive of events ranging in size from base-pair substitutions at the aprt locus to apparent loss of chromosome 8. Large mutational events, defined by loss of the remaining aprt allele, were found to predominate in spontaneous mutants and those induced by ionizing radiation (Turker et al., Mutation Res., 329, 97–105, 1995). In this study we have used a PCR based assay to screen for loss of heterozygosity at microsatellite loci both proximal and distal to aprt in 137Cs-induced and spontaneous aprt mutants. This approach allowed us to distinguish apparent interstitial deletional events from apparent recombinational events. Significantly, 32.5% (26 of 80) of the mutational events induced by 137Cs appeared to be interstitial deletions as compared with 7.7% (6 of 78) in the spontaneous group. This difference was statistically significant (p<0.0001) suggesting that exposure to 137Cs caused a significant number of deletion mutations. Most 137Cs-induced interstitial deletions were larger than 6 cM, whereas none of the spontaneous deletions were larger than 6 cM. These results provide further support for the notion that ionizing radiation induces deletion mutations and validate the use of the P19H22 cell line for the study of events induced by ionizing radiation.

Region- and cell type-specific de novo DNA methylation in cultured mammalian cells

A region upstream of the mouse adenine phosphoribosyltransferase (aprt) gene has a well characterized methylation pattern for HpaII/MspI sites. When an unmethylated plasmid construct containing this region was transfected into P19 mouse teratocarcinoma stem cells appropriate de novo methylation was observed. However, de novo methylation was significantly reduced when this plasmid was introduced into a differentiated derivative of the P19 stem cell line. Finally, a position effect for de novo methylation was shown by demonstrating methylation of a normally unmethylated HpaII/MspI site when it was placed in this upstream region. This system should prove useful for elucidating DNA signals for de novo methylation and changes in DNA methyltransferase activities that occur during cellular differentiation.

High Frequency "Switching" at the Adenine Phosphoribosyltransferase Locus in Multipotent Mouse Teratocarcinoma Stem Cells

Clones of multipotent mouse tetratocarcinoma stem cells, presumptively heterozygous at the adenine phosphoribosyltransferase (APRT) locus (EC 2.4.2.7), were selected for partial resistance to the purine analog 2′,6′-diaminopurine (DAP). All had approximately 50% APRT activity as compared to the parental line and were found to segregate homozygous deficient cells at a high frequency (∼10−2). Homozygous deficient cells were isolated from one of the heterozygotes and were found to fall into a single class characterized by residual activity and the segregation of revertants at an equally high frequency. The revertants in turn gave rise to full mutants at comparably high frequencies. Chromosomal changes detectable with the light microscope were not associated with these transitions. Physical characterization of the APRT enzymes derived from mutant, revertant, and wild-type cells did not reveal any differences. We conclude that the reversible “switching” between heterozygosity and homozygosity is attributable to some form of gene inactivation and reactivation rather than to classical mutational events.

Spontaneous and ionizing radiation induced mutations involve large events when selecting for loss of an autosomal locus

The mouse P19H22 embryonal carcinoma cell line contains two distinct chromosome 8 homologs, one derived from Mus musculus domesticus (M. domesticus) and the other derived from Mus musculus musculus (M. musculus). It also contains a deletion for the M. musculus aprt allele, which is located on chromosome 8. In this study, cells with spontaneous or induced aprt deficiencies were isolated from P19H22 and examined to determine the nature of the mutational events that had occurred. Ultraviolet radiation (UV), ethyl methanesulfonate (EMS), and two forms of ionizing radiation, 137Cs and 252Cf, were used for mutation induction. DNA preparations from the aprt deficient cells were initially screened with a Southern blot analysis and separated into two broad classes: those that had lost the M. domesticus aprt allele and those that had retained it. The overwhelming majority (> 95%) of the spontaneous and ionizing radiation-induced mutants exhibited aprt gene loss, indicating that relatively large events had occurred and that homozygosity for the deleted region was not a lethal event. Loss of heterozygosity for syntenic markers was found to be a common event in cells exhibiting aprt gene loss. In contrast, a majority of the UV-induced mutants (61%) and a substantial minority of the EMS-induced mutants (38%) retained the aprt gene. A sequence analysis confirmed that base-pair substitutions were responsible for this class of mutation. Gene inactivation associated with hypermethylation of the promoter region was found to be a rare event and was not induced by any of the mutagenic agents tested. The results demonstrate the suitability of the P19H22 cell line for mutational studies, particularly those that are large in nature.

Region-specific rates of molecular evolution: A fourfold reduction in the rate of accumulation of “Silent” mutations in transcribed versus nontranscribed regions of homologous DNA fragments derived from two closely related mouse species

SummaryWe have sequenced homologous DNA fragments of 2.7 and 2.8 kbp derived from the closely related mouse speciesMus musculus domesticus (M. domesticus) andMus musculus musculus (M. musculus), respectively. These two species diverged approximately 1 million years ago. Each DNA fragment contains 1.35 kbp of the 3′ end of the constitutively expressed 2.2-kbpaprt (adenine phosphoribosyltransferase) gene and a similarly sized nontranscribed region downstream of theaprt gene. Theaprt gene region contains protein coding sequences (0.35 kbp), intronic sequences (0.75 kbp), and a 3′ nontranslated sequence (0.25 kbp). Both theM. domesticus andM. musculus downstream regions share three partial copies of the B1 repetitive element with theM. musculus downstream region containing an additional complete copy of this element. A comparison of the 2.7-and 2.8-kbp DNA fragments revealed a total of 63 molecular alterations (i.e., mutations) that were approximately fourfold more abundant in the nontranscribed downstream region than in the transcribedaprt gene. Of the 11 mutations observed in the transcribed region, 7 were found in introns, 3 in the 3′ untranslated sequence, and 1 was a synonymous change in an exon. A comparison of the human andM. domesticus aprt genes has previously revealed no homology in either the intronic or 3′ nontranslated regions with the exception of a 26-bp sequence in intron 3 and sequences at the exon/intron boundaries necessary for correct mRNA splicing (Broderick et al.,Proc. Natl. Acad. Sci. USA, 84:3349, 1987). Therefore, there does not appear to be selective pressure for sequences within these regions. We conclude that there is a lower rate of accumulation of “silent” mutations in the transcribed mouseaprt gene than in a contiguous nontranscribed downstream region. A possible molecular mechanism involving preferential DNA repair for the transcribed region is discussed.