No abstract

A DNA segment of approximately 200 base pairs (bp) from Crithidia fasciculata kinetoplast minicircles was previously shown by electron microscopy (EM) to bend into a small circle due to its unique nucleotide sequence containing repeated blocks of 4-6 A's. When this segment was flanked by 207 bp of plasmid DNA on one side and 460 bp on the other, the resulting 890-bp DNA was found to appear either relatively straight or extremely bent as visualized by EM. The bend was located one-third the distance from one end. The fraction of molecules with the most extreme bend increased from approximately 2% to 50-60% following incubation of the DNA with increasing concentrations of Zn2+, Co2+, Ba2+, and Mn2+. These observations suggest that sequence-directed bending in DNA is an inducible and not a static phenomenon. Possible roles of transitions between the bent and straight conformations in the control of gene expression are discussed.

The study of immortalization and other alterations associated with neoplastic transformation of endometrial stromal cells is important to understanding the development of uterine sarcomas and mixed tumors. Because stromal cells are important regulators of associated epithelial cells, alterations in the regulation of stromal cell proliferation that influence epithelial cells may also contribute to the development of endometrial carcinomas. To study immortalization and associated phenotypic and genetic alterations of human endometrial stromal cells, cultures were transfected with a plasmid containing an ori-, temperature-sensitive mutant SV40, A209 (tsSV40). Morphologically transformed colonies were selected and propagated at the permissive temperature until they entered 'crisis'. In contrast to human fibroblasts, every clone tested was immortalization competent. The frequency of immortalization was approximately 1 x 10(-6). One uncloned and six cloned cell lines escaped from crisis and appear to be immortal. Two clones, M4 and B10T1, were selected for further study. Immortalization is conditional; proliferative arrest occurs at the restrictive temperature for large T antigen function. Furthermore, withdrawal of the large T antigen results in expression of the senescent phenotype of enlarged, flattened cells. Colony-forming efficiency at the restrictive temperature was undetectable. Immortalization is also associated with several genetic alterations. The DNA content of tsSV40 transfected cells was either diploid or tetraploid in the precrisis stage of proliferation, but became aneuploid upon immortalization. Several structural rearrangements of chromosomes were detected in the immortalized stromal cells which differ from those found in SV40 immortalized fibroblasts. Although their capacity for anchorage-independent proliferation (AIP) is variable, tsSV40-immortalized endometrial stromal cells have a higher capacity for AIP than their tsSV40-transfected progenitor cells in the period of proliferation prior to 'crisis'.

Duplications and deletions of the same gene loci or chromosome regions are known to produce different clinical manifestations and are significant factors in human morbidity and mortality. Extensive cytogenetic and molecular cytogenetic studies with cosmid and YAC probes in two patients with unique mosaicism for reciprocal duplication-deletion allowed us to further understand the origin of these abnormalities. The first patient’s mosaic karyotype was 46,XX,inv dup(11) (q23q13)/46,XX,del(11)(q13q23). The second patient had a 46,XY,dup(7)(p11.2p13)/46,XY,del(7)(p11.2p13)/46,XY karyotype. Fluorescence in situ hybridization studies on the first patient placed the two breakpoints near the folate-sensitive fragile sites FRA11A and FRA11B. The presence of repeated sequences responsible for these fragile sites may have been involved in the patient’s duplication-deletion. Our investigation leads us to conclude that, in addition to known mechanisms (such as unequal crossovers between homologs, unequal sister chromatid exchanges, excision of intrachromatid loops, and meiotic recombination within a single chromatid), duplication-deletion can also arise by the formation of an overlying loop followed by an uneven crossover at the level of the DNA strand.