Kenneth S. Krauter

A second-generation YAC contig map of human chromosome 12.

Breakthrough: The Quest to Isolate the Gene for Hereditary Breast Cancer.By Kevin Davies and Michael White. Mac-millan: 1995. Pp. 370. £16.99. To be published in the United States by Wiley.

Translocation breakpoints upstream of the HMGIC gene in uterine leiomyomata suggest dysregulation of this gene by a mechanism different from that in lipomas

Uterine leiomyomata are the most common pelvic tumors in women and are the indication for more than 200,000 hysterectomies annually in the United States. Rearrangement of chromosome 12 in bands q14‐q15 is characteristic of uterine leiomyomata and other benign mesenchymal tumors, and we identified a yeast artificial chromosome (YAC) spanning chromosome 12 translocation breakpoints in a uterine leiomyoma, a pulmonary chondroid hamartoma, and a lipoma. Recently, we demonstrated that HMGIC, which is an architectural factor mapping within the YAC, is disrupted in lipomas, resulting in novel fusion transcripts. Here, we report on the localization of translocation breakpoints in seven uterine leiomyomata from 10 to > 100 kb upstream of HMGIC by use of fluorescence in situ hybridization. Our findings suggest a different pathobiologic mechanism in uterine leiomyomata from that in lipomas. HMGIC is the first gene identified in chromosomal rearrangements in uterine leiomyomata and has important implications for an understanding of benign mesenchymal proliferation and differentiation. Genes Chromosom Cancer 17:1–6 (1996).

Identification of a YAC spanning the translocation breakpoints in uterine leiomyomata, pulmonary chondroid hamartoma, and lipoma : physical mapping of the 12q14-q15 breakpoint region in uterine leiomyomata

Uterine leiomyomata are the most common tumors in women and can cause abnormal uterine bleeding, pelvic pain, and infertility. Approximately 200,000 hysterectomies are performed annually in the U.S. to relieve patients of the medical sequelae of these benign neoplasms. Our efforts have focused on cloning the t(12;14)(q14-q15;q23-q24) breakpoint in uterine leiomyoma to further our understanding of the biology of these tumors. Thirty-nine YACs and six cosmids mapping to 12q14-q15 have been mapped by fluorescence in situ hybridization to tumor metaphase chromosomes containing a t(12;14). One YAC spanned the translocation breakpoint and was mapped to tumor metaphases from a pulmonary chondroid hamartoma containing a t(12;14)(q14-q15;q23-q24) and a lipoma containing a t(12;15)(q15;q24); this YAC also spanned the breakpoint in these two tumors, suggesting that the same gene on chromosome 12 may be involved in the pathobiology of these distinct benign neoplasms.

Unco-ordinate regulation of ribosomal RNA and ribosomal protein synthesis during L6E9 myoblast differentiation

Abstract Using the rat myoblast L6E9 cell line, we have shown previously that a five- to tenfold reduction in ribosome synthesis occurs concomitant with the differentiation of myoblasts into myotubes (Krauter et al., 1979). Regulation of ribosome synthesis, which occurs under physiological conditions, has been shown to occur at the transcriptional level. This cell line provides an excellent model with which to study whether all ribosomal components (RNAs and protein) are co-ordinately or independently regulated. The rate of synthesis and degradation of individual ribosomal proteins has been studied by a combination of pulse labeling and two-dimensional gel electrophoresis. We have found that ribosomal protein synthesis is unco-ordinately regulated with respect to the rate of ribosomal RNA synthesis. In spite of the five- to tenfold decrease in rRNA synthesis in myotubes, ribosomal proteins are synthesized at the same rate in myoblasts and myotubes. The rate of ribosomal protein accumulation, however, reflects the rate of rRNA synthesis. Myotubes accumulated ribosomal proteins fivefold more slowly than myoblasts. The ribosomal proteins synthesized in excess of rRNA in myotubes are degraded with half-lives of one hour or less. On the contrary, the proteins which assemble onto mature ribosomes are stable for at least 24 hours. From these results, we conclude that in L6E9 cells the limiting step in ribosome assembly is the transcription of the 45 S precursor rRNA.

Transcriptional regulation of ribosomal RNA accumulation during L6E9 myoblast differentiation.

The rat myoblast cell line L6E9 under appropriate culture conditions is a uniform population of cycling cells which can be induced to differentiate into pure populations of myotubes. These myotubes have irreversibly withdrawn from the cell cycle but continue to synthesize messenger RNA and protein at the pre-fusion level. We have used this cell line to study the regulation of ribosome synthesis during differentiation of the growing myoblasts into non-growing myotubes. We have found that ribosome accumulation is reduced five- to tenfold in the myofibers as compared to the myoblasts. Pulse-labeling with [3H]uridine and pulsechase experiments with either [3H]uridine or [3H]methyl-labeled methionine, combined with the measurement of the specific activity of the nucleoside triphosphate pool, demonstrate that this decrease in ribosomal RNA accumulation is produced by a similar five- to tenfold decrease in rRNA synthesis in the myotubes. This regulation is primarily at the level of transcription of 45 S precursor rRNA. We have found no evidence for turnover of mature cytoplasmic rRNA, “wastage” of rRNA precursors during processing, or rapid degradation of the newly synthesized 45 S precursor rRNA in L6E9 myoblasts or myotubes. We conclude that in L6E9 cells the regulation of rRNA accumulation during differentiation is completely accounted for by the measured changes of rRNA synthesis at the transcriptional level.