J. Zimmer

Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9

A human autosomal XY sex reversal locus, SRA1, associated with the skeletal malformation syndrome campomelic dysplasia (CMPD1), has been placed at distal 17q. The SOX9 gene, a positional candidate from the chromosomal location and expression pattern reported for mouse Sox9, was isolated and characterized. SOX9 encodes a putative transcription factor structurally related to the testis-determining factor SRY and is expressed in many adult tissues, and in fetal testis and skeletal tissue. Inactivating mutations on one SOX9 allele identified in nontranslocation CMPD1-SRA1 cases point to haploinsufficiency for SOX9 as the cause for both campomelic dysplasia and autosomal XY sex reversal. The 17q breakpoints in three CMPD1 translocation cases map 50 kb or more from SOX9.

Unscheduled DNA synthesis after partial UV irradiation of the cell nucleus: Distribution in interphase and metaphase

Cells of an euploid strain of the Chinese hamster synchronized in the G1 phase were microirradiated in the nucleus with a laser UV microbeam (λ = 257 nm) and pulse-labelled with [3H]thymidine. In autoradiographs of cells fixed immediately after the pulse unscheduled DNA synthesis (UDS) was found restricted to the microirradiated part of the nucleus. The rate of UDS varied with the UV energy applied and the post-irradiation incubation time. In other experiments chromosome preparations were established after an additional chase and a subsequent growth period. In 28 mitotic cells autoradiographic label was found concentrated on a few chromosomes which lay adjacent to each other in one part of the metaphase plate. The distribution of label on the chromosomes could clearly be distinguished from patterns which originate from semi-conservative DNA synthesis within S phase. The label on chromosomes of microirradiated cells thus represents UDS. Our findings support the following ideas on the arrangement of interphase chromosomes: (1) Decondensed interphase chromosomes may occupy rather compact territories. (2) Chromosomes do not necessarily exhibit a close and permanent association with their respective homologues.

A somatic cell hybrid panel for distal 17q: GDIA1 maps to 17q25.3.

A somatic cell hybrid panel was constructed consisting of seven hybrids with translocation breakpoints spanning the region 17q23-->q25. Hybrid clones carrying the longarm derivative of chromosome 17 in the absence of the normal chromosome 17 and of the derivative 17 were initially identified by PCR typing for a proximal and distal 17q marker. The translocation breakpoints of the hybrids were then mapped in more detail by PCR analysis for a number of microsatellite markers from chromosome 17q as well as for five gene loci (CACNLG, GH1, SOX9, TIMP2, TK1) previously mapped to the region 17q23-->q25. In addition, the locus for GDIA1 was mapped by FISH to 17q25.3 and fine mapped with the help of the hybrid panel. These seven new hybrids complement the existing somatic cell hybrid panel for the long arm of chromosome 17q.

Formation of viable cell fragments by treatment with colchicine

Time-lapse cinematography of human fibroblasts revealed that mitotic cells separated into numerous cell fragments containing varying amounts of chromatin and cytoplasm when treated with colchicine. As cell fragments were very loosely attached to the surface of the culture vessel during their formation, they could be easily detached like mitotic cells by gently shaking the vessel and thus separated from normal interphase cells. Fragments obtained by this procedure were able to exclude trypan blue indicating, therefore, an intact cell membrane. When placed into Petri dishes many of them attached to and even spread out on the surface. Five hours later the majority of the attached fragments incorporated [3H]leucine. Time-lapse films showed that fragments were able to extend and retract pseudopodia at least for several hours after their formation. Although the fragments degenerated within a few days, in the present experiments the possibility was not excluded that fragments which had lost only a very small amount of chromatin and cytoplasm survived for longer periods of time. The observations clearly indicate viability of many newly formed fragments.