Processing of endogenous pre-mRNAs in association with SC-35 domains is gene specific.

Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript "trees" of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional "tracks" of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism.

X microchromosome with additional chromosome anomalies found in Ullrich-Turner syndrome.

Using standard cytogenetic methods coupled with molecular techniques, the following karyotype mos 45,X/46,XXq+/46,X+mar (X)/47,XXq+,+mar(X), was identified in a patient with Ullrich-Turner syndrome (UTS). High-resolution banding (n = 650) of the metaphase chromosomes yielded a breakpoint at q28 on the Xq+ rearranged chromosome. FISH was used to determine the presence of Y-containing DNA in the Xq+ and the mar(X) chromosomes. The following molecular probes were used: DYZ1, DYZ3, and spectrum orange WCP Y. The lack of specific hybridization of these probes was interpreted as a low risk of gonadoblastoma in this patient. Using X-chromosome- and centromere-specific probes, FISH demonstrated the presence of hybridizing material on both rearranged chromosomes, the Xq+ and mar(X). Finally, we determined that the mar(X) and Xq+ chromosomes contained telomeres in the absence of any interstitial telomeric hybridizing material. A micro-X chromosome is present in this UTS patient. Delineation of events leading toward the mechanisms responsible for the multiple DNA rearrangements required to generate the micro-X and Xq+ chromosomes awaits future studies.

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures.

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.

Chromosomal assignment of human nuclear envelope protein genes LMNA, LMNB1, and LBR by fluorescence in situ hybridization.

We have used fluorescence in situ hybridization to establish precise chromosomal localizations for three human genes encoding four different nuclear envelope proteins. Lamin A/C (LMN1, HGMW-approved symbol LMNA) mapped to 1q21.2-q21.3, with a most probable gene assignment to 1q21.3; lamin B receptor (LBR) was localized to 1q42.1; and lamin B1 (LMNB1) was mapped to the interface of bands 5q23.3-q31.1. Assignments were determined by direct placement of signals relative to high-resolution DAPI or G-bands. Comparison of these results of band positions predicted from fractional length measurements to signal placement indicated that more accurate predictions are made using Francke idiograms and that measurement strategy avoids variance due to polymorphic chromosome segments.

E2F-4, a new member of the E2F transcription factor family, interacts with p107.

The E2F family of transcription factors has been implicated in the regulation of cell proliferation, and E2F-binding sites are present in the promoters of several growth-regulating genes. E2F family members are functionally regulated, in part, by complex formation with one or more members of the nuclear pocket protein family, RB, p107, and p130. Pocket protein regulation of E2F likely contributes to normal cellular growth control. While the three cloned species of E2F, E2F-1, E2F-2, and E2F-3, are known to be targets of RB interaction, no E2F species has yet been shown to be a specific p107 or p130 target. Here, we describe the cloning of a new member of the E2F family, E2F-4, which forms heterodimers with a member(s) of the DP family and, unlike some family members, is present throughout the cell cycle and appears to be a differentially phosphorylated p107-binding partner. p107 binding not only can be linked to the regulation of E2F-4 transcriptional activity, but also to suppression of the ability of E2F-4 to transform an immortalized rodent cell line.

Genomic analysis of a new mammalian distal-less gene: Dlx7.

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt gene NvHBox-5. The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouse Dlx7 upstream of the homeodomain that may account for some of this divergence. We have mapped human DLX7 distal to the 5' end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.

In situ hybridization to chromosomes stabilized in gel microdrops.

Conventional chromosome in situ hybridization procedures rely on fixation to glass slides followed by microscopic evaluation. This report describes the development of a microdrop in situ hybridization to chromosomes in suspension. Chromosomes encapsulated in gel microdrops (GMDs) composed of an agarose matrix withstood stringent hybridization and denaturation conditions. Because of the increased stability, hybridization to encapsulated chromosomes was detected by flow cytometry as well as conventional microscopy. Thus, the MISH method offers a means for chromosome hybridization without slides and may enable identification and isolation of chromosome using hybridization rather than nucleic acid binding dyes.