Eukaryotic viruses can maintain latency in dividing cells as extrachromosomal nuclear plasmids. Segregation and nuclear retention of DNA is, therefore, a key issue in retaining copy number. The E2 enhancer protein of the papillomaviruses is required for viral DNA replication and transcription. Viral mutants that prevent phosphorylation of the bovine papillomavirus type 1 (BPV) E2 protein are transformation-defective, despite normal viral gene expression and replication function. Cell colonies harboring such mutants show sectoring of viral DNA and are unable to maintain the episome. We find that transforming viral DNA attaches to mitotic chromosomes, in contrast to the mutant genome encoding the E2 phosphorylation mutant. Second-site suppressor mutations were uncovered in both E1 and E2 genes that allow for transformation, maintenance, and chromosomal attachment. E2 protein was also found to colocalize to mitotic chromosomes, whereas the mutant did not, suggesting a direct role for E2 in viral attachment to chromosomes. Such viral hitch-hiking onto cellular chromosomes is likely to provide a general mechanism for maintaining nuclear plasmids.

When linear DNAs are injected into Xenopus laevis eggs, they are converted into several different kinds of recombination products. Some molecules undergo homologous recombination by a resection-annealing mechanism; some ends are precisely ligated; and some ends are joined by illegitimate means. The homologous and illegitimate products are also generated in nuclear extracts from stage VI Xenopus oocytes. In order to gain insight into the mechanism(s) of illegitimate end joining, we amplified, cloned and sequenced a number of junctions from eggs and from oocyte extracts. The egg junctions fell into three categories: some with no homology at the join point that may have been produced by blunt-end ligation; some based on small, but significant homologies (5-10 bp); and some with matches of only 1 or 2 nucleotides at the joint. Junctions made in oocyte extracts were largely of the latter type. In the extracts, formation of illegitimate joints required the addition of all four deoxyribonucleoside triphosphates and was inhibited by aphidicolin. This indicates that this process involves DNA synthesis, and mechanisms incorporating this feature are considered. The spectrum of recombination products formed in Xenopus eggs is very reminiscent of those produced from DNA introduced into mammalian cells.

TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

Xenopus laevis oocytes efficiently recombine linear DNA injected into their nuclei (germinal vesicles). This process.requires homologous sequences at or near the molecular ends. Here we report that a cell-free extract made from germinal vesicles is capable of accomplishing the complete recombination reaction in vitro. Like the in vivo process, the extract converts the overlapping ends of linear substrate molecules into covalently closed products. Establishment of this cell-free system has allowed examination of the cofactors required for recombination. The first step involves a 5' -+ 3' exonuclease activity that requires a divalent cation but not NTPs. Completion of recombination requires a hydrolyzable NTP; maximal product formation occurs in the presence of millimolar levels of ATP or dATP. At submillimolar levels of all four dNTPs, homologous recombination is inefficient, and a side reaction produces end-joined products. This cell-free system should facilitate a step-by-step understanding of an homologous recombination pathway that operates not only in Xenopus laevis oocytes but also in cells from a wide variety of organisms.Homologous genetic recombination is crucial for meiosis and also occurs in mitotic cells. Because of its role in chromosome segregation, allele redistribution, and DNA repair, recombination has attracted the attention of a wide variety of investigators (for reviews, see refs. 1-3). A focus for these studies recently has been the molecular mechanisms of recombination reactions. Much of our current knowledge is derived from the analysis of fungal recombination products and the characterization of phage and bacterial recombination enzymes (4-6). Further progress would be facilitated by the development of in vitro systems that can support complete recombination events. A number of organisms have been used for this purpose. Extracts and partially purified components capable of catalyzing at least partial events have been prepared from Escherichia coli (7,8), Ustilago maydis (9), Saccharomyces cerevisiae (10, 11), and mammalian cells (12-15); however, recombination products were generated only at low levels, which have usually required amplification by bacterial transformation.We have been studying the very efficient homologous recombination of DNA molecules injected into Xenopus laevis oocyte nuclei (16)(17)(18)(19)(20) Our efforts to establish oocyte extracts capable of performing homologous recombination were encouraged by the above studies. We show here that homogenates of manually isolated oocyte nuclei support complete recombination events in vitro, and we report some requirements of the process. MATERIALS AND METHODSGerminal Vesicle (GV) Extract Preparation. X. laevis ovary segments were surgically removed (36); oocytes were isolated by collagenase digestion (16), rinsed extensively in OR-2 medium (37), and kept at 19'C in OR-2 medium until dissected (a few hours to 2 days). Healthy stage VI oocytes (38) were selected and rinsed immediately before use in supplemented isolation bu...

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