A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. transActing AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.

A recombinant plasmid carrying an infectious adeno-associated viral genome was constructed that differs in several key respects from previously described recombinants. First, the vector is pEMBL8(+), which allows isolation of viral plus and minus strands. Second, the inserted viral sequences contain two XbaI cleavage sites that flank the viral coding domain. These inserts do not affect replication of the virus, and they allow nonviral sequences to be easily inserted between the cis-acting terminal repeats of adeno-associated virus. Third, the viral genome is flanked by PvuII cleavage sites that allow the entire, infectious viral chromosome to be excised from plasmid sequences in vitro. Viral DNA was replicated more efficiently within adenovirus-infected 293 cells if it was excised from the vector with PvuII before transfection. Presumably, the increased efficiency reflects bypass of the excision step which must normally precede replication when a recombinant plasmid enters the nucleus. The ability to bypass the excision step was exploited to search for a viral function required specifically for excision of the viral genome from the integrated state. None of the mutants tested identified a gene product required for excision that was not also essential for replication. The ability to produce pure populations of viral plus and minus strands was used to demonstrate that both strands are infectious.

SUMMARY Germline mutations in the RAS/ERK signaling pathway underlie several related developmental disorders collectively termed neuro-cardio-facial-cutaneous (NCFC) syndromes. Patients with these disorders manifest varying degrees of cognitive impairment, but the developmental basis of their brain abnormalities remains largely unknown. Among NCFC syndromes, neurofibromatosis type 1 (NF1) is an exception, as it is caused by loss-of-function heterozygous mutations. Here, we show that bi-allelic Nf1 inactivation promotes Erk-dependent, ectopic Olig2 expression specifically in transit-amplifying progenitors, leading to increased gliogenesis at the expense of neurogenesis in neonatal and adult subventricular zone (SVZ). Nf1-deficient brains exhibit enlarged corpus callosum - a structural brain defect recently linked to severe learning deficits in NF1 patients. Strikingly, these NF1-associated developmental defects are rescued by transient treatment with an MEK/ERK pathway inhibitor during neonatal stages. These studies reveal a critical role for Nf1 in maintaining postnatal SVZ-derived neurogenesis, and identify a potential therapeutic window for treating NF1-associated brain abnormalities.

Amplification/overexpression of the human neu protooncogene has been frequently found in human primary breast and ovarian cancers and is correlated with the number of axillary lymph nodes positive for metastasis in breast cancer patients. Identification of the factors controlling transcription of the neu gene is essential for understanding the mechanisms of neu gene regulation and its role in tumorigenicity. The adenovirus early region 1A (E1A) gene products are pleiotropic transcription regulators of viral and cellular genes and have been identified as a viral suppressor gene for metastasis. Here we demonstrate that transcription of neu can be strongly repressed by the ElA gene products. The 13S and 12S products of ElA gene are effective at repressing neu transcription and the transcriptional repression requires the conserved region 2 of the ElA proteins. The target for ElA repression was localized within a 139-base-pair DNA fragment in the upstream region of the neu promoter. In addition, competition experiments suggest that the sequence TGGAATG, within the 139-base-pair fragment, is an important element for the E1A-induced repression. These results indicate that ElA negatively regulates neu gene expression at the transcriptional level by means of a specific DNA element.The neu (also called murine c-erbB-2) oncogene was first identified by transfection studies in which NIH 3T3 cells were transformed with DNA from ethylnitrosourea-induced rat neuro/glioblastomas (1). Structural and functional analysis between the transforming neu oncogene and its normal cellular counterpart, neu protooncogene, revealed that a subtle structural alteration-namely, a single point mutation-is sufficient to convert the neu protooncogene into a transforming neu oncogene (2, 3). The neu gene encodes a 185-kDa transmembrane protein (p185) that is related to, but distinct from, the epidermal growth factor receptor (4). The transforming p185 is associated with an increased tyrosine kinase activity (4-6). Interestingly, a structurally divergent group of oncogenes encoding protein kinases has been shown to induce the metastatic phenotype (7). Evidence linking this kinase oncogene to the induction or progression of human malignancies comes from recent observations that the human homologue of the rat neu oncogene (human gene symbol NGL for neuro/glioblastoma-derived; has been called ERBB2, HER-2, human c-erbB-2, or TKRJ) is amplified/overexpressed in 25-30o of human primary breast cancers and ovarian cancers, notably in breast cancer patients with more than three axillary lymph nodes positive for metastasis (8-10).It has also been noted that some human breast cancer cell lines overexpress human neu mRNA, while the neu gene is not amplified (11). Together, these studies suggest that regulation of the neu gene may play an important role in malignant transformation and metastasis.The primary function of the adenovirus early region 1A (ElA) gene is to activate other adenoviral genes during a permissive viral infection by modifying the ho...