Selected regions of cloned EcoRI fragments of the chicken ovalbumin gene have been sequenced. The positions where the sequences coding for ovalbumin mRNA (ovmRNA) are interrupted in the genome have been determined, and a previously unreported interruption in the DNA sequences coding for the 5' nontranslated region of the messenger has been discovered. Because directly repeated sequences are found at exon-intron boundaries, the nucleotide sequence alone cannot define unique excision-ligation points for the processing of a possible ov-mRNA precursor. However, the sequences in these boundary regions share common features; this leads to the proposal that there are, in fact, unique excision-ligation points common to all boundaries. It has been shown (1-4) that the chicken ovalbumin gene is split into seven ovalbumin messenger coding sequences (exons; see ref. 5) separated by six intervening sequences (introns; see ref.5). The respective locations in the chicken genome of the seven exons (numbered 1-7) and of the six introns (designated by the letters B-G) are shown in Fig. lb. These positions have been deduced from restriction enzyme mapping of chicken DNA using appropriate ovalbumin gene probes (1, 3, 4) and from electron microscopy of the cloned EcoRI DNA fragments "a," "b," "c," and "d" (Fig. lb) which contain all of the ovalbumin exons and introns (2, 3). Electron microscopy did not reveal any evidence for a long (150-200 nucleotides) virus-like leader sequence (for review, see refs. 7 and 8) that could be spliced at the 5' end of the ovalbumin mRNA (ov-mRNA) (3, 4). By comparison with viruses, we use the term "leader" to describe a nontranslated RNA sequence present at the 5' end of a given mRNA and encoded by DNA sequences physically separated from those coding for the protein. However, the possible existence of a leader sequence shorter than 50-100 nucleotides was not excluded by our electron microscopy studies (3, 4). As discussed previously (1,4), the split organization of the ovalbumin gene raises the possibility that the primary transcript of the gene could be longer than mature ov-mRNA and contain transcripts of both exons and introns. Maturation of ov-mRNA might then involve the looping out of intron transcripts for excision and the concomitant splicing of exon transcripts. Whatever the detailed mechanisms involved in such processing, it was generally postulated that the nature of the DNA sequences at the intron-exon junctions or in their immediate vicinity should play a role in the recognition of the intron-exon boundaries and in the excision-splicing events.In the present paper we report the result of sequence analyses carried out both on the cloned double-stranded cDNA containing the sequences complementary to ov-mRNA (ov-dscDNA; see ref. 9) and on cloned cellular DNA fragments. These studies have led to the discovery of a short leader sequence at the 5' end of ov-mRNA and have revealed some interesting features in the DNA sequences at exon-intron boundaries.MATERIALS AND METHODS Plasmid pCR1 ...

Nonviral gene transfer into the central nervous system (CNS) offers the prospect of providing safe therapies for neurological disorders and manipulating gene expression for studying neuronal function. However, results reported so far have been disappointing. We show that the cationic polymer polyethylenimine (PEI) provides unprecedentedly high levels of transgene expression in the mature mouse brain. Three different preparations of PEI (25-, 50-, and 800-kD) were compared for their transfection efficiencies in the brains of adult mice. The highest levels of transfection were obtained with the 25-kD polymer. With this preparation, DNA/PEI complexes bearing mean ionic charge ratios closest to neutrality gave the best results. Under such conditions, and using a cytomegalovirus (CMV)-luciferase construction, we obtained up to 0.4 10(6) RLU/microgram DNA (equivalent to 0.4 ng of luciferase), which is close to the values obtained using PEI to transfect neuronal cultures and the more easily transfected newborn mouse brain (10(6) RLU/microgram DNA). Widespread expression (over 6 mm3) of marker (luciferase) or functional genes (bcl2) was obtained in neurons and glia after injection into the cerebral cortex, hippocampus, and hypothalamus. Transgene expression was found more than 3 months post-injection in cortical neurons. No morbidity was observed with any of the preparations used. Thus, PEI, a low-toxicity vector, appears to have potential for fundamental research and genetic therapy of the brain.

Generally, cationic vector-based intravenous delivery of at a ratio of 4 nitrogen equivalents per DNA phosphate. DNA is hindered by interactions of positively charged comLower levels of transfection were found in the heart, plexes with serum proteins. However, if optimally formuspleen, liver and kidney. Expression was dose-and timelated, cationic vectors can provide reasonable levels of dependent in all tissues examined. In the lung, ␤-galactotransfection in the lung either by intravenous or intrapulsidase staining showed transgene expression in clusters monary routes. We investigated the in vivo transfection of 10 or more pulmonary cells including the alveolar endocapacity of a cationic polymer: linear, 22 kDa polyethylenithelium, squamous and great alveolar epithelial cells (type mine. PEI/DNA complexes were formulated in 5% glucose I and II pneumocytes) and septal cells. These findings indiand delivered into adult mice through the tail vein. Two cate that the complexes pass the capillary barrier in the marker genes were used, ␤-galactosidase and luciferase.lung. Although the delivery mechanism requires eluciHigh levels of luciferase expression (10 7 RLU/mg protein) dation, linear PEI has promise as a vector for intravenous were found in the lung when DNA was complexed with PEI transfer of therapeutic genes.Keywords: cationic polymers; pneumocytes; plasmid DNA; nonviral; gene therapy A number of generations of cationic vectors have been synthetised and tested in a variety of in vivo models and some have been taken to clinical trials. Most of these vectors are either monocationic or polycationic lipids, but there has been more recent interest in cationic polymers. Indeed, we showed that the branched cationic polymer polyethylenimine (PEI) can provide high levels of transfection in vivo. [1][2][3] In particular the lowest molecular weight preparation then commercially available, the 25 kDa preparation from Aldrich, was shown to be a versatile and efficient vector in the mammalian brain. 2 In a more recent study, 3 we chose to examine the effect of formulation procedures (glucose or saline solutions) on the size and in vivo transfection activity of a mixture of linear polymers with a mean MW of 22 kDa (Exgene 500; Euromedex, Souffleweyersheim, France). We found that the complexes formed in glucose were an order of magnitude smaller than those formed in saline and these complexes provided high levels of gene transfer following dilution into a physiological medium, the cerebrospinal fluid. In the light of these findings we chose to examine the effects of injecting complexes of plasmid DNA formulated with 22 kDa PEI in 5% glucose directly into the blood system and to examine transgene expression in a variety of organs.PEI-DNA complexes with different ratios of PEI nitro- gen to DNA phosphate (N/P ratio) were prepared in 5% glucose using a CMV-Luc plasmid 4 and the 22 kDa linear PEI. This PEI is synthesised to a degree of polymerisation of 510 units. Earlier experiments carried out with the branched 25 kDa PEI (Aldri...

A clone which contains the complete chicken ovalbumin gene, including its leader coding sequences, has been isolated. From electron microscopic analysis of this DNA we conclude that the minimal size of the transcriptional unit for ovalbumin is 7.7 kilobases. The DNA sequence of the region surrounding the 5' end of the ovalbumin gene is presented. Comparison of this sequence with those of other eukaryotic genes reveals striking similarities, possibly related to a promoter region, approximately 30 base pairs upstream from the site coding for the 5' end of the mRNA.