Corrected splicing of a chicken ovalbumin gene transcript in mouse L cells.

Abstract

Mouse thymidine-kinase-negative L cells were transformed with a cloned chicken ovalbumin gene linked to the cloned thymidine kinase gene from herpes simplex virus type 1. Most thymidine-kinase-positive clones contained one or more copies of the ovalbumin gene, which were stably maintained for at least 6 months during continuous culture under selective conditions. Transcription of the ovalbumin gene was detected at a low level, producing RNA molecules that were correctly spliced and polyadenylylated by comparison with genuine ovalbumin mRNA. However, the 5' end of these RNA molecules does not correspond to that of ovalbumin mRNA. Molecular cloning has made possible the determination of the structure of the chicken ovalbumin gene (1), which is expressed under hormonal control in the oviduct tubular gland cells (2). By comparison with the structure of other genes, we have identified several conserved sequences that may play a role in the regulation of the expression of the ovalbumin gene and in the splicing of its primary transcript. However, for the true significance of these sequences to be evaluated, they, or mutants derived from them, must be tested in appropriate in vitro or in vivo systems (3, 4). We describe here our first approach to the development of such an in vivo biological system, which consists of introducing the ovalbumin gene into a heterologous cultured eukaryotic cell. Previous work (5-7) has indeed shown that it is possible to introduce foreign DNA into thymidinekinase-negative (TK-) mouse L cells by cotransformation with a thymidine kinase gene from herpes simplex virus type 1. Using this approach, we have introduced the chicken ovalbumin gene into L cells. MATERIALS AND METHODSPlasmids and Enzymes. pBovl and pBov2 were constructed as described in the legends to Figs. la and 3b and in the text. Other plasmids and enzymes have been described elsewhere (8-12). Ovalbumin mRNA (ov-mRNA) was a gift from M. LeMeur and A. Krust. Cell Culture and Transformation. LMTK-cells were grown in monolayer cultures as described (5). LMTK+ cells were maintained in the presence of hypoxanthine/aminopterin/ thymidine medium. Six micrograms of pBovl and 1 ,g of TK-M4 plasmid DNA (7,8,13) were cleaved at their single Sal I sites, extracted with phenol, and precipitated with ethanol.They were then ligated with Biolabs T4 DNA ligase (1 Al) in a volume of 50 Ml for 3 hr before phenol extraction and ethanol precipitation. The heterologous concatenates were used to transform LMTK-cells on five 100-mm petri dishes as described (5). The TK+ phenotype was selected as described (5), and 10 isolated colonies of cells were picked by using the sterile cylinder technique.Analysis of DNA. DNA was isolated from confluent cultures. After digestion with proteinase K (100 ,g/ml), phenol/chloroform extraction, and ethanol precipitation, the DNA was digested with a restriction enzyme by using adenovirus DNA as digestion marker (10). Electrophoresis, blotting, and hybridization to nick-translation probes were as described (10 (...

Introduction and expression of a fetal human globin gene in mouse fibroblasts.

“…Furthermore, the fact that 9S y-globin RNA is produced in transfectants in which the y-gene is inserted in either transcriptional orientation with respect to the TK gene strongly indicates that transcription is initiated within the human DNA segment, most likely at the y-gene promoter. This view is supported by the finding that in three transfectants analyzed, the level of y- (3). We have also observed differences in the level of expression, but in general the variation is not large.…”

Yeast is unable to excise foreign intervening sequences from hybrid gene transcripts.

“…2. ic cells is supported by the high sequence conservation of the exon-intron junctions and by the observation that transcripts of many vertebrate genes are correctly spliced in nonhomologous mammalian cells (4)(5)(6)(7)(8).…”

“…Experiments supporting this notion have shown that, for instance, mouse and rat genes inserted into simian virus 40 (SV40) vectors are transcribed in monkey cells and their transcripts are properly spliced (4,5) and that transcripts ofrabbit and chicken genes are correctly spliced in mouse cells (6,7). Even a hybrid intervening sequence with the 5' splice region from the mouse ,3-globin gene was properly excised from a chimeric transcript in monkey cells (8), although the splice junction sequences of the two donor genes do not exhibit obvious similarities, except for the conserved G-T and A-G dinucleotides.…”

Le contrôle de la synthèse protéique dans les cellules des eucaryotes

“…Le présent rapport ne saurait prétendre considérer la totalité des données relatives à ces questions dont la masse est écrasante. Ce sont préférentiellement certains aspects ayant reçu un intense développement ces dernières années que nous envisagerons en nous limitant volontairement aux étapes précédant l'assemblage des acides aminés en polypeptides qui est traité conjointement dans un autre rapport (Pétrissant, 1981 (Breathnach et al, 1977 ;Jeffryes et Flavell, 1977 ;Gilbert, 1978 ;Crick, 1979 ;Kolata, 1980 (Wigler et al,1977(Wigler et al, , 1978(Wigler et al, , 1979Pellicer et al, 1978 ;Wold et al, 1979 ;Scangos et al, 1979 ;Mantei et al, 1979 ;Hamer et al, 1979 ;Mulligan et al, 1979 ; Leder, 19796 ;Lai et al, 1980 ;Breathnach et al, 1980 (Knapp et al, 1978 ;Melton et al, 1980). Le fait que des introns n'aient jamais pu être mis en évidence dans les gènes des procaryotes a conduit à postuler que les introns (Kolata, 1980 ;Gilbert, 1978) étaient apparus au cours de l'évolution, ou qu'au contraire deux voies relativement indépen-dantes auraient été suivies par les procaryotes et les eucaryotes (Darnell, 1978 ;Doolittle, 1978 Ross, 1976 ;Kinninburgh et al, 1978 ;Horowitz et al, 1978 ;Roop et al, 1978Roop et al, , 1980Harpold et al, 1979 ;Scherrer et al, 1979 ;Ryffel et al, 1980).…”