No more than seven interruptions in the ovalbumin gene: comparison of genomic and double-stranded cDNA sequences

O’Hare, Kevin; Breathnach, Richard; Benoist, C; Chambón, P.

doi:10.1093/nar/7.2.321

Cited by 46 publications

(24 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As in the reverse transcriptase model presented in Fig. 4, the DNA polymerase model of O'Hare et al (10) have reported the existence of a similar, although shorter, inverted repeat at the end of a cloned cDNA derived from ovalbumin mRNA. They suggest similar mechanisms for generating inverted repeats of this nature.…”

Section: Panel Bmentioning

confidence: 72%

“…The alternative to these possibilities is that the inverted repeat structures of the pFN200 and pFN600 inserts were generated by duplication and rearrangement of sequences present only once in the fibronectin mRNA. We prefer this second alternative, since precedents exist for such nonsequential transcription (10,11,12). This could occur during synthesis of the first strand by AMV reverse transcriptase, during synthesis of the second strand by E. coli DNA polymerase I, or possibly during replication of the plasmid.…”

Section: Panel Bmentioning

confidence: 99%

See 1 more Smart Citation

Sequence rearrangement and duplication of double stranded fibronectin cDNA probably occurring during cDNA synthesis by AMV reverse transcriptase and Escherichia coli DNA polymerase I

1980

View full text Add to dashboard Cite

Two cloned cDNAs derived from the mRNA for cell fibronectin have been sequenced, providing evidence that transcription with AMV reverse transcriptase or Escherichia coli DNA polymerase I may not always result in double stranded cDNA that is exactly homologous with its mRNA template. Instead, the sequences of these cloned cDNAs are consistent with the duplication and rearrangement of sequences during synthesis of double stranded cDNA. INTRODUCTIONWe have constructed a set of recombinant plasmids with inserts complementary to the fibronectin mRNA (1). Cell fibronectin plays important roles in cell adhesion and in maintaining normal cell morphology (2-4).

show abstract

Section: Panel Bmentioning

confidence: 72%

Section: Panel Bmentioning

confidence: 99%

Sequence rearrangement and duplication of double stranded fibronectin cDNA probably occurring during cDNA synthesis by AMV reverse transcriptase and Escherichia coli DNA polymerase I

1980

View full text Add to dashboard Cite

show abstract

“…Total RNA was isolated from the L cell ovalbumin clones and analyzed by a modification (20) of the S1 nuclease mapping method of Berk and Sharp (15) by using pCRlov2.1, a double-stranded (ds) cDNA plasmid that contains an almost full-length copy of ovmRNA, lacking only the 14 nucleotides corresponding to the 5' end of ov-mRNA (9,17). The plasmid was linearized with Xba I (Fig.…”

mentioning

confidence: 99%

“…If the Lov cell transcripts of exon 7 terminate at the same site as ov-mRNA and have the intron G transcript spliced out, then the transcripts would be complementary to pBov2 over the length of exon 7 only (1042 bp, see ref. 17). The initial product of SI digestion would then be a 1042-bp DNA-RNA hybrid containing a single nick on the DNA strand at the Xba I site.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Breathnach¹,

Mantei²,

Chambon³

1980

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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 (...

show abstract

Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

Sun¹,

Xu²,

Bellard³

et al. 1986

The EMBO Journal

Self Cite

View full text Add to dashboard Cite

The structure of the chicken adult beta‐globin gene chromatin in immature and mature erythrocyte nuclei has been analysed using micrococcal nuclease digestion. The resulting DNA fragments were blotted onto DBM‐papers and probed with labelled DNA fragments spanning the adult beta‐globin gene and its 5′‐ and 3′‐flanking regions. The structure of the nucleosomes within and in the regions flanking the adult beta‐globin gene appears to be altered in at least two ways in erythrocyte chromatin, when compared with either bulk or inactive ovalbumin gene chromatin. First, oligomeric DNA fragments containing the beta‐globin gene are released faster than those of either bulk or ovalbumin gene chromatin. Second, although the difference in size of the liberated oligomeric DNA fragments is similar to the nucleosomal repeat length of bulk and ovalbumin gene chromatin, the individual oligomers are approximately 100 bp shorter than their bulk or ovalbumin gene counterparts, most noticeably when the nuclease digestion is performed at 37 degrees C. This results in an atypical ladder of approximately 300, 500, 700, 900 bp instead of the canonical chicken erythrocyte ladder which is an integral multiple of 207 bp. The same ladder was obtained from immature erythrocytes, in which the beta‐globin gene is actively transcribed, and from mature erythrocytes, in which it is considered to be inactive with RNA polymerase molecules clustered in the 5′ moiety of the gene. This indicates that the alteration of the nucleosomal structure is not due to transcription per se.(ABSTRACT TRUNCATED AT 250 WORDS)

show abstract

No more than seven interruptions in the ovalbumin gene: comparison of genomic and double-stranded cDNA sequences

Cited by 46 publications

References 20 publications

Sequence rearrangement and duplication of double stranded fibronectin cDNA probably occurring during cDNA synthesis by AMV reverse transcriptase and Escherichia coli DNA polymerase I

Sequence rearrangement and duplication of double stranded fibronectin cDNA probably occurring during cDNA synthesis by AMV reverse transcriptase and Escherichia coli DNA polymerase I

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

Digestion of the chicken beta-globin gene chromatin with micrococcal nuclease reveals the presence of an altered nucleosomal array characterized by an atypical ladder of DNA fragments.

Contact Info

Product

Resources

About