Partial purification and characterization of the messenger RNA for cell fibronectin

Fagan, John B.; Yamada, Kenneth M.; Crombrugghe, Benoít de; Pastan, Ira

doi:10.1093/nar/6.11.3471

Cited by 14 publications

(13 citation statements)

References 32 publications

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“…The total exon length of 8 kb estimated by R-loop analysis is similar to the length of mRNA for fibronectin determined by gel electrophoresis (7,9) minus a poly(A) tail. The coding regions of the gene are divided into at least 48 exons by introns ofwidely different lengths.…”

supporting

confidence: 70%

“…A 600-bp cDNA fragment carried in, pBR322 (pFN600) has been characterized previously and corresponds to a 3'-terminal untranslated region ofchicken cellular fibronectin mRNA (7)(8)(9). A 188-bp Taq I/HindIll fragment was isolated from this plasmid as described (9), except that Taq I was substituted for Hinfl to facilitate separation of the probe from other DNA fragments during gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

“…taining cDNA for fibronectin (7)(8)(9). Using a cloned cDNA probe, we found that levels of mRNA for fibronectin were decreased to 10-13% of normal levels after oncogenic transformation, accounting for much of the known decrease in fibronectin in these cells after transformation (9).…”

mentioning

confidence: 99%

“…Our initial probe was a previously characterized cDNA fragment from the 3'-noncoding region of fibronectin mRNA (7)(8)(9). After identifying clones hybridizing to a32P-labeled probe from this clone, the clone with the longest 5'-proximal region was identified by restriction mapping.…”

mentioning

confidence: 99%

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Isolation of genomic DNA clones spanning the entire fibronectin gene.

Hirano¹,

Yamada²,

Sullivan³

et al. 1983

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

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Overlapping recombinant clones that appear toencompass the entire fibronectin gene have been isolated by stepwise screening of a library of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned fibronectin cDNA hybridization probe. The remaining clones were obtained by using defined fragments of this and successive genomic clones as probes. Their relationships and overlaps were determined by electron microscopy, restriction mapping, and heteroduplex analysis. Based on electron microscopic analysis of hybrids between these clones and fibronectin mRNA, the gene is approximately 48 kilobases long, more than 5 times larger than the corresponding mRNA. This large gene contains at least 48 exons interrupted by introns of highly variable size. The total exon size as estimated by R-loop analysis is 8 kilobases, similar to the mRNA for fibronectin. With the exception of the 3'-and 5'-terminal exons, the exons are small and roughly similar in size. The average exon size is 147 ± 37 base pairs, corresponding to a protein unit of50 amino acids. The nucleotide sequence of one of these exons was determined. The deduced amino acid sequence has marked homologies with one type of repetitive protein sequence unit known to exist in bovine fibronectin. These results suggest that the gene for fibronectin may have arisen by multiple gene duplications of a primordial gene or genes 150 base pairs long.Cellular fibronectin is a glycoprotein of subunit Mr 220,000 present on the cell surface of many cells; a similar but not identical protein termed plasma fibronectin is a major glycoprotein in plasma. The fibronectins are involved in a number of biological events, including cell adhesion, migration, and differentiation-(reviewed in refs. 1-4).. The fibronectin molecule is composed of a series of structural and functional domains mediating.adhesive interactions with the cell surface, collagen, fibrin, and several other-macromolecules (1-4).Quantities of fibronectin are altered in many diseases', including malignancy (reviewed-in ref. 5). To examine the regulation of fibronectin, we previously isolated the mRNA for chicken cellular fibronectin (6, 7) and constructed plasmids containing cDNA for fibronectin (7-9). Using a cloned cDNA probe, we found that levels of mRNA for fibronectin were decreased to 10-13% of normal levels after oncogenic transformation, accounting for much of the known decrease in fibronectin in these cells after transformation (9).We now describe the isolation and partial characterization of a series of overlapping DNA clones from a phage library of genomic DNA fragments. These clones span approximately 59 kilobases (kb) ofcontiguous DNA sequence and appear to cover the entire 48-kb gene. The gene for cellular fibronectin has an unusual structure consisting ofa series ofat least 48 exons, most of which are close to 150 base pairs (bp) long. MATERIALS AND METHODSHybridization Probes. A 600-bp cDNA fragment carried in, pBR322 (pFN600) has been characterized previously and correspon...

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supporting

confidence: 70%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Isolation of genomic DNA clones spanning the entire fibronectin gene.

Hirano¹,

Yamada²,

Sullivan³

et al. 1983

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

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“…In short, single stranded cDNA was transcribed from a fibronectin mRNA template, purified from CEF as reported (5 The cDNA inserts of the plasmids pFN200 and pFN600 were excised from the plasmids with Hlind III and isolated by centrifugation of 120 pg of DNA through 5-20 percent sucrose gradients in 10 mM Tris-Cl pH 7.5, 1 mM Na-EDTA for 14 hours at 26.5 x 103 rpm at 40C in a Beckman SW 27 rotor.…”

Section: Plasmid Constructionmentioning

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

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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).

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