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Biomedical subjects

A R Kornblihtt

Publications and source records attributed to A R Kornblihtt.

At least 37 records · Page 2Linked to original sources
PubMed1994

Binding of nuclear factors to a satellite DNA of retroviral origin with marked differences in copy number among species of the rodent Ctenomys.

The major satellite DNA of the subterranean rodent Ctenomys, named RPCS, contains several consensus sequences characteristic of the U3 region of retroviral long terminal repeats (LTRs), such as a polypurine tract, CCAAT boxes, binding sites for the CCAAT/enhancer-binding protein (C/EBP), a TATA box and putative polyadenylation signals. RPCS presents an enormous variation in abundance between species of the same genus: while C. australis or C. talarum have approximately 3 x 10(6) copies per genome, C. opimus has none. A sequence (RPCS-I) with identity to the SV40-enhancer core element, present in all the repeating units of the satellite is specifically protected in DNase I footprintings. Competitions of band-shift assays with different transcription factor binding sites indicate that binding to RPCS-I is specific and involves CCAAT proteins related to NF-1, but not to C/EBP. By the use of quantitative protein/DNA binding assays we determined that, despite of their conspicuous difference in RPCS copy number, C. talarum and C. opimus have equivalent amounts and identical quality of RPCS-binding proteins. These results are consistent with the observation, by in situ hybridization, that RPCS is clustered in heterochromatic regions, where it might have restricted accessibility to transcription factors in vivo. This is the first report of the binding of transcription factors to a satellite DNA of retroviral origin.

Animals↗
PubMed1993

The CRE-binding factor ATF-2 facilitates the occupation of the CCAAT box in the fibronectin gene promoter.

The cAMP response element (CRE) and the CCAAT box of the fibronectin gene promoter are separated by only twenty base pairs. A specific factor that binds the CRE interacts cooperatively with the protein which binds to the adjacent CCAAT box, stimulating transcription [1992, J. Biol. Chem. 267, 12767-12774]. Here we show that the CRE factor is an heterodimer between a 43 kDa and the '73 kDa' CRE-binding proteins and we identify the latter as ATF-2 (also named CRE-BPI), a protein implicated in recruiting transcriptional activators to promoters, able to form heterodimers with Jun and for which a sequence-deduced MW of 55 kDa had been previously reported.

Activating Transcription Factors↗
PubMed1993

A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding.

The inclusion of the 270-nucleotide human fibronectin ED1 exon in HeLa cells requires the presence of a centrally located 81-nucleotide exon sequence. We have conducted a series of in vitro experiments aimed at understanding the structural and functional features associated with this splicing enhancer (SE). Using hybrid model pre-mRNA substrates, we show that the SE element markedly stimulates the use of the 3' splice site of ED1. Deletion and replacement analysis identifies the stimulating sequences as a purine-rich stretch of 9 nucleotides (GAAGAAGAC). The SE element stimulates splicing to the ED1 3' splice site from various positions within the exon except when placed beyond 293 nucleotides downstream from that 3' splice site. The action of the enhancer is not limited to the ED1 acceptor site because the SE element stimulates human beta-globin splicing and also induces the use of a 3' splice site in a prokaryotic sequence in vitro. We have explored the mechanism of action of the fibronectin splicing enhancer and found that the SE element is required for efficient assembly of early splicing complexes, allowing a more efficient interaction of the U2 snRNP with branch site sequences. In competition experiments, an RNA containing mainly SE sequences specifically abolished the action of the SE element, suggesting that factors bind the enhancer element to mediate stimulation of splicing. Using RNA mobility shift assays we show that SR proteins interact specifically with the SE element. Our results demonstrate that exon sequences lying in the SE element play a crucial role in specifying splice site recognition through interactions with factors binding to the 3' splice site.

Alternative Splicing↗
PubMed1993

Retroviral-like features in the monomer of the major satellite DNA from the South American rodents of the genus Ctenomys.

It is well known that uninfected mammalian cells contain DNA sequences which are closely related to retroviral genomic segments. However, these sequences seldom (if ever) have been found associated to highly repetitive (satellite) DNA. RPCS is a 348 bp monomer of a major satellite DNA from the South American rodents of the genus Ctenomys. It was found that RPCS contains several elements which are typical of the U3 region of retroviral LTRs. These elements are: a) a polypurine tract; b) two enhancer core sequences; c) two NF1 binding sites; d) two C/EBP binding sites; e) two CCAAT-motifs; f) a TATA box, and g) two putative polyadenylation motifs. Furthermore, the relative positions of these elements are as in the U3 retroviral regions.

Animals↗
PubMed1992

Interaction of the -170 cyclic AMP response element with the adjacent CCAAT box in the human fibronectin gene promoter.

In the fibronectin gene promoter the cAMP response element (CRE) and the CCAAT box are separated by only 20 base pairs (bp), i.e. two turns of double helix. Binding of nuclear proteins to these elements, assessed by DNase I footprinting, differs in the different cell types. While in a variety of cells tested (HeLa, granulosa, brain, and adenocarcinoma) only CRE binding activity is observed, liver extracts show both CRE and CCAAT binding activities. Competitions with CRE oligonucleotides were able to prevent the binding of both liver factors, while competitions with CCAAT oligonucleotides only abolished the binding to the CCAAT box. Consistently, the occupation of the CCAAT box was reduced when the distance between the CRE and CCAAT elements was increased in a series of spacing mutants in which DNA fragments of 20, 28, or 44 bp were inserted, and in a construct where the CRE sequence was deleted. Furthermore, the mutants are less efficient than the wild type as templates for in vitro transcription elicited by liver nuclear extracts. Transcriptional activity decreases with the 20- and 28-bp insertions but is partially recovered with the 44-bp insertion. Partial purification of liver CRE- and CCAAT-binding proteins by high performance liquid chromatography on a Mono Q column and recombination of column fractions showed that a novel 73-kDa CRE-binding protein facilitates the association of the CCAAT-binding protein to the CCAAT site of the fibronectin gene.

Animals↗
PubMed1991

Generation of full-length cDNA recombinant vectors for the transient expression of human fibronectin in mammalian cell lines.

In order to study the roles of the different alternatively spliced variants of human fibronectin (FN) as well as of its binding sites and structural domains in the processes of extracellular matrix assembly, cell adhesion, and cell migration, we constructed expression vectors coding for different human full-length FN polypeptides and deleted versions of these constructs. We expressed them transiently in mammalian cells by calcium phosphate transfection and microinjection techniques. While the deleted recombinants were expressed by both transfection and microinjection, the full-length recombinants could be only expressed by microinjection. Calcium phosphate transfection leads to the accumulation of recombinant FN in cytoplasmic vesicles of both matrix-forming and nonforming cells. On the contrary, microinjection leads to the accumulation of recombinant FN in cytoplasmic vesicles in cells that do not form a matrix, but to the rapid incorporation into the extracellular matrix of matrix-forming cells in addition to a cytoplasmic localization. Identical results were obtained when the FN signal and propeptides were replaced by those of E-cadherin.

Animals↗
PubMed1991

Inverse correlation between fibronectin mRNA levels and the metastatic potential of two murine mammary adenocarcinomas.

The ability of cells to metastasize is thought to be related with the loss of the capacity to synthesize and secrete fibronectin (FN). We found that primary cultures from 2 murine adenocarcinoma tumors, M3 of moderate metastasizing ability and the highly metastasizing MM3, had a dramatic difference in the immunohistochemical expression of FN. It was shown, as assayed by the S1 mapping technique that, while total FN mRNA was fairly abundant in M3 cells, it was almost undetectable in MM3 cells. This difference is not due to a significant deletion of the FN gene in MM3 cells.

Adenocarcinoma↗
PubMed1990

Cyclic AMP inhibits fibronectin gene expression in a newly developed granulosa cell line by a mechanism that suppresses cAMP-responsive element-dependent transcriptional activation.

The main role of the ovarian granulosa cells is to nurse the oocyte and to produce estradiol and progesterone upon stimulation by gonadotropins. In fact, follicle-stimulating hormone (FSH) and luteinizing hormone control the expression of several genes during granulosa cell differentiation via cyclic AMP-dependent phosphorylations. Cyclic AMP stimulates transcription of genes that carry the cAMP-responsive element (CRE,5'TGACGTCA3') in their promoters. The fibronectin (FN) gene contains one CRE sequence at position -170. However, gonadotropins and cAMP inhibit FN gene expression in granulosa cells. To study the mechanism of the inhibition we developed a bovine granulosa cell line (BGC-1) that synthesizes estradiol in response to FSH and in which FSH and dibutyryl cAMP specifically decrease FN synthesis and its mRNA levels. The inhibitory effect (a) is not due to an alteration in FN mRNA stability, (b) requires upstream sequences other than CRE, located between positions -510 and -223, that are able to bind granulosa cell nuclear proteins, (c) is entirely dependent on the synthesis of intermediate proteins induced and or phosphorylated by cAMP, and (d) effectively suppresses the CRE-dependent transcriptional activation.

Animals↗
PubMed1988

Cellular fibronectin is induced by epidermal growth factor, but not by dexamethasone or cyclic AMP in rat liver epithelial cells.

Epidermal growth factor (EGF) induces fibronectin (FN) and FN mRNA in rat liver epithelial cells, under conditions where the factor also induces the cells to migrate. Newly synthesized protein is secreted into the medium and deposited as substratum-bound extracellular matrix. The levels of mRNA and the amount of protein synthesized are not influenced by cyclic AMP or dexamethasone, factors that have been found to modulate FN expression in other cells. However, the cells are sensitive to the factors, suggesting a cell-specific regulation. The EGF-induced RNA contains the sequences EIIIA and EIIIB characteristic of cellular fibronectin.

Animals↗
PubMed1987

Identification of a third region of cell-specific alternative splicing in human fibronectin mRNA.

We describe here a third region of variability in human fibronectin (FN) due to alternative RNA splicing. Two other positions of alternative splicing have been reported previously (ED and IIICS). The third region involves a 273-nucleotide exon encoding exactly one 91-amino acid repeat of type III homology, located between the DNA- and the cell-binding domains of FN, which is either included in or excluded from FN mRNA. The two mRNA variants arising by an exon-skipping mechanism are present in cells known to synthesize the cellular form of FN. However, liver cells, which are the source of plasma FN, produce only messengers without the extra type III sequence. Therefore, the region described here resembles, both structurally and functionally, the previously described ED (for extra domain) region, located toward the C terminus of the molecule, between the cell- and heparin- (hep 2) binding domains. We conclude that both the extra type III repeat (named EDII) and ED represent sequences restricted to cellular FN. Combination of all the possible patterns of splicing in the three regions described to date may generate up to 20 distinct FN polypeptides from a single gene.

Amino Acid Sequence↗
PubMed1985

Isolation and characterization of cDNA clones for human liver fibronectin.

Cellular and plasma fibronectin dimers are constituted by similar but not identical polypeptides. Their differences are the consequence of internal primary sequence variability due to alternative splicing in at least 2 regions (ED and IIICS) of the pre mRNAs [1-8]. A detailed analysis of human liver fibronectin mRNA in these regions was carried out by isolating cDNA clones and determining their nucleotide sequence. A novel type of IIICS segment (coding for 64 amino acids) was present in the two cDNA clones studied and, as expected from previous S1 mapping studies [6], the ED segment was absent in both.

Base Composition↗
PubMed1985

Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene.

Cellular and plasma fibronectins are heterodimers consisting of similar but not identical polypeptides. The differences between fibronectin subunits are due in part to the variability of internal primary sequences. This results from alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA. The complete primary structure of human fibronectin, including most of the internal variations, has been determined by sequencing a series of overlapping cDNA clones. In total, they covered 7692 nucleotides and represented the mRNA sequence coding from the amino terminus of the mature protein to the poly(A) tail. The deduced amino acid sequence of fibronectin has been analysed in terms of the arrangement of internal homologies and the different binding domains.

Amino Acid Sequence↗
PubMed1984

Human fibronectin: cell specific alternative mRNA splicing generates polypeptide chains differing in the number of internal repeats.

The nucleotide sequence of five independent cDNA clones, which cover 4843 nucleotides from the poly(A) addition site of human fibronectin (FN) mRNA was determined. The deduced amino acid sequence (1383 residues) covers the COOH-terminal 60% of human FN, spanning the C-terminus, fibrin-, heparin- and cell-binding domains, and shows the exact location of the only two free sulphydryl groups present in each subunit chain. We have recently reported two different FN mRNA species; one of them containing an additional 270 nucleotide insert (ED) that encodes exactly one of the homology type III repeats of the protein. The two mRNAs arise by alternative splicing of a common precursor. S1 nuclease mapping of cDNA/RNA hybrids shows that the expression of the two mRNAs is cell specific. Liver only produces the mRNA without the ED, whereas hepatoma cells, breast tumor cells and normal fibroblasts produce both forms of mRNA. Another area of alternative splicing generating three different FN mRNAs in rat liver has been reported by Schwarzbauer et al (16). We here provide evidence for the existence in human cells of a fourth mRNA species different from the three described in rat liver.

Amino Acid Sequence↗