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F S Jones

Publications and source records attributed to F S Jones.

At least 37 records · Page 2Linked to original sources
PubMed1996

Embryonic expression patterns of the neural cell adhesion molecule gene are regulated by homeodomain binding sites.

During development of the vertebrate nervous system, the neural cell adhesion molecule (N-CAM) is expressed in a defined spatiotemporal pattern. We have proposed that the expression of N-CAM is controlled, in part, by proteins encoded by homeobox genes. This hypothesis has been supported by previous in vitro experiments showing that products of homeobox genes can both bind to and transactivate the N-CAM promoter via two homeodomain binding sites, HBS-I and HBS-II. We have now tested the hypothesis that the N-CAM gene is a target of homeodomain proteins in vivo by using transgenic mice containing native and mutated N-CAM promoter constructs linked to a beta-galactosidase reporter gene. Segments of the 5' flanking region of the mouse N-CAM gene were sufficient to direct expression of the reporter gene in the central nervous system in a pattern consistent with that of the endogenous N-CAM gene. For example, at embryonic day (E) 11, beta-galactosidase staining was found in postmitotic neurons in dorsolateral and ventrolateral regions of the spinal cord; at E14.5, staining was seen in these neurons throughout the spinal cord. In contrast, mice carrying an N-CAM promoter-reporter construct with mutations in both homeodomain binding sites (HBS-I and HBS-II) showed altered expression patterns in the spinal cord. At E11, beta-galactosidase expression was seen in the ventrolateral spinal cord, but was absent in the dorsolateral areas, and at E 14.5, beta-galactosidase expression was no longer detected in any cells of the cord. Homeodomain binding sites found in the N-CAM promoter thus appear to be important in determining specific expression patterns of N-CAM along the dorsoventral axis in the developing spinal cord. These experiments suggest that the N-CAM gene is an in vivo target of homeobox gene products in vertebrates.

Animals↗
PubMed1995

Developmental control of N-CAM expression by Hox and Pax gene products.

A mounting body of evidence suggests that cell-cell adhesion molecules (CAMs) play critical roles in morphogenetic patterning. Perturbations of CAM binding can lead to altered tissue pattern and interruption of tissue interactions can lead to altered patterns of CAM expression. These observations focus attention on the factors responsible for the place-dependent expression of adhesion molecules such as N-CAM, the neural cell adhesion molecule. Our recent experiments in vitro indicate that transcription factors encoded by Hox and Pax genes bind to specific DNA sequences in the N-CAM promoter and activate that promoter. In particular, a region of the N-CAM promoter designated the homeodomain binding site (HBS) interacts with a variety of different homeodomain proteins. A different region of the N-CAM promoter binds to the paired domain of Pax proteins. These transcription factors differentially regulate the N-CAM gene. Such in vitro studies suggest that the N-CAM gene may be an in vivo target for homeobox and Pax gene products. Recent experiments on transgenic mice carrying normal and mutated segments of the N-CAM promoter linked to a lacZ reporter gene suggest that the N-CAM regulation observed in vitro actually has counterparts in vivo. The significance of these observations is that they connect gene products capable of morphoregulation (such as CAMs) to pattern-forming genes.

Animals↗
PubMed1995

Silencer elements modulate the expression of the gene for the neuron-glia cell adhesion molecule, Ng-CAM.

The combined factors that regulate the expression of cell adhesion molecules (CAMs) during development of the nervous system are largely unknown. To identify such factors for Ng-CAM, the neuron-glia CAM, constructs containing portions of the 5' end of the Ng-CAM gene were examined for activity after transfection into N2A neuroblastoma and NIH3T3 cells. Positive regulatory elements active in both cell types included an Ng-CAM proximal promoter with SP1 and cAMP response element motifs extending 447 base pairs upstream of a single RNA start site and a region within the first exon corresponding to 5'-untranslated sequences. Negative regulatory elements included five neuron-restrictive silencer elements (NRSEs) and a binding site for Pax gene products in a 305-base pair segment of the first intron. Constructs containing the promoter together with the entire first intron were active in N2A cells but were silenced in NIH3T3 cells. This silencer activity was mapped to the NRSEs. In contrast, the Pax motif inhibited activity of Ng-CAM constructs in both cell types. The DNA elements defined in these transfection experiments were examined for their ability to bind nuclear factors. The region within the first exon formed a DNA-protein complex after exposure to nuclear extracts prepared from both NIH3T3 and N2A cells. The NRSE region formed a more prominent complex with proteins prepared from NIH3T3 cells than it did with extracts from N2A cells. A member of the Pax protein family, Pax-3 bound to the Pax motif. Mutations introduced within the Pax motif in its ATTA sequence eliminated this binding whereas mutations in its GTTCC sequence did not, suggesting that paired homeodomain interactions are important for the recognition of Pax-3 by this DNA target sequence. The combined data suggest that negative regulation by NRSEs and Pax proteins may play a key role in the place-dependent expression patterns of Ng-CAM during development.

3T3 Cells↗
PubMed1995

Structural and functional similarities between the promoters for mouse tenascin and chicken cytotactin.

Cytotactin/tenascin is an extracellular matrix glycoprotein expressed in a restricted anteroposterior pattern during vertebrate development and is reexpressed in the adult during wound healing, tumorigenesis, and nerve regeneration. Previously, we have characterized the chicken cytotactin promoter and have shown its regulation by homeobox gene products in vitro. We have now isolated the promoter for the mouse tenascin gene in order to determine whether common or different DNA regulatory elements control the expression of this gene in these two species. Like the chicken cytotactin gene, the mouse tenascin gene has a single RNA start site that lies 27 bp downstream of a TATA box. A 4028-bp region of DNA upstream of the mouse tenascin gene was sequenced and examined for regulatory motifs in common with the upstream sequence from the chicken cytotactin promoter. Two hundred thirty base pairs of the proximal promoter regions from both genes had an extended sequence similarity and contained common regulatory motifs such as two tracts of homopolymeric dA.dT sequence, an octamer motif, an ATTA (TAAT) motif which is a common core sequence for binding of homeodomain transcription factors, and a TATA-box/cap-site region. Reporter gene constructs with various 5' deletions of the mouse tenascin upstream sequence were tested in transient transfections of mouse NIH 3T3 and chicken embryo fibroblasts. The conserved proximal promoter region of tenascin was responsible for most of the positive regulatory activity. In addition, an upstream region (-2478 to -247) repressed proximal promoter activity in mouse fibroblasts and also in chicken embryo fibroblasts. These data indicate that both the structure and function of the cytotactin/tenascin proximal promoters have remained conserved over 250 million years.

3T3 Cells↗
PubMed1994

Pax-3 contains domains for transcription activation and transcription inhibition.

Pax-3 is a member of the Pax family of transcription factors involved in transcriptional control events during embryonic development. Here we report a functional dissection of the Pax-3 protein and describe the protein domains which are responsible for different activities. A transcription inhibition activity is located in the first 90 N-terminal amino acids and includes part of the paired domain. Furthermore, the C terminus of Pax-3 is able to confer transcriptional activation of basal promoters. Pax-3 can utilize both transcription modulating functions and activates transcription over a narrow range of protein concentration in the presence of promoter elements containing functional binding sites.

Amino Acid Sequence↗
PubMed1994

Binding and activation of the promoter for the neural cell adhesion molecule by Pax-8.

The neural cell adhesion molecule (N-CAM), is expressed in definite spatiotemporal patterns during development. To identify factors that may influence place-dependent n-cam gene expression, we have studied the binding and activation of the n-cam promoter by Pax-8, a member of the Pax family of transcription factors. Pax-8 increased n-cam promoter activity 13.4-fold in cellular co-transfection experiments, and a short segment of the promoter (-143 to -15) mediated the response. This region of the n-cam promoter produced a DNA-protein complex when incubated with either extracts from COS-7 cells transfected with the Pax-8 expression vector or a Pax-8/GST fusion protein. Pax-8 bound to the n-cam promoter through two TGCTCC motifs (designated PBS-1 and PBS-2) that resemble paired domain binding sites. Mutation of PBS-1 and PBS-2 eliminated Pax-8 activation of the n-cam promoter. Transfection of N2A neuroblastoma cells with the Pax-8 expression vector resulted in a 5-fold increase in the transcription of the endogenous n-cam gene. The combined results suggest that Pax-8 activates transcription of the n-cam gene through binding of sequences resembling paired domain binding sites in the n-cam promoter. The data raise the possibility that the n-cam promoter may be regulated by other members of the Pax gene family.

3T3 Cells↗
PubMed1994

Regulation in vitro of an L-CAM enhancer by homeobox genes HoxD9 and HNF-1.

Previous studies have shown that in vitro expression of the neural cell adhesion molecule (N-CAM) can be regulated by the products of homeobox genes HoxB9, -B8, and -C6. N-CAM is a Ca(2+)-independent immunoglobulin-related CAM that plays an important role in neural development. In the present study, we investigated whether the liver cell adhesion molecule (L-CAM) a member of the Ca(2+)-dependent CAM family (cadherins) is also regulated by homeobox-containing genes. In transient cotransfection experiments of NIH 3T3 cells, we observed that both HoxD9 and liver-enriched POU-homeodomain transcription factor, HNF-1, activated chloramphenicol acetyltransferase gene reporter constructs containing the L-CAM promoter and an enhancer present in the second intron of the chicken L-CAM gene. Using electrophoretic mobility-shift assays, we found that components of cell extracts from NIH 3T3 cells transfected with HoxD9 bound to a small region of the L-CAM enhancer having a consensus sequence that is a putative binding site for HNF-1. Components of extracts from the chicken hepatoma cell line LMH that had been transfected with an HNF-1 expression vector also bound to this same site. In nuclear run-on experiments with nuclei from LMH cells that were transfected with expression vectors for HoxD9 or HNF-1, L-CAM RNA levels were increased 33-fold and 4-fold respectively. Using the same run-on procedure, it was confirmed that nuclei prepared from normal embryonic chicken liver cells expressed the RNAs for HoxD9, HNF-1, and L-CAM. Taken together with previous observations, these data raise the possibility that homeobox-containing genes will have a widespread role in the place-dependent expression of CAMs belonging both to immunoglobulin-related and to cadherin families.

3T3 Cells↗
PubMed1993

Identification of the promoter and a transcriptional enhancer of the gene encoding L-CAM, a calcium-dependent cell adhesion molecule.

L-CAM is a calcium-dependent cell adhesion molecule that is expressed in a characteristic place-dependent pattern during development. Previous studies of ectopic expression of the chicken L-CAM gene under the control of heterologous promoters in transgenic mice suggested that cis-acting sequences controlling the spatiotemporal expression patterns of L-CAM were present within the gene itself. We have now examined the L-CAM gene for sequences that control its expression and have found an enhancer within the second intron of the gene. A 2.5-kb Kpn I-EcoRI fragment from the intron acted as an enhancer of a simian virus 40 minimal promoter driving a chloramphenicol acetyltransferase (CAT) reporter gene and produced 14.0-fold induction of CAT activity in MDCK cells. To narrow down the region responsible for enhancer activity and to determine whether the enhancer could function in a cell type-specific manner, a number of smaller restriction fragments from the intron were tested for activity in two chicken cell lines, the LMH hepatoma line, which produces high levels of L-CAM, and the SL-29 fibroblast line, which produces little, if any, L-CAM. Four L-CAM enhancer plasmids containing shorter segments derived from the intron showed enhanced CAT activity levels (between 9.4- and 16.5-fold) in extracts from transfected LMH cells but not from SL-29 cells. DNA sequence analysis of the L-CAM enhancer region revealed putative binding sites for the transcription factors SP1, E2A, and AP-2. In addition, LE-9, the smallest L-CAM enhancer segment (310 bp), contained a consensus binding site for the liver-enriched POU-homeodomain transcription factor, HNF-1. Tests of upstream sequences showed that a 630-bp fragment, corresponding to nearly the entire intergenic region between L-CAM and its neighboring CAM gene, K-CAM, could function as a promoter. In combination with the L-CAM enhancer, this fragment directed cell type-specific expression of the CAT reporter gene in LMH cells at a level comparable to that observed with enhancer constructs using the simian virus 40 minimal promoter. These combined observations define a promoter and an enhancer for the chicken L-CAM gene. They raise the possibility that these cis-acting regulatory sequences may be instrumental in directing specific place-dependent expression of the L-CAM gene in the chicken.

Animals↗
PubMed1993

Binding and transcriptional activation of the promoter for the neural cell adhesion molecule by HoxC6 (Hox-3.3).

Scores of homeobox gene-encoded transcription factors are expressed in a definite spatiotemporal pattern during embryogenesis and regulate a series of as yet unidentified target genes to help coordinate the morphogenetic process. We have suggested that homeobox gene products modulate the expression of adhesion molecule genes and have shown in cotransfection experiments that the promoters for the neural cell adhesion molecule (N-CAM) and cytotactin/tenascin genes respond to cues from different homeobox-containing genes. In this study, we show that the HoxC6 (Hox-3.3)-encoded homeoprotein binds to a DNA sequence in the N-CAM promoter CCTAATTATTAA, designated homeodomain binding site I (HBS-I). To test whether HoxC6 regulated N-CAM promoter activity, we cotransfected the Long and Short reading frame variants of Xenopus HoxC6 (CMV-HoxC6-L and CMV-HoxC6-S) driven by the human cytomegalovirus (CMV) promoter together with a chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse N-CAM promoter (N-CAM-Pro-CAT). Cotransfection of NIH 3T3 cells with either of the CMV-HoxC6 expression vectors stimulated N-CAM promoter-driven CAT expression. A 47-bp region from the N-CAM promoter that included HBS-I and an adjacent potential HBS, HBS-II, conferred HoxC6 regulation on a simian virus 40 minimal promoter. HBS-I was sufficient for transactivation of the minimal promoter by CMV-HoxC6-S. However, transcriptional activation by CMV-HoxC6-L required both HBS-I and HBS-II, inasmuch as mutation of either HBS-I, HBS-II, or both motifs abolished the response. These studies suggest that HBS-I is a target site for binding and transcriptional control of the N-CAM promoter by homeoproteins, although accessory DNA sequences (such as HBS-II) may also be required. Together with previous studies, these results support the notion that N-CAM gene expression may be controlled by different combinations of homeoproteins that appear in a place-dependent manner during embryogenesis.

3T3 Cells↗
PubMed1992

Cell adhesion molecules as targets for Hox genes: neural cell adhesion molecule promoter activity is modulated by cotransfection with Hox-2.5 and -2.4.

In an effort to determine whether homeobox genes modulate the activity of the promoter of the mouse neural cell adhesion molecule (N-CAM) gene, we have carried out a series of cotransfection experiments using NIH 3T3 cells. Plasmids were constructed containing Xenopus laevis Hox-2.5 and -2.4 coding sequences linked to a human cytomegalovirus promoter (CMV-Hox-2.5 and CMV-Hox-2.4). A 4.9-kilobase DNA fragment containing 5' flanking and first exon sequences of the mouse N-CAM gene was linked to a chloramphenicol acetyltransferase (CAT) reporter gene (N-CAM-Pro-CAT). Cotransfection with CMV-Hox-2.5 and N-CAM-Pro-CAT resulted in a strong induction of CAT activity. The N-CAM promoter contained two potential homeodomain binding sites (sites I and II) within a 47-base-pair segment (512-559 base pairs upstream of the ATG codon in the first exon of the N-CAM gene). This segment was linked to a minimal promoter (simian virus 40 early) and a downstream CAT gene. Although this construct was transcriptionally active at a low level in NIH 3T3 cells, cotransfection of CMV-Hox-2.5 resulted in CAT activity that was greatly elevated. Mutational studies revealed that it was the homeodomain binding site II sequence that was required for this regulation. In contrast, cotransfection with CMV-Hox-2.4 eliminated the CAT activity that was driven by the CMV-Hox-2.5 construct. Thus, the products of two related Hox genes, which are located adjacent to each other in the Hox-2 complex, can differentially modulate transcription from the promoter of a cell adhesion molecule gene. The results suggest that the N-CAM gene is likely to be a target for regulation by Hox gene products.

3T3 Cells↗
PubMed1992

Activation of the cytotactin promoter by the homeobox-containing gene Evx-1.

Cytotactin is a morphoregulatory molecule of the extracellular matrix affecting cell shape, division, and migration that appears in a characteristic and complex site-restricted pattern during embryogenesis. The promoter region of the gene that encodes chicken cytotactin contains a variety of potential regulatory sequences. These include putative binding sites for homeodomain proteins and a phorbol 12-O-tetradecanoate 13-acetate response element (TRE)/AP-1 element, a potential target for transcription factors thought to be involved in growth-factor signal transduction. To determine the effects of homeobox-containing genes on cytotactin promoter activity, we conducted a series of cotransfection experiments on NIH 3T3 cells using cytotactin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs and plasmids driving the expression of mouse homeobox genes Evx-1 and Hox-1.3. cotransfection with Evx-1 stimulated cytotactin promoter activity whereas cotransfection in control experiments with Hox-1.3 had no effect. To localize the sequences required for Evx-1 activation, we tested a series of deletions in the cytotactin promoter. An 89-base-pair region containing a consensus TRE/AP-1 element was found to be required for activation. An oligonucleotide segment containing this TRE/AP-1 site was found to confer Evx-1 inducibility on a simian virus 40 minimal promoter; mutation of the TRE/AP-1 site abolished this activity. To explore the potential role of growth factors in cytotactin promoter activation, chicken embryo fibroblasts, which are known to synthesize cytotactin, were first transfected with cytotactin promoter constructs and cultured under minimal conditions in 1% fetal bovine serum. Although the cells exhibited only low levels of CAT activity under these conditions, cells exposed for 12 h to 10% (vol/vol) fetal bovine serum showed a marked increase in CAT activity. Cotransfection with Evx-1 and cytotactin promoter constructs of cells cultured in 1% fetal bovine serum was sufficient, however, to produce high levels of CAT activity. These findings are consistent with the hypothesis that Evx-1, a homeobox-containing gene, may activate the cytotactin promoter by a mechanism involving a growth-factor signal transduction pathway. More generally, the results support the hypothesis that the place-dependent expression of morphoregulatory molecules may depend upon local cues provided by homeobox genes and their encoded proteins.

3T3 Cells↗
PubMed1992

Cytotactin: a morphoregulatory molecule and a target for regulation by homeobox gene products.

Two major lines of research in developmental biology should help us to understand the bases of morphogenesis. The first is the analysis of the morphogenetic effects of local expression of various adhesion molecules. The second is the analysis of cascades of regulatory genes that interact during development. Of particular significance are regulatory interactions involving homeobox-containing genes which are expressed in a place-dependent manner in the embryo. Success in connecting these two lines of research would help to resolve the puzzle of how species-specific tissue patterns can arise and be maintained. In this article, we focus on cytotactin, a morphoregulatory molecule of the extracellular matrix that exhibits sharply restricted spatiotemporal patterns of expression during development. Recent experiments indicate the promoter of the cytotactin gene contains target regions that appear to respond to homeodomain proteins. These observations, and those on other morphoregulatory molecules, suggest a possible connection between their effects on cell patterning and control by homeobox-containing genes.

Animals↗
PubMed1990

Expression of adhesion molecules and the establishment of boundaries during embryonic and neural development.

Evidence is accumulating that molecules involved in cell-cell and cell-substratum interactions are important in the establishment and maintenance of borders between cell groups during development. In this report, we review evidence supporting this conclusion, particularly in regard to the role of adhesion molecules in the formation of cell collectives and in the modulation of cell and neurite movements.

Animals↗
PubMed1990

Identification and characterization of the promoter for the cytotactin gene.

The extracellular glycoprotein cytotactin is expressed in a characteristic and complex spatiotemporal sequence during development of the chicken embryo. To identify the various control elements underlying its expression, the promoter region of the cytotactin gene has been isolated and characterized. Clones were isolated from genomic libraries by using a fragment near the 5' end of the cDNA sequence. The sequence of this cDNA fragment was found to be distributed over two exons separated by a large first intron. The site of transcription initiation was determined by S1 nuclease and primer-extension mapping. Sequencing of a 4.3-kilobase (kb) genomic DNA clone that contains 3986 base pairs (bp) upstream of the RNA start site, the first exon, and part of the first intron revealed a number of sequence motifs implicated in the regulation and expression of eukaryotic genes. These included CCAAT boxes, phorbol ester-responsive elements, enhancer elements, and a consensus TATA sequence located 24 bp upstream of the major RNA cap site. The flanking sequence also contained a number of regions of dyad symmetry and direct repeats unique to cytotactin, as well as an array of A + T-rich sequences that resemble engrailed elements. Constructs containing fragments of the upstream region of the cytotactin gene fused to a promoterless gene for chloramphenicol acetyltransferase were transiently transfected into chicken embryo fibroblasts to define functional promoter sequences. Although sequences from -721 to +121 exhibited minimal promoter activity, the entire region between -3986 to +374 was required to yield maximal expression in chicken embryo fibroblasts. Transfection of the -3986/+374 chloramphenicol acetyltransferase plasmid into the human U251MG astrocytoma cells but not HT1080 fibrosarcoma cells resulted in chloramphenicol acetyltransferase expression, consistent with the observed synthesis of cytotactin protein only by the U251MG cell line. These data indicate that the chicken cytotactin promoter can control expression in a cell type-specific fashion within cells of another species. These studies provide a basis for the dissection of cis elements and trans factors that govern the developmental expression of the cytotactin gene.

Animals↗
PubMed1990

Localization during development of alternatively spliced forms of cytotactin mRNA by in situ hybridization.

Cytotactin, an extracellular glycoprotein found in neural and nonneural tissues, influences a variety of cellular phenomena, particularly cell adhesion and cell migration. Northern and Western blot analysis and in situ hybridization were used to determine localization of alternatively spliced forms of cytotactin in neural and nonneural tissues using a probe (CT) that detected all forms of cytotactin mRNA, and one (VbVc) that detected two of the differentially spliced repeats homologous to the type III repeats of fibronectin. In the brain, the levels of mRNA and protein increased from E8 through E15 and then gradually decreased until they were barely detectable by P3. Among the three cytotactin mRNAs (7.2, 6.6, and 6.4 kb) detected in the brain, the VbVc probe hybridized only to the 7.2-kb message. In isolated cerebella, the 220-kD polypeptide and 7.2-kb mRNA were the only cytotactin species present at hatching, indicating that the 220-kD polypeptide is encoded by the 7.2-kb message that contains the VbVc alternatively spliced insert. In situ hybridization showed cytotactin mRNA in glia and glial precursors in the ventricular zone throughout the central nervous system. In all regions of the nervous system, cytotactin mRNAs were more transient and more localized than the polypeptides. For example, in the radial glia, cytotactin mRNA was observed in the soma whereas the protein was present externally along the glial fibers. In the telencephalon, cytotactin mRNAs were found in a narrow band at the edge of a larger region in which the protein was wide-spread. Hybridization with the VbVc probe generally overlapped that of the CT probe in the spinal cord and cerebellum, consistent with the results of Northern blot analysis. In contrast, in the outermost tectal layers, differential hybridization was observed with the two probes. In nonneural tissues, hybridization with the CT probe, but not the VbVc probe, was detected in chondroblasts, tendinous tissues, and certain mesenchymal cells in the lung. In contrast, hybridization with both probes was observed in smooth muscle and lung epithelium. Both epithelium and mesenchyme expressed cytotactin mRNA in varying combinations: in the choroid plexus, only epithelial cells expressed cytotactin mRNA; in kidney, only mesenchymal cells; and in the lung, both of these cell types contained cytotactin mRNA. These spatiotemporal changes during development suggest that the synthesis of the various alternatively spliced cytotactin mRNAs is responsive to tissue-specific local signals and prompt a search for functional differences in the various molecular forms of the protein.

Animals↗
PubMed1989

A detailed structural model of cytotactin: protein homologies, alternative RNA splicing, and binding regions.

A combination of cDNA sequencing of the complete coding region, protein comparisons, binding site mapping, and electron microscopic imaging has permitted the formulation of a structural model of cytotactin. Cytotactin is a large extracellular matrix glycoprotein that displays a restricted tissue distribution during development. Although there appears to be a single cytotactin gene, multiple cytotactin polypeptides and mRNAs are detected in a variety of tissues. We report here the sequences and relationships of cDNAs that encode the complete amino acid sequences of two cytotactin polypeptides in chicken brain. The translated cDNA sequences agree with those obtained by direct analysis of cytotactin and fragments of the molecule. All regions of the polypeptides appear to be identical except for a 273 amino acid segment found in the larger but not in the smaller. At their amino termini, both polypeptides contain a cysteine-rich segment that probably includes those residues that link monomers into hexamers. This segment is followed by 13 epidermal growth factor-like (EGFL) repeats and then 8 consecutive segments that each resemble the type III repeats found in fibronectin. At their carboxyl termini, the polypeptides are similar to the beta and gamma chains of fibrinogen, including a calcium-binding segment. The additional sequence in the large polypeptide is inserted after the fifth type III repeat and includes three additional type III repeats. On RNA transfer blot analyses, cytotactin cDNA probes detected a 6.4-kilobase (kb) component in both brain and gizzard and larger mRNAs in both tissues, but those in gizzard were larger by about 1 kb than those in brain. A probe specific to the insert did not hybridize to the 6.4-kb mRNA in either tissue but detected the larger mRNAs in both tissues. At least a portion of the insert is thus present in both tissues, but there may be additional inserts in the gizzard mRNAs. The proposed model of cytotactin specifies the orientation of the polypeptides, the localization of interchain disulfide bonds, the structural elements constituting the thin and thick segments (EGFL repeats and type III repeats, respectively), the terminal fibrinogen-like nodular region, and the relative location of the cell-binding region.

Amino Acid Sequence↗