Murine GBP-2: a new IFN-gamma-induced member of the GBP family of GTPases isolated from macrophages.

We have cloned a new member of the interferon (IFN)-induced guanylate-binding protein (GBP) family of GTPases, murine GBP-2 (mGBP-2), from bone marrow-derived macrophages. mGBP-2 is located on murine chromosome 3, where it is linked to mGBP-1. With the identification of mGBP-2 there are now two human and two murine GBPs. Like other GBPs, mGBP-2 RNA and protein are induced by IFN-gamma. In addition, mGBP-2 shares with the other GBPs important structural features that distinguish this family from other GTPases. First, mGBP-2 contains only two of the three consensus sequences for nucleotide binding found within the classic GTP binding regions of other GTPases. A second amino acid motif found in mGBP-2 is a potential C-terminal site for isoprenoid modification, called a CaaX sequence. mGBP-2 is prenylated, as detected by [3H]mevalonate incorporation, when expressed in COS cells and preferentially incorporates the C-20 isoprenoid geranylgeraniol. Surprisingly, despite having a functional CaaX sequence, mGBP-2 is primarily cytosolic. GBP proteins are very abundant in IFN-exposed cells, but little is known about their function. mGBP-2 is expressed by IFN-gamma-treated cells from C57Bl/6 mice, whereas mGBP-1 is not. Thus, the identification of mGBP-2 makes possible the study of GBP function in the absence of a second family member.

Organization, 5'-flanking sequence and promoter activity of the rat GPC1 gene.

Glypicans are a member of a family of glycosylphosphatidylinositol anchored heparan sulfate proteoglycans that are expressed in cell and development specific patterns. Rat GPC1 cDNA probes were used to screen rat genomic libraries. Three overlapping genomic clones that contained the entire rat GPC1 gene were isolated. The rat GPC1 gene is approximately 15kb in length and consists of eight exons interrupted by introns of varying lengths. Two of the introns are quite short, with lengths of 41 and 43 base pairs. Each exon-intron splice junction exhibited the consensus splice site sequence. Exon 1 encodes the putative signal peptide and the serine residue of the first putative heparan sulfate attachment site. The last exon encodes the cluster of three potential COOH-terminal heparan sulfate attachment sites, the putative GPI anchor and polypeptide cleavage site, and the 3'-untranslated region including the polyadenylation signal. One of the genomic clones extended approximately 2.8 kb 5' of the exon 1 coding sequence, and is thus likely to contain sequences that regulate GPC1 gene expression. Sequence analysis of the 5'-flanking sequence revealed a lack of consensus TATA and CAAT boxes. A search for potential transcription factor binding sites revealed a number of such motifs, including Sp1 (GC box), NF-kappaB, and MyoD (E-box). This region of the rat GPC1 gene shows significant sequence homology to the 5'-flanking region of the human GPC3 gene. Functional promoter activity of the rat GPC1 sequence was demonstrated by its ability to drive the expression of a luciferase reporter gene in several cell types.

Phosphorylation of recombinant N-syndecan (syndecan 3) core protein.

The cytoplasmic domain of the syndecan family of heparan sulfate proteoglycans is punctuated by the presence of four regularly spaced tyrosine residues. In this report, we explore the possibility of whether the four tyrosine residues in the cytoplasmic domain of N-syndecan (Syndecan 3) are potential substrates for phosphorylation by a tyrosine kinase. Bacterially expressed elk kinase was used to phosphorylate a series of bacterially expressed N-syndecan fusion proteins. Our results clearly demonstrate that the tyrosine residues in the cytoplasmic domain of N-syndecan can be phosphorylated by a tyrosine-specific kinase, and that all four tyrosine residues are capable of being phosphorylated.

Identification of glypican as a dual modulator of the biological activity of fibroblast growth factors.

Heparan sulfate moieties of cell-surface proteoglycans modulate the biological responses to fibroblast growth factors (FGFs). We have reported previously that cell-associated heparan sulfates inhibit the binding of the keratinocyte growth factor (KGF), but enhance the binding of acidic FGF to the KGF receptor, both in keratinocytes, which naturally express this receptor, and in rat myoblasts, which ectopically express it (Reich-Slotky, R., Bonneh-Barkay, D., Shaoul, E., Berman, B., Svahn, C. M., and Ron, D. (1994) J. Biol. Chem. 269, 32279-32285). The proteoglycan bearing these modulatory heparan sulfates was purified to homogeneity from salt extracts of rat myoblasts by anion-exchange and FGF affinity chromatography and was identified as rat glypican. Affinity-purified glypican augmented the binding of acidic FGF and basic FGF to human FGF receptor-1 in a cell-free system. This effect was abolished following digestion of glypican by heparinase. Addition of purified soluble glypican effectively replaced heparin in supporting basic FGF-induced cellular proliferation of heparan sulfate-negative cells expressing recombinant FGF receptor-1. In keratinocytes, glypican strongly inhibited the mitogenic response to KGF while enhancing the response to acidic FGF. Taken together, these findings demonstrate that glypican plays an important role in regulating the biological activity of fibroblast growth factors and that, for different growth factors, glypican can either enhance or suppress cellular responsiveness.

Developmental and cell-type-specific expression of cell surface heparan sulfate proteoglycans in the rat heart.

The expression of cell surface heparan sulfate proteoglycans in rat heart was investigated by Northern blot analysis with specific cDNA probes. In adult heart syndecan-3 and glypican mRNAs were abundantly expressed. Lower levels of syndecan-2 mRNA and very low levels of syndecan-1 mRNA were also detected. Analysis of RNA isolated from hearts of rats of various ages revealed that syndecan-3 and glypican mRNAs levels increased dramatically at birth, and continued to be expressed at high levels in adult animals. To determine which of these proteoglycans was expressed in cardiomyocytes, primary cultures of cardiomyocytes and nonmyocytes isolated from neonatal rat hearts were analyzed for proteoglycan expression. Glypican mRNA was localized almost exclusively to cardiomyocytes. Syndecan-3 mRNA was not detected in myocytes, but was detected in the nonmyocyte cells. Biochemical characterization of cardiomyocyte glypican revealed that it was a phosphatidylinositol-anchored heparan sulfate proteoglycan. Results of immunofluorescent staining of rat hearts with anti-glypican antibodies were consistent with the Northern blot data, and localized glypican to the lateral regions of myocyte plasma membrane that contact the basement membrane, as well as sites of myocyte adhesion junctions. At the latter site glypican colocalized with vinculin. Visualization of basic fibroblast growth factor binding sites by means of a tissue slice overlay assay also revealed colocalization with glypican. These results demonstrate developmental and cell-type-specific expression of membrane heparan sulfate proteoglycans in the heart. They also show that glypican is a major heparan sulfate proteoglycan expressed on the cardiomyocyte plasma membrane.

cDNA cloning, genomic organization, and in vivo expression of rat N-syndecan.

The amino acid sequence of rat N-syndecan core protein was deduced from the cloned cDNA sequence. The sequence predicts a core protein of 442 amino acids with six structural domains: an NH2-terminal signal peptide, a membrane distal glycosaminoglycan attachment domain, a mucin homology domain, a membrane proximal glycosaminoglycan attachment domain, a single transmembrane domain, and a noncatalytic COOH-terminal cytoplasmic domain. Transfection of human 293 cells resulted in the expression of N-syndecan that was modified by heparan sulfate chain addition. Heparitinase digestion of the expressed proteoglycan produced a core protein that migrated on SDS-polyacrylamide gels at an apparent molecular weight of 120, 000, identical to N-syndecan synthesized by neonatal rat brain or Schwann cells. Rat genomic DNA coding for N-syndecan was isolated by hybridization screening. The rat N-syndecan gene is comprised of five exons. Each exon corresponds to a specific core protein structural domain, with the exception of the fifth exon, which contains the coding information for both the transmembrane and cytoplasmic domains as well as the 3'-untranslated region of the mRNA. The first intron is large, with a length of 22 kilobases. The expression of N-syndecan was investigated in late embryonic, neonatal, and adult rats by immunoblotting and Northern blotting analysis. Among the tissues and developmental stages studied, high levels of N-syndecan expression were restricted to the early postnatal nervous system. N-syndecan was expressed in all regions of the nervous system, including cortex, midbrain, spinal cord, and peripheral nerve. Immunohistochemical staining revealed high levels of N-syndecan expression in all brain regions and fiber tract areas.

Rat p67 GBP is induced by interferon-gamma and isoprenoid-modified in macrophages.

The guanylate binding proteins, GBPs, are a family of interferon-induced GTP-binding proteins that include the rat p67. We report here that rat p67, for which interferon regulation had not previously been demonstrated, is induced by IFN-gamma and also by LPS in both cultured bone marrow-derived macrophages and microglia. The basal level of rat p67 in macrophages is low but increases dramatically between 2 and 4 hours after treating cells with either IFN-gamma or LPS. It then remains elevated over the next 24 hours. Rat p67 is isoprenoid modified. The isoprenoid modification was detected in p67 isolated both from primary IFN-gamma-activated macrophages and when the gene for p67 was transfected into COS cells. This is the first demonstration of in vivo prenylation of a GBP. The interferon regulation and prenylation of rat p67 point toward this protein being significant in the functions of both activated macrophages and microglia.

Self-association of N-syndecan (syndecan-3) core protein is mediated by a novel structural motif in the transmembrane domain and ectodomain flanking region.

We expressed domains of the core protein of the transmembrane heparan sulfate proteoglycan N-syndecan (syndecan-3) either individually or as maltose-binding protein fusion proteins. Biochemical characterization of the purified proteins revealed that some of them were capable of self-association and formed stable, noncovalent multimeric complexes. The formation of N-syndecan core protein complexes was also demonstrated in mammalian cells by in situ cross-linking. Identification of structural motifs in the core protein of N-syndecan responsible for the formation of these complexes was accomplished by analyzing a series of constructs comprising different regions of the protein as well as site-directed mutants. Self-association was assayed by SDS-polyacrylamide gel electrophoresis, glutaraldehyde cross-linking, and size-exclusion high pressure liquid chromatography. Our results indicated that (i) the transmembrane domain of the N-syndecan core protein was required but not sufficient for the formation of stable complexes; (ii) the minimal amino acid sequence that conferred the ability of the N-syndecan core protein to form multimeric complexes included the last four amino acids (ERKE) of the extracellular domain plus the transmembrane domain; (iii) point mutations that changed the basic residues in this sequence to alanine residues either partially or completely abolished the ability of the N-syndecan core protein to form complexes; and (iv) replacement of conserved glycine residues in the transmembrane domain with leucines abolished complex formation. This property is similar to the oligomerization activity of other transmembrane receptors and suggests that regulated self-association may be important for the biological activity of transmembrane proteoglycans.

Molecular cloning and characterization of an isoprenylated 67 kDa protein.

The cDNA coding for a 67 kDa protein (p67) was isolated from a rat Schwann cell library. A recombinant form of p67 expressed in bacteria was used to produce polyclonal anti-p67 antibodies. By immunoblot analysis p67 was found to be expressed in most tissues and cell lines examined. Inspection of the deduced amino acid sequence revealed a COOH-terminal consensus sequence for isoprenylation. Consistent with this finding, p67 was a substrate for isoprenylation in vitro by geranylgeranylpyrophosphate. p67 was associated predominantly with the particulate fraction of rat smooth muscle cells. The rat p67 sequence was highly homologous to a family of recently described human and mouse gamma-interferon inducible, guanine nucleotide binding proteins.

Syndecan-1 expressed in Schwann cells causes morphological transformation and cytoskeletal reorganization and associates with actin during cell spreading.

To investigate the biological functions of transmembrane proteoglycans we have produced clonal cell lines of rat Schwann cells that express the hybrid proteoglycan syndecan-1. This was done by transfection of newborn rat Schwann cells with a plasmid vector bearing the rat syndecan-1 cDNA sequence under transcriptional control of the constitutively active cytomegalovirus promoter, and a neomycin resistance gene. Stably expressing cells were selected by growth in G418. Expression of syndecan-1 was verified by Northern and immunoblot analysis and immunoprecipitation of 35SO4-labeled proteoglycans. The syndecan-1 expressing cells exhibited significantly enhanced spreading on several different substrata, including fibronectin and laminin, and an altered morphology. The enhanced spreading appeared to result from the presence of syndecan-1, based on the observation that anti-syndecan-1 antibodies inhibited the enhanced substratum spreading. There was also a reorganization of cytoskeletal structures and formation of focal adhesions, visualized by anti-vinculin staining, which were absent from control Schwann cells. There was no apparent stable association of cell surface syndecan-1 with focal contact sites, as determined by dual staining with anti-syndecan-1 and anti-vinculin antibodies. Colocalization of patches of cell surface syndecan-1 with actin was observed, but only during cell spreading. These findings provide evidence for a role of transmembrane proteoglycans in cellular morphogenesis, and suggest that transient association of syndecans with microfilaments may be an important aspect of their biological function.

Processing and subcellular distribution of the Schwann cell lipid-anchored heparan sulfate proteoglycan and identification as glypican.

We previously identified a phosphatidylinositol-specific phospholipase c (PI-PLC)-releasable heparan sulfate proteoglycan (HSPG) on the surface of rat Schwann cells (D. J. Carey and R. C. Stahl, J. Cell Biol. 111, 2053-2062, 1990). The present study was carried out to investigate the localization and processing of this proteoglycan. The HSPG was synthesized as a PI-PLC-releasable form that was shed into the culture medium with a T1/2 of 17 h. Degradation of the HSPG was negligible. The HSPG was present on the surface of Schwann cells on small (100-200 nm diameter) cylindrical membrane extensions that resembled filopodia. In neonatal peripheral nerve, brain, heart, and striated muscle the HSPG was found to be localized principally to regions of the cell surface that were in contact with basement membranes. Northern blot analysis with cDNA coding for rat glypican (a previously described human fibroblast HSPG) demonstrated abundant expression of glypican mRNA in Schwann cells. Antibodies made against recombinant rat glypican core protein immunoprecipitated the Schwann cell PI-PLC-releasable HSPG. These data demonstrate that the Schwann cell HSPG is rat glypican and support the hypothesis that this proteoglycan functions in cell-extracellular matrix interactions.

Molecular characterization of vascular smooth muscle decorin: deduced core protein structure and regulation of gene expression.

Two overlapping clones containing sequences homologous to bovine, human and chicken decorin have been recovered from poly A+ RNA isolated from rat vascular smooth muscle cells (VSMC) using cDNA cloning and reverse transcription-polymerase chain reaction (PCR) methodologies. Results of nucleotide sequence analysis performed on these clones demonstrated that they encode the complete mature rat decorin protein expressed by VSMC. Within the coding region, rat decorin exhibits 76% nucleotide sequence homology to human and bovine decorin, and 69% homologous to chicken decorin indicating a significant level of conservation among these species. This level of conservation among species was also maintained at the protein level with rat decorin being 77% homologous to its human, bovine and chicken homologues. As previously observed its human homologue, rat decorin, is made up of seven, tandem, leucine-rich repeat sequences. Furthermore, within the core of these repeats was the consensus protein sequence NKISK which has been proposed to be the fibronectin binding region of decorin (G. Schmidt et al., Biochem. J. 280, 411-414 (1991)). The vast majority of amino acid substitutions within rat decorin were of the conservative type. The highest frequency of amino acid substitutions were found to be localized within a hypervariable region located near the amino terminus of the decorin core protein. Unlike rat biglycan, rat decorin mRNA levels were found to increase significantly in density-arrested VSMC cultures. In contrast to rat biglycan gene expression, no quantitative differences in rat decorin mRNA levels were observed between proliferating VSMC and VSMC made quiescent through serum depletion. Finally, specific extracellular matrix (ECM) proteins were able to regulate the expression of decorin at the mRNA level in a slightly different manner than previously observed for biglycan.

Regulated expression of syndecan in vascular smooth muscle cells and cloning of rat syndecan core protein cDNA.

cDNA encoding the core protein of rat syndecan was cloned from a neonatal rat aortic cDNA library by polymerase chain reaction amplification. Expression of syndecan mRNA in rat aortic vascular smooth muscle (VSM) cells was demonstrated by reverse transcriptase-linked polymerase chain reaction amplification of syndecan sequences using total RNA from rat aortic VSM cells as templates. Polyclonal antibodies against rat syndecan core protein were produced by immunizing rabbits with a recombinant fusion protein containing a fragment of the extracellular domain. The anti-syndecan antibodies immunoprecipitated a large 35SO4-labeled molecule synthesized by cultured rat aortic VSM cells. The immunoprecipitated molecule was identified as a hybrid proteoglycan, based on results of alkaline, nitrous acid, and chondroitinase ABC digestions. On immunoblots the antibodies recognized a proteoglycan of greater than 200 kDa, with a core protein size after deglycosylation of approximately 50 kDa. The anti-syndecan antibodies stained cultured rat aortic VSM cells as well as tissue sections of neonatal and adult rat aortas in the medial, smooth muscle layer. On Northern blots of RNA isolated from cultured VSM cells, a syndecan cDNA probe hybridized to a major RNA species of 2.6 kilobases. Quantitative Northern blot analysis of total RNA isolated from VSM cells harvested at different cell densities revealed a decrease in syndecan mRNA levels with increased cell density. These results demonstrate the regulated synthesis of syndecan by rat VSM cells.

Molecular cloning and characterization of N-syndecan, a novel transmembrane heparan sulfate proteoglycan.

A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.

Effect of anti-Ig on rabbit B cell function and Ig expression.

We have examined the effect which xenogeneic anti-Ig has on rabbit B cell function and Ig expression in an effort to understand the phenomenon of antibody mediated suppression. Treatment of rabbit lymphocyte cultures with xenogeneic anti-rabbit Ig causes 2.5-3.8 fold decrease in the level of Ig secreting cells with little or no long-term effect on surface Ig. This suppression in B cell secretory function is not the result of suppression of Ig gene expression since xenogeneic anti-rabbit Ig treatment causes a 1.7-2.7 fold increase in Ig L and H chain mRNA levels. Collectively, these data are consistent with the hypothesis that antibody mediated suppression of B cell function occurs at a post-transcriptional level involving either the secretory pathway of Ig expression and/or blockage in B cell differentiation.

Vascular smooth muscle biglycan represents a highly conserved proteoglycan within the arterial wall.

Two overlapping cDNA clones containing sequences homologous to human bone biglycan were isolated from a rat vascular smooth muscle (VSM) cell cDNA library. Nucleotide sequence analysis demonstrated that these clones encoded the rat VSM biglycan complete core protein sequence. A high degree of genetic conservation was observed for biglycan since nucleotide sequence homology comparisons revealed an 88% homology occurring between rat and human biglycan cDNA coding regions. The deduced core protein for the secreted form of VSM biglycan was found to be composed of ten leucine-rich repeating units whose consensus sequence was similar to that present in human bone and bovine cartilage biglycans. Protein homology comparisons revealed that the mature rat VSM biglycan core protein was 97% homologous to both human bone and bovine cartilage biglycan core proteins. Most of the amino acid substitutions in the secreted form of rat VSM biglycan are of the conservative type thereby maintaining the hydrophobic property of this proteoglycan within the arterial wall. The highest frequency of amino acid substitutions occurring between rat, bovine and human biglycan core proteins was found to reside within a small hypervariable region located near the N-terminus of these proteoglycans. Rat VSM biglycan was found to contain dinucleotide repeat elements located in the 3' untranslated region of its mRNA which may influence the expression of this transcript. Biglycan mRNA levels were not found to vary significantly as a function of VSM cell density. However, biglycan transcript levels did vary significantly as a function of VSM cell growth state. Similar studies revealed that collagen, fibronectin, and laminin were able to influence biglycan mRNA levels in quiescent VSM cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of large cDNA inserts within crude lambda gt11 lysates: a rapid and sensitive method.

A method is presented for the isolation of bacteriophage lambda DNA and the rapid identification of large cDNA inserts within crude phage lysates. The primary screening of a lambda gt11 cDNA library with a 32P-radiolabeled cDNA probe yielded 21 putative positive clones. A phage "spot-blot" analysis was employed to quickly screen these potential recombinants. This eliminated 9 of the 21 clones as the result of false positive signals. The remaining 12 recombinant phage were amplified on agarose-based media, and phage DNA was isolated using a modified plate lysate procedure. The DNA thus obtained from these crude lysates could be easily digested with EcoRI and examined by Southern blot analysis. The resulting blot was hybridized with the same cDNA probe used in the initial screening of the library. Thus, two clones harboring the longest cDNA insert were identified from a mixed phage population and were subsequently plaque purified. The procedure is rapid, sensitive, reproducible, inexpensive and allows the processing of several clones at once without sacrificing the quality or yields of the DNA preparation. Furthermore, the method obviates the need for plaque purifying all the positives obtained from the initial screening of a cDNA library.

Rabbit Ig kappa 1b6 gene structure.

Previous studies employing Southern blot analyses have detected multiple kappa-homologous sequences within EcoRI-digested DNA isolated from kappa 1b6 homozygous rabbits and kappa 1b6 L chain secreting RMH H158 cell line. These results are very unexpected because the published partial protein sequence for the kappa 1b6 C region is incompatible with an EcoRI restriction endonuclease recognition sequence at the nucleotide level for this allotype. To determine their identity, the kappa-homologous sequences were isolated from DNA extracted from a kappa 1b6 L chain secreting RMH H158 cell line by molecular cloning. Structural analyses demonstrated these sequences to contain genetic information encoding the majority of the kappa 1b6 L chain gene locus. The protein sequence deduced from the kappa 1b6 C region gene was shown to differ from the published partial kappa 1b6 C region protein sequence at five amino acid positions. One of these differences results in a glycine to serine interchange that introduces an EcoRI restriction endonuclease recognition site within the kappa 1b6 C region gene. Subsequent genomic Southern blot analyses confirmed this structural assignment. Based on these data, the EcoRI-sensitive kappa-homologous fragments present within the genomes of the RMH H158 cell line and kappa 1b6 homozygous rabbits represent the nominal kappa 1 gene and not an alternative kappa isotype or kappa pseudogene. Rabbit Ig kappa 1 allelic nucleotide sequence homology comparisons have shown the isolated kappa 1b6 J-C gene locus to display common structural features previously identified in other kappa 1 alleles.

Characterization of extracellular matrix proteoglycan transcripts expressed by vascular smooth muscle cells.

Northern blot analysis using intraspecies and cross-species cDNA probes encoding a variety of proteoglycan (PG) core proteins was employed to examine in vitro and in vivo vascular smooth muscle cell (VSMC) extracellular matrix (ECM) PG gene expression. Similar studies were performed using a cDNA probe encoding the rat chondrosarcoma link protein. VSMCs maintained in vitro as well as in vivo expressed a 1.8 kb transcript homologous to a cDNA encoding the bovine bone small PGII. Densitometric analysis revealed an 11-fold increase in the in vivo level of this transcript when compared to an equivalent amount of RNA extracted from rat VSMCs maintained in vitro. Rat aorta medial tissue contained approximately a 10-fold higher level of the bovine bone PGII homologous transcript when compared to rat chondrosarcoma tissue. A 2.9 kb transcript homologous to a cDNA encoding the human bone small PGI (biglycan) was detected in poly(A)+ RNA isolated from rat VSMCs maintained in vitro as well as within rat thoracic aorta medial tissue. Densitometric analysis revealed a 0.55-fold increase in the in vitro level of the PGI transcript when compared to in vivo levels. Rat chondrosarcoma expressed approximately a 3-fold higher level of a human bone PGI homologous transcript when compared to rat aorta medial tissue. A 7.3 kb and an 8.4 kb transcript homologous to a cDNA encoding the rat chondrosarcoma large hyaluronic acid (HA) binding chondroitin-sulfate (CS) PG core protein was detected in poly(A)+ RNA extracted from rat VSMCs maintained in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical synthesis of a biologically active gene for human immune interferon-gamma. Prospect for site-specific mutagenesis and structure-function studies.

A 453-base pair DNA duplex consisting of a gene coding for human interferon-gamma and initiation and termination signals plus appropriate restriction enzyme sites for plasmid insertion has been totally synthesized. The synthesis involved preparation of 66 oligodeoxynucleotides by a modified, solid phase phosphite procedure and enzymatic ligation of the oligonucleotides. The gene, when inserted into a previously constructed expression vector, was expressed in Escherichia coli, demonstrating functional activity for the synthetic gene. Several strategically located restriction cleavage sites have been introduced into the sequence. This provides a convenient system for site-specific mutagenesis for structure-function studies.