Cortical thickness and oscillatory phase resetting: a proposed mechanism of salience network dysfunction in schizophrenia.

Schizophrenia is characterised by both electrophysiological abnormalities and consistent changes in the structure of cortical grey matter. But the relationship between these two observations is largely unknown. Structural changes reported in schizophrenia include reduced grey matter volume, thickness and surface area in several cortical regions, but most frequently in the insula and anterior cingulate cortex. These two regions together constitute an intrinsic brain circuit known as the "Salience Network", which has a key role in stimulus processing. During stimulus processing tasks, evoked activity is noted using electroencephalography (EEG). Phase resetting of ongoing oscillations contributes to this evoked activity. Neuronal oscillations play a crucial role in cerebral recruitment during cognitive tasks, and influencing the oscillatory phase can modulate cortical excitability and the transition between various cognitive states. At a network level, such a transition or switch is thought to be enabled by the Salience Network. In this study, we investigated the relationship between the cortical thickness in the Salience Network (measured using MRI) and the degree of phase resetting observed during an oddball task (measured using EEG) in 18 medicated male patients in a clinically stable phase of schizophrenia and 20 age and gender matched healthy controls. We obtained a measure of partial phase resetting after a stimulus is presented, and a second measure representing mean evoked activity, using the methods proposed by Martinez-Montes. Using MRI analysis, we have firstly shown that there is a significant loss of cortical thickness of regions that constitute the Salience Network in patients with schizophrenia. EEG analysis revealed that in healthy controls, the expected relationship between phase resetting and evoked electrical activity is observed, but in patients with schizophrenia the theta phase resetting is a weak predictor of the activity evoked by attending to a target stimulus, though the difference between the groups did not reach statistical significance in the present sample. Furthermore, in patients with schizophrenia the reduced thickness in the Salience Network is associated with the inefficient phase resetting of theta oscillations. Our findings suggest that the grey matter reduction seen in the Salience Network in patients with schizophrenia has substantial functional consequences. In particular, the structural defect of the insula that is seen in schizophrenia is likely to be associated with less efficient recruitment of brain circuits for processing information. This implies a possible mechanism by which disruptions in the intrinsic Salience Network can result in a general disturbance in salience detection seen in schizophrenia.

Reduced event-related low frequency EEG activity in schizophrenia during an auditory oddball task.

This study examines EEG low frequency characteristics which have been linked to specific cognitive functions such as stimulus encoding and attention during an auditory oddball task in schizophrenia patients and healthy controls. EEG data was recorded from 17 young schizophrenia patients in a stable phase of their illness and 17 healthy controls performing an auditory oddball task. Evoked and induced delta and theta activity, N100, P300 amplitude were computed. Between 200-500 ms after a stimulus was presented, patients displayed significantly reduced P300, less evoked and induced delta and theta activity than controls. We conclude that the well known finding of P300 reduction in schizophrenia can be linked to reductions in delta and theta activity, which are a manifestation of impaired stimulus evaluation, memory retrieval, and a lack of sustained attention.

The influence of MHC class II molecules containing the rheumatoid arthritis shared epitope on the immune response to aggrecan G1 and its peptides.

Aggrecan has been implied as an autoantigen in rheumatoid arthritis (RA). Immunization with aggrecan induces arthritis in BALB/c (H-2(d)) mice but not in other strains of mice [e.g. C57BL/6 (H-2(b))]. In humans, the strongest genetic association with RA is to the shared epitope (SE), and aggrecan peptides are predicted to bind to the SE. Therefore, we hypothesized that C57BL/6 mice transgenic (tg) for the RA SE (DR4 tg mice) may be susceptible to aggrecan-induced arthritis. C57BL/6 and DR4 tg mice were immunized with a mixture of SE-binding aggrecan peptides and tested for immune responses to the corresponding peptides as well as aggrecan. Sustained T- and B-cell immune responses to aggrecan and several of its peptides were detected in DR4 tg mice. C57BL/6 mice showed only transient T-cell responses to different immunizing peptides and little B-cell response. Therefore, an immune response to peptides of aggrecan can be induced experimentally in DR4 tg mice as anticipated from the predicted and actual binding affinities of these peptides for the RA SE. Failure to induce arthritis in these DR4 tg mice may be due to a lack of appropriate non-MHC genes.

Calreticulin, PDI, Grp94 and BiP chaperone proteins are associated with retained COMP in pseudoachondroplasia chondrocytes.

Cartilage oligomeric matrix protein (COMP), a large pentameric glycoprotein and member of the thrombospondin (TSP) group of extracellular proteins, is found in the territorial matrix surrounding chondrocytes. More than 50 unique COMP mutations have been identified as causing two skeletal dysplasias: pseudoachondroplasia (PSACH); and multiple epiphyseal dysplasia (EDM1). Recent studies suggest that calcium-binding and calcium-induced protein folding differ between wild type and mutant proteins, and abnormal processing of the mutant COMP protein contributes to the characteristic enlarged lamellar appearing rER cisternae in PSACH and EDMI chondrocytes in vivo and in vitro. Towards the goal of delineating the pathogenesis of PSACH and EDM1, in-vivo PSACH growth plate and in-vitro PSACH chondrocytes cultured in alginate beads were examined to identify and localize the chaperone proteins participating in the processing of the retained extracellular matrix proteins in the PSACH rER. Aggrecan was localized to both the rER cisternae and matrix while COMP and type IX collagen were only found in the rER. Type II collagen was solely found in the ECM suggesting that it is processed and transported differently from other retained ECM proteins. Five chaperone proteins: BiP (Grp78); calreticulin (CRT); protein disulfide (PDI); ERp72; and Grp94, demonstrated immunoreactivity in the enlarged PSACH cisternae and the short rER channels of chondrocytes from both in-vivo and in-vitro samples. The chaperone proteins cluster around the electron dense material within the enlarged rER cisternae. CRT, PDI and GRP94 AB-gold particles appear to be closely associated with COMP. Immunoprecipitation and Western blot, and Fluorescence Resonance Energy Transfer (FRET) analyses indicate that CRT, PDI and GRP94 are in close proximity to normal and mutant COMP and BiP to mutant COMP. These results suggest that these proteins play a role in the processing and transport of wild type COMP in normal chondrocytes and in the retention of mutant COMP in PSACH chondrocytes.

The intermediates of aggrecanase-dependent cleavage of aggrecan in rat chondrosarcoma cells treated with interleukin-1.

We have examined the abundance and structure of intermediates in the chondrocyte-mediated degradation of aggrecan by aggrecanase(s). Degradation products were identified by Western-blot analysis with antibodies to cleavage-site neoepitopes and to peptides within the globular domains. Rat chondrosarcoma tumour contained full-length aggrecan and all of the individual peptides expected from single independent cleavages at each of the four aggrecanase sites in the chondroitin sulphate (CS) domain. Kinetic analysis of the products present in rat chondrosarcoma cell cultures treated with interleukin-1b showed that the first aggrecanase-mediated cleavages occurred at the four sites within the CS attachment region to generate two stable intermediates, Val(1)-Glu(1459) and Val(1)-Glu(1274). These species were subsequently cleaved at the Glu(373) site in the interglobular domain to form the terminal products, Val(1)-Glu(373), Ala(374)-Glu(1274) and Ala(374)-Glu(1459). It therefore appears that the aggrecanase-mediated processing of native aggrecan by chondrocytes in situ is initiated within the CS-attachment region and completed by cleavage within the interglobular domain. Since it has been shown that digestion of aggrecan monomer in solution with recombinant ADAMTS-4 [Tortorella, Pratta, Liu, Austin, Ross, Abbaszade, Burn and Arner (2000) Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4). J. Biol. Chem. 275, 18566-18573] exhibits similar kinetics, it appears that preferential proteinase cleavage in the CS-rich region is determined by properties inherent in the aggrecan monomer itself, such as preferred peptide sequences for enzyme binding or enhanced accessibility to the core protein at these sites.

Recombinant human aggrecan G1-G2 exhibits native binding properties and substrate specificity for matrix metalloproteinases and aggrecanase.

A recombinant human aggrecan G1-G2 fragment comprising amino acids Val(1)-Arg(656) has been expressed in Sf21 cells using a baculovirus expression system. The recombinant G1-G2 (rG1-G2) was purified to homogeneity by hyaluronan-Sepharose affinity chromatography followed by high performance liquid chromatography gel filtration, and gave a single band of M(r) 90,000-95,000 by silver stain or immunoblotting with monoclonal antibody 1-C-6. The expressed G1-G2 bound to both hyaluronan and link protein indicating that the immunoglobulin-fold motif and proteoglycan tandem repeat loops of the G1 domain were correctly folded. Further analysis of secondary structure by rotary shadowing electron microscopy confirmed a double globe appearance, but revealed that the rG1-G2 was more compact than its native counterpart. The size of rG1-G2 by SDS-polyacrylamide gel electorphoresis was unchanged following digestion with keratanase and keratanase II and reduced by only 2-5 kDa following digestion with either O-glycosidase or N-glycosidase F. Recombinant G1-G2 was digested with purified matrix metalloproteinases (MMP), isolated aggrecanase, purified atrolysin C, or proteinases present in conditioned medium from cartilage explant cultures, and the products analyzed on SDS gels by silver stain and immunoblotting. Neoepitope antibodies recognizing the N-terminal F(342)FGVG or C-terminal DIPEN(341) sequences were used to confirm MMP cleavage at the Asn(341) downward arrow Phe bond, while neoepitope antibodies recognizing the N-terminal A(374)RGSV or C-terminal ITEGE(373) sequences were used to confirm aggrecanase cleavage at the Glu(373) downward arrow Ala bond. Cleavage at the authentic MMP and aggrecanase sites revealed that these proteinases have the same specificity for rG1-G2 as for native aggrecan. Incubation of rG1-G2 with conditioned medium from porcine cartilage cultures revealed that active soluble aggrecanase but no active MMPs, was released following stimulation with interleukin-1alpha or retinoic acid. Atrolysin C, which cleaves native bovine aggrecan at both the aggrecanase and MMP sites, efficiently cleaved rG1-G2 at the aggrecanase site but failed to cleave at the MMP site. In contrast, native glycosylated G1-G2 with or without keratanase treatment was cleaved by atrolysin C at both the aggrecanase and MMP sites. The results suggest that the presence or absence per se of keratan sulfate on native G1-G2 does not affect the activity of atrolysin C toward the two sites.

An association between an aggrecan polymorphic allele and bilateral hand osteoarthritis in elderly white men: data from the Baltimore Longitudinal Study of Aging (BLSA).

OBJECTIVE: The aggrecan proteoglycan is a major component of articular cartilage and supports the biomechanical function of this tissue. A variable number tandem repeat (VNTR) polymorphism has been discovered recently in a region of the human aggrecan gene that codes for the chondroitin sulfate attachment sites. We examined whether alleles of this polymorphism displayed a non-random association with bilateral hand or knee osteoarthritis (OA) in men from the Baltimore Longitudinal Study of Aging (BLSA). DESIGN: DNA was obtained from 93 Caucasian men, aged 60 and above, who had bilateral hand and standing knee radiographs read for changes of OA. The DNA was analyzed by polymerase chain reaction (PCR) and/or Southern blotting for the presence of the VNTR alleles. RESULTS: Bilateral hand OA and knee OA were present in 46 and 30% of the men respectively. The following distribution of alleles was observed: allele 33 (0.5%), 29 (2.2%), 28 (31.7%), 27 (43.0%), 26 (16.7%), 25 (3.2%), 22 (2.2%) and 19 (0.5%). This distribution was similar to that detected in a random population of individuals from a separate study. In multiple logistic regression analysis, adjusting for age and body mass index, the presence of allele 27 was associated with bilateral hand OA with an odds ratio (OR) = 3.23 (95% confidence intervals (CI): 1.24-8.41). No other alleles showed an association with bilateral hand OA and the association between allele 27 and bilateral knee OA was not statistically significant (OR = 1.14; 95% CI: 0.45-2.88). CONCLUSIONS: These data demonstrate the first association between a human aggrecan gene polymorphic allele and hand OA. This finding supports the concept that genetic factors may play a role in the development and/or progression of some forms of age-onset OA.

Non-glycosaminoglycan bearing domains of perlecan and aggrecan influence the utilization of sites for heparan and chondroitin sulfate synthesis.

Perlecan and aggrecan are proteoglycans that receive primarily heparan sulfate and chondroitin sulfate side chains, respectively. Their large multidomained core proteins have little or no homology to each other and their glycosaminoglycan (GAG) attachment sites are restricted to certain domains only. We examined the involvement of the non-GAG bearing domains in designating the GAG type added to the GAG attachment domain by preparing cDNA constructs that expressed perlecan/aggrecan chimeras as recombinant products in COS-7 cells and then determining the size and GAG composition of the recombinant products. The results showed that domain I of perlecan receives primarily (73-81%) heparan sulfate when coupled with domain II and III of perlecan, but when coupled with the G3 domain of aggrecan, it receives primarily (59-63%) chondroitin sulfate. Furthermore, the chondroitin sulfate attachment region of aggrecan received GAG side chains more readily when coupled to the G3 domain of aggrecan than when coupled to domains II and III of perlecan. The GAG side chains on all these recombinant products were small and similar in size. These findings indicate that the utilization of attachment sites for heparan and chondroitin sulfate or the sulfation of these GAGs can be influenced, in part, by non-GAG bearing domains.

A human-specific polymorphism in the coding region of the aggrecan gene. Variable number of tandem repeats produce a range of core protein sizes in the general population.

Aggrecan, one of the major structural genes of cartilage, encodes a proteoglycan core protein composed of an extended central glycosaminoglycan-bearing domain, flanked by globular domains at each end. The central region consists of long stretches of repeating amino acids that serve as attachment sites for glycosaminoglycans such as chondroitin and keratan sulfate; the terminal globular domains interact with other cartilage components. The glycosaminoglycan attachment region is encoded in several species by a single large exon, within which are several different types of repeating sequences. Several species show within this exon a similar block of conserved repeats for attachment of chondroitin sulfate, but in humans this group of repeats is particularly well conserved. Examination of genomic DNA from a population of unrelated individuals by polymerase chain reaction or Southern blot assays shows this block of repeat sequences exists in multiple allelic forms, which differ by the number of repeats at this site in each allele. Thirteen different alleles have been identified, with repeat numbers ranging from 13 to 33. This is an unusual example of an expressed variable number of tandem repeat polymorphism. This polymorphism is apparently restricted to humans, of several species examined. This polymorphism results in individuals with differing length aggrecan core proteins, bearing different numbers of potential attachment sites for chondroitin sulfate. The possibility exists for a molecular understanding of biological variation in cartilage functional properties.

Mouse aggrecan, a large cartilage proteoglycan: protein sequence, gene structure and promoter sequence.

Seven genomic clones for mouse aggrecan core protein have been isolated including 3 kb of 5'- and 7 kb of 3'-flanking sequences. All exon sequences and their intron boundary sequences in these clones were identified and mapped by DNA sequencing. The gene spans at least 61 kb and contains 18 exons. Exon 1 encodes 5'-untranslated sequence and exon 2 contains a translation start codon, methionine. The coding sequence is 6545 bp for a 2132-amino-acid protein with calculated M(r) = 259,131 including an 18-amino-acid signal peptide. There is a strong correlation between structural domains and exons. Notably, the chondroitin sulphate domain consisting of 1161 amino acids is encoded by a single exon of 3.6 kb. Although link protein has similar structural domains and subdomains, the sequence identity and the organization of exons encoding the subdomains B and B' of G1 and G2 domains revealed a strong similarity of mouse aggrecan to both human versican and rat neurocan. Primer extension analysis identified four transcription start sites which are close together. The promoter sequence showed high G/C content (65%) and contained several consensus binding motifs for transcription factors including Sp-1 and the glucocorticoid receptor. There are stretches of sequences similar to the promoter region of both the type-II collagen and link protein genes. These sequences may be important for cartilage gene expression.

Alternative exon splicing within the amino-terminal nontriple-helical domain of the rat pro-alpha 1(XI) collagen chain generates multiple forms of the mRNA transcript which exhibit tissue-dependent variation.

Type XI collagen is an integral, although minor component of cartilage collagen fibrils. We have established that alternative exon usage is a mechanism for increasing structural diversity within the amino-terminal nontriple helical domain of the pro-alpha 1(XI) collagen gene. cDNA clones spanning the amino-terminal domain were selected from a rat chondrosarcoma library, and were shown to contain two major sequence differences from the previously reported human sequence. The first difference was the replacement of sequence encoding an acidic domain of 39 amino acids in length by a sequence encoding a 51-amino acid basic domain with a predicted pI of 11.9. The second difference was the absence of a sequence that would translate into a highly acidic 85-amino acid sequence downstream from the first variation. These two changes, expressed together, result in the replacement of most of the acidic domain with one that is smaller and basic. These two sequence differences serve to identify subdomains of a variable region, designated V1 and V2, respectively. V1a is defined as the acidic 39-amino acid sequence element and V1b is defined as the 51-amino acid basic sequence. Analysis of genomic DNA revealed that both V1a and V1b are encoded by separate adjacent exons in the rat genome and V2 is also encoded in a single exon downstream. Analysis of mRNA from cartilage-derived sources revealed a complex pattern of alpha 1(XI) transcript expression due to differential exon usage. In non-cartilage sources, the pattern is less complex; the most prevalent form is the one containing the two acidic sequences, V1a and V2.

The structure of the rat aggrecan gene and preliminary characterization of its promoter.

Aggrecan is a major structural component of cartilage extracellular matrix and a specific gene product of differentiated chondrocytes. cDNA clones have been used to isolate rat aggrecan genomic clones from phage and cosmid libraries, producing over 80 kilobases (kb) of overlapping DNA containing the complete rat aggrecan gene, including 12 kb of 5'- and 8 kb of 3'-flanking DNA. DNA sequencing shows 18 exons, most of which encode structural or functional modules; exceptions are domains G1-B and G2-B, which are split into two exons and the G3 lectin domain, which is encoded by three exons. There is one expressed epidermal growth factor-like exon and in addition a non-expressed "pseudo-exon" encoding a heavily mutated epidermal growth factor-like domain. Intron sizes have been determined by restriction mapping and inter-exon polymerase chain reaction; a 30-kb intron separates exons 1 and 2. Exon 1 has been mapped by primer extension and S1 nuclease protection; it encodes 381 base pairs (bp) of 5'-untranslated sequence. There is a minor promoter which initiates transcription an additional 68 bp 5' of the major promoter start site. DNA sequence is reported for a 529-bp fragment encompassing exon 1, including 120 bp of 5'-flanking DNA comprising the promoter. This promoter is lacking the TATAA or CCAAT elements but has several putative binding sites for transcription factors. A 922-bp DNA fragment with 640-bp 5'-flanking DNA and 282-bp exon 1 sequence showed higher promoter activity in transfected chondrocytes than in fibroblasts, is completely inactive in the reverse orientation, and is strongly enhanceable in the forward direction by the SV40 enhancer.

Characterization of type II and type XI collagen synthesis by an immortalized rat chondrocyte cell line (IRC) having a low level of type II collagen mRNA expression.

The biosynthesis of type XI and type II collagens was examined using a stable rat chondrocyte cell line established by W. E. Horton et al. (1988, Exp. Cell Res. 178, 457-468.). These cells (IRC; immortalized rat chondrocytes) were created by transformation with a murine retrovirus carrying the v-myc and v-raf oncogenes. They grow in suspension culture as multicellular aggregates and synthesize typical cartilage proteins, aggrecan and link protein. Type II collagen is absent or synthesized at severely reduced levels, as shown by Northern analysis of mRNA. Thus, this cell type represents a unique model in which to study cartilage matrix protein interactions in the absence of type II collagen. A more detailed look at the proteins secreted into the medium by metabolically labeled IRC cells revealed the presence of collagenase-sensitive bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands were identified as the alpha 1, alpha 2, and alpha 3 chains of heterotrimeric type XI collagen by electrophoretic migration after pepsin digestion, by CNBr peptide mapping, and by immunoprecipitation with antibodies to rat alpha 1(XI). mRNA for all three chains was detected by Northern blot analysis. The data indicate that the low level of alpha 1(II) mRNA previously detected in these cells is translated into pro alpha 3(XI) polypeptide chains which are incorporated into molecules of type XI. Under normal culture conditions, homotrimers of type II collagen were not detected. The carboxyl propeptide domain of the fibrillar collagens directs chain selection and molecular assembly of the trimeric molecules. The sequence of the carboxyl propeptide domain from pro alpha 3(XI) of IRC cells was found to be identical to this domain from pro alpha 1(II) of swarm rat chondrosarcoma, supporting previous evidence that pro alpha 3(XI) and pro alpha 1(II) have the same primary structure. When cultured in the presence of 50 mM arginine, IRC cells could be induced to synthesize pro alpha 1(II) chains in excess over pro alpha 1(XI) and pro alpha 2(XI). Only under these conditions were type II collagen molecules detected, suggesting a preferential association of pro alpha 1(II) with the pro alpha 1 and/or pro alpha 2 chains of type XI collagen.

Complete coding sequence, deduced primary structure, chromosomal localization, and structural analysis of murine aggrecan.

We have isolated and sequenced overlapping cDNA clones encoding the entire core protein of aggrecan (the large aggregating chondroitin sulfate/keratan sulfate proteoglycan of cartilage) from three chondrocyte cDNA libraries of BALB/c mice and localized the aggrecan gene in mouse chromosome 7. We determined 7386 bp of the cDNA sequence, including 132 and 854 nucleotides of 5' and 3' untranslated regions, respectively. The core protein precursor is 2132 amino acids long (M(r) 222,008), including a 19-residue secretory signal peptide. The overall amino acid sequence of the mouse aggrecan shows 91.6% identity to rat and 72.5% to human aggrecan. Comparison of the amino acid sequences of various domains and subdomain structures of mouse aggrecan to known sequences of other species and related proteins (versican, neurocan, link protein, and lymphocyte homing receptor CD44) revealed high levels of identity of the G1, G2, and G3 globular domains and relatively less conserved structures in the interglobular and glycosaminoglycan-attachment regions. Epidermal growth factor (EGF)-like module was detected in only a minor fraction of aggrecan clones, while the complement regulatory protein (CRP)-like domain was regularly expressed in all samples.

Alternate exon usage is a commonly used mechanism for increasing coding diversity within genes coding for extracellular matrix proteins.

Extracellular matrix proteins are a diverse family of secreted proteins and glycoproteins that are responsible for a variety of critical functions in different tissues. A large number of multiexon genes encode these proteins of the extracellular matrix. Over the last few years, it has become evident that the processing of the pre-mRNA from several of these genes involves alternative splicing. This review summarizes the known examples of alternative splicing in genes coding for the extracellular matrix and attempts to relate the increase in coding diversity generated by alternate exon usage to the function(s) of individual extracellular matrix proteins.

Expression and structure of cartilage proteins.

Cartilage has unique physical characteristics attributable to the presence of an unusually high content of proteoglycan embedded in the network of collagen fibrils. Advances in understanding the structure of these components and how their synthesis is regulated have been greatly assisted by the application of molecular biology. For example, an immortalized rat chondrocyte cell line was obtained by infection with a recombinant retrovirus encoding the myc gene product. Several positive and negative DNA regulatory elements of the collagen II gene have been identified that appear to be important in the regulation of this gene in chondrocytes. The complete primary structure of the cartilage proteoglycan (aggrecan) core protein deduced from cDNA sequence displays a complex multidomain structure including numerous repeats of Ser-Gly sequences and sequence homologies with link protein and animal lectins. Such studies advance our understanding of normal morphogenetic events and lay the groundwork for determining the basis of molecular and genetic defects.

Characterization of the promoter for the rat and human link protein gene.

We have isolated the 5'-end of the gene for the rat and human link protein by screening genomic libraries with oligonucleotides corresponding to the 5'-cDNA sequence. Several overlapping clones were isolated for the human link protein gene, while only one clone was obtained for the rat. All the clones contained a single exon of which the sequence was identical to the most 5'-end of the rat and human cDNAs. Transcription initiation sites for the rat link gene were identified by primer extension and S1 protection analysis using total RNA from the rat Swarm chondrosarcoma. Transcriptional initiation sites for the human link gene were determined by specific primer extension of RNA from human fetal cartilage. Comparison of 1500 bp of 5'-flanking sequence between the rat and human link protein genes showed strong sequence conservation near the start site of transcription with 80% overall identity. Analysis of the 5'-flanking regions also revealed a large inverted repeat consisting of repeating purine-pyrimidine, which has the potential to form left-handed Z-DNA. Transcriptional regulation of the link protein gene was studied by coupling either 7.0 kb or 0.85 kb of 5'-flanking rat DNA to the chloramphenicol acetyltransferase (CAT) gene followed by transfection into chick embryonic chondrocytes (CEC) and HeLa cells. Both constructs had considerable CAT activity in CEC cells and less activity in HeLa cells. Furthermore, inclusion of a DNA fragment from the first intron increased relative CAT activity in both of these cell types. The increased activity from the first intron was shown to be orientation independent in CEC. These results indicate the presence of positive cisacting regulatory elements in both the promoter and first intron of the rat gene for link protein.

Analysis of the chondroitin sulfate proteoglycan core protein (CSPGCP) gene in achondroplasia and pseudoachondroplasia.

Achondroplasia and pseudoachondroplasia are autosomal dominant skeletal dysplasias resulting in short-limbed dwarfism. Histologic and ultrastructural studies of the cartilage in pseudoachondroplasia and in homozygous achondroplasia have suggested a structural abnormality in chondroitin sulfate proteoglycan (CSPG), a major structural protein in the extra-cellular matrix. The gene encoding CSPG core protein (CSPGCP) is thus a logical "candidate gene" for analysis in these conditions. cDNA probes encoding CSPGCP were used to identify restriction fragment length polymorphisms (RFLPs) in DNA from a panel of control individuals. No gross alterations at the CSPGCP locus were noted in DNA from 37 individuals with achondroplasia and 5 individuals with pseudoachondroplasia. In addition, allelic frequencies of the RFLPs were not significantly different among controls and patients with either condition. In one three-generation family with achondroplasia, close linkage of the CSPGCP locus and the skeletal dysplasia was excluded using a Bgl II polymorphism. Similarly, in a three-generation family with pseudoachondroplasia, the CSPGCP gene was not tightly linked to the disease phenotype. These results indicate that mutations at the chondroitin sulfate proteoglycan core protein locus do not cause achondroplasia or pseudoachondroplasia in these families.

Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. Human-specific repeats, and additional alternatively spliced forms.

We have obtained the complete coding sequence of the large aggregating chondroitin sulfate proteoglycan of human cartilage (aggrecan) from a combination of cDNA and genomic exon sequencing. We screened a human costal chondrocyte cDNA library, using rat aggrecan cDNA probes, and obtained three nonoverlapping clones totaling 6.2 kilobases in length. These clones were sequenced, and the sequence of the gaps between clones was obtained from genomic exon fragments and polymerase chain reaction-amplified cDNA. The composite sequence is 7137 nucleotides long, encoding 2316 amino acids. The human and rat aggrecan amino acid sequences are about 75% identical, with domains ranging from 100% to about 60% of conserved amino acids. The human sequence contains two regions of highly conserved repeats not found in rat aggrecan: 11 repeats of a hexameric sequence in the keratan sulfate attachment domain, E-E-P-(S,F)-P-S; and a 19-amino acid sequence reiterated 19 times, in the CS-1 portion of the serine-glycine-containing region. There are at least three forms of aggrecan transcripts, generated by alternative exon usage, and the form reported here is the shortest and also the most prevalent, lacking both the epidermal growth factor-like domain, and the complement regulatory protein-like sequence.