Structure of the chicken link protein gene: exons correlate with the protein domains.

The structure of the chicken link protein gene has been determined from a series of genomic clones that cover the entire coding region as well as the complete 3'-untranslated region and a small portion of the 5'-untranslated region. The gene is greater than 80 kilobase pairs long and is present in a single copy in the chicken genome. The link protein gene contains at least five exons with four encoding the entire protein. The domain of link protein that has homologies with immunoglobulin-like proteins and the tandemly repeated hyaluronic acid binding domains are each encoded by separate exons. The exon-intron structure indicates that the link protein gene may have arisen by exon duplication and exon shuffling.

Structural features of cartilage matrix protein deduced from cDNA.

cDNAs encoding the Mr 54,000 chicken cartilage matrix protein (CMP) were selected from a cartilage cDNA expression library by immunological means. Antibodies elicited against insert-encoded protein purified from one of the clones reacted specifically with chicken CMP in immunoblots of total cartilage extract, providing positive identification of the cDNA clones isolated. The cDNAs detect a 3.4-kilobase transcript that was present in sternal cartilage and in cartilaginous but not in precartilaginous embryonic limb tissues. The cDNAs code for 416 amino acids of the chicken CMP, including its COOH terminus. There are two striking features in the deduced CMP amino acid sequence: first, it contains a region with significant homologies to repeat sequences in the precursor for epidermal growth factor; and second, it is made up of two large homologous repeat sequences. These results provide the first detailed structural information on the CMP and establish it as a developmentally regulated marker of cartilage differentiation.

Complete amino acid sequence of chicken cartilage link protein deduced from cDNA clones.

cDNA clones coding for chicken cartilage link protein were isolated and sequenced. The DNA sequence for the entire core polypeptide of the mature link protein and the predicted signal peptide consists of 1065 nucleotides. The deduced primary translation product (355 amino acids) has a molecular mass of 40.7 kDa; the calculated molecular mass of the mature link protein core polypeptide (340 amino acids) is 39.06 kDa. The DNA sequence contains two tandemly arranged repeat sequences that may code for repeated functional domains of link protein involved in binding to hyaluronic acid. The mRNAs for chicken link protein are 6.0, 5.8, and 3.0 kilobase pairs, and the difference between the sizes of the RNA species lies in the 3' untranslated region.

Primary structure of the telopeptide and a portion of the helical domain of chicken type II procollagen as determined by DNA sequence analysis.

A comparison of the nucleotide sequences of three new cDNA clones for chicken type II procollagen with the sequences of the other three types of chicken fibrillar procollagens reveals that the most conserved regions correlate with the positions of hydroxyproline, hydroxylysine, cysteine and lysine residues. On the basis of replacement-site-divergence calculations it is concluded that alpha 1(II) and alpha 1(I) procollagens diverged later than alpha 1(I) and alpha 2(I) procollagens.

Preparation and characterization of an antiserum against purified proteoglycan link proteins from avian cartilage.

Link proteins have been purified from avian xyphoid process. Cartilage was extracted in 4.0 M guanidine hydrochloride and a link fraction (A1D5) was obtained by sequential cesium chloride centrifugation. Link proteins were separated from low buoyant density proteoglycans by chromatography on Sephacryl S-200 and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The presence of contaminating proteoglycans at various purification steps was monitored in an enzyme-linked immunosorbent assay using antiserum against avian cartilage proteoglycan monomer (anti A1D1-1400 Vo). Antiserum generated against this purified link preparation (anti link[SDS]) was characterized for its ability to bind link proteins, proteoglycan monomer, and aggregate. The serum was specific only for link proteins when tested by an enzyme-linked immunosorbent assay. Some reactivity against proteoglycan monomer was observed in a Farr-type assay.

Physical and immunochemical characterization of proteoglycans synthesized during chondrogenesis in the chick embryo.

Chick limb buds from 4- to 7-day-old embryos and sterna from 14-day-old embryos were labeled with [35S]sulfate, and the sulfated proteoglycans (PGS) were extracted either with associative (0.5 M guanidine . HCl) or dissociative (4.0 M guanidine . HCl) solvents. The existence of several populations of PGS is revealed in each age group when the limb extracts are analyzed on sucrose density gradients under dissociative conditions. The major component of 7-day limbs has a faster sedimentation rate than does that of 4-day limb buds. These major components of 4- and 7-day extracts chromatograph in the void volume of a controlled-pore glass (CPG) 1400 column. They differ from each other immunologically. The CPG 1400 void volume material from 4-day limb mesenchyme (PGS(LM)-I) shows no cross-reactivity with antiserum against PGS from juvenile cartilage (A1-D1-1400 V0). In contrast, the CPG 1400 void volume material from 7-day limbs, which contain cartilage [PGS(LC)-I], gave a cross-reaction of 70%. When PGS(LM)-I is chromatographed on CPG 2500, 45% of the labeled material chromatographs in the void volume. When PGS(LM)-I is sedimented in a cesium chloride gradient under dissociative conditions, it can be shown that the PGS in the bottom fraction (D1) has a monomer-aggregate relationship. In the absence of hyaluronic acid, this fraction is included on CPG 2500, whereas in the presence of hyaluronic acid it is excluded. Limb mesenchyme, therefore, synthesizes a PGS molecule which can interact with hyaluronic acid to form an aggregate. The endogenous material of a 4-day limb mesenchyme which causes the aggregation of PGS cannot be separated from PGS by chromatography on CPG 240 or 1400 under dissociative conditions. In contrast, the aggregating material from sterna can be separated from PGS under these conditions. These observations are interpreted to mean that the aggregating material of limb mesenchyme is larger than is that of cartilage.

Quantitative determination of the human immune response to immunization with meningococcal vaccines.

Radioactive antigen binding tests have been developed to measure quantitatively the antibody response of 167 adults, 84 children, and 51 infants to several different preparations of group A and group C meningococcal polysaccharides. Almost all the adults injected responded and the geometric mean responses were approximately 15 mug/ml of antibody protein in individuals vaccinated subcutaneously with two preparations of group A vaccine. The geometric mean antibody concentration after immunization with two preparations of group C vaccine was approximately 35 mug/ml. Most children aged 7 yr responded to immunization with two group A vaccines, and their mean response was only slightly less than that seen in adults. There was no difference between the subcutaneous and the intradermal route if both were given with jet gun. The majority of infants aged 6-13 months responded to a preparation of group A vaccine and the geometric mean titer was approximately 1.2 mug/ml. Adults, children, and infants responded significantly less to a preparation of group A polysaccharide which was of low molceular weight.