A B S T R A C T 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 /Ag/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 Ag/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 ug/ml. Adults, children, and infants responded significantly less to a preparation of group A polysaccharide which was of low molceular weight. INTRODUCTIONCurrently there are under development two meningococcal vaccines based upon the use of high molecular weight group-specific polysaccharides. Immunization of military recruits with the group C polysaccharide has been shown to be effective in preventing meningococcal disease (1), and in lowering the rate of acquisition of the nasopharyngeal carrier stage by group C meningococci (2). Such information has not yet been obtained for the group A vaccine, in part because these strains are exceedingly rare in the U. S. military population. The efficacy of this vaccine will probably need to be tested in the African "Meningitis belt" where disease is caused primarily by group A organisms (3).Received for publication 12 July 1971.Administration of either the group A or the group C vaccine to adults induces the formation of antibodies belonging to the three major immunoglobulin classes which have both bactericidal (4) and opsonic properties (5). To date quantitative data is available on the response of children.' There is no published data on the immune response of infants to these vaccines. Inasmuch as this age group stands to benefit most from meningococcal immunoprophylaxis (6), quantitative data of their serological response is vital.The present report will describe the immune response of adults, children, and infants to immunization with several preparations of meningococcal polysaccharides measured by means of quantitative radioactive antigen binding tests (7). METHODSAntigens. Group A meningococcal vaccines lots A-5, A-7, V-1, and group C meningococcal vaccines lots C-6 and C-7 were prepared as described by Gotschlich, Liu, and Artenstein (8). The group A polysaccharide contained in lots V-4 and V-5 was prepared by a modified procedure, employing cold phenol extraction to remove protein contamination.2 A-5 and C-6 were prepared at the Walter Reed Army Institute of Research; lots A-7 and C-7 by E. R. Squ...
ABSTRACTcDNAs 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.The importance of the extracellular matrix in developmental processes has been established from studies on morphogenesis (1, 2), tissue interactions (3, 4), and cell migration (5). The specific mode of action of the extracellular matrix components is now known to be mediated through specialized molecular domains that permit these macromolecules to interact with other matrix components or with the cell surface. In cartilage, such macromolecular interactions have been documented for the interaction between proteoglycan monomer and link protein, and the interaction between these two molecules and hyaluronic acid in the formation of link protein stabilized proteoglycan aggregates (6). To be structurally integrated in the cartilage matrix, these proteoglycan aggregates must interact, directly or indirectly and in an as yet unknown manner, with other cartilage matrix macromolecules. Some of these are: type II collagen (7), several minor collagenous proteins (8), a number of low molecular weight (8-10) and low buoyant density proteoglycans (11), chondronectin (12), and cartilage matrix protein (13). The highly structured extracellular matrix that results from these macromolecular interactions is important for the growth and morphogenesis of cartilaginous rudiments.Recent reports on the protein chemistry (14) or the molecular cloning (15, 16) of link protein have provided structural information that suggest a molecular basis for the interaction of link protein with hyaluronic acid. As the structural details of the various components of the cartilaginous matrix become known, it will be possible to elucidate the molecular basis of their interactions and to begin to understand their role in growth and morphogenesis.Cartilage matrix protein (CMP) is an abundant noncollagenous extracellular matrix protein of which relatively little is known. The protein was first identified in bovine tracheal cartilage (13). The molecular weight of the intact bovine protein is 148,000; upon reduction, the protein yields ...
ABSTRACTcDNA 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.Proteoglycan monomers of bovine (1-3) and chicken (4) hyaline cartilage and the Swarm rat chondrosarcoma (5) can interact with hyaluronic acid to form macromolecular aggregates. This interaction is stabilized by one or more link proteins (6)(7)(8)(9)(10). Isolated link protein affects proteoglycan aggregate structure (11) and it can bind to either hyaluronic acid (12) or proteoglycan monomer (13).Link proteins vary in size (14-18), in part as a result of differences in glycosylation (14,18). Partial amino acid sequences from Swarm rat chondrosarcoma link protein are homologous to those of bovine link protein (19,20). Whereas no amino acid sequence has been published for chicken cartilage link protein, it has been reported to be closely related to bovine link protein (21). Here we provide the complete amino acid sequence of chicken link protein deduced from cDNA sequences. MATERIALS AND METHODSIsolation of cDNA Clones. The isolation of poly(A)+ RNA from 14-day-old chicken embryo sternal cartilage, synthesis, and cloning of cDNA has been described (22). Two cDNA libraries constructed in either pUC8 or pUC9 vectors (at the Sal I-EcoRI sites) were screened with a synthetic oligonucleotide probe (GARGCNGARCARGCNAARGT) that was deduced from the amino acid sequence of link protein identical in bovine cartilage (19) and rat chondrosarcoma (20) link proteins (Glu-Ala-Glu-Gln-Ala-Lys-Val). The oligonucleotide was synthesized by the phosphoramidite method using a Microsyn (Systec, Minneapolis, MN) DNA synthesizer and was end-labeled by using [y-32P]ATP and T4 polynucleotide kinase. Filters were prehybridized at 37°C in 0.9 M NaCl/90 mM sodium citrate, pH 7/0.1% NaDod-S04/0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin/0.05% sodium pyrophosphate/10% dextran sulfate. After 6 hr, 2 x 106 cpm (20 ng) of end-labeled probe was added per 2 ml of prehybridization mixture per filter. After overnight hybridization the filters were washed four times (20 min per wash) at 370C and twice (30 min per wash) at 450C in 0.9 M NaCl/90 mM sodium citrate, pH 7/0.05% sodium pyrophosphate.DNA Sequencing. The nucleotide sequence was determined by using the method of Sanger et al. (23). Inserts of cDNA clones were subcloned in M13 phage vectors (24). The cDNA clones were linearized at...
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 >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. We have reported (3) the isolation of cDNA clones encoding the entire chicken cartilage link protein (LP) and deduced the amino acid sequence from the nucleotide sequence. A comparison between the complete amino acid sequence of rat LP (4) and chicken LP (3) shows a very high degree of homology. The analysis of the amino acid sequence of LPs reveals that the protein can be divided into several domains. The N-terminal half of LP has homology with the repeating units of the human free secretory component, the 'y chain of the T-cell receptor, and three types of immunoglobulin variable regions (5). The C-terminal half contains tandemly repeated sequences that may be the hyaluronic acid binding regions of LP (3,4,6).Here we report on the isolation of chicken LP genomic clones covering the entire protein coding region as well as the complete 3'-untranslated region of the mRNA. The structure of the gene is compared to the domain structure of the protein. MATERIALS AND METHODSConstruction and Screening of the Chicken Genomic Library. High molecular weight DNA, isolated from 9-day chicken embryos, was partially digested with Sau3AI and inserted into the BamHI site of XEMBL3 vector DNA as described by Frischauf et al. (7). Ligation mixtures were packaged in vitro, and 0.5-1 x 106 recombinant phages were amplified in Q359 host cells.The library was screened according to the method of Benton and Davis (8) using '2P-labeled LP cDNA probes (3). Duplicate filters were hybridized as described by Maniatis et al. (9). Phage DNA was isolated (10), and restriction maps were established based on the results of single and double digests of phage DNA. Restriction fragments were subcloned in pUC8/pUC9 (11) or in M13mp8/Ml3mp9 (12). These genomic fragments as well as different LP cDNA fragments were used as probes in Southern hybridizations (13) to confirm the restriction map.DNA fragments were labeled with 32P by random oligonucleotide priming (14). Hybridization and washing conditions of Southern blots of restriction digests of genomic DNA were carried out according to Jeffreys and Flavell (15).Electron Microscopy. Separated complementary DNA strands of genomic clones were isolated from agarose gels as described (16). Double heteroduplexes were prepared by incubating the complementary DNA strands of appropriate phages with 3-5 ,ug of chicken sternal total RNA in 80% (vol/vol) formamide and 0.1 M Hepes (pH 8.3), 0.4 M NaCl, 0.01 M EDTA (17) in a final volume of 25 ,ul at a DNA concentration of 5 uzg/ml. Incubation was done for 15 hr at 45°C. Excess RNA was removed by chromatography on a Sephacryl S-1000 column (2 x 45 mm...
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